The Eukaryotic Linear Motif resource for
Functional Sites in Proteins

Articles used during ELM annotation

Currently 2981 articles annotated:

Pellegrini,2016 (27889209) Pellegrini E, Palencia A, Braun L, Kapp U, Bougdour A, Belrhali H, Bowler MW, Hakimi MA "Structural Basis for the Subversion of MAP Kinase Signaling by an Intrinsically Disordered Parasite Secreted Agonist." Structure 2016 Nov 27
The causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38alpha (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38alpha has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway. An adapted kinase interaction motif (KIM) forms a highly stable complex that competes with cytoplasmic regulatory partners. In addition, the recombinant complex forms a powerful in vitro tool to evaluate the specificity and effectiveness of p38alpha inhibitors that have advanced to clinical trials, as it provides a hitherto unavailable stable and highly active form of p38alpha.
Davey,2016 (27716480) Davey NE, Morgan DO "Building a Regulatory Network with Short Linear Sequence Motifs: Lessons from the Degrons of the Anaphase-Promoting Complex." Mol Cell 2016 Oct 07
The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase that polyubiquitinates specific substrates at precise times in the cell cycle, thereby triggering the events of late mitosis in a strict order. The robust substrate specificity of the APC/C prevents the potentially deleterious degradation of non-APC/C substrates and also averts the cell-cycle errors and genomic instability that could result from mistimed degradation of APC/C targets. The APC/C recognizes short linear sequence motifs, or degrons, on its substrates. The specific and timely modification and degradation of APC/C substrates is likely to be modulated by variations in degron sequence and context. We discuss the extensive affinity, specificity, and selectivity determinants encoded in APC/C degrons, and we describe some of the extrinsic mechanisms that control APC/C-substrate recognition. As an archetype for protein motif-driven regulation of cell function, the APC/C-substrate interaction provides insights into the general properties of post-translational regulatory systems.
Hertz,2016 (27453045) Hertz EP, Kruse T, Davey NE, Lopez-Mendez B, Sigurethsson JO, Montoya G, Olsen JV, Nilsson J "A Conserved Motif Provides Binding Specificity to the PP2A-B56 Phosphatase." Mol Cell 2016 Aug 20
Dynamic protein phosphorylation is a fundamental mechanism regulating biological processes in all organisms. Protein phosphatase 2A (PP2A) is the main source of phosphatase activity in the cell, but the molecular details of substrate recognition are unknown. Here, we report that a conserved surface-exposed pocket on PP2A regulatory B56 subunits binds to a consensus sequence on interacting proteins, which we term the LxxIxE motif. The composition of the motif modulates the affinity for B56, which in turn determines the phosphorylation status of associated substrates. Phosphorylation of amino acid residues within the motif increases B56 binding, allowing integration of kinase and phosphatase activity. We identify conserved LxxIxE motifs in essential proteins throughout the eukaryotic domain of life and in human viruses, suggesting that the motifs are required for basic cellular function. Our study provides a molecular description of PP2A binding specificity with broad implications for understanding signaling in eukaryotes.
Rogers,2016 (27417119) Rogers S, McCloy R, Watkins DN, Burgess A "Mechanisms regulating phosphatase specificity and the removal of individual phosphorylation sites during mitotic exit." Bioessays 2016 Jul 15
Entry into mitosis is driven by the activity of kinases, which phosphorylate over 7000 proteins on multiple sites. For cells to exit mitosis and segregate their genome correctly, these phosphorylations must be removed in a specific temporal order. This raises a critical and important question: how are specific phosphorylation sites on an individual protein removed? Traditionally, the temporal order of dephosphorylation was attributed to decreasing kinase activity. However, recent evidence in human cells has identified unique patterns of dephosphorylation during mammalian mitotic exit that cannot be fully explained by the loss of kinase activity. This suggests that specificity is determined in part by phosphatases. In this review, we explore how the physicochemical properties of an individual phosphosite and its surrounding amino acids can affect interactions with a phosphatase. These positive and negative interactions in turn help determine the specific pattern of dephosphorylation required for correct mitotic exit.
Wang,2016 (27350047) Wang J, Wang Z, Yu T, Yang H, Virshup DM, Kops GJ, Lee SH, Zhou W, Li X, Xu W, Rao Z "Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization." Protein Cell 2016 Jul 04
Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.
Wei,2016 (27212118) Wei Y, Xu X "UFMylation: A Unique & Fashionable Modification for Life." Genomics Proteomics Bioinformatics 2016 Jun 04
Ubiquitin-fold modifier 1 (UFM1) is one of the newly-identified ubiquitin-like proteins. Similar to ubiquitin, UFM1 is conjugated to its target proteins by a three-step enzymatic reaction. The UFM1-activating enzyme, ubiquitin-like modifier-activating enzyme 5 (UBA5), serves as the E1 to activate UFM1; UFM1-conjugating enzyme 1 (UFC1) acts as the E2 to transfer the activated UFM1 to the active site of the E2; and the UFM1-specific ligase 1 (UFL1) acts as the E3 to recognize its substrate, transfer, and ligate the UFM1 from E2 to the substrate. This process is called ufmylation. UFM1 chains can be cleaved from its target proteins by UFM1-specific proteases (UfSPs), suggesting that the ufmylation modification is reversible. UFM1 cascade is conserved among nearly all of the eukaryotic organisms, but not in yeast, and associated with several cellular activities including the endoplasmic reticulum stress response and hematopoiesis. Furthermore, the UFM1 cascade is closely related to a series of human diseases. In this review, we summarize the molecular details of this reversible modification process, the recent progress of its functional studies, as well as its implication in tumorigenesis and potential therapeutic targets for cancer.
Sanchez-Barcelo,2016 (27121162) Sanchez-Barcelo EJ, Mediavilla MD, Vriend J, Reiter RJ "Constitutive photomorphogenesis protein 1 (COP1) and COP9 signalosome, evolutionarily conserved photomorphogenic proteins as possible targets of melatonin." J Pineal Res 2016 Jul 09
The ubiquitin proteasome system has been proposed as a possible mechanism involved in the multiple actions of melatonin. COP1 (constitutive photomorphogenesis protein 1), a RING finger-type ubiquitin E3 ligase formerly identified in Arabidopsis, is a central switch for the transition from plant growth underground in darkness (etiolation) to growth under light exposure (photomorphogenesis). In darkness, COP1 binds to photomorphogenic transcription factors driving its degradation via the 26S proteasome; blue light, detected by cryptochromes, and red and far-red light detected by phytochromes, negatively regulate COP1. Homologues of plant COP1 containing all the structural features present in Arabidopsis as well as E3 ubiquitin ligase activity have been identified in mice and humans. Substrates for mammalian (m) COP1 include p53, AP-1 and c-Jun, p27(Kip1), ETV1, MVP, 14-3-3sigma, C/EBPalpha, MTA1, PEA3, ACC, TORC2 and FOXO1. This mCOP1 target suggests functions related to tumorigenesis, gluconeogenesis, and lipid metabolism. The role of mCOP1 in tumorigenesis (either as a tumor promoter or tumor suppressor), as well as in glucose metabolism (inhibition of gluconeogenesis) and lipid metabolism (inhibition of fatty acid synthesis), has been previously demonstrated. COP1, along with numerous other ubiquitin ligases, is regulated by the COP9 signalosome; this protein complex is associated with the oxidative stress sensor Keap1 and the deubiquitinase USP15. The objective of this review was to provide new information on the possible role of COP1 and COP9 as melatonin targets. The hypothesis is based on common functional aspects of melatonin and COP1 and COP9, including their dependence on light, regulation of the metabolism, and their control of tumor growth.
Hesbacher,2016 (27121059) Hesbacher S, Pfitzer L, Wiedorfer K, Angermeyer S, Borst A, Haferkamp S, Scholz CJ, Wobser M, Schrama D, Houben R "RB1 is the crucial target of the Merkel cell polyomavirus Large T antigen in Merkel cell carcinoma cells." Oncotarget 2016 Apr 28
The pocket protein (PP) family consists of the three members RB1, p107 and p130 all possessing tumor suppressive properties. Indeed, the PPs jointly control the G1/S transition mainly by inhibiting E2F transcription factors. Notably, several viral oncoproteins are capable of binding and inhibiting PPs. Merkel cell polyomavirus (MCPyV) is considered as etiological factor for Merkel cell carcinoma (MCC) with expression of the viral Large T antigen (LT) harboring an intact PP binding domain being required for proliferation of most MCC cells. Therefore, we analyzed the interaction of MCPyV-LT with the PPs. Co-IP experiments indicate that MCPyV-LT binds potently only to RB1. Moreover, MCPyV-LT knockdown-induced growth arrest in MCC cells can be rescued by knockdown of RB1, but not by p107 or p130 knockdown. Accordingly, cell cycle arrest and E2F target gene repression mediated by the single PPs can only in the case of RB1 be significantly reverted by MCPyV-LT expression. Moreover, data from an MCC patient indicate that loss of RB1 rendered the MCPyV-positive MCC cells LT independent. Thus, our results suggest that RB1 is the dominant tumor suppressor PP in MCC, and that inactivation of RB1 by MCPyV-LT is largely sufficient for its growth supporting function in established MCPyV-positive MCC cells.
Senda,2016 (27116701) Senda Y, Murata-Kamiya N, Hatakeyama M "C-terminal Src kinase-mediated EPIYA phosphorylation of Pragmin creates a feed-forward C-terminal Src kinase activation loop that promotes cell motility." Cancer Sci 2016 Jul 16
Pragmin is one of the few mammalian proteins containing the Glu-Pro-Ile-Tyr-Ala (EPIYA) tyrosine-phosphorylation motif that was originally discovered in the Helicobacter pylori CagA oncoprotein. Following delivery into gastric epithelial cells by type IV secretion and subsequent tyrosine phosphorylation at the EPIYA motifs, CagA serves as an oncogenic scaffold/adaptor that promiscuously interacts with SH2 domain-containing mammalian proteins such as the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) and the C-terminal Src kinase (Csk), a negative regulator of Src family kinases. Like CagA, Pragmin also forms a physical complex with Csk. In the present study, we found that Pragmin directly binds to Csk by the tyrosine-phosphorylated EPIYA motif. The complex formation potentiates kinase activity of Csk, which in turn phosphorylates Pragmin on tyrosine-238 (Y238), Y343, and Y391. As Y391 of Pragmin comprises the EPIYA motif, Pragmin-Csk interaction creates a feed-forward regulatory loop of Csk activation. Together with the finding that Pragmin and Csk are colocalized to focal adhesions, these observations indicate that the Pragmin-Csk interaction, triggered by Pragmin EPIYA phosphorylation, robustly stimulates the kinase activity of Csk at focal adhesions, which direct cell-matrix adhesion that regulates cell morphology and cell motility. As a consequence, expression of Pragmin and/or Csk in epithelial cells induces an elongated cell shape with elevated cell scattering in a manner that is mutually dependent on Pragmin and Csk. Deregulation of the Pragmin-Csk axis may therefore induce aberrant cell migration that contributes to tumor invasion and metastasis.
Uljon,2016 (27041596) Uljon S, Xu X, Durzynska I, Stein S, Adelmant G, Marto JA, Pear WS, Blacklow SC "Structural Basis for Substrate Selectivity of the E3 Ligase COP1." Structure 2016 May 05
COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPalpha degradation. Here we report structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed beta propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the beta propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPalpha for degradation.
Habisov,2016 (26929408) Habisov S, Huber J, Ichimura Y, Akutsu M, Rogova N, Loehr F, McEwan DG, Johansen T, Dikic I, Doetsch V, Komatsu M, Rogov VV, Kirkin V "Structural and Functional Analysis of a Novel Interaction Motif within UFM1-activating Enzyme 5 (UBA5) Required for Binding to Ubiquitin-like Proteins and Ufmylation." J Biol Chem 2016 Apr 30
The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/gamma-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
Stevers,2016 (26888287) Stevers LM, Lam CV, Leysen SF, Meijer FA, van Scheppingen DS, de Vries RM, Carlile GW, Milroy LG, Thomas DY, Brunsveld L, Ottmann C "Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR." Proc Natl Acad Sci U S A 2016 Mar 02
Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics.
Li,2016 (26820724) Li Y, Burclaff J, Anderson JT "Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro." PLoS One 2016 Jan 29
RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.
Kilisch,2016 (26743085) Kilisch M, Lytovchenko O, Arakel EC, Bertinetti D, Schwappach B "A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1." J Cell Sci 2016 Feb 24
The transport of the K(+) channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic.
Maertens,2016 (26657642) Maertens GN "B'-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase." Nucleic Acids Res 2016 Jan 09
To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to delta-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B' family of the regulatory subunits. B'-PP2A and HTLV-1 IN display nuclear co-localization, and the B' subunit stimulates concerted strand transfer activity of delta-retroviral INs in vitro. The protein-protein interaction interface maps to a patch of highly conserved residues on B', which when mutated render B' incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity.
Fox,2016 (26612606) Fox MJ, Mosley AL "Rrp6: Integrated roles in nuclear RNA metabolism and transcription termination." Wiley Interdiscip Rev RNA 2016 Jan 15
The yeast RNA exosome is a eukaryotic ribonuclease complex essential for RNA processing, surveillance, and turnover. It is comprised of a barrel-shaped core and cap as well as a 3'-5' ribonuclease known as Dis3 that contains both endo- and exonuclease domains. A second exonuclease, Rrp6, is added in the nucleus. Dis3 and Rrp6 have both shared and distinct roles in RNA metabolism, and this review will focus primarily on Rrp6 and the roles of the RNA exosome in the nucleus. The functions of the nuclear exosome are modulated by cofactors and interacting partners specific to each type of substrate. Generally, the cofactor TRAMP (Trf4/5-Air2/1-Mtr4 polyadenylation) complex helps unwind unstable RNAs, RNAs requiring processing such as rRNAs, tRNAs, or snRNAs or improperly processed RNAs and direct it toward the exosome. In yeast, Rrp6 interacts with Nrd1, the cap-binding complex, and RNA polymerase II to aid in nascent RNA processing, termination, and polyA tail length regulation. Recent studies have shown that proper termination and processing of short, noncoding RNAs by Rrp6 is particularly important for transcription regulation across the genome and has important implications for regulation of diverse processes at the cellular level. Loss of proper Rrp6 and exosome activity may contribute to various pathologies such as autoimmune disease, neurological disorders, and cancer. WIREs RNA 2016, 7:91-104. doi: 10.1002/wrna.1317 For further resources related to this article, please visit the WIREs website.
Aouacheria,2015 (26541461) Aouacheria A, Combet C, Tompa P, Hardwick JM "Redefining the BH3 Death Domain as a 'Short Linear Motif'." Trends Biochem Sci 2015 Nov 25
B cell lymphoma-2 (BCL-2)-related proteins control programmed cell death through a complex network of protein-protein interactions mediated by BCL-2 homology 3 (BH3) domains. Given their roles as dynamic linchpins, the discovery of novel BH3-containing proteins has attracted considerable attention. However, without a clearly defined BH3 signature sequence the BCL-2 family has expanded to include a nebulous group of nonhomologous BH3-only proteins, now justified by an intriguing twist. We present evidence that BH3s from both ordered and disordered proteins represent a new class of short linear motifs (SLiMs) or molecular recognition features (MoRFs) and are diverse in their evolutionary histories. The implied corollaries are that BH3s have a broad phylogenetic distribution and could potentially bind to non-BCL-2-like structural domains with distinct functions.
Zeke,2015 (26538579) Zeke A, Bastys T, Alexa A, Garai A, Meszaros B, Kirsch K, Dosztanyi Z, Kalinina OV, Remenyi A "Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases." Mol Syst Biol 2015 Nov 05
Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.
Wang,2015 (26442585) Wang Y, Zheng Z, Zhang J, Kong R, Liu J, Zhang Y, Deng H, Du X, Ke Y "A Novel Retinoblastoma Protein (RB) E3 Ubiquitin Ligase (NRBE3) Promotes RB Degradation and Is Transcriptionally Regulated by E2F1 Transcription Factor." J Biol Chem 2015 Nov 21
Retinoblastoma protein (RB) plays critical roles in tumor suppression and is degraded through the proteasomal pathway. However, E3 ubiquitin ligases responsible for proteasome-mediated degradation of RB are largely unknown. Here we characterize a novel RB E3 ubiquitin ligase (NRBE3) that binds RB and promotes RB degradation. NRBE3 contains an LXCXE motif and bound RB in vitro. NRBE3 interacted with RB in cells when proteasome activity was inhibited. NRBE3 promoted RB ubiquitination and degradation via the ubiquitin-proteasome pathway. Importantly, purified NRBE3 ubiquitinated recombinant RB in vitro, and a U-box was identified as essential for its E3 activity. Surprisingly, NRBE3 was transcriptionally activated by E2F1/DP1. Consequently, NRBE3 affected the cell cycle by promoting G1/S transition. Moreover, NRBE3 was up-regulated in breast cancer tissues. Taken together, we identified NRBE3 as a novel ubiquitin E3 ligase for RB that might play a role as a potential oncoprotein in human cancers.
Kristensen,2015 (26410532) Kristensen O "Crystal structure of the G3BP2 NTF2-like domain in complex with a canonical FGDF motif peptide." Biochem Biophys Res Commun 2015 Oct 16
The crystal structure of the NTF2-like domain of the human Ras GTPase SH3 Binding Protein (G3BP), isoform 2, was determined at a resolution of 2.75 A in complex with a peptide containing a FGDF sequence motif. The overall structure of the protein is highly similar to the homodimeric N-terminal domains of the G3BP1 and Rasputin proteins. Recently, a subset of G3BP interacting proteins was recognized to share a common sequence motif, FGDF. The most studied binding partners, USP10 and viral nsP3, interfere with essential G3BP functions related to assembly of cellular stress granules. Reported molecular modeling suggested that FGDF-motif containing peptides bind in an extended conformation into a hydrophobic groove on the surface of the G3BP NTF2-like domain in a manner similar to the known binding of FxFG nucleoporin repeats. The results in this paper provide evidence for a different binding mode. The FGDF peptide binds and changes conformation of the protruding N-terminal residues by providing hydrophobic interactions to a symmetry related molecule that facilitated crystallization of the G3BP2 isoform.
Richards,2016 (26385761) Richards KF, Guastafierro A, Shuda M, Toptan T, Moore PS, Chang Y "Merkel cell polyomavirus T antigens promote cell proliferation and inflammatory cytokine gene expression." J Gen Virol 2016 Jun 02
Merkel cell polyomavirus (MCV) is clonally integrated in over 80 % of Merkel cell carcinomas and mediates tumour development through the expression of viral oncoproteins, the large T (LT) and small T antigens (sT). Viral integration is associated with signature mutations in the T-antigen locus that result in deletions of C-terminal replicative functions of the LT antigen. Despite these truncations, the LT LXCXE retinoblastoma (Rb) pocket protein family binding domain is retained, and the entire sT isoform is maintained intact. To investigate the ability of MCV oncoproteins to regulate host gene expression, we performed microarray analysis on cells stably expressing tumour-derived LT, tumour-derived LT along with sT, and tumour-derived LT with a mutated Rb interaction domain. Gene expression alterations in the presence of tumour-derived LT could be classified into three main groups: genes that are involved in the cell cycle (specifically the G1/S transition), genes involved in DNA replication and genes involved in cellular movement. The LXCXE mutant LT largely reversed gene expression alterations detected with the WT tumour-derived LT, while co-expression of sT did not significantly affect these patterns of gene expression. LXCXE-dependent upregulation of cyclin E and CDK2 correlated with increased proliferation in tumour-derived LT-expressing cells. Tumour-derived LT and tumour-derived LT plus sT increased expression of multiple cytokines and chemokines, which resulted in elevated levels of secreted IL-8. We concluded that, in human fibroblasts, the LXCXE motif of tumour-derived LT enhances cellular proliferation and upregulates cell cycle and immune signalling gene transcription.
Fros,2015 (26384002) Fros JJ, Geertsema C, Zouache K, Baggen J, Domeradzka N, van Leeuwen DM, Flipse J, Vlak JM, Failloux AB, Pijlman GP "Mosquito Rasputin interacts with chikungunya virus nsP3 and determines the infection rate in Aedes albopictus." Parasit Vectors 2015 Sep 19
BACKGROUND: Chikungunya virus (CHIKV) is an arthritogenic alphavirus (family Togaviridae), transmitted by Aedes species mosquitoes. CHIKV re-emerged in 2004 with multiple outbreaks worldwide and recently reached the Americas where it has infected over a million individuals in a rapidly expanding epidemic. While alphavirus replication is well understood in general, the specific function (s) of non-structural protein nsP3 remain elusive. CHIKV nsP3 modulates the mammalian stress response by preventing stress granule formation through sequestration of G3BP. In mosquitoes, nsP3 is a determinant of vector specificity, but its functional interaction with mosquito proteins is unclear. METHODS: In this research we studied the domains required for localization of CHIKV nsP3 in insect cells and demonstrated its molecular interaction with Rasputin (Rin), the mosquito homologue of G3BP. The biological involvement of Rin in CHIKV infection was investigated in live Ae. albopictus mosquitoes. RESULTS: In insect cells, nsP3 localized as cytoplasmic granules, which was dependent on the central domain and the C-terminal variable region but independent of the N-terminal macrodomain. Ae. albopictus Rin displayed a diffuse, cytoplasmic localization, but was effectively sequestered into nsP3-granules upon nsP3 co-expression. Site-directed mutagenesis showed that the Rin-nsP3 interaction involved the NTF2-like domain of Rin and two conserved TFGD repeats in the C-terminal variable domain of nsP3. Although in vitro silencing of Rin did not impact nsP3 localization or CHIKV replication in cell culture, Rin depletion in vivo significantly decreased the CHIKV infection rate and transmissibility in Ae.albopictus. CONCLUSIONS: We identified the nsP3 hypervariable C-terminal domain as a critical factor for granular localization and sequestration of mosquito Rin. Our study offers novel insight into a conserved virus-mosquito interaction at the molecular level, and reveals a strong proviral role for G3BP homologue Rin in live mosquitoes, making the nsP3-Rin interaction a putative target to interfere with the CHIKV transmission cycle.
Schrama,2016 (26383606) Schrama D, Hesbacher S, Angermeyer S, Schlosser A, Haferkamp S, Aue A, Adam C, Weber A, Schmidt M, Houben R "Serine 220 phosphorylation of the Merkel cell polyomavirus large T antigen crucially supports growth of Merkel cell carcinoma cells." Int J Cancer 2016 Jan 15
Merkel cell polyomavirus (MCPyV) is regarded as a major causal factor for Merkel cell carcinoma (MCC). Indeed, tumor cell growth of MCPyV-positive MCC cells is dependent on the expression of a truncated viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. Here we determined the phosphorylation pattern of a truncated MCPyV-LT characteristically for MCC by mass spectrometry revealing MCPyV-LT as multi-phospho-protein phosphorylated at several serine and threonine residues. Remarkably, disruption of most of these phosphorylation sites did not affect its ability to rescue knockdown of endogenous T antigens in MCC cells indicating that phosphorylation of the respective amino acids is not essential for the growth promoting function of MCPyV-LT. However, alteration of serine 220 to alanine completely abolished the ability of MCPyV-LT to support proliferation of MCC cells. Conversely, mimicking the phosphorylated state by mutation of serine 220 to glutamic acid resulted in a fully functional LT. Moreover, MCPyV-LT(S220A) demonstrated reduced binding to RB in co-immunoprecipitation experiments as well as weaker induction of RB target genes in MCC cells. In conclusion, we provide evidence that phosphorylation of serine 220 is required for efficient RB inactivation in MCC and may therefore be a potential target for future therapeutic approaches.
Bardwell,2015 (26370088) Bardwell AJ, Bardwell L "Two hydrophobic residues can determine the specificity of mitogen-activated protein kinase docking interactions." J Biol Chem 2015 Oct 31
MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.
Chen,2015 (26344567) Chen HC, Kanai M, Inoue-Yamauchi A, Tu HC, Huang Y, Ren D, Kim H, Takeda S, Reyna DE, Chan PM, Ganesan YT, Liao CP, Gavathiotis E, Hsieh JJ, Cheng EH "An interconnected hierarchical model of cell death regulation by the BCL-2 family." Nat Cell Biol 2015 Sep 30
Multidomain pro-apoptotic BAX and BAK, once activated, permeabilize mitochondria to trigger apoptosis, whereas anti-apoptotic BCL-2 members preserve mitochondrial integrity. The BH3-only molecules (BH3s) promote apoptosis by either activating BAX-BAK or inactivating anti-apoptotic members. Here, we present biochemical and genetic evidence that NOXA is a bona fide activator BH3. Using combinatorial gain-of-function and loss-of-function approaches in Bid(-/-)Bim(-/-)Puma(-/-)Noxa(-/-) and Bax(-/-)Bak(-/-) cells, we have constructed an interconnected hierarchical model that accommodates and explains how the intricate interplays between the BCL-2 members dictate cellular survival versus death. BID, BIM, PUMA and NOXA directly induce stepwise, bimodal activation of BAX-BAK. BCL-2, BCL-XL and MCL-1 inhibit both modes of BAX-BAK activation by sequestering activator BH3s and 'BH3-exposed' monomers of BAX-BAK, respectively. Furthermore, autoactivation of BAX and BAK can occur independently of activator BH3s through downregulation of BCL-2, BCL-XL and MCL-1. Our studies lay a foundation for targeting the BCL-2 family for treating diseases with dysregulated apoptosis.
Choi,2015 (26254224) Choi HH, Phan L, Chou PC, Su CH, Yeung SC, Chen JS, Lee MH "COP1 enhances ubiquitin-mediated degradation of p27Kip1 to promote cancer cell growth." Oncotarget 2015 Aug 26
p27 is a critical CDK inhibitor involved in cell cycle regulation, and its stability is critical for cell proliferation. Constitutive photomorphogenic 1 (COP1) is a RING-containing E3 ubiquitin ligase involved in regulating important target proteins for cell growth, but its biological activity in cell cycle progression is not well characterized. Here, we report that p27Kip1 levels are accumulated in G1 phase, with concurrent reduction of COP1 levels. Mechanistic studies show that COP1 directly interacts with p27 through a VP motif on p27 and functions as an E3 ligase of p27 to accelerate the ubiquitin-mediated degradation of p27. Also, COP1-p27 axis deregulation is involved in tumorigenesis. These findings define a new mechanism for posttranslational regulation of p27 and provide insight into the characteristics of COP1-overexpressing cancers.
Zhao,2015 (26183396) Zhao Y, Yang X "Regulation of sensitivity of tumor cells to antitubulin drugs by Cdk1-TAZ signalling." Oncotarget 2015 Sep 22
Antitubulin drugs are commonly used for the treatment of numerous cancers. However, either the intrinsic or acquired resistances of patients to these drugs result in the failure of the treatment and high mortality of cancers. Therefore, identifying genes or signalling pathways involved in antitubulin drug resistances is critical for future successful treatment of cancers.TAZ (Transcriptional coactivator with PDZ-binding motif), which is a core component of the Hippo pathway, is overexpressed in various cancers. We have recently shown that high levels of TAZ in cancer cells result in Taxol resistance through up-regulation of downstream targets Cyr61 and CTGF. However, how TAZ is regulated in response to Taxol is largely unknown. In this study, we found that Cdk1 (Cyclin-dependent kinase 1) directly phosphorylated TAZ on six novel sites independent of the Hippo pathway, which further resulted in TAZ degradation through proteasome system. Phosphorylation-mimicking TAZ mutant was unstable, and therefore abolished TAZ-induced antitubulin drug resistances. This study provides first evidence that Cdk1 is a novel kinase phosphorylating and regulating TAZ stability and suggests that Cdk1-TAZ signalling is a critical regulator of antitubulin drug response in cancer cells and may be a potential target for the treatment of antitubulin-drug resistant cancer patients.
Vleugel,2015 (26148513) Vleugel M, Hoek TA, Tromer E, Sliedrecht T, Groenewold V, Omerzu M, Kops GJ "Dissecting the roles of human BUB1 in the spindle assembly checkpoint." J Cell Sci 2015 Aug 16
Mitotic chromosome segregation is initiated by the anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 (forming APC/C(CDC20)). APC/C(CDC20) is inhibited by the spindle assembly checkpoint (SAC) when chromosomes have not attached to spindle microtubules. Unattached kinetochores catalyze the formation of a diffusible APC/C(CDC20) inhibitor that comprises BUBR1 (also known as BUB1B), BUB3, MAD2 (also known as MAD2L1) and a second molecule of CDC20. Recruitment of these proteins to the kinetochore, as well as SAC activation, rely on the mitotic kinase BUB1, but the molecular mechanism by which BUB1 accomplishes this in human cells is unknown. We show that kinetochore recruitment of BUBR1 and BUB3 by BUB1 is dispensable for SAC activation. Unlike its yeast and nematode orthologs, human BUB1 does not associate stably with the MAD2 activator MAD1 (also known as MAD1L1) and, although required for accelerating the loading of MAD1 onto kinetochores, BUB1 is dispensable for the maintenance of steady-state levels of MAD1 there. Instead, we identify a 50-amino-acid segment that harbors the recently reported ABBA motif close to a KEN box as being crucial for the role of BUB1 in SAC signaling. The presence of this segment correlates with SAC activity and efficient binding of CDC20 but not of MAD1 to kinetochores.
McInerney,2015 (26101899) McInerney GM "FGDF motif regulation of stress granule formation." DNA Cell Biol 2015 Sep 03
RNA stress granules (SGs) represent a cell-intrinsic antiviral defense mechanism. The assembly of SGs in response to viral infection is coordinated by the cellular protein G3BP, which is targeted by many viruses to block SG formation. We recently showed that proteins containing the short linear motif Phe-Gly-Asp-Phe (FGDF), bind G3BP in a hydrophobic groove on the surface of the nuclear transport factor-2-like domain. Binding in this manner blocks the ability of G3BP to form SGs and allows efficient replication of viruses carrying this motif.
Pfoh,2015 (26046769) Pfoh R, Lacdao IK, Georges AA, Capar A, Zheng H, Frappier L, Saridakis V "Crystal Structure of USP7 Ubiquitin-like Domains with an ICP0 Peptide Reveals a Novel Mechanism Used by Viral and Cellular Proteins to Target USP7." PLoS Pathog 2015 Jun 06
Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.
Wu,2015 (26025930) Wu H, Leng RP "MDM2 mediates p73 ubiquitination: a new molecular mechanism for suppression of p73 function." Oncotarget 2015 May 30
The protein p73, a homologue of the tumor suppressor protein p53, is capable of inducing apoptosis and cell cycle arrest. MDM2 is transcriptionally activated by p73 and represses the functions of p73, including p73-dependent transactivation and growth suppression. However, the molecular mechanism of this repression is unknown. In this study, we show that MDM2 mediates p73 ubiquitination. MDM2 mainly utilizes K11, K29 and K63-linked chains to mediate p73 ubiquitination in vivo and in vitro. However, MDM2 is unable to promote p73 degradation in most tested cell lines. Surprisingly, we observe that overexpression of Mdm2 promotes p73 degradation mainly through Itch in Mdm2-null MEFs. We further find that Itch interacts with the transfected Mdm2 in Mdm2-null cells. Moreover, our findings reveal that the E3 ligase activity of MDM2 is required to repress p73-dependent apoptosis and cell cycle arrest but not p73-dependent transcriptional activity. Furthermore, the data suggest a link between p73 ubiquitination/MDM2 E3 ligase activity and p73 biological functions.
Cheng,2015 (25960197) Cheng J, Yang H, Fang J, Ma L, Gong R, Wang P, Li Z, Xu Y "Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation." Nat Commun 2015 May 11
DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 A resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1-USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1-USP7 interaction surface for therapeutic applications.
Killoran,2015 (25909186) Killoran RC, Fan J, Yang D, Shilton BH, Choy WY "Structural Analysis of the 14-3-3zeta/Chibby Interaction Involved in Wnt/beta-Catenin Signaling." PLoS One 2015 Apr 27
The partially disordered Chibby (Cby) is a conserved nuclear protein that antagonizes the Wnt/beta-catenin signaling pathway. By competing with the Tcf/Lef family proteins for binding to beta-catenin, Cby abrogates the beta-catenin-mediated transcription of Wnt signaling genes. Additionally, upon phosphorylation on S20 by the kinase Akt, Cby forms a complex with 14-3-3 to facilitate the nuclear export of beta-catenin, which represents another crucial mechanism for the regulation of Wnt signaling. To obtain a mechanistic understanding of the 14-3-3/Cby interaction, we have extensively characterized the complex using X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and isothermal titration calorimetry (ITC). The crystal structure of the human 14-3-3zeta/Cby protein-peptide complex reveals a canonical binding mode; however the residue at the +2 position from the phosphorylated serine is shown to be uniquely oriented relative to other solved structures of 14-3-3 complexes. Our ITC results illustrate that although the phosphorylation of S20 is essential for Cby to recognize 14-3-3, residues flanking the phosphorylation site also contribute to the binding affinity. However, as is commonly observed in other 14-3-3/phosphopeptide crystal structures, residues of Cby flanking the 14-3-3 binding motif lack observable electron density. To obtain a more detailed binding interface, we have completed the backbone NMR resonance assignment of 14-3-3zeta. NMR titration experiments reveal that residues outside of the 14-3-3 conserved binding cleft, namely a flexible loop consisting of residues 203-210, are also involved in binding Cby. By using a combined X-ray and NMR approach, we have dissected the molecular basis of the 14-3-3/Cby interaction.
Selvaraj,2015 (25885538) Selvaraj N, Kedage V, Hollenhorst PC "Comparison of MAPK specificity across the ETS transcription factor family identifies a high-affinity ERK interaction required for ERG function in prostate cells." Cell Commun Signal 2015 Apr 18
BACKGROUND: The RAS/MAPK signaling pathway can regulate gene expression by phosphorylating and altering the function of some, but not all, ETS transcription factors. ETS family transcription factors bind similar DNA sequences and can compete for genomic binding sites. However, MAPK regulation varies across the ETS family. Therefore, changing the ETS factor bound to a cis-regulatory element can alter MAPK regulation of gene expression. To understand RAS/MAPK regulated gene expression programs, comprehensive knowledge of the ETS family members that are MAPK targets and relative MAPK targeting efficiency across the family is needed. RESULTS: An in vitro kinase assay was used to rank-order 27 human ETS family transcription factors based on phosphorylation by ERK2, JNK1, and p38alpha. Many novel MAPK targets and specificities were identified within the ETS family, including the identification of the prostate cancer oncoprotein ERG as a specific target of ERK2. ERK2 phosphorylation of ERG S215 required a DEF docking domain and was necessary for ERG to activate transcription of cell migration genes and promote prostate cell migration. The ability of ERK2 to bind ERG with higher affinity than ETS1 provided a potential molecular explanation for why ERG overexpression drives migration of prostate cells with low levels of RAS/ERK signaling, while ETS1 has a similar function only when RAS/ERK signaling is high. CONCLUSIONS: The rank ordering of ETS transcription factors as MAPK targets provides an important resource for understanding ETS proteins as mediators of MAPK signaling. This is emphasized by the difference in rank order of ERG and ETS1, which allows these factors to have distinct roles based on the level of RAS/ERK signaling present in the cell.
Brown,2015 (25833379) Brown JS, Jackson SP "Ubiquitylation, neddylation and the DNA damage response." Open Biol 2015 Apr 02
Failure of accurate DNA damage sensing and repair mechanisms manifests as a variety of human diseases, including neurodegenerative disorders, immunodeficiency, infertility and cancer. The accuracy and efficiency of DNA damage detection and repair, collectively termed the DNA damage response (DDR), requires the recruitment and subsequent post-translational modification (PTM) of a complex network of proteins. Ubiquitin and the ubiquitin-like protein (UBL) SUMO have established roles in regulating the cellular response to DNA double-strand breaks (DSBs). A role for other UBLs, such as NEDD8, is also now emerging. This article provides an overview of the DDR, discusses our current understanding of the process and function of PTM by ubiquitin and NEDD8, and reviews the literature surrounding the role of ubiquitylation and neddylation in DNA repair processes, focusing particularly on DNA DSB repair.
Nie,2015 (25752541) Nie J, Xu C, Jin J, Aka JA, Tempel W, Nguyen V, You L, Weist R, Min J, Pawson T, Yang XJ "Ankyrin repeats of ANKRA2 recognize a PxLPxL motif on the 3M syndrome protein CCDC8." Structure 2015 Apr 12
Peptide motifs are often used for protein-protein interactions. We have recently demonstrated that ankyrin repeats of ANKRA2 and the paralogous bare lymphocyte syndrome transcription factor RFXANK recognize PxLPxL/I motifs shared by megalin, three histone deacetylases, and RFX5. We show here that that CCDC8 is a major partner of ANKRA2 but not RFXANK in cells. The CCDC8 gene is mutated in 3M syndrome, a short-stature disorder with additional facial and skeletal abnormalities. Two other genes mutated in this syndrome encode CUL7 and OBSL1. While CUL7 is a ubiquitin ligase and OBSL1 associates with the cytoskeleton, little is known about CCDC8. Binding and structural analyses reveal that the ankyrin repeats of ANKRA2 recognize a PxLPxL motif at the C-terminal region of CCDC8. The N-terminal part interacts with OBSL1 to form a CUL7 ligase complex. These results link ANKRA2 unexpectedly to 3M syndrome and suggest novel regulatory mechanisms for histone deacetylases and RFX7.
Cairns,2015 (25742493) Cairns J, Peng Y, Yee VC, Lou Z, Wang L "Bora downregulation results in radioresistance by promoting repair of double strand breaks." PLoS One 2015 Mar 06
Following DNA double-strand breaks cells activate several DNA-damage response protein kinases, which then trigger histone H2AX phosphorylation and the accumulation of proteins such as MDC1, p53-binding protein 1, and breast cancer gene 1 at the damage site to promote DNA double-strand breaks repair. We identified a novel biomarker, Bora (previously called C13orf34), that is associated with radiosensitivity. In the current study, we set out to investigate how Bora might be involved in response to irradiation. We found a novel function of Bora in DNA damage repair response. Bora down-regulation increased colony formation in cells exposed to irradiation. This increased resistance to irradiation in Bora-deficient cells is likely due to a faster rate of double-strand breaks repair. After irradiation, Bora-knockdown cells displayed increased G2-M cell cycle arrest and increased Chk2 phosphorylation. Furthermore, Bora specifically interacted with the tandem breast cancer gene 1 C-terminal domain of MDC1 in a phosphorylation dependent manner, and overexpression of Bora could abolish irradiation induced MDC1 foci formation. In summary, Bora may play a significant role in radiosensitivity through the regulation of MDC1 and DNA repair.
Alexa,2015 (25730857) Alexa A, Gogl G, Glatz G, Garai A, Zeke A, Varga J, Dudas E, Jeszenoi N, Bodor A, Hetenyi C, Remenyi A "Structural assembly of the signaling competent ERK2-RSK1 heterodimeric protein kinase complex." Proc Natl Acad Sci U S A 2015 Mar 04
Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK-->MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.
Leite,2015 (25681743) Leite F, Way M "The role of signalling and the cytoskeleton during Vaccinia Virus egress." Virus Res 2015 Nov 02
Viruses are obligate intracellular parasites that are critically dependent on their hosts to replicate and generate new progeny. To achieve this goal, viruses have evolved numerous elegant strategies to subvert and utilise the different cellular machineries and processes of their unwilling hosts. Moreover, they often accomplish this feat with a surprisingly limited number of proteins. Among the different systems of the cell, the cytoskeleton is often one of the first to be hijacked as it provides a convenient transport system for viruses to reach their site of replication with relative ease. At the latter stages of their replication cycle, the cytoskeleton also provides an efficient means for newly assembled viral progeny to reach the plasma membrane and leave the infected cell. In this review we discuss how Vaccinia virus takes advantage of the microtubule and actin cytoskeletons of its host to promote the spread of infection into neighboring cells. In particular, we highlight how analysis of actin-based motility of Vaccinia has provided unprecedented insights into how a phosphotyrosine-based signalling network is assembled and functions to stimulate Arp2/3 complex-dependent actin polymerization. We also suggest that the formin FHOD1 promotes actin-based motility of the virus by capping the fast growing ends of actin filaments rather than directly promoting filament assembly. We have come a long way since 1976, when electron micrographs of vaccinia-infected cells implicated the actin cytoskeleton in promoting viral spread. Nevertheless, there are still many unanswered questions concerning the role of signalling and the host cytoskeleton in promoting viral spread and pathogenesis.
Di Fiore,2015 (25669885) Di Fiore B, Davey NE, Hagting A, Izawa D, Mansfeld J, Gibson TJ, Pines J "The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators." Dev Cell 2015 Feb 11
The anaphase-promoting complex or cyclosome (APC/C) is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the spindle assembly checkpoint (SAC). How the APC/C recognizes its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in cyclin A, BUBR1, BUB1, and Acm1, and we show that it binds to the APC/C coactivator CDC20. The ABBA motif in cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC.
Roskoski R,2015 (25662515) Roskoski R Jr "Src protein-tyrosine kinase structure, mechanism, and small molecule inhibitors." Pharmacol Res 2015 Apr 04
The physiological Src proto-oncogene is a protein-tyrosine kinase that plays key roles in cell growth, division, migration, and survival signaling pathways. From the N- to C-terminus, Src contains a unique domain, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a regulatory tail. The chief phosphorylation sites of human Src include an activating pTyr419 that results from phosphorylation in the kinase domain by an adjacent Src molecule and an inhibitory pTyr530 in the regulatory tail that results from phosphorylation by C-terminal Src kinase (Csk) or Chk (Csk homologous kinase). The oncogenic Rous sarcoma viral protein lacks the equivalent of Tyr530 and is constitutively activated. Inactive Src is stabilized by SH2 and SH3 domains on the rear of the kinase domain where they form an immobilizing and inhibitory clamp. Protein kinases including Src contain hydrophobic regulatory and catalytic spines and collateral shell residues that are required to assemble the active enzyme. In the inactive enzyme, the regulatory spine contains a kink or a discontinuity with a structure that is incompatible with catalysis. The conversion of inactive to active Src is accompanied by electrostatic exchanges involving the breaking and making of distinct sets of kinase domain salt bridges and hydrogen bonds. Src-catalyzed protein phosphorylation requires the participation of two Mg(2+) ions. Although nearly all protein kinases possess a common K/E/D/D signature, each enzyme exhibits its unique variations of the protein-kinase reaction template. Bosutinib, dasatinib, and ponatinib are Src/multikinase inhibitors that are approved by the FDA for the treatment of chronic myelogenous leukemia and vandetanib is approved for the treatment of medullary thyroid cancer. The Src and BCR-Abl inhibitors saracatinib and AZD0424, along with the previous four drugs, are in clinical trials for a variety of solid tumors including breast and lung cancers. Both ATP and targeted therapeutic Src protein kinase inhibitors such as dasatinib and ponatinib make hydrophobic contacts with catalytic spine residues and form hydrogen bonds with hinge residues connecting the small and large kinase lobes.
Panas,2015 (25658430) Panas MD, Schulte T, Thaa B, Sandalova T, Kedersha N, Achour A, McInerney GM "Viral and cellular proteins containing FGDF motifs bind G3BP to block stress granule formation." PLoS Pathog 2015 Feb 09
The Ras-GAP SH3 domain-binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP.
Sun,2015 (25640033) Sun Y, Stine JM, Atwater DZ, Sharmin A, Ross JB, Briknarova K "Structural and functional characterization of the acidic region from the RIZ tumor suppressor." Biochemistry 2015 Feb 17
RIZ (retinoblastoma protein-interacting zinc finger protein), also denoted PRDM2, is a transcriptional regulator and tumor suppressor. It was initially identified because of its ability to interact with another well-established tumor suppressor, the retinoblastoma protein (Rb). A short motif, IRCDE, in the acidic region (AR) of RIZ was reported to play an important role in the interaction with the pocket domain of Rb. The IRCDE motif is similar to a consensus Rb-binding sequence LXCXE (where X denotes any amino acid) that is found in several viral Rb-inactivating oncoproteins. To improve our understanding of the molecular basis of binding of Rb to RIZ, we investigated the interaction between purified recombinant AR and the pocket domain of Rb using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and fluorescence anisotropy experiments. We show that AR is intrinsically disordered and that it binds the pocket domain with submicromolar affinity. We also demonstrate that the interaction between AR and the pocket domain is mediated primarily by the short stretch of residues containing the IRCDE motif and that the contribution of other parts of AR to the interaction with the pocket domain is minimal. Overall, our data provide clear evidence that RIZ is one of the few cellular proteins that can interact directly with the LXCXE-binding cleft on Rb.
Shin,2015 (25591003) Shin JS, Ha JH, Lee DH, Ryu KS, Bae KH, Park BC, Park SG, Yi GS, Chi SW "Structural convergence of unstructured p53 family transactivation domains in MDM2 recognition." Cell Cycle 2015 Feb 21
The p53, p63, and p73 proteins belong to the p53 family of transcription factors, which play key roles in tumor suppression. Although the transactivation domains (TADs) of the p53 family are intrinsically disordered, these domains are commonly involved in the regulatory interactions with mouse double minute 2 (MDM2). In this study, we determined the solution structure of the p73TAD peptide in complex with MDM2 using NMR spectroscopy and biophysically characterized the interactions between the p53 family TAD peptides and MDM2. In combination with mutagenesis data, the complex structures revealed remarkably close mimicry of the MDM2 recognition mechanism among the p53 family TADs. Upon binding with MDM2, the intrinsically disordered p73TAD and p63TAD peptides adopt an amphipathic alpha-helical conformation, which is similar to the conformation of p53TAD, although the alpha-helical content induced by MDM2 binding varies. With isothermal titration calorimetry (ITC) and circular dichroism (CD) data, our biophysical characterization showed that p73TAD resembles p53TAD more closely than p63TAD in terms of helical stability, MDM2 binding affinity, and phosphorylation effects on MDM2 binding. Therefore, our structural information may be useful in establishing alternative anticancer strategies that exploit the activation of the p73 pathway against human tumors bearing p53 mutations.
Losh,2015 (25589546) Losh JS, King AK, Bakelar J, Taylor L, Loomis J, Rosenzweig JA, Johnson SJ, van Hoof A "Interaction between the RNA-dependent ATPase and poly(A) polymerase subunits of the TRAMP complex is mediated by short peptides and important for snoRNA processing." Nucleic Acids Res 2015 Apr 02
The RNA exosome is one of the main 3' to 5' exoribonucleases in eukaryotic cells. Although it is responsible for degradation or processing of a wide variety of substrate RNAs, it is very specific and distinguishes between substrate and non-substrate RNAs as well as between substrates that need to be 3' processed and those that need to be completely degraded. This specificity does not appear to be determined by the exosome itself but rather by about a dozen other proteins. Four of these exosome cofactors have enzymatic activity, namely, the nuclear RNA-dependent ATPase Mtr4, its cytoplasmic paralog Ski2 and the nuclear non-canonical poly(A) polymerases, Trf4 and Trf5. Mtr4 and either Trf4 or Trf5 assemble into a TRAMP complex. However, how these enzymes assemble into a TRAMP complex and the functional consequences of TRAMP complex assembly remain unknown. Here, we identify an important interaction site between Mtr4 and Trf5, and show that disrupting the Mtr4/Trf interaction disrupts specific TRAMP and exosome functions, including snoRNA processing.
Doerflinger,2015 (25565426) Doerflinger M, Glab JA, Puthalakath H "BH3-only proteins: a 20-year stock-take." FEBS J 2015 Mar 20
BH3-only proteins are the sentinels of cellular stress, and their activation commits cells to apoptosis. Since the discovery of the first BH3-only protein BAD almost 20 years ago, at least seven more BH3-only proteins have been identified in mammals. They are regulated by a variety of environmental stimuli or by developmental cues, and play a crucial role in cellular homeostasis. Some are considered to be tumor suppressors, and also play a significant role in other pathologies. Their non-apoptotic functions are controversial, but there is broad consensus emerging regarding their role in apoptosis, which may help in designing better therapeutic agents for treating a variety of human diseases.
Wilson,2014 (25516977) Wilson MH, Holzbaur EL "Nesprins anchor kinesin-1 motors to the nucleus to drive nuclear distribution in muscle cells." Development 2014 Dec 17
During skeletal muscle development, nuclei move dynamically through myotubes in a microtubule-dependent manner, driven by the microtubule motor protein kinesin-1. Loss of kinesin-1 leads to improperly positioned nuclei in culture and in vivo. Two models have been proposed to explain how kinesin-1 functions to move nuclei in myotubes. In the cargo model, kinesin-1 acts directly from the surface of the nucleus, whereas in an alternative model, kinesin-1 moves nuclei indirectly by sliding anti-parallel microtubules. Here, we test the hypothesis that an ensemble of Kif5B motors acts from the nuclear envelope to distribute nuclei throughout the length of syncytial myotubes. First, using an inducible dimerization system, we show that controlled recruitment of truncated, constitutively active kinesin-1 motors to the nuclear envelope is sufficient to prevent the nuclear aggregation resulting from depletion of endogenous kinesin-1. Second, we identify a conserved kinesin light chain (KLC)-binding motif in the nuclear envelope proteins nesprin-1 and nesprin-2, and show that recruitment of the motor complex to the nucleus via this LEWD motif is essential for nuclear distribution. Together, our findings demonstrate that the nucleus is a kinesin-1 cargo in myotubes and that nesprins function as nuclear cargo adaptors. The importance of achieving and maintaining proper nuclear position is not restricted to muscle fibers, suggesting that the nesprin-dependent recruitment of kinesin-1 to the nuclear envelope through the interaction of a conserved LEWD motif with kinesin light chain might be a general mechanism for cell-type-specific nuclear positioning during development.
Diaz-Martinez,2015 (25505175) Diaz-Martinez LA, Tian W, Li B, Warrington R, Jia L, Brautigam CA, Luo X, Yu H "The Cdc20-binding Phe box of the spindle checkpoint protein BubR1 maintains the mitotic checkpoint complex during mitosis." J Biol Chem 2015 Jan 27
The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms.
Cappadocia,2015 (25497731) Cappadocia L, Mascle XH, Bourdeau V, Tremblay-Belzile S, Chaker-Margot M, Lussier-Price M, Wada J, Sakaguchi K, Aubry M, Ferbeyre G, Omichinski JG "Structural and Functional Characterization of the Phosphorylation-Dependent Interaction between PML and SUMO1." Structure 2015 Jan 08
PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.
Akamatsu,2015 (25472445) Akamatsu R, Ishida-Kitagawa N, Aoyama T, Oka C, Kawaichi M "BNIP-2 binds phosphatidylserine, localizes to vesicles, and is transported by kinesin-1." Genes Cells 2015 Jan 28
BNIP-2 shows high homology with the Cayman ataxia protein, caytaxin, which functions as a kinesin-1 adapter bridging cargos and kinesin light chains (KLCs). BNIP-2 is known to induce cell shape changes when over-expressed in culture cells, but its physiological functions are mostly unknown. BNIP-2 interacts with KLC through the conserved WED motif in the N-terminal region of BNIP-2. Interaction with KLC and transportation by kinesin-1 are essential for over-expressed BNIP-2 to elongate cells and induce cellular processes. Endogenous BNIP-2 localizes to the Golgi apparatus, early and recycling endosomes and mitochondria, aligned with microtubules, and moves at a speed compatible with kinesin-1 transportation. The CRAL-TRIO domain of BNIP-2 specifically interacts with phosphatidylserine, and the vesicular localization of BNIP-2 requires interaction with this phospholipid. BNIP-2 mutants which do not bind phosphatidylserine do not induce morphological changes in cells. These data show that similar to caytaxin, BNIP-2 is a kinesin-1 adapter involved in vesicular transportation in the cytoplasm and that association with cargos depends on interaction of the CRAL-TRIO domain with membrane phosphatidylserine.
Garcia,2014 (25461409) Garcia JD, Dewey EB, Johnston CA "Dishevelled binds the Discs large 'Hook' domain to activate GukHolder-dependent spindle positioning in Drosophila." PLoS One 2014 Dec 03
Communication between cortical cell polarity cues and the mitotic spindle ensures proper orientation of cell divisions within complex tissues. Defects in mitotic spindle positioning have been linked to various developmental disorders and have recently emerged as a potential contributor to tumorigenesis. Despite the importance of this process to human health, the molecular mechanisms that regulate spindle orientation are not fully understood. Moreover, it remains unclear how diverse cortical polarity complexes might cooperate to influence spindle positioning. We and others have demonstrated spindle orientation roles for Dishevelled (Dsh), a key regulator of planar cell polarity, and Discs large (Dlg), a conserved apico-basal cell polarity regulator, effects which were previously thought to operate within distinct molecular pathways. Here we identify a novel direct interaction between the Dsh-PDZ domain and the alternatively spliced "I3-insert" of the Dlg-Hook domain, thus establishing a potential convergent Dsh/Dlg pathway. Furthermore, we identify a Dlg sequence motif necessary for the Dsh interaction that shares homology to the site of Dsh binding in the Frizzled receptor. Expression of Dsh enhanced Dlg-mediated spindle positioning similar to deletion of the Hook domain. This Dsh-mediated activation was dependent on the Dlg-binding partner, GukHolder (GukH). These results suggest that Dsh binding may regulate core interdomain conformational dynamics previously described for Dlg. Together, our results identify Dlg as an effector of Dsh signaling and demonstrate a Dsh-mediated mechanism for the activation of Dlg/GukH-dependent spindle positioning. Cooperation between these two evolutionarily-conserved cell polarity pathways could have important implications to both the development and maintenance of tissue homeostasis in animals.
Malinauskas,2014 (25460271) Malinauskas T, Jones EY "Extracellular modulators of Wnt signalling." Curr Opin Struct Biol 2014 Dec 16
Wnt morphogens are secreted signalling proteins that play leading roles in embryogenesis and tissue homeostasis throughout life. Wnt signalling is controlled by multiple mechanisms, including posttranslational modification of Wnts, antagonist binding (to Wnts or their receptors), and regulation of the availability of Wnt receptors. Recent crystallographic, structure-guided biophysical and cell-based studies have advanced our understanding of how Wnt signalling is regulated at the cell surface. Structures include Wnt in complex with the cysteine-rich domain (CRD) of Frizzled, extracellular fragments of Wnt co-receptor LRP6, LRP6-binding antagonists Dickkopf and Sclerostin, antagonists 5T4/WAIF1 and Wnt inhibitory factor 1 (WIF-1), as well as Frizzled-ubiquitin ligases ZNRF3/RNF43 (in isolation and in complexes with Wnt signalling promoters R-spondins and LGR5). We review recent discoveries and remaining questions.
Schuch,2014 (25319414) Schuch B, Feigenbutz M, Makino DL, Falk S, Basquin C, Mitchell P, Conti E "The exosome-binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase." EMBO J 2014 Dec 02
The exosome is a conserved multi-subunit ribonuclease complex that functions in 3' end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10-subunit core complex of the exosome (Exo-10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo-10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors. Crystallographic analyses reveal that the N-terminal domains of Rrp6 and Rrp47 form a highly intertwined structural unit. Rrp6 and Rrp47 synergize to create a composite and conserved surface groove that binds the N-terminus of Mtr4. Mutation of conserved residues within Rrp6 and Mtr4 at the structural interface disrupts their interaction and inhibits growth of strains expressing a C-terminal GFP fusion of Mtr4. These studies provide detailed structural insight into the interaction between the Rrp6-Rrp47 complex and Mtr4, revealing an important link between Mtr4 and the core exosome.
Chen,2014 (25301550) Chen WK, Yeap YY, Bogoyevitch MA "The JNK1/JNK3 interactome--contributions by the JNK3 unique N-terminus and JNK common docking site residues." Biochem Biophys Res Commun 2014 Dec 19
The c-Jun N-terminal Kinases (JNKs) play important roles in cell responses to stress or growth factor stimulation. The JNK1alpha1 isoform shares >90% identity with a predominantly neuronal JNK3alpha1 isoform, but JNK3alpha1 also includes a distinctive 38 amino acid N-terminal sequence. To address the outstanding question of the potential for these JNK isoforms to have different binding partners that mediate different biological actions, the work presented here refined the yeast two-hybrid approach to identify and categorize binding partners for JNK1alpha1 and JNK3alpha1. Specifically, site-directed mutagenesis of the JNK1alpha1 common docking (CD) domain that mediates typical JNK-binding domain (JBD)-dependent interactions, truncation of the distinctive JNK3 N-terminal domain (i.e. DeltaN JNK3alpha1), and interaction evaluation in the yeast two-hybrid system defined the interacting partners as either JNK1-specific interactors (ATF7, FUS, KCNE4, PIAS1, SHANK1, TKT), typical JBD-dependent interactors shared by JNK1alpha1 and JNK3alpha1 (AKAP6, BMPR2, EEF1A1, GFAP, GRIP2, GTF2F1, HDAC2, MAP1B, MYO9B, PTPN2, RABGAP1, RUSC2, SUMO1, SYPL1, TOPBP1, ZNF668), or JNK3-specific partners (ATXN1, NNAT, PTGDS) dependent on interaction with the JNK3 N-terminal extension. The interacting partners ATF7, AKAP6, and ATXN1 were explored further as representatives of these different classes. Two potential JBDs were identified in ATF7 as important for its interaction with JNK1alpha1, but additionally an interaction between ATF7 and DeltaN JNK3alpha1 was shown to be JBD-dependent, suggesting that the JNK3alpha1 N-terminus prevents interaction with some proteins. For the shared partner AKAP6, one of the multiple potential JBDs predicted by sequence analysis was important for the AKAP6-JNK interaction in the yeast screening system as well as in mammalian cells. Finally, the ATXN1-JNK3alpha1 interaction was dependent on the JNK3alpha1 N-terminus in a mammalian cell context. These studies therefore highlight a diversity of potential JNK-interacting partners with both JBD-dependent as well as JBD-independent modes of interaction.
Lu,2014 (25287299) Lu D, Hsiao JY, Davey NE, Van Voorhis VA, Foster SA, Tang C, Morgan DO "Multiple mechanisms determine the order of APC/C substrate degradation in mitosis." J Cell Biol 2014 Oct 14
The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/C(Cdc20) substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1-Cks1 complex and the presence of a Cdc20-binding "ABBA motif" in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/C(Cdc20) substrate destruction.
Cumming,2015 (25255283) Cumming JG, Debreczeni JE, Edfeldt F, Evertsson E, Harrison M, Holdgate GA, James MJ, Lamont SG, Oldham K, Sullivan JE, Wells SL "Discovery and characterization of MAPK-activated protein kinase-2 prevention of activation inhibitors." J Med Chem 2015 Jan 09
Two structurally distinct series of novel, MAPK-activated kinase-2 prevention of activation inhibitors have been discovered by high throughput screening. Preliminary structure-activity relationship (SAR) studies revealed substructural features that influence the selective inhibition of the activation by p38alpha of the downstream kinase MK2 in preference to an alternative substrate, MSK1. Enzyme kinetics, surface plasmon resonance (SPR), 2D protein NMR, and X-ray crystallography were used to determine the binding mode and the molecular mechanism of action. The compounds bind competitively to the ATP binding site of p38alpha but unexpectedly with higher affinity in the p38alpha-MK2 complex compared with p38alpha alone. This observation is hypothesized to be the origin of the substrate selectivity. The two lead series identified are suitable for further investigation for their potential to treat chronic inflammatory diseases with improved tolerability over previously studied p38alpha inhibitors.
Hendriks,2014 (25218447) Hendriks IA, D'Souza RC, Yang B, Verlaan-de Vries M, Mann M, Vertegaal AC "Uncovering global SUMOylation signaling networks in a site-specific manner." Nat Struct Mol Biol 2014 Oct 08
SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution MS, we have studied global SUMOylation in human cells in a site-specific manner, identifying a total of >4,300 SUMOylation sites in >1,600 proteins. To our knowledge, this is the first time that >1,000 SUMOylation sites have been identified under standard growth conditions. We quantitatively studied SUMOylation dynamics in response to SUMO protease inhibition, proteasome inhibition and heat shock. Many SUMOylated lysines have previously been reported to be ubiquitinated, acetylated or methylated, thus indicating cross-talk between SUMO and other post-translational modifications. We identified 70 phosphorylation and four acetylation events in proximity to SUMOylation sites, and we provide evidence for acetylation-dependent SUMOylation of endogenous histone H3. SUMOylation regulates target proteins involved in all nuclear processes including transcription, DNA repair, chromatin remodeling, precursor-mRNA splicing and ribosome assembly.
Falk,2014 (25175027) Falk S, Weir JR, Hentschel J, Reichelt P, Bonneau F, Conti E "The molecular architecture of the TRAMP complex reveals the organization and interplay of its two catalytic activities." Mol Cell 2014 Sep 20
The TRAMP complex is involved in the nuclear surveillance and turnover of noncoding RNAs and intergenic transcripts. TRAMP is associated with the nuclear exosome and consists of a poly(A)polymerase subcomplex (Trf4-Air2) and a helicase (Mtr4). We found that N-terminal low-complexity regions of Trf4 and Air2 bind Mtr4 in a cooperative manner. The 2.4 A resolution crystal structure of the corresponding ternary complex reveals how Trf4 and Air2 wrap around the DExH core of the helicase. Structure-based mutations on the DExH core impair binding to Trf4 and Air2, and also to Trf5 and Air1. The combination of structural, biochemical, and biophysical data suggests that the poly(A)polymerase core of Trf4-Air2 is positioned below the base of the helicase, where the unwound 3' end of an RNA substrate is expected to emerge. The results reveal conceptual similarities between the two major regulators of the exosome, the nuclear TRAMP and cytoplasmic Ski complexes.
Lampard,2014 (25172143) Lampard GR, Wengier DL, Bergmann DC "Manipulation of mitogen-activated protein kinase kinase signaling in the Arabidopsis stomatal lineage reveals motifs that contribute to protein localization and signaling specificity." Plant Cell 2014 Sep 26
When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four "stomatal" MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.
Lu,2014 (25117710) Lu G, Zhang Q, Huang Y, Song J, Tomaino R, Ehrenberger T, Lim E, Liu W, Bronson RT, Bowden M, Brock J, Krop IE, Dillon DA, Gygi SP, Mills GB, Richardson AL, Signoretti S, Yaffe MB, Kaelin WG Jr "Phosphorylation of ETS1 by Src family kinases prevents its recognition by the COP1 tumor suppressor." Cancer Cell 2014 Aug 13
Oncoproteins and tumor suppressors antagonistically converge on critical nodes governing neoplastic growth, invasion, and metastasis. We discovered that phosphorylation of the ETS1 and ETS2 transcriptional oncoproteins at specific serine or threonine residues creates binding sites for the COP1 tumor suppressor protein, which is an ubiquitin ligase component, leading to their destruction. In the case of ETS1, however, phosphorylation of a neighboring tyrosine residue by Src family kinases disrupts COP1 binding, thereby stabilizing ETS1. Src-dependent accumulation of ETS1 in breast cancer cells promotes anchorage-independent growth in vitro and tumor growth in vivo. These findings expand the list of potential COP1 substrates to include proteins whose COP1-binding sites are subject to regulatory phosphorylation and provide insights into transformation by Src family kinases.
Impens,2014 (25114211) Impens F, Radoshevich L, Cossart P, Ribet D "Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli." Proc Natl Acad Sci U S A 2014 Aug 27
SUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and high-throughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease.
Tudek,2014 (25066235) Tudek A, Porrua O, Kabzinski T, Lidschreiber M, Kubicek K, Fortova A, Lacroute F, Vanacova S, Cramer P, Stefl R, Libri D "Molecular basis for coordinating transcription termination with noncoding RNA degradation." Mol Cell 2014 Aug 09
The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.
Uyar,2014 (25057855) Uyar B, Weatheritt RJ, Dinkel H, Davey NE, Gibson TJ "Proteome-wide analysis of human disease mutations in short linear motifs: neglected players in cancer?" Mol Biosyst 2014 Aug 27
Disease mutations are traditionally thought to impair protein functionality by disrupting the folded globular structure of proteins. However, 22% of human disease mutations occur in natively unstructured segments of proteins known as intrinsically disordered regions (IDRs). This therefore implicates defective IDR functionality in various human diseases including cancer. The functionality of IDRs is partly attributable to short linear motifs (SLiMs), but it remains an open question how much defects in SLiMs contribute to human diseases. A proteome-wide comparison of the distribution of missense mutations from disease and non-disease mutation datasets revealed that, in IDRs, disease mutations are more likely to occur within SLiMs than neutral missense mutations. Moreover, compared to neutral missense mutations, disease mutations more frequently impact functionally important residues of SLiMs, cause changes in the physicochemical properties of SLiMs, and disrupt more SLiM-mediated interactions. Analysis of these mutations resulted in a comprehensive list of experimentally validated or predicted SLiMs disrupted in disease. Furthermore, this in-depth analysis suggests that 'prostate cancer pathway' is particularly enriched for proteins with disease-related SLiMs. The contribution of mutations in SLiMs to disease may currently appear small when compared to mutations in globular domains. However, our analysis of mutations in predicted SLiMs suggests that this contribution might be more substantial. Therefore, when analysing the functional impact of mutations on proteins, SLiMs in proteins should not be neglected. Our results suggest that an increased focus on SLiMs in the coming decades will improve our understanding of human diseases and aid in the development of targeted treatments.
Park,2014 (24998779) Park JY, Zhang F, Andreassen PR "PALB2: the hub of a network of tumor suppressors involved in DNA damage responses." Biochim Biophys Acta 2014 Aug 21
PALB2 was first identified as a partner of BRCA2 that mediates its recruitment to sites of DNA damage. PALB2 was subsequently found as a tumor suppressor gene. Inherited heterozygosity for this gene is associated with an increased risk of cancer of the breast and other sites. Additionally, biallelic mutation of PALB2 is linked to Fanconi anemia, which also has an increased risk of developing malignant disease. Recent work has identified numerous interactions of PALB2, suggesting that it functions in a network of proteins encoded by tumor suppressors. Notably, many of these tumor suppressors are related to the cellular response to DNA damage. The recruitment of PALB2 to DNA double-strand breaks at the head of this network is via a ubiquitin-dependent signaling pathway that involves the RAP80, Abraxas and BRCA1 tumor suppressors. Next, PALB2 interacts with BRCA2, which is a tumor suppressor, and with the RAD51 recombinase. These interactions promote DNA repair by homologous recombination (HR). More recently, PALB2 has been found to bind the RAD51 paralog, RAD51C, as well as the translesion polymerase pol eta, both of which are tumor suppressors with functions in HR. Further, an interaction with MRG15, which is related to chromatin regulation, may facilitate DNA repair in damaged chromatin. Finally, PALB2 interacts with KEAP1, a regulator of the response to oxidative stress. The PALB2 network appears to mediate the maintenance of genome stability, may explain the association of many of the corresponding genes with similar spectra of tumors, and could present novel therapeutic opportunities.
Van Roey,2014 (24926813) Van Roey K, Uyar B, Weatheritt RJ, Dinkel H, Seiler M, Budd A, Gibson TJ, Davey NE "Short linear motifs: ubiquitous and functionally diverse protein interaction modules directing cell regulation." Chem Rev 2014 Jul 09
Xie,2014 (24910198) Xie S, Lu Y, Jakoncic J, Sun H, Xia J, Qian C "Structure of RPA32 bound to the N-terminus of SMARCAL1 redefines the binding interface between RPA32 and its interacting proteins." FEBS J 2014 Jul 29
Replication protein A subunit RPA32 contains a C-terminal domain that interacts with a variety of DNA damage response proteins including SMARCAL1, Tipin, UNG2 and XPA. We have solved the high-resolution crystal structure of RPA32 C-terminal domain (RPA32C) in complex with a 26-amino-acid peptide derived from the N-terminus of SMARCAL1 (SMARCAL1N). The RPA32C-SMARCAL1N structure reveals a 1 : 1 binding stoichiometry and displays a well-ordered binding interface. SMARCAL1N adopts a long alpha-helical conformation with the highly conserved 11 residues aligned on one face of the alpha-helix showing extensive interactions with the RPA32C domain. Extensive mutagenesis experiments were performed to corroborate the interactions observed in crystal structure. Moreover, the alpha1/alpha2 loop of the RPA32C domain undergoes a conformational rearrangement upon SMARCAL1N binding. NMR study has further confirmed that the RPA32C-SMARCAL1N interaction induces conformational changes in RPA32C. Isothermal titration calorimetry studies have also demonstrated that the conserved alpha-helical motif defined in the current study is required for sufficient binding of RPA32C. Taken together, our study has provided convincing structural information that redefines the common recognition pattern shared by RPA32C interacting proteins. DATABASE: The atomic coordinates of RPA32C in complex with 26-aa SMARCAL1 (SMARCAL1N) peptide have been deposited at the Protein Data Bank with accession code 4MQV. STRUCTURED DIGITAL ABSTRACT: RPA32 and SMARCAL1 bind by isothermal titration calorimetry(1, 2, 3, 4, 5, 6, 7, 8, 9) RPA32 and SMARCAL1 bind by molecular sieving (View interaction) RPA32 and SMARCAL1 bind by x-ray crystallography (View interaction) Tipin and RPA32 bind by isothermal titration calorimetry (1, 2) RPA32 and UNG2 bind by isothermal titration calorimetry (1, 2, 3) SMARCAL1 and RPA32 bind by nuclear magnetic resonance (View interaction) UNG2 and RPA32 bind by nuclear magnetic resonance (View interaction) Tipin and RPA32 bind by nuclear magnetic resonance (View interaction).
Dodd,2014 (24902122) Dodd DA, Worth RG, Rosen MK, Grinstein S, van Oers NS, Hansen EJ "The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase." MBio 2014 Jun 06
Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcgamma receptor (FcgammaR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. IMPORTANCE: Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for phagocytic activity. Haemophilus ducreyi, a sexually transmitted pathogen, secretes a 4,153-amino-acid (aa) protein (LspA1) that effectively inhibits FcgammaR-mediated phagocytic activity. In this study, we show that a 294-aa domain within this bacterial protein binds to C-terminal Src kinase (Csk) and stimulates its catalytic activity, resulting in a significant attenuation of Src kinase activity and consequent inhibition of phagocytosis. The ability to inhibit phagocytosis via Csk is not unique to H. ducreyi, because we found that the Helicobacter pylori CagA protein also inhibits phagocytosis in a Csk-dependent manner. Harnessing Csk to subvert the FcgammaR-mediated phagocytic pathway represents a new bacterial effector mechanism for circumventing the innate immune response.
Zhao,2014 (24880689) Zhao Q, Xie Y, Zheng Y, Jiang S, Liu W, Mu W, Liu Z, Zhao Y, Xue Y, Ren J "GPS-SUMO: a tool for the prediction of sumoylation sites and SUMO-interaction motifs." Nucleic Acids Res 2014 Jul 03
Small ubiquitin-like modifiers (SUMOs) regulate a variety of cellular processes through two distinct mechanisms, including covalent sumoylation and non-covalent SUMO interaction. The complexity of SUMO regulations has greatly hampered the large-scale identification of SUMO substrates or interaction partners on a proteome-wide level. In this work, we developed a new tool called GPS-SUMO for the prediction of both sumoylation sites and SUMO-interaction motifs (SIMs) in proteins. To obtain an accurate performance, a new generation group-based prediction system (GPS) algorithm integrated with Particle Swarm Optimization approach was applied. By critical evaluation and comparison, GPS-SUMO was demonstrated to be substantially superior against other existing tools and methods. With the help of GPS-SUMO, it is now possible to further investigate the relationship between sumoylation and SUMO interaction processes. A web service of GPS-SUMO was implemented in PHP+JavaScript and freely available at
Ben-Johny,2014 (24863929) Ben-Johny M, Yue DT "Calmodulin regulation (calmodulation) of voltage-gated calcium channels." J Gen Physiol 2014 May 27
Calmodulin regulation (calmodulation) of the family of voltage-gated CaV1-2 channels comprises a prominent prototype for ion channel regulation, remarkable for its powerful Ca(2+) sensing capabilities, deep in elegant mechanistic lessons, and rich in biological and therapeutic implications. This field thereby resides squarely at the epicenter of Ca(2+) signaling biology, ion channel biophysics, and therapeutic advance. This review summarizes the historical development of ideas in this field, the scope and richly patterned organization of Ca(2+) feedback behaviors encompassed by this system, and the long-standing challenges and recent developments in discerning a molecular basis for calmodulation. We conclude by highlighting the considerable synergy between mechanism, biological insight, and promising therapeutics.
Dias,2014 (24788516) Dias J, Van Nguyen N, Georgiev P, Gaub A, Brettschneider J, Cusack S, Kadlec J, Akhtar A "Structural analysis of the KANSL1/WDR5/KANSL2 complex reveals that WDR5 is required for efficient assembly and chromatin targeting of the NSL complex." Genes Dev 2014 May 05
The subunits of the nonspecific lethal (NSL) complex, which include the histone acetyltransferase MOF (males absent on the first), play important roles in various cellular functions, including transcription regulation and stem cell identity maintenance and reprogramming, and are frequently misregulated in disease. Here, we provide the first biochemical and structural insights into the molecular architecture of this large multiprotein assembly. We identified several direct interactions within the complex and show that KANSL1 acts as a scaffold protein interacting with four other subunits, including WDR5, which in turn binds KANSL2. Structural analysis of the KANSL1/WDR5/KANSL2 subcomplex reveals how WDR5 is recruited into the NSL complex via conserved linear motifs of KANSL1 and KANSL2. Using structure-based KANSL1 mutants in transgenic flies, we show that the KANSL1-WDR5 interaction is required for proper assembly, efficient recruitment of the NSL complex to target promoters, and fly viability. Our data clearly show that the interactions of WDR5 with the MOF-containing NSL complex and MLL/COMPASS histone methyltransferase complexes are mutually exclusive. We propose that rather than being a shared subunit, WDR5 plays an important role in assembling distinct histone-modifying complexes with different epigenetic regulatory roles.
Tammsalu,2014 (24782567) Tammsalu T, Matic I, Jaffray EG, Ibrahim AF, Tatham MH, Hay RT "Proteome-wide identification of SUMO2 modification sites." Sci Signal 2014 Apr 30
Posttranslational modification with small ubiquitin-like modifiers (SUMOs) alters the function of proteins involved in diverse cellular processes. SUMO-specific enzymes conjugate SUMOs to lysine residues in target proteins. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteomic scale are lacking. We developed a method that enabled proteome-wide identification of sumoylated lysines that involves the expression of polyhistidine (6His)-tagged SUMO2 with Thr(90) mutated to Lys. Endoproteinase cleavage with Lys-C of 6His-SUMO2(T90K)-modified proteins from human cell lysates produced a diGly remnant on SUMO2(T90K)-conjugated lysines, enabling immunoprecipitation of SUMO2(T90K)-modified peptides and producing a unique mass-to-charge signature. Mass spectrometry analysis of SUMO-enriched peptides revealed more than 1000 sumoylated lysines in 539 proteins, including many functionally related proteins involved in cell cycle, transcription, and DNA repair. Not only can this strategy be used to study the dynamics of sumoylation and other potentially similar posttranslational modifications, but also, these data provide an unprecedented resource for future research on the role of sumoylation in cellular physiology and disease.
Bieging,2014 (24739573) Bieging KT, Mello SS, Attardi LD "Unravelling mechanisms of p53-mediated tumour suppression." Nat Rev Cancer 2014 Apr 24
p53 is a crucial tumour suppressor that responds to diverse stress signals by orchestrating specific cellular responses, including transient cell cycle arrest, cellular senescence and apoptosis, which are all processes associated with tumour suppression. However, recent studies have challenged the relative importance of these canonical cellular responses for p53-mediated tumour suppression and have highlighted roles for p53 in modulating other cellular processes, including metabolism, stem cell maintenance, invasion and metastasis, as well as communication within the tumour microenvironment. In this Opinion article, we discuss the roles of classical p53 functions, as well as emerging p53-regulated processes, in tumour suppression.
Bhandari,2014 (24736845) Bhandari D, Raisch T, Weichenrieder O, Jonas S, Izaurralde E "Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression." Genes Dev 2014 Apr 16
The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4-NOT deadenylase complex. Here, we show that the three human Nanos paralogs (Nanos1-3) interact with the CNOT1 C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1-3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1-3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. Our findings provide the structural basis for the recruitment of the CCR4-NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4-NOT complex as the main effector complex for Nanos function.
Stamos,2014 (24642411) Stamos JL, Chu ML, Enos MD, Shah N, Weis WI "Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6." Elife 2014 Mar 19
Glycogen synthase kinase-3 (GSK-3) is a key regulator of many cellular signaling pathways. Unlike most kinases, GSK-3 is controlled by inhibition rather than by specific activation. In the insulin and several other signaling pathways, phosphorylation of a serine present in a conserved sequence near the amino terminus of GSK-3 generates an auto-inhibitory peptide. In contrast, Wnt/beta-catenin signal transduction requires phosphorylation of Ser/Pro rich sequences present in the Wnt co-receptors LRP5/6, and these motifs inhibit GSK-3 activity. We present crystal structures of GSK-3 bound to its phosphorylated N-terminus and to two of the phosphorylated LRP6 motifs. A conserved loop unique to GSK-3 undergoes a dramatic conformational change that clamps the bound pseudo-substrate peptides, and reveals the mechanism of primed substrate recognition. The structures rationalize target sequence preferences and suggest avenues for the design of inhibitors selective for a subset of pathways regulated by GSK-3. DOI:
Schumacher,2014 (24641320) Schumacher FR, Sorrell FJ, Alessi DR, Bullock AN, Kurz T "Structural and biochemical characterization of the KLHL3-WNK kinase interaction important in blood pressure regulation." Biochem J 2014 May 14
WNK1 [with no lysine (K)] and WNK4 regulate blood pressure by controlling the activity of ion co-transporters in the kidney. Groundbreaking work has revealed that the ubiquitylation and hence levels of WNK isoforms are controlled by a Cullin-RING E3 ubiquitin ligase complex (CRL3KLHL3) that utilizes CUL3 (Cullin3) and its substrate adaptor, KLHL3 (Kelch-like protein 3). Loss-of-function mutations in either CUL3 or KLHL3 cause the hereditary high blood pressure disease Gordon's syndrome by stabilizing WNK isoforms. KLHL3 binds to a highly conserved degron motif located within the C-terminal non-catalytic domain of WNK isoforms. This interaction is essential for ubiquitylation by CRL3KLHL3 and disease-causing mutations in WNK4 and KLHL3 exert their effects on blood pressure by disrupting this interaction. In the present study, we report on the crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif. This reveals an intricate web of interactions between conserved residues on the surface of the Kelch domain beta-propeller and the WNK4 degron motif. Importantly, many of the disease-causing mutations inhibit binding by disrupting critical interface contacts. We also present the structure of the WNK4 degron motif in complex with KLHL2 that has also been reported to bind WNK4. This confirms that KLHL2 interacts with WNK kinases in a similar manner to KLHL3, but strikingly different to how another KLHL protein, KEAP1 (Kelch-like enoyl-CoA hydratase-associated protein 1), binds to its substrate NRF2 (nuclear factor-erythroid 2-related factor 2). The present study provides further insights into how Kelch-like adaptor proteins recognize their substrates and provides a structural basis for how mutations in WNK4 and KLHL3 lead to hypertension.
Panas,2014 (24623412) Panas MD, Ahola T, McInerney GM "The C-terminal repeat domains of nsP3 from the Old World alphaviruses bind directly to G3BP." J Virol 2014 Apr 23
The Old World alphaviruses block stress granule assembly by sequestration of RasGAP SH3-domain binding protein (G3BP). Here, we show that the proline-rich sequences in the hypervariable domain of nonstructural protein 3 (nsP3) of both Semliki Forest virus and Chikungunya virus were dispensable for binding to G3BP. nsP3 variants with or without this domain colocalized with G3BP. Furthermore, we show that the C-terminal repeat motifs of nsP3 were sufficient for G3BP binding.
Beale,2014 (24528869) Beale R, Wise H, Stuart A, Ravenhill BJ, Digard P, Randow F "A LC3-interacting motif in the influenza A virus M2 protein is required to subvert autophagy and maintain virion stability." Cell Host Microbe 2014 Feb 17
Autophagy recycles cellular components and defends cells against intracellular pathogens. While viruses must evade autophagocytic destruction, some viruses can also subvert autophagy for their own benefit. The ability of influenza A virus (IAV) to evade autophagy depends on the Matrix 2 (M2) ion-channel protein. We show that the cytoplasmic tail of IAV M2 interacts directly with the essential autophagy protein LC3 and promotes LC3 relocalization to the unexpected destination of the plasma membrane. LC3 binding is mediated by a highly conserved LC3-interacting region (LIR) in M2. The M2 LIR is required for LC3 redistribution to the plasma membrane in virus-infected cells. Mutations in M2 that abolish LC3 binding interfere with filamentous budding and reduce virion stability. IAV therefore subverts autophagy by mimicking a host short linear protein-protein interaction motif. This strategy may facilitate transmission of infection between organisms by enhancing the stability of viral progeny.
Rogov,2014 (24462201) Rogov V, Dotsch V, Johansen T, Kirkin V "Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy." Mol Cell 2014 Jan 27
Selective autophagy ensures recognition and removal of various cytosolic cargoes. Hence, aggregated proteins, damaged organelles, or pathogens are enclosed into the double-membrane vesicle, the autophagosome, and delivered to the lysosome for degradation. This process is mediated by selective autophagy receptors, such as p62/SQSTM1. These proteins recognize autophagic cargo and, via binding to small ubiquitin-like modifiers (UBLs)--Atg8/LC3/GABARAPs and ATG5--mediate formation of selective autophagosomes. Recently, it was found that UBLs can directly engage the autophagosome nucleation machinery. Here, we review recent findings on selective autophagy and propose a model for selective autophagosome formation in close proximity to cargo.
Paiardini,2014 (24408864) Paiardini A, Aducci P, Cervoni L, Cutruzzola F, Di Lucente C, Janson G, Pascarella S, Rinaldo S, Visconti S, Camoni L "The phytotoxin fusicoccin differently regulates 14-3-3 proteins association to mode III targets." IUBMB Life 2014 Feb 06
Modulation of the interaction of regulatory 14-3-3 proteins to their physiological partners through small cell-permeant molecules is a promising strategy to control cellular processes where 14-3-3s are engaged. Here, we show that the fungal phytotoxin fusicoccin (FC), known to stabilize 14-3-3 association to the plant plasma membrane H(+) -ATPase, is able to stabilize 14-3-3 interaction to several client proteins with a mode III binding motif. Isothermal titration calorimetry analysis of the interaction between 14-3-3s and different peptides reproducing a mode III binding site demonstrated the FC ability to stimulate 14-3-3 the association. Moreover, molecular docking studies provided the structural rationale for the differential FC effect, which exclusively depends on the biochemical properties of the residue in peptide C-terminal position. Our study proposes FC as a promising tool to control cellular processes regulated by 14-3-3 proteins, opening new perspectives on its potential pharmacological applications.
Borchert,2014 (24371076) Borchert S, Czech-Sioli M, Neumann F, Schmidt C, Wimmer P, Dobner T, Grundhoff A, Fischer N "High-affinity Rb binding, p53 inhibition, subcellular localization, and transformation by wild-type or tumor-derived shortened Merkel cell polyomavirus large T antigens." J Virol 2014 Feb 25
Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in approximately 90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE: MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential.
Fukutomi,2014 (24366543) Fukutomi T, Takagi K, Mizushima T, Ohuchi N, Yamamoto M "Kinetic, thermodynamic, and structural characterizations of the association between Nrf2-DLGex degron and Keap1." Mol Cell Biol 2014 Feb 10
Transcription factor Nrf2 (NF-E2-related factor 2) coordinately regulates cytoprotective gene expression, but under unstressed conditions, Nrf2 is degraded rapidly through Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination. Nrf2 harbors two Keap1-binding motifs, DLG and ETGE. Interactions between these two motifs and Keap1 constitute a key regulatory nexus for cellular Nrf2 activity through the formation of a two-site binding hinge-and-latch mechanism. In this study, we determined the minimum Keap1-binding sequence of the DLG motif, the low-affinity latch site, and defined a new DLGex motif that covers a sequence much longer than that previously defined. We have successfully clarified the crystal structure of the Keap1-DC-DLGex complex at 1.6 A. DLGex possesses a complicated helix structure, which interprets well the human-cancer-derived loss-of-function mutations in DLGex. In thermodynamic analyses, Keap1-DLGex binding is characterized as enthalpy and entropy driven, while Keap1-ETGE binding is characterized as purely enthalpy driven. In kinetic analyses, Keap1-DLGex binding follows a fast-association and fast-dissociation model, while Keap1-ETGE binding contains a slow-reaction step that leads to a stable conformation. These results demonstrate that the mode of DLGex binding to Keap1 is distinct from that of ETGE structurally, thermodynamically, and kinetically and support our contention that the DLGex motif serves as a converter transmitting environmental stress to Nrf2 induction as the latch site.
Wild,2013 (24345374) Wild P, McEwan DG, Dikic I "The LC3 interactome at a glance." J Cell Sci 2013 Dec 31
Continuous synthesis of all cellular components requires their constant turnover in order for a cell to achieve homeostasis. To this end, eukaryotic cells are endowed with two degradation pathways - the ubiquitin-proteasome system and the lysosomal pathway. The latter pathway is partly fed by autophagy, which targets intracellular material in distinct vesicles, termed autophagosomes, to the lysosome. Central to this pathway is a set of key autophagy proteins, including the ubiquitin-like modifier Atg8, that orchestrate autophagosome initiation and biogenesis. In higher eukaryotes, the Atg8 family comprises six members known as the light chain 3 (LC3) or gamma-aminobutyric acid (GABA)-receptor-associated protein (GABARAP) proteins. Considerable effort during the last 15 years to decipher the molecular mechanisms that govern autophagy has significantly advanced our understanding of the functioning of this protein family. In this Cell Science at a Glance article and the accompanying poster, we present the current LC3 protein interaction network, which has been and continues to be vital for gaining insight into the regulation of autophagy.
Boschert,2013 (24312339) Boschert V, van Dinther M, Weidauer S, van Pee K, Muth EM, Ten Dijke P, Mueller TD "Mutational analysis of sclerostin shows importance of the flexible loop and the cystine-knot for Wnt-signaling inhibition." PLoS One 2013 Dec 06
The cystine-knot containing protein Sclerostin is an important negative regulator of bone growth and therefore represents a promising therapeutic target. It exerts its biological task by inhibiting the Wnt (wingless and int1) signaling pathway, which participates in bone formation by promoting the differentiation of mesenchymal stem cells to osteoblasts. The core structure of Sclerostin consists of three loops with the first and third loop (Finger 1 and Finger 2) forming a structured beta-sheet and the second loop being unstructured and highly flexible. Biochemical data showed that the flexible loop is important for binding of Sclerostin to Wnt co-receptors of the low-density lipoprotein related-protein family (LRP), by interacting with the Wnt co-receptors LRP5 or -6 it inhibits Wnt signaling. To further examine the structural requirements for Wnt inhibition, we performed an extensive mutational study within all three loops of the Sclerostin core domain involving single and multiple mutations as well as truncation of important regions. By this approach we could confirm the importance of the second loop and especially of amino acids Asn92 and Ile94 for binding to LRP6. Based on a Sclerostin variant found in a Turkish family suffering from Sclerosteosis we generated a Sclerostin mutant with cysteines 84 and 142 exchanged thereby removing the third disulfide bond of the cystine-knot. This mutant binds to LRP6 with reduced binding affinity and also exhibits a strongly reduced inhibitory activity against Wnt1 thereby showing that also elements outside the flexible loop are important for inhibition of Wnt by Sclerostin. Additionally, we examined the effect of the mutations on the inhibition of two different Wnt proteins, Wnt3a and Wnt1. We could detect clear differences in the inhibition of these proteins, suggesting that the mechanism by which Sclerostin antagonizes Wnt1 and Wnt3a is fundamentally different.
Cherry,2013 (24311597) Cherry AL, Finta C, Karlstrom M, Jin Q, Schwend T, Astorga-Wells J, Zubarev RA, Del Campo M, Criswell AR, de Sanctis D, Jovine L, Toftgard R "Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation." Acta Crystallogr D Biol Crystallogr 2013 Dec 06
Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.
Collins,2014 (24290140) Collins KJ, Yuan Z, Kovall RA "Structure and function of the CSL-KyoT2 corepressor complex: a negative regulator of Notch signaling." Structure 2014 Jan 13
Notch refers to a highly conserved cell-to-cell signaling pathway with essential roles in embryonic development and tissue maintenance. Dysfunctional signaling causes human disease, highlighting the importance of pathway regulation. Notch signaling ultimately results in the activation of target genes, which is regulated by the nuclear effector CSL (CBF-1/RBP-J, Su(H), Lag-1). CSL dually functions as an activator and a repressor of transcription through differential interactions with coactivator or corepressor proteins, respectively. Although the structures of CSL-coactivator complexes have been determined, the structures of CSL-corepressor complexes are unknown. Here, using a combination of structural, biophysical, and cellular approaches, we characterize the structure and function of CSL in complex with the corepressor KyoT2. Collectively, our studies provide molecular insights into how KyoT2 binds CSL with high affinity and competes with coactivators, such as Notch, for binding CSL. These studies are important for understanding how CSL functions as both an activator and a repressor of transcription of Notch target genes.
Neuhaus,2014 (24235149) Neuhaus A, Kooshapur H, Wolf J, Meyer NH, Madl T, Saidowsky J, Hambruch E, Lazam A, Jung M, Sattler M, Schliebs W, Erdmann R "A novel Pex14 protein-interacting site of human Pex5 is critical for matrix protein import into peroxisomes." J Biol Chem 2014 Jan 06
Protein import into peroxisomes relies on the import receptor Pex5, which recognizes proteins with a peroxisomal targeting signal 1 (PTS1) in the cytosol and directs them to a docking complex at the peroxisomal membrane. Receptor-cargo docking occurs at the membrane-associated protein Pex14. In human cells, this interaction is mediated by seven conserved diaromatic penta-peptide motifs (WXXX(F/Y) motifs) in the N-terminal half of Pex5 and the N-terminal domain of Pex14. A systematic screening of a Pex5 peptide library by ligand blot analysis revealed a novel Pex5-Pex14 interaction site of Pex5. The novel motif composes the sequence LVAEF with the evolutionarily conserved consensus sequence LVXEF. Replacement of the amino acid LVAEF sequence by alanines strongly affects matrix protein import into peroxisomes in vivo. The NMR structure of a complex of Pex5-(57-71) with the Pex14-N-terminal domain showed that the novel motif binds in a similar alpha-helical orientation as the WXXX(F/Y) motif but that the tryptophan pocket is now occupied by a leucine residue. Surface plasmon resonance analyses revealed 33 times faster dissociation rates for the LVXEF ligand when compared with a WXXX(F/Y) motif. Surprisingly, substitution of the novel motif with the higher affinity WXXX(F/Y) motif impairs protein import into peroxisomes. These data indicate that the distinct kinetic properties of the novel Pex14-binding site in Pex5 are important for processing of the peroxisomal targeting signal 1 receptor at the peroxisomal membrane. The novel Pex14-binding site may represent the initial tethering site of Pex5 from which the cargo-loaded receptor is further processed in a sequential manner.
Zhang,2013 (24217340) Zhang Y, Fu L, Qi X, Zhang Z, Xia Y, Jia J, Jiang J, Zhao Y, Wu G "Structural insight into the mutual recognition and regulation between Suppressor of Fused and Gli/Ci." Nat Commun 2013 Nov 12
Hedgehog (Hh) signalling regulates embryonic development and adult tissue homoeostasis. Mutations of its pathway components including Suppressor of Fused (Sufu) and Gli/Ci predispose to cancers and congenital anomalies. The Sufu-Gli protein complex occupies a central position in the vertebrate Hh signalling pathway, especially in mammals. Here structures of full-length human and Drosophila Sufu, the human Sufu-Gli complex, along with normal mode analysis and FRET measurement results, reveal that Sufu alternates between 'open' and 'closed' conformations. The 'closed' form of Sufu is stabilized by Gli binding and inhibited by Hh treatment, whereas the 'open' state of Sufu is promoted by Gli-dissociation and Hh signalling. Mutations of critical interface residues disrupt the Sufu-Gli complex and prevent Sufu from repressing Gli-mediated transcription, tethering Gli in the cytoplasm and protecting Gli from the 26S proteasome-mediated degradation. Our study thus provides mechanistic insight into the mutual recognition and regulation between Sufu and Gli/Ci.
Warfel,2013 (24189531) Warfel NA, Dolloff NG, Dicker DT, Malysz J, El-Deiry WS "CDK1 stabilizes HIF-1alpha via direct phosphorylation of Ser668 to promote tumor growth." Cell Cycle 2013 Dec 06
Hypoxia-inducible factor 1 (HIF-1) is a major mediator of tumor physiology, and its activation is correlated with tumor progression, metastasis, and therapeutic resistance. HIF-1 is activated in a broad range of solid tumors due to intratumoral hypoxia or genetic alterations that enhance its expression or inhibit its degradation. As a result, decreasing HIF-1alpha expression represents an attractive strategy to sensitize hypoxic tumors to anticancer therapies. Here, we show that cyclin-dependent kinase 1 (CDK1) regulates the expression of HIF-1alpha, independent of its known regulators. Overexpression of CDK1 and/or cyclin B1 is sufficient to stabilize HIF-1alpha under normoxic conditions, whereas inhibition of CDK1 enhances the proteasomal degradation of HIF-1alpha, reducing its half-life and steady-state levels. In vitro kinase assays reveal that CDK1 directly phosphorylates HIF-1alpha at a previously unidentified regulatory site, Ser668. HIF-1alpha is stabilized under normoxic conditions during G 2/M phase via CDK1-mediated phosphorylation of Ser668. A phospho-mimetic construct of HIF-1alpha at Ser668 (S668E) is significantly more stable under both normoxic and hypoxic conditions, resulting in enhanced transcription of HIF-1 target genes and increased tumor cell invasion and migration. Importantly, HIF-1alpha (S668E) displays increased tumor angiogenesis, proliferation, and tumor growth in vivo compared with wild-type HIF-1alpha. Thus, we have identified a novel link between CDK1 and HIF-1alpha that provides a potential molecular explanation for the elevated HIF-1 activity observed in primary and metastatic tumors, independent of hypoxia, and offers a molecular rationale for the clinical translation of CDK inhibitors for use in tumors with constitutively active HIF-1.
McGrath,2013 (24186063) McGrath DA, Balog ER, Koivomagi M, Lucena R, Mai MV, Hirschi A, Kellogg DR, Loog M, Rubin SM "Cks confers specificity to phosphorylation-dependent CDK signaling pathways." Nat Struct Mol Biol 2013 Dec 05
Cks is an evolutionarily conserved protein that regulates cyclin-dependent kinase (CDK) activity. Clarifying the underlying mechanisms and cellular contexts of Cks function is critical because Cks is essential for proper cell growth, and its overexpression has been linked to cancer. We observe that budding-yeast Cks associates with select phosphorylated sequences in cell cycle-regulatory proteins. We characterize the molecular interactions responsible for this specificity and demonstrate that Cks enhances CDK activity in response to specific priming phosphosites. Identification of the binding consensus sequence allows us to identify putative Cks-directed CDK substrates and binding partners. We characterize new Cks-binding sites in the mitotic regulator Wee1 and discover a new role for Cks in regulating CDK activity at mitotic entry. Together, our results portray Cks as a multifunctional phosphoadaptor that serves as a specificity factor for CDK activity.
Koivomagi,2013 (24186061) Koivomagi M, Ord M, Iofik A, Valk E, Venta R, Faustova I, Kivi R, Balog ER, Rubin SM, Loog M "Multisite phosphorylation networks as signal processors for Cdk1." Nat Struct Mol Biol 2013 Dec 05
The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, distribution of serines and threonines as phosphoacceptors and positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phosphoadaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within networks of phosphorylation sites in different targets provides a wide range of possibilities for differential amplification of Cdk1 signals, thus providing a mechanism to generate a wide range of thresholds in the cell cycle.
Klebba,2013 (24184097) Klebba JE, Buster DW, Nguyen AL, Swatkoski S, Gucek M, Rusan NM, Rogers GC "Polo-like kinase 4 autodestructs by generating its Slimb-binding phosphodegron." Curr Biol 2013 Nov 22
Polo-like kinase 4 (Plk4) is a conserved master regulator of centriole assembly. Previously, we found that Drosophila Plk4 protein levels are actively suppressed during interphase. Degradation of interphase Plk4 prevents centriole overduplication and is mediated by the ubiquitin-ligase complex SCF(Slimb/betaTrCP). Since Plk4 stability depends on its activity, we studied the consequences of inactivating Plk4 or perturbing its phosphorylation state within its Slimb-recognition motif (SRM). Mass spectrometry of in-vitro-phosphorylated Plk4 and Plk4 purified from cells reveals that it is directly responsible for extensively autophosphorylating and generating its Slimb-binding phosphodegron. Phosphorylatable residues within this regulatory region were systematically mutated to determine their impact on Plk4 protein levels and centriole duplication when expressed in S2 cells. Notably, autophosphorylation of a single residue (Ser293) within the SRM is critical for Slimb binding and ubiquitination. Our data also demonstrate that autophosphorylation of numerous residues flanking S293 collectively contribute to establishing a high-affinity binding site for SCF(Slimb). Taken together, our findings suggest that Plk4 directly generates its own phosphodegron and can do so without the assistance of an additional kinase(s).
Park,2014 (24141787) Park JY, Singh TR, Nassar N, Zhang F, Freund M, Hanenberg H, Meetei AR, Andreassen PR "Breast cancer-associated missense mutants of the PALB2 WD40 domain, which directly binds RAD51C, RAD51 and BRCA2, disrupt DNA repair." Oncogene 2014 Oct 02
Heterozygous carriers of germ-line mutations in the BRCA2/FANCD1, PALB2/FANCN and RAD51C/FANCO DNA repair genes have an increased lifetime risk of developing breast, ovarian and other cancers; bi-allelic mutations in these genes clinically manifest as Fanconi anemia (FA). Here, we demonstrate that RAD51C is part of a novel protein complex that contains PALB2 and BRCA2. Further, the PALB2 WD40 domain can directly and independently bind RAD51C and BRCA2. To understand the role of these homologous recombination (HR) proteins in DNA repair, we functionally characterize effects of missense mutants of the PALB2 WD40 domain that have been reported in breast cancer patients. In contrast to large truncations of PALB2, which display a complete loss of interaction, the L939W, T1030I and L1143P missense mutants/variants of the PALB2 WD40 domain are associated with altered patterns of direct binding to the RAD51C, RAD51 and BRCA2 HR proteins in biochemical assays. Further, the T1030I missense mutant is unstable, whereas the L939W and L1143P proteins are stable but partially disrupt the PALB2-RAD51C-BRCA2 complex in cells. Functionally, the L939W and L1143P mutants display a decreased capacity for DNA double-strand break-induced HR and an increased cellular sensitivity to ionizing radiation. As further evidence for the functional importance of the HR complex, RAD51C mutants that are associated with cancer susceptibility and FA also display decreased complex formation with PALB2. Together, our results suggest that three different cancer susceptibility and FA proteins function in a DNA repair pathway based upon the PALB2 WD40 domain binding to RAD51C and BRCA2.
Boland,2013 (24121232) Boland A, Chen Y, Raisch T, Jonas S, Kuzuoglu-Ozturk D, Wohlbold L, Weichenrieder O, Izaurralde E "Structure and assembly of the NOT module of the human CCR4-NOT complex." Nat Struct Mol Biol 2013 Nov 07
The CCR4-NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4-NOT complex to mRNA targets.
Dees,2014 (24118232) Dees C, Distler JH "Canonical Wnt signalling as a key regulator of fibrogenesis - implications for targeted therapies?" Exp Dermatol 2014 Jan 17
Canonical Wnt signalling belongs to the so-called morphogen pathways and plays essential roles in development and tissue homeostasis. Being such a crucial regulatory pathway, Wnt signalling is tightly controlled at different levels. However, uncontrolled activation of canonical Wnt signalling has been implicated into the pathogenesis of various human disorders. In the last years, aberrant Wnt signalling has been demonstrated in fibrotic diseases including systemic sclerosis (SSc). In this review, we will discuss the current state of research on canonical Wnt signalling in SSc. Activation of canonical Wnt signalling induces fibroblast activation with subsequent myofibroblast differentiation and excessive collagen release resulting in tissue fibrosis. Genetic or pharmacological blockade of Wnt activation ameliorates experimental fibrosis in different preclinical models. These findings have direct translational implications because several small molecule inhibitors of Wnt signalling are currently evaluated in clinical trials and some already showed first promising results.
Fahraeus,2014 (24096477) Fahraeus R, Olivares-Illana V "MDM2's social network." Oncogene 2014 Aug 28
MDM2 is considered a hub protein due to its capacity to interact with a large number of different partners of which p53 is most well described. MDM2 is an E3 ubiquitin ligase, and many, but not all, of its interactions relate directly to this activity, such as substrates, adaptors or bridges, promoters, inhibitors or complementary factors. Some interactions serve regulatory functions that in response to cellular stresses control the localisation and functions of MDM2 including protein kinases, ribosomal proteins and proteases. Moreover, interactions with nucleotides serve other functions such as mRNA to regulate protein synthesis and DNA to control transcription. To perform such a pleiotropic panorama of different functions, MDM2 is subjected to a multitude of post-translational modifications and is expressed in different isoforms. The large and diverse interactome is made possible due to the plasticity of MDM2 and in this review we have listed the MDM2 interactions until now and we will discuss how this multifaceted protein can interact with such a variety of substrates to provide a key intermediary role in different signalling pathways.
Sasaki-Osugi,2013 (24078636) Sasaki-Osugi K, Imoto C, Takahara T, Shibata H, Maki M "Nuclear ALG-2 protein interacts with Ca2+ homeostasis endoplasmic reticulum protein (CHERP) Ca2+-dependently and participates in regulation of alternative splicing of inositol trisphosphate receptor type 1 (IP3R1) pre-mRNA." J Biol Chem 2013 Nov 18
The intracellular Ca(2+) signaling pathway is important for the control of broad cellular processes from fertilization to cell death. ALG-2 is a Ca(2+)-binding protein that contains five serially repeated EF-hand motifs and interacts with various proteins in a Ca(2+)-dependent manner. Although ALG-2 is present both in the cytoplasm and in the nucleus, little is known about its nuclear function. Ca(2+) homeostasis endoplasmic reticulum protein (CHERP) was first identified as an endoplasmic reticulum protein that regulates intracellular Ca(2+) mobilization in human cells, but recent proteomics data suggest an association between CHERP and spliceosomes. Here, we report that CHERP, containing a Pro-rich region and a phosphorylated Ser/Arg-rich RS-like domain, is a novel Ca(2+)-dependent ALG-2-interactive target in the nucleus. Immunofluorescence microscopic analysis revealed localization of CHERP to the nucleoplasm with prominent accumulation at nuclear speckles, which are the sites of storage and modification for pre-mRNA splicing factors. Live cell time-lapse imaging showed that nuclear ALG-2 was recruited to the CHERP-localizing speckles upon Ca(2+) mobilization. Results of co-immunoprecipitation assays revealed binding of CHERP to a phosphorylated form of RNA polymerase II. Knockdown of CHERP or ALG-2 in HT1080 cells resulted in generation of alternatively spliced isoforms of the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) pre-mRNA that included exons 41 and 42 in addition to the major isoform lacking exons 40-42. Furthermore, binding between CHERP and IP3R1 RNA was detected by an RNA immunoprecipitation assay using a polyclonal antibody against CHERP. These results indicate that CHERP and ALG-2 participate in regulation of alternative splicing of IP3R1 pre-mRNA and provide new insights into post-transcriptional regulation of splicing variants in Ca(2+) signaling pathways.
Braun,2013 (24043761) Braun L, Brenier-Pinchart MP, Yogavel M, Curt-Varesano A, Curt-Bertini RL, Hussain T, Kieffer-Jaquinod S, Coute Y, Pelloux H, Tardieux I, Sharma A, Belrhali H, Bougdour A, Hakimi MA "A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation." J Exp Med 2013 Sep 24
Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite that resides inside a parasitophorous vacuole. During infection, Toxoplasma actively remodels the transcriptome of its hosting cells with profound and coupled impact on the host immune response. We report that Toxoplasma secretes GRA24, a novel dense granule protein which traffics from the vacuole to the host cell nucleus. Once released into the host cell, GRA24 has the unique ability to trigger prolonged autophosphorylation and nuclear translocation of the host cell p38alpha MAP kinase. This noncanonical kinetics of p38alpha activation correlates with the up-regulation of the transcription factors Egr-1 and c-Fos and the correlated synthesis of key proinflammatory cytokines, including interleukin-12 and the chemokine MCP-1, both known to control early parasite replication in vivo. Remarkably, the GRA24-p38alpha complex is defined by peculiar structural features and uncovers a new regulatory signaling path distinct from the MAPK signaling cascade and otherwise commonly activated by stress-related stimuli or various intracellular microbes.
De Nicola,2013 (24037507) De Nicola GF, Martin ED, Chaikuad A, Bassi R, Clark J, Martino L, Verma S, Sicard P, Tata R, Atkinson RA, Knapp S, Conte MR, Marber MS "Mechanism and consequence of the autoactivation of p38alpha mitogen-activated protein kinase promoted by TAB1." Nat Struct Mol Biol 2013 Oct 07
p38alpha mitogen-activated protein kinase (p38alpha) is activated by a variety of mechanisms, including autophosphorylation initiated by TGFbeta-activated kinase 1 binding protein 1 (TAB1) during myocardial ischemia and other stresses. Chemical-genetic approaches and coexpression in mammalian, bacterial and cell-free systems revealed that mouse p38alpha autophosphorylation occurs in cis by direct interaction with TAB1(371-416). In isolated rat cardiac myocytes and perfused mouse hearts, TAT-TAB1(371-416) rapidly activates p38 and profoundly perturbs function. Crystal structures and characterization in solution revealed a bipartite docking site for TAB1 in the p38alpha C-terminal kinase lobe. TAB1 binding stabilizes active p38alpha and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif, allowing autophosphorylation in cis. Interference with p38alpha recognition by TAB1 abolishes its cardiac toxicity. Such intervention could potentially circumvent the drawbacks of clinical pharmacological inhibitors of p38 catalytic activity.
Ran,2013 (24027329) Ran X, Bian X, Ji Y, Yan X, Yang F, Li F "White spot syndrome virus IE1 and WSV056 modulate the G1/S transition by binding to the host retinoblastoma protein." J Virol 2013 Nov 04
DNA viruses often target cellular proteins to modulate host cell cycles and facilitate viral genome replication. However, whether proliferation of white spot syndrome virus (WSSV) requires regulation of the host cell cycle remains unclear. In the present study, we show that two WSSV paralogs, IE1 and WSV056, can interact with Litopenaeus vannamei retinoblastoma (Rb)-like protein (lv-RBL) through the conserved LxCxE motif. Further investigation revealed that IE1 and WSV056 could also bind to Drosophila retinoblastoma family protein 1 (RBF1) in a manner similar to how they bind to lv-RBL. Using the Drosophila RBF-E2F pathway as a model system, we demonstrated that both IE1 and WSV056 could sequester RBF1 from Drosophila E2F transcription factor 1 (E2F1) and subsequently activate E2F1 to stimulate the G1/S transition. Our findings provide the first evidence that WSSV may regulate cell cycle progression by targeting the Rb-E2F pathway.
Gordon,2013 (23981301) Gordon EA, Whisenant TC, Zeller M, Kaake RM, Gordon WM, Krotee P, Patel V, Huang L, Baldi P, Bardwell L "Combining docking site and phosphosite predictions to find new substrates: identification of smoothelin-like-2 (SMTNL2) as a c-Jun N-terminal kinase (JNK) substrate." Cell Signal 2013 Oct 21
Specific docking interactions between mitogen-activated protein kinases (MAPKs), their regulators, and their downstream substrates, are crucial for efficient and accurate signal transmission. To identify novel substrates of the c-Jun N-terminal kinase (JNK) family of MAPKs, we searched the human genome for proteins that contained (1), a predicted JNK-docking site (D-site); and (2), a cluster of putative JNK target phosphosites located close to the D-site. Here we describe a novel JNK substrate that emerged from this analysis, the functionally uncharacterized protein smoothelin-like 2 (SMTNL2). SMTNL2 protein bound with high-affinity to multiple MAPKs including JNK1-3 and ERK2; furthermore, the identity of conserved amino acids in the predicted docking site (residues 180-193) was necessary for this high-affinity binding. In addition, purified full-length SMTNL2 protein was phosphorylated by JNK1-3 in vitro, and this required the integrity of the D-site. Using mass spectrometry and mutagenesis, we identified four D-site-dependent phosphoacceptor sites in close proximity to the docking site, at S217, S241, T236 and T239. A short peptide comprised of the SMTNL2 D-site inhibited JNK-mediated phosphorylation of the ATF2 transcription factor, showing that SMTNL2 can compete with other substrates for JNK binding. Moreover, when transfected into HEK293 cells, SMTNL2 was phosphorylated by endogenous JNK in a D-site dependent manner, on the same residues identified in vitro. SMTNL2 protein was expressed in many mammalian tissues, with a notably high expression in skeletal muscle. Consistent with the hypothesis that SMTNL2 has a function in skeletal muscle, SMTNL2 protein expression was strongly induced during the transition from myoblasts to myotubes in differentiating C2C12 cells.
Esteban,2013 (23933132) Esteban V, Martin MJ, Blanco L "The BRCT domain and the specific loop 1 of human Polmu are targets of Cdk2/cyclin A phosphorylation." DNA Repair (Amst) 2013 Oct 14
Human family X polymerases contribute both to genomic stability and variability through their specialized functions in DNA repair. Polmu participates in the repair of spontaneous double strand breaks (DSB) by non homologous end-joining (NHEJ), and also in the V(D)J recombination process after programmed DSBs. Polmu plays this dual role due to its template-dependent and terminal transferase (template-independent) polymerization activities. In this study we evaluated if Polmu could be regulated by Cdk phosphorylation along the cell cycle. In vitro kinase assays showed that the S phase-associated Cdk2/cyclin A complex was able to phosphorylate Polmu. We identified Ser12, Thr21 (located in the BRCT domain) and Ser372 (located in loop1) as the target residues. Mutation of these residues to alanine indicated that Ser372 is the main phosphorylation site. Mobilization of loop1, which mediates DNA end micro-synapsis, is crucial both for terminal transferase and NHEJ. Interestingly, the phospho-mimicking S372E mutation specifically impaired these activities. Our evidences suggest that Polmu could be regulated in vivo by phosphorylation of the BRCT domain (Ser12/Thr21) and of Ser372, affecting the function of loop1. Consequently, Polmu's most distinctive activities would be turned off at specific cell-cycle phases (S and G2), when these promiscuous functions might be harmful to the cell.
Francis,2013 (23932588) Francis DM, Kumar GS, Koveal D, Tortajada A, Page R, Peti W "The differential regulation of p38alpha by the neuronal kinase interaction motif protein tyrosine phosphatases, a detailed molecular study." Structure 2013 Sep 09
The MAP kinase p38alpha is essential for neuronal signaling. To better understand the molecular regulation of p38alpha we used atomistic and molecular techniques to determine the structural basis of p38alpha regulation by the two neuronal tyrosine phosphatases, PTPSL/PTPBR7 (PTPRR) and STEP (PTPN5). We show that, despite the fact that PTPSL and STEP belong to the same family of regulatory proteins, they interact with p38alpha differently and their distinct molecular interactions explain their different catalytic activities. Although the interaction of PTPSL with p38alpha is similar to that of the previously described p38alpha:HePTP (PTPN7) complex, STEP binds and regulates p38alpha in an unexpected manner. Using NMR and small-angle X-ray scattering data, we generated a model of the p38alpha:STEP complex and define molecular differences between its resting and active states. Together, these results provide insights into molecular regulation of p38alpha by key regulatory proteins.
Okumura,2013 (23924735) Okumura M, Katsuyama AM, Shibata H, Maki M "VPS37 isoforms differentially modulate the ternary complex formation of ALIX, ALG-2, and ESCRT-I." Biosci Biotechnol Biochem 2013 Aug 27
The endosomal sorting complex required for transport (ESCRT) system comprises a series of protein complexes that play essential roles in multivesicular body (MVB) sorting of ubiquitylated membrane proteins, enveloped RNA virus budding, and cytokinesis in mammalian cells. The complex, named ESCRT-I, consists of four subunits (TSG101, VPS28, VPS37, and MVB12). There are four VPS37 isoforms. We have reported that ALIX (an ALG-2-interacting protein and accessory protein in the ESCRT system) is physically linked with TSG101 by ALG-2 in a Ca(2)(+)-dependent manner, but the role of ALG-2 as an adaptor protein for the ESCRT-I complex remains unknown. To characterize this adaptor function, initially we investigated the binding of ALG-2 to ESCRT-I complexes containing each one of the four different VPS37 isoforms by two approaches: first, Far-Western blot analysis with biotin-labeled ALG-2 probe, and second, a pulldown assay to determine the binding of the four recombinant ESCRT-I complexes to Strep-tagged ALG-2 after co-expression in HEK293T cells. VPS37B and VPS37C appeared to interact with ALG-2 in a stronger manner than TSG101 does. The results of in vitro binding assays using purified recombinant proteins indicated that ALG-2 functions as a Ca(2)(+)-dependent adaptor protein that bridges ALIX and ESCRT-I to form a ternary complex, ESCRT-I/ALIX/ALG-2.
Birgisdottir,2013 (23908376) Birgisdottir AB, Lamark T, Johansen T "The LIR motif - crucial for selective autophagy." J Cell Sci 2013 Aug 02
(Macro)autophagy is a fundamental degradation process for macromolecules and organelles of vital importance for cell and tissue homeostasis. Autophagy research has gained a strong momentum in recent years because of its relevance to cancer, neurodegenerative diseases, muscular dystrophy, lipid storage disorders, development, ageing and innate immunity. Autophagy has traditionally been thought of as a bulk degradation process that is mobilized upon nutritional starvation to replenish the cell with building blocks and keep up with the energy demand. This view has recently changed dramatically following an array of papers describing various forms of selective autophagy. A main driving force has been the discovery of specific autophagy receptors that sequester cargo into forming autophagosomes (phagophores). At the heart of this selectivity lies the LC3-interacting region (LIR) motif, which ensures the targeting of autophagy receptors to LC3 (or other ATG8 family proteins) anchored in the phagophore membrane. LIR-containing proteins include cargo receptors, members of the basal autophagy apparatus, proteins associated with vesicles and of their transport, Rab GTPase-activating proteins (GAPs) and specific signaling proteins that are degraded by selective autophagy. Here, we comment on these new insights and focus on the interactions of LIR-containing proteins with members of the ATG8 protein family.
Cino,2013 (23892546) Cino EA, Killoran RC, Karttunen M, Choy WY "Binding of disordered proteins to a protein hub." Sci Rep 2013 Jul 29
A small number of proteins, called hubs, have high connectivity and are essential for interactome functionality and integrity. Keap1 is a crucial hub in the oxidative stress response and apoptosis. The Kelch domain of Keap1 preferentially binds to disordered regions of its partners, which share similar binding motifs, but have a wide range of binding affinities. Isothermal titration calorimetry (ITC) and multi-microsecond molecular dynamics (MD) simulations were used to determine the factors that govern the affinity of all currently known disordered binding partners to Kelch. Our results show that the affinities to this hub are largely determined by the extent of preformed bound state-like conformation in the free state structures of these disordered targets. Based on our findings, we have designed a high-affinity peptide that can specifically disrupt the Keap1-NRF2 interaction and has the potential for therapeutic applications.
Andersen,2013 (23881912) Andersen OM, Dagil R, Kragelund BB "New horizons for lipoprotein receptors: communication by beta-propellers." J Lipid Res 2013 Sep 12
The lipoprotein receptor (LR) family constitutes a large group of structurally closely related receptors with broad ligand-binding specificity. Traditionally, ligand binding to LRs has been anticipated to involve merely the complement type repeat (CR)-domains omnipresent in the family. Recently, this dogma has transformed with the observation that beta-propellers of some LRs actively engage in complex formation too. Based on an in-depth decomposition of current structures and sequences, we suggest that exploitation of the beta-propellers as binding targets depends on receptor subgroups. In particular, we highlight the shutter mechanism of beta-propellers as a general recognition motif for NxI-containing ligands, and we present indications that the generalized beta-propeller-induced ligand release mechanism is not applicable for the larger LRs. For the giant LR members, we present evidence that their beta-propellers may also actively engage in ligand binding. We therefore advocate for an increased focus on solving the structure-function relationship of this group of important biological receptors.
LaConte,2013 (23863172) LaConte L, Mukherjee K "Structural constraints and functional divergences in CASK evolution." Biochem Soc Trans 2013 Jul 18
CASK (Ca2+/calmodulin-activated serine kinase) is a synaptic protein that interacts with the cytosolic tail of adhesion molecules such as neurexins, syncam and syndecans. It belongs to the MAGUK (membrane-associated guanylate kinase) family of scaffolding proteins which are known to decorate cell-cell junctions. CASK is an essential gene in mammals, critical for neurodevelopment. Mutations in the CASK gene in humans result in phenotypes that range from intellectual disability to lethality. Despite its importance, CASK has a single genetic isoform located in the short arm of the X chromosome near an evolutionary breakpoint. Surprisingly, CASK is a non-essential gene in invertebrates and displays functional divergence. In the present article, we describe the phylogenetic differences in existing CASK orthologues. The CASK gene has undergone a huge expansion in size (~55-fold). Almost all of this expansion is a direct result of an increase in the size of the introns. The coding region of CASK orthologues, and hence the protein, exhibit a high degree of evolutionary conservation. Within the protein, domain arrangement is completely conserved and substitution rates are higher in the connecting loop regions [L27 (Lin2, Lin7)] than within the domain. Our analyses of single residue substitutions and genotype-phenotype relationships suggest that, other than intronic expansion, the dramatic functional changes of CASK are driven by subtle (non-radical) primary structure changes within the CASK protein and concomitant changes in its protein interactors.
Yin,2013 (23850291) Yin Q, Sester DP, Tian Y, Hsiao YS, Lu A, Cridland JA, Sagulenko V, Thygesen SJ, Choubey D, Hornung V, Walz T, Stacey KJ, Wu H "Molecular mechanism for p202-mediated specific inhibition of AIM2 inflammasome activation." Cell Rep 2013 Jul 29
Mouse p202 containing two hemopoietic expression, interferon inducibility, nuclear localization (HIN) domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found that only HIN1 of p202 binds double-stranded DNA (dsDNA), while HIN2 forms a homotetramer. Crystal structures of HIN1 revealed that dsDNA is bound on face opposite the site used in AIM2 and IFI16. The structure of HIN2 revealed a dimer of dimers, the face analogous to the HIN1 dsDNA binding site being a dimerization interface. Electron microscopy imaging showed that HIN1 is flexibly linked to HIN2 in p202, and tetramerization provided enhanced avidity for dsDNA. Surprisingly, HIN2 of p202 interacts with the AIM HIN domain. We propose that this results in a spatial separation of the AIM2 pyrin domains, and indeed p202 prevented the dsDNA-dependent clustering of apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) and AIM2 inflammasome activation. We hypothesize that while p202 was evolutionarily selected to limit AIM2-mediated inflammation in some mouse strains, the same mechanism contributes to increased interferon production and lupus susceptibility.
Takahashi,2013 (23838290) Takahashi D, Mori T, Wakabayashi M, Mori Y, Susa K, Zeniya M, Sohara E, Rai T, Sasaki S, Uchida S "KLHL2 interacts with and ubiquitinates WNK kinases." Biochem Biophys Res Commun 2013 Aug 01
Mutations in the WNK1 and WNK4 genes result in an inherited hypertensive disease, pseudohypoaldosteronism type II (PHAII). Recently, the KLHL3 and Cullin3 genes were also identified as responsible genes for PHAII. Although we have reported that WNK4 is a substrate for the KLHL3-Cullin3 E3 ligase complex, it is not clear whether all of the WNK isoforms are regulated only by KLHL3. To explore the interaction of WNKs and other Kelch-like proteins, we focused on KLHL2 (Mayven), a human homolog of Drosophila Kelch that shares the highest similarity with KLHL3. We found that KLHL2, as well as KLHL3, was co-immunoprecipitated with all four WNK isoforms. The direct interaction of KLHL2 with WNKs was confirmed on fluorescence correlation spectroscopy. Co-expression of KLHL2 and Cullin3 decreased the abundance of WNK1, WNK3 and WNK4 within HEK293T cells, and a significant increase of WNK4 ubiquitination by KLHL2 and Cullin3 was observed both in HEK293T cells and in an in vitro ubiquitination assay. These results suggest that KLHL2-Cullin3 also functions as an E3-ligase for WNK isoforms within the body.
Molzan,2013 (23808890) Molzan M, Kasper S, Roglin L, Skwarczynska M, Sassa T, Inoue T, Breitenbuecher F, Ohkanda J, Kato N, Schuler M, Ottmann C "Stabilization of physical RAF/14-3-3 interaction by cotylenin A as treatment strategy for RAS mutant cancers." ACS Chem Biol 2013 Sep 23
One-third of all human cancers harbor somatic RAS mutations. This leads to aberrant activation of downstream signaling pathways involving the RAF kinases. Current ATP-competitive RAF inhibitors are active in cancers with somatic RAF mutations, such as BRAF(V600) mutant melanomas. However, they paradoxically promote the growth of RAS mutant tumors, partly due to the complex interplay between different homo- and heterodimers of A-RAF, B-RAF, and C-RAF. Based on pathway analysis and structure-guided compound identification, we describe the natural product cotylenin-A (CN-A) as stabilizer of the physical interaction of C-RAF with 14-3-3 proteins. CN-A binds to inhibitory 14-3-3 interaction sites of C-RAF, pSer233, and pSer259, but not to the activating interaction site, pSer621. While CN-A alone is inactive in RAS mutant cancer models, combined treatment with CN-A and an anti-EGFR antibody synergistically suppresses tumor growth in vitro and in vivo. This defines a novel pharmacologic strategy for treatment of RAS mutant cancers.
Rogov,2013 (23805866) Rogov VV, Suzuki H, Fiskin E, Wild P, Kniss A, Rozenknop A, Kato R, Kawasaki M, McEwan DG, Lohr F, Guntert P, Dikic I, Wakatsuki S, Dotsch V "Structural basis for phosphorylation-triggered autophagic clearance of Salmonella." Biochem J 2013 Oct 10
Selective autophagy is mediated by the interaction of autophagy modifiers and autophagy receptors that also bind to ubiquitinated cargo. Optineurin is an autophagy receptor that plays a role in the clearance of cytosolic Salmonella. The interaction between receptors and modifiers is often relatively weak, with typical values for the dissociation constant in the low micromolar range. The interaction of optineurin with autophagy modifiers is even weaker, but can be significantly enhanced through phosphorylation by the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor kappaB activator]-binding kinase 1}. In the present study we describe the NMR and crystal structures of the autophagy modifier LC3B (microtubule-associated protein light chain 3 beta) in complex with the LC3 interaction region of optineurin either phosphorylated or bearing phospho-mimicking mutations. The structures show that the negative charge induced by phosphorylation is recognized by the side chains of Arg(1)(1) and Lys(5)(1) in LC3B. Further mutational analysis suggests that the replacement of the canonical tryptophan residue side chain of autophagy receptors with the smaller phenylalanine side chain in optineurin significantly weakens its interaction with the autophagy modifier LC3B. Through phosphorylation of serine residues directly N-terminally located to the phenylalanine residue, the affinity is increased to the level normally seen for receptor-modifier interactions. Phosphorylation, therefore, acts as a switch for optineurin-based selective autophagy.
Xu,2013 (23789096) Xu P, Raetz EA, Kitagawa M, Virshup DM, Lee SH "BUBR1 recruits PP2A via the B56 family of targeting subunits to promote chromosome congression." Biol Open 2013 Jun 21
BUBR1 is a mitotic phosphoprotein essential for the maintenance of chromosome stability by promoting chromosome congression and proper kinetochore-microtubule (K-fiber) attachment, but the underlying mechanism(s) has remained elusive. Here we identify BUBR1 as a binding partner of the B56 family of Protein Phosphatase 2A regulatory subunits. The interaction between BUBR1 and the B56 family is required for chromosome congression, since point mutations in BUBR1 that block B56 binding abolish chromosome congression. The BUBR1:B56-PP2A complex opposes Aurora B kinase activity, since loss of the complex can be reverted by inhibiting Aurora B. Importantly, we show that the failure of BUBR1 to recruit B56-PP2A also contributes to the chromosome congression defects found in cells derived from patients with the Mosaic Variegated Aneuploidy (MVA) syndrome. Together, we propose that B56-PP2A is a key mediator of BUBR1's role in chromosome congression and functions by antagonizing Aurora B activity at the kinetochore for establishing stable kinetochore-microtubule attachment at the metaphase plate.
McCubrey,2014 (23778311) McCubrey JA, Steelman LS, Bertrand FE, Davis NM, Abrams SL, Montalto G, D'Assoro AB, Libra M, Nicoletti F, Maestro R, Basecke J, Cocco L, Cervello M, Martelli AM "Multifaceted roles of GSK-3 and Wnt/beta-catenin in hematopoiesis and leukemogenesis: opportunities for therapeutic intervention." Leukemia 2014 Jan 08
Glycogen synthase kinase-3 (GSK-3) is well documented to participate in a complex array of critical cellular processes. It was initially identified in rat skeletal muscle as a serine/threonine kinase that phosphorylated and inactivated glycogen synthase. This versatile protein is involved in numerous signaling pathways that influence metabolism, embryogenesis, differentiation, migration, cell cycle progression and survival. Recently, GSK-3 has been implicated in leukemia stem cell pathophysiology and may be an appropriate target for its eradication. In this review, we will discuss the roles that GSK-3 plays in hematopoiesis and leukemogenesis as how this pivotal kinase can interact with multiple signaling pathways such as: Wnt/beta-catenin, phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR), Ras/Raf/MEK/extracellular signal-regulated kinase (ERK), Notch and others. Moreover, we will discuss how targeting GSK-3 and these other pathways can improve leukemia therapy and may overcome therapeutic resistance. In summary, GSK-3 is a crucial regulatory kinase interacting with multiple pathways to control various physiological processes, as well as leukemia stem cells, leukemia progression and therapeutic resistance. GSK-3 and Wnt are clearly intriguing therapeutic targets.
Qian,2013 (23746640) Qian J, Beullens M, Lesage B, Bollen M "Aurora B defines its own chromosomal targeting by opposing the recruitment of the phosphatase scaffold Repo-Man." Curr Biol 2013 Jun 21
Aurora B is the catalytic subunit of the chromosomal passenger complex (CPC), which coordinates mitotic processes through phosphorylation of key regulatory proteins. In prometaphase, the CPC is enriched at the centromeres to regulate the spindle checkpoint and kinetochore-microtubule interactions. Centromeric CPC binds to histone H3 that is phosphorylated at T3 (H3T3ph) by Aurora B-stimulated Haspin. PP1/Repo-Man acts antagonistically to Haspin and dephosphorylates H3T3ph at the chromosome arms but is somehow prevented from causing a net dephosphorylation of centromeric H3T3ph during prometaphase. Here, we show that Aurora B phosphorylates Repo-Man at S893, preventing its recruitment by histones. We also identify PP2A as a mitotic interactor of Repo-Man that dephosphorylates S893 and thereby promotes the targeting of Repo-Man to chromosomes and the dephosphorylation of H3T3ph by PP1. Thus, Repo-Man-associated PP1 and PP2A collaborate to oppose the chromosomal targeting of Aurora B. We propose that the reciprocal feedback regulation of Haspin and Repo-Man by Aurora B generates a robust bistable response that culminates in the centromeric targeting of the CPC during prometaphase.
He,2013 (23707760) He J, Chao WC, Zhang Z, Yang J, Cronin N, Barford D "Insights into degron recognition by APC/C coactivators from the structure of an Acm1-Cdh1 complex." Mol Cell 2013 Jun 10
The anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs--predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor.
Dhanoa,2013 (23676014) Dhanoa BS, Cogliati T, Satish AG, Bruford EA, Friedman JS "Update on the Kelch-like (KLHL) gene family." Hum Genomics 2013 May 21
The Kelch-like (KLHL) gene family encodes a group of proteins that generally possess a BTB/POZ domain, a BACK domain, and five to six Kelch motifs. BTB domains facilitate protein binding and dimerization. The BACK domain has no known function yet is of functional importance since mutations in this domain are associated with disease. Kelch domains form a tertiary structure of beta-propellers that have a role in extracellular functions, morphology, and binding to other proteins. Presently, 42 KLHL genes have been classified by the HUGO Gene Nomenclature Committee (HGNC), and they are found across multiple human chromosomes. The KLHL family is conserved throughout evolution. Phylogenetic analysis of KLHL family members suggests that it can be subdivided into three subgroups with KLHL11 as the oldest member and KLHL9 as the youngest. Several KLHL proteins bind to the E3 ligase cullin 3 and are known to be involved in ubiquitination. KLHL genes are responsible for several Mendelian diseases and have been associated with cancer. Further investigation of this family of proteins will likely provide valuable insights into basic biology and human disease.
Wu,2013 (23665031) Wu G, Peng JB "Disease-causing mutations in KLHL3 impair its effect on WNK4 degradation." FEBS Lett 2013 Jun 14
Mutations in with-no-lysine (K) kinase 4 (WNK4) and a ubiquitin E3 ligase complex component kelch-like 3 (KLHL3) both cause pseudohypoaldosteronism II (PHAII), a hereditary form of hypertension. We determined whether WNK4 or its effector is regulated by KLHL3 in Xenopus oocytes. KLHL3 inhibited the positive effect of WNK4 on Na(+)-Cl(-) cotransporter (NCC) by decreasing WNK4 protein abundance without decreasing that of NCC and the downstream kinase OSR1 directly. Ubiquitination and degradation of WNK4 were induced by KLHL3. The effect of KLHL3 on WNK4 degradation was blocked by a dominant negative form of cullin 3. All five PHAII mutations of KLHL3 tested disrupted the regulation on WNK4. We conclude that KLHL3 is a substrate adaptor for WNK4 in a ubiquitin E3 ligase complex.
Wang,2013 (23636324) Wang C, Wu H, Katritch V, Han GW, Huang XP, Liu W, Siu FY, Roth BL, Cherezov V, Stevens RC "Structure of the human smoothened receptor bound to an antitumour agent." Nature 2013 May 16
The smoothened (SMO) receptor, a key signal transducer in the hedgehog signalling pathway, is responsible for the maintenance of normal embryonic development and is implicated in carcinogenesis. It is classified as a class frizzled (class F) G-protein-coupled receptor (GPCR), although the canonical hedgehog signalling pathway involves the GLI transcription factors and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure of the transmembrane domain of the human SMO receptor bound to the small-molecule antagonist LY2940680 at 2.5 A resolution. Although the SMO receptor shares the seven-transmembrane helical fold, most of the conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulphide bonds. The ligand binds at the extracellular end of the seven-transmembrane-helix bundle and forms extensive contacts with the loops.
Taiakina,2013 (23626724) Taiakina V, Boone AN, Fux J, Senatore A, Weber-Adrian D, Guillemette JG, Spafford JD "The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels." PLoS One 2013 Apr 29
NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca(2+) concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca(2+)-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.
Anders,2013 (23601647) Anders C, Higuchi Y, Koschinsky K, Bartel M, Schumacher B, Thiel P, Nitta H, Preisig-Muller R, Schlichthorl G, Renigunta V, Ohkanda J, Daut J, Kato N, Ottmann C "A semisynthetic fusicoccane stabilizes a protein-protein interaction and enhances the expression of K+ channels at the cell surface." Chem Biol 2013 Apr 22
Small-molecule stabilization of protein-protein interactions is an emerging field in chemical biology. We show how fusicoccanes, originally identified as fungal toxins acting on plants, promote the interaction of 14-3-3 proteins with the human potassium channel TASK-3 and present a semisynthetic fusicoccane derivative (FC-THF) that targets the 14-3-3 recognition motif (mode 3) in TASK-3. In the presence of FC-THF, the binding of 14-3-3 proteins to TASK-3 was increased 19-fold and protein crystallography provided the atomic details of the effects of FC-THF on this interaction. We also tested the functional effects of FC-THF on TASK channels heterologously expressed in Xenopus oocytes. Incubation with 10 muM FC-THF was found to promote the transport of TASK channels to the cell membrane, leading to a significantly higher density of channels at the surface membrane and increased potassium current.
Tidow,2013 (23601118) Tidow H, Nissen P "Structural diversity of calmodulin binding to its target sites." FEBS J 2013 Oct 16
Calmodulin (CaM) is a ubiquitous, highly conserved, eukaryotic protein that binds to and regulates a number of diverse target proteins involved in different functions such as metabolism, muscle contraction, apoptosis, memory, inflammation and the immune response. In this minireview, we analyze the large number of CaM-complex structures deposited in the Protein Data Bank (i.e. crystal and nuclear magnetic resonance structures) to gain insight into the structural diversity of CaM-binding sites and mechanisms, such as those for CaM-activated protein kinases and phosphatases, voltage-gated Ca(2+)-channels and the plasma membrane Ca(2+)-ATPase.
Jia,2013 (23598156) Jia L, Kim S, Yu H "Tracking spindle checkpoint signals from kinetochores to APC/C." Trends Biochem Sci 2013 May 27
Accurate chromosome segregation during mitosis is critical for maintaining genomic stability. The kinetochore--a large protein assembly on centromeric chromatin--functions as the docking site for spindle microtubules and a signaling hub for the spindle checkpoint. At metaphase, spindle microtubules from opposing spindle poles capture each pair of sister kinetochores, exert pulling forces, and create tension across sister kinetochores. The spindle checkpoint detects improper kinetochore-microtubule attachments and translates these defects into biochemical activities that inhibit the anaphase-promoting complex or cyclosome (APC/C) throughout the cell to delay anaphase onset. A deficient spindle checkpoint leads to premature sister-chromatid separation and aneuploidy. Here, we review recent progress on the generation, propagation, transmission, and silencing of the spindle checkpoint signals from kinetochores to APC/C.
Primorac,2013 (23589490) Primorac I, Musacchio A "Panta rhei: the APC/C at steady state." J Cell Biol 2013 Apr 16
The anaphase-promoting complex or cyclosome (APC/C) is a conserved, multisubunit E3 ubiquitin (Ub) ligase that is active both in dividing and in postmitotic cells. Its contributions to life are especially well studied in the domain of cell division, in which the APC/C lies at the epicenter of a regulatory network that controls the directionality and timing of cell cycle events. Biochemical and structural work is shedding light on the overall organization of APC/C subunits and on the mechanism of substrate recognition and Ub chain initiation and extension as well as on the molecular mechanisms of a checkpoint that seizes control of APC/C activity during mitosis. Here, we review how these recent advancements are modifying our understanding of the APC/C.
McCullough,2013 (23527693) McCullough J, Colf LA, Sundquist WI "Membrane Fission Reactions of the Mammalian ESCRT Pathway." Annu Rev Biochem 2013 Mar 26
The endosomal sorting complexes required for transport (ESCRT) pathway was initially defined in yeast genetic screens that identified the factors necessary to sort membrane proteins into intraluminal endosomal vesicles. Subsequent studies have revealed that the mammalian ESCRT pathway also functions in a series of other key cellular processes, including formation of extracellular microvesicles, enveloped virus budding, and the abscission stage of cytokinesis. The core ESCRT machinery comprises Bro1 family proteins and ESCRT-I, ESCRT-II, ESCRT-III, and VPS4. Site-specific adaptors recruit these soluble factors to assemble on different cellular membranes, where they carry out membrane fission reactions. ESCRT-III proteins form filaments that draw membranes together from the cytoplasmic face, and mechanistic models have been advanced to explain how ESCRT-III filaments and the VPS4 ATPase can work together to catalyze membrane fission. Expected final online publication date for the Annual Review of Biochemistry Volume 82 is June 02, 2013. Please see for revised estimates.
Gogl,2013 (23519423) Gogl G, Toro I, Remenyi A "Protein-peptide complex crystallization: a case study on the ERK2 mitogen-activated protein kinase." Acta Crystallogr D Biol Crystallogr 2013 Mar 22
Linear motifs normally bind with only medium binding affinity (Kd of approximately 0.1-10 microM) to shallow protein-interaction surfaces on their binding partners. The crystallization of proteins in complex with linear motif-containing peptides is often challenging because the energy gained upon crystal packing between symmetry mates in the crystal may be on a par with the binding energy of the protein-peptide complex. Furthermore, for extracellular signal-regulated kinase 2 (ERK2) the protein-peptide docking surface is comprised of a small hydrophobic surface patch that is often engaged in the crystal packing of apo ERK2 crystals. Here, a rational surface-engineering approach is presented that involves mutating protein surface residues that are distant from the peptide-binding ERK2 docking groove to alanines. These ERK2 surface mutations decrease the chance of `unwanted' crystal packing of ERK2 and the approach led to the structure determination of ERK2 in complex with new docking peptides. These findings highlight the importance of negative selection in crystal engineering for weakly binding protein-peptide complexes.
Pernigo,2013 (23519214) Pernigo S, Lamprecht A, Steiner RA, Dodding MP "Structural basis for kinesin-1:cargo recognition." Science 2013 Apr 19
Kinesin-mediated cargo transport is required for many cellular functions and plays a key role in pathological processes. Structural information on how kinesins recognize their cargoes is required for a molecular understanding of this fundamental and ubiquitous process. Here, we present the crystal structure of the tetratricopeptide repeat domain of kinesin light chain 2 in complex with a cargo peptide harboring a "tryptophan-acidic" motif derived from SKIP (SifA-kinesin interacting protein), a critical host determinant in Salmonella pathogenesis and a regulator of lysosomal positioning. Structural data together with biophysical, biochemical, and cellular assays allow us to propose a framework for intracellular transport based on the binding by kinesin-1 of W-acidic cargo motifs through a combination of electrostatic interactions and sequence-specific elements, providing direct molecular evidence of the mechanisms for kinesin-1:cargo recognition.
Slupe,2013 (23486469) Slupe AM, Merrill RA, Flippo KH, Lobas MA, Houtman JC, Strack S "A calcineurin docking motif (LXVP) in dynamin-related protein 1 contributes to mitochondrial fragmentation and ischemic neuronal injury." J Biol Chem 2013 Apr 29
Fission and fusion events dynamically control the shape and function of mitochondria. The activity of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) is finely tuned by several post-translational modifications. Phosphorylation of Ser-656 by cAMP-dependent protein kinase (PKA) inhibits Drp1, whereas dephosphorylation by a mitochondrial protein phosphatase 2A isoform and the calcium-calmodulin-dependent phosphatase calcineurin (CaN) activates Drp1. Here, we identify a conserved CaN docking site on Drp1, an LXVP motif, which mediates the interaction between the phosphatase and mechanoenzyme. We mutated the LXVP motif in Drp1 to either increase or decrease similarity to the prototypical LXVP motif in the transcription factor NFAT, and assessed stability of the mutant Drp1-CaN complexes by affinity precipitation and isothermal titration calorimetry. Furthermore, we quantified effects of LXVP mutations on Drp1 dephosphorylation kinetics in vitro and in intact cells. With tools for bidirectional control of the CaN-Drp1 signaling axis in hand, we demonstrate that the Drp1 LXVP motif shapes mitochondria in neuronal and non-neuronal cells, and that CaN-mediated Drp1 dephosphorylation promotes neuronal death following oxygen-glucose deprivation. These results point to the CaN-Drp1 complex as a potential target for neuroprotective therapy of ischemic stroke.
Grigoriu,2013 (23468591) Grigoriu S, Bond R, Cossio P, Chen JA, Ly N, Hummer G, Page R, Cyert MS, Peti W "The molecular mechanism of substrate engagement and immunosuppressant inhibition of calcineurin." PLoS Biol 2013 Mar 07
Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca(2+)/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 A structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, "LxVP." The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that "LxVP" sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent "LxVP"-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.
Schmidt,2013 (23417976) Schmidt K, Butler JS "Nuclear RNA surveillance: role of TRAMP in controlling exosome specificity." Wiley Interdiscip Rev RNA 2013 Feb 20
The advent of high-throughput sequencing technologies has revealed that pervasive transcription generates RNAs from nearly all regions of eukaryotic genomes. Normally, these transcripts undergo rapid degradation by a nuclear RNA surveillance system primarily featuring the RNA exosome. This multimeric protein complex plays a critical role in the efficient turnover and processing of a vast array of RNAs in the nucleus. Despite its initial discovery over a decade ago, important questions remain concerning the mechanisms that recruit and activate the nuclear exosome. Specificity and modulation of exosome activity requires additional protein cofactors, including the conserved TRAMP polyadenylation complex. Recent studies suggest that helicase and RNA-binding subunits of TRAMP direct RNA substrates for polyadenylation, which enhances their degradation by Dis3/Rrp44 and Rrp6, the two exosome-associated ribonucleases. These findings indicate that the exosome and TRAMP have evolved highly flexible functions that allow recognition of a wide range of RNA substrates. This flexibility provides the nuclear RNA surveillance system with the ability to regulate the levels of a broad range of coding and noncoding RNAs, which results in profound effects on gene expression, cellular development, gene silencing, and heterochromatin formation. This review summarizes recent findings on the nuclear RNA surveillance complexes, and speculates upon possible mechanisms for TRAMP-mediated substrate recognition and exosome activation.
Song,2013 (23413029) Song D, Li LS, Heaton-Johnson KJ, Arsenault PR, Master SR, Lee FS "Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway." J Biol Chem 2013 Apr 08
Prolyl hydroxylase domain protein 2 (PHD2, also known as Egg Laying Defective Nine homolog 1) is a key oxygen-sensing protein in metazoans. In an oxygen-dependent manner, PHD2 site-specifically prolyl hydroxylates the master transcription factor of the hypoxic response, hypoxia-inducible factor-alpha (HIF-alpha), thereby targeting HIF-alpha for degradation. In this report we show that the heat shock protein 90 (HSP90) co-chaperones p23 and FKBP38 interact via a conserved Pro-Xaa-Leu-Glu motif (where Xaa = any amino acid) in these proteins with the N-terminal Myeloid Nervy and DEAF-1 (MYND)-type zinc finger of PHD2. Knockdown of p23 augments hypoxia-induced HIF-1alpha protein levels and HIF target genes. We propose that p23 recruits PHD2 to the HSP90 machinery to facilitate HIF-1alpha hydroxylation. These findings identify a link between two ancient pathways, the PHD:HIF and the HSP90 pathways, and suggest that this link was established concurrent with the emergence of the PHD:HIF pathway in evolution.
Davids,2013 (23410971) Davids MS, Letai A "ABT-199: taking dead aim at BCL-2." Cancer Cell 2013 Feb 15
ABT-199 is a new selective small molecule inhibitor of BCL-2 that appears to spare platelets while achieving potent antitumor activity. Assays that can predict the efficacy of ABT-199 in individual tumors will be critical in determining how best to incorporate this promising agent into the armamentarium of cancer therapies.
Ohta,2013 (23387299) Ohta A, Schumacher FR, Mehellou Y, Johnson C, Knebel A, Macartney TJ, Wood NT, Alessi DR, Kurz T "The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction." Biochem J 2013 Mar 15
The WNK (with no lysine kinase)-SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin-RING E3 ubiquitin ligase complexes. To explore how a CUL3-KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na(+)/Cl(-) co-transporter)/NKCC1 (Na(+)/K(+)/2Cl(-) co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3-KLHL3 E3 ligase complex, but not a disease-causing CUL3-KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479-667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479-667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3-KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.
Repetto,2013 (23383002) Repetto D, Aramu S, Boeri Erba E, Sharma N, Grasso S, Russo I, Jensen ON, Cabodi S, Turco E, Di Stefano P, Defilippi P "Mapping of p140Cap phosphorylation sites: the EPLYA and EGLYA motifs have a key role in tyrosine phosphorylation and Csk binding, and are substrates of the Abl kinase." PLoS One 2013 Feb 05
Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.
Hast,2013 (23382044) Hast BE, Goldfarb D, Mulvaney KM, Hast MA, Siesser PF, Yan F, Hayes DN, Major MB "Proteomic analysis of ubiquitin ligase KEAP1 reveals associated proteins that inhibit NRF2 ubiquitination." Cancer Res 2013 Apr 04
Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting the hypothesis that alternative mechanisms of pathway activation exist. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2, and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Like WTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an "ETGE" motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1-interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA overexpression of the DPP3 gene in tumors with high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 overexpression in the presence of these mutants further promotes NRF2 activation. Collectively, our findings further support the competition model of NRF2 activation and suggest that "ETGE"-containing proteins such as DPP3 contribute to NRF2 activity in cancer.
Kateb,2013 (23372760) Kateb F, Perrin H, Tripsianes K, Zou P, Spadaccini R, Bottomley M, Franzmann TM, Buchner J, Ansieau S, Sattler M "Structural and functional analysis of the DEAF-1 and BS69 MYND domains." PLoS One 2013 Feb 01
DEAF-1 is an important transcriptional regulator that is required for embryonic development and is linked to clinical depression and suicidal behavior in humans. It comprises various structural domains, including a SAND domain that mediates DNA binding and a MYND domain, a cysteine-rich module organized in a Cys(4)-Cys(2)-His-Cys (C4-C2HC) tandem zinc binding motif. DEAF-1 transcription regulation activity is mediated through interactions with cofactors such as NCoR and SMRT. Despite the important biological role of the DEAF-1 protein, little is known regarding the structure and binding properties of its MYND domain.Here, we report the solution structure, dynamics and ligand binding of the human DEAF-1 MYND domain encompassing residues 501-544 determined by NMR spectroscopy. The structure adopts a betabetaalpha fold that exhibits tandem zinc-binding sites with a cross-brace topology, similar to the MYND domains in AML1/ETO and other proteins. We show that the DEAF-1 MYND domain binds to peptides derived from SMRT and NCoR corepressors. The binding surface mapped by NMR titrations is similar to the one previously reported for AML1/ETO. The ligand binding and molecular functions of the related BS69 MYND domain were studied based on a homology model and mutational analysis. Interestingly, the interaction between BS69 and its binding partners (viral and cellular proteins) seems to require distinct charged residues flanking the predicted MYND domain fold, suggesting a different binding mode. Our findings demonstrate that the MYND domain is a conserved zinc binding fold that plays important roles in transcriptional regulation by mediating distinct molecular interactions with viral and cellular proteins.
Canning,2013 (23349464) Canning P, Cooper CD, Krojer T, Murray JW, Pike AC, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Ayinampudi V, Marsden BD, Gileadi O, Knapp S, von Delft F, Bullock AN "Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases." J Biol Chem 2013 Mar 18
Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein "3-box" motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.
Kruse,2013 (23345399) Kruse T, Zhang G, Larsen MS, Lischetti T, Streicher W, Kragh Nielsen T, Bjorn SP, Nilsson J "Direct binding between BubR1 and B56-PP2A phosphatase complexes regulate mitotic progression." J Cell Sci 2013 Apr 26
BubR1 is a central component of the spindle assembly checkpoint that inhibits progression into anaphase in response to improper kinetochore-microtubule interactions. In addition, BubR1 also helps stabilize kinetochore-microtubule interactions by counteracting the Aurora B kinase but the mechanism behind this is not clear. Here we show that BubR1 directly binds to the B56 family of protein phosphatase 2A (PP2A) regulatory subunits through a conserved motif that is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and polo-like kinase 1 (Plk1). Two highly conserved hydrophobic residues surrounding the serine 670 Cdk1 phosphorylation site are required for B56 binding. Mutation of these residues prevents the establishment of a proper metaphase plate and delays cells in mitosis. Furthermore, we show that phosphorylation of serines 670 and 676 stimulates the binding of B56 to BubR1 and that BubR1 targets a pool of B56 to kinetochores. Our data suggest that BubR1 counteracts Aurora B kinase activity at improperly attached kinetochores by recruiting B56-PP2A phosphatase complexes.
McDonald,2013 (23334917) McDonald CB, El Hokayem J, Zafar N, Balke JE, Bhat V, Mikles DC, Deegan BJ, Seldeen KL, Farooq A "Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner." J Mol Recognit 2013 Jan 21
Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi-protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non-overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2-Sos1 and Grb2-Gab1 binary signaling complexes in concert in lieu of a composite Sos1-Grb2-Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2-Sos1 and Grb2-Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery.
Khan,2013 (23318954) Khan H, Cino EA, Brickenden A, Fan J, Yang D, Choy WY "Fuzzy complex formation between the intrinsically disordered prothymosin alpha and the Kelch domain of Keap1 involved in the oxidative stress response." J Mol Biol 2013 Mar 04
Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription. Prothymosin alpha (ProTalpha), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, we have characterized the interaction between ProTalpha and the Kelch domain of Keap1 by using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, peptide array analysis, site-directed mutagenesis, and molecular dynamic simulations. The results of nuclear magnetic resonance chemical shift mapping, amide hydrogen exchange, and spin relaxation measurements revealed that ProTalpha retains a high level of flexibility, even in the bound state with Kelch. This finding is in agreement with the observations from the molecular dynamic simulations of the ProTalpha-Kelch complex. Mutational analysis of ProTalpha, guided by peptide array data and isothermal titration calorimetry, further pinpointed that the region (38)NANEENGE(45) of ProTalpha is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding.
Sedgwick,2013 (23288039) Sedgwick GG, Hayward DG, Di Fiore B, Pardo M, Yu L, Pines J, Nilsson J "Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex." EMBO J 2013 Jan 23
The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.
Saito-Diaz,2013 (23256519) Saito-Diaz K, Chen TW, Wang X, Thorne CA, Wallace HA, Page-McCaw A, Lee E "The way Wnt works: components and mechanism." Growth Factors 2013 Feb 08
The canonical Wnt/beta-catenin pathway is an ancient and evolutionarily conserved signaling pathway that is required for the proper development of all metazoans, from the basal demosponge Amphimedon queenslandica to humans. Misregulation of Wnt signaling is implicated in many human diseases, making this pathway an intense area of research in industry as well as academia. In this review, we explore our current understanding of the molecular steps involved in the transduction of a Wnt signal. We will focus on how the critical Wnt pathway component, beta-catenin, is in a "futile cycle" of constant synthesis and degradation and how this cycle is disrupted upon pathway activation. We describe the role of the Wnt pathway in major human cancers and in the control of stem cell self-renewal in the developing organism and in adults. Finally, we describe well-accepted criteria that have been proposed as evidence for the involvement of a molecule in regulating the canonical Wnt pathway.
Zhu,2013 (23209295) Zhu Y, Massen S, Terenzio M, Lang V, Chen-Lindner S, Eils R, Novak I, Dikic I, Hamacher-Brady A, Brady NR "Modulation of serines 17 and 24 in the LC3-interacting region of Bnip3 determines pro-survival mitophagy versus apoptosis." J Biol Chem 2013 Jan 14
BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-x(L) positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.
Monda,2013 (23201271) Monda JK, Scott DC, Miller DJ, Lydeard J, King D, Harper JW, Bennett EJ, Schulman BA "Structural conservation of distinctive N-terminal acetylation-dependent interactions across a family of mammalian NEDD8 ligation enzymes." Structure 2013 Jan 14
Little is known about molecular recognition of acetylated N termini, despite prevalence of this modification among eukaryotic cytosolic proteins. We report that the family of human DCN-like (DCNL) co-E3s, which promote ligation of the ubiquitin-like protein NEDD8 to cullin targets, recognizes acetylated N termini of the E2 enzymes UBC12 and UBE2F. Systematic biochemical and biophysical analyses reveal 40- and 10-fold variations in affinities among different DCNL-cullin and DCNL-E2 complexes, contributing to varying efficiencies of different NEDD8 ligation cascades. Structures of DCNL2 and DCNL3 complexes with N-terminally acetylated peptides from UBC12 and UBE2F illuminate a common mechanism by which DCNL proteins recognize N-terminally acetylated E2s and how selectivity for interactions dependent on N-acetyl-methionine are established through side chains recognizing distal residues. Distinct preferences of UBC12 and UBE2F peptides for inhibiting different DCNLs, including the oncogenic DCNL1 protein, suggest it may be possible to develop small molecules blocking specific N-acetyl-methionine-dependent protein interactions.
Aouacheria,2013 (23199982) Aouacheria A, Rech de Laval V, Combet C, Hardwick JM "Evolution of Bcl-2 homology motifs: homology versus homoplasy." Trends Cell Biol 2013 Feb 26
Bcl-2 family proteins regulate apoptosis in animals. This protein family includes several homologous proteins and a collection of other proteins lacking sequence similarity except for a Bcl-2 homology (BH)3 motif. Thus, membership in the Bcl-2 family requires only one of the four BH motifs. On this basis, a growing number of diverse BH3-only proteins are being reported. Although compelling cell biological and biophysical evidence validates many BH3-only proteins, claims of significant BH3 sequence similarity are often unfounded. Computational and phylogenetic analyses suggest that only some BH3 motifs arose by divergent evolution from a common ancestor (homology), whereas others arose by convergent evolution or random coincidence (homoplasy), challenging current assumptions about which proteins constitute the extended Bcl-2 family.
Xu,2013 (23178170) Xu Q, Chang A, Tolia A, Minor DL Jr "Structure of a Ca(2+)/CaM:Kv7.4 (KCNQ4) B-helix complex provides insight into M current modulation." J Mol Biol 2013 Jan 08
Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M currents and cardiac and auditory I(KS) currents and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca(2+)/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved in both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca(2+)/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca(2+)/CaM:Kv7.4 B segment complex shows that Ca(2+)/CaM wraps around the Kv7.4 B segment, which forms an alpha-helix, in an antiparallel orientation that embodies a variation of the classic 1-14 Ca(2+)/CaM interaction motif. Taken together with the context of prior studies, our data suggest a model for modulation of neuronal Kv7 channels involving a calcium-dependent conformational switch from an apo-CaM form that bridges the A and B segments to a Ca(2+)/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease-causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function.
Zhang,2012 (23175388) Zhang P, Bergamin E, Couture JF "The many facets of MLL1 regulation." Biopolymers 2012 Nov 23
In the last 20 years, we have witnessed an exponential number of evidences linking the human mixed lineage leukemia-1 (MLL1) gene to several acute and myelogenous leukemias. MLL1 is one of the founding members of the SET1 family of lysine methyltransferases and is key for the proper control of developmentally regulated gene expression. MLL1 is a structurally complex protein composed of several functional domains. These domains play pivotal roles for the recruitment of regulatory proteins. These MLL1 regulatory proteins (MRPs) dynamically interact with MLL1 and consequently control gene expression. In this review, we summarize recent structural and functional studies of MRPs and discuss emergent structural paradigms for the control of MLL1 activity. (c) 2012 Wiley Periodicals, Inc. Biopolymers 99: 136-145, 2013.
Hickey,2012 (23175280) Hickey CM, Wilson NR, Hochstrasser M "Function and regulation of SUMO proteases." Nat Rev Mol Cell Biol 2012 Nov 23
Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins is highly dynamic, and both SUMO-protein conjugation and cleavage can be regulated. Protein desumoylation is carried out by SUMO proteases, which control cellular mechanisms ranging from transcription and cell division to ribosome biogenesis. Recent advances include the discovery of two novel classes of SUMO proteases, insights regarding SUMO protease specificity, and revelations of previously unappreciated SUMO protease functions in several key cellular pathways. These developments, together with new connections between SUMO proteases and the recently discovered SUMO-targeted ubiquitin ligases (STUbLs), make this an exciting period to study these enzymes.
Stamos,2013 (23169527) Stamos JL, Weis WI "The beta-catenin destruction complex." Cold Spring Harb Perspect Biol 2013 Jan 03
The Wnt/beta-catenin pathway is highly regulated to insure the correct temporal and spatial activation of its target genes. In the absence of a Wnt stimulus, the transcriptional coactivator beta-catenin is degraded by a multiprotein "destruction complex" that includes the tumor suppressors Axin and adenomatous polyposis coli (APC), the Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and the E3-ubiquitin ligase beta-TrCP. The complex generates a beta-TrCP recognition site by phosphorylation of a conserved Ser/Thr-rich sequence near the beta-catenin amino terminus, a process that requires scaffolding of the kinases and beta-catenin by Axin. Ubiquitinated beta-catenin is degraded by the proteasome. The molecular mechanisms that underlie several aspects of destruction complex function are poorly understood, particularly the role of APC. Here we review the molecular mechanisms of destruction complex function and discuss several potential roles of APC in beta-catenin destruction.
Laughlin,2012 (23142346) Laughlin JD, Nwachukwu JC, Figuera-Losada M, Cherry L, Nettles KW, LoGrasso PV "Structural mechanisms of allostery and autoinhibition in JNK family kinases." Structure 2012 Dec 11
c-Jun N-terminal (JNK) family kinases have a common peptide-docking site used by upstream activating kinases, substrates, scaffold proteins, and phosphatases, where the ensemble of bound proteins determines signaling output. Although there are many JNK structures, little is known about mechanisms of allosteric regulation between the catalytic and peptide-binding sites, and the activation loop, whose phosphorylation is required for catalytic activity. Here, we compare three structures of unliganded JNK3 bound to different peptides. These were compared as a class to structures that differ in binding of peptide, small molecule ligand, or conformation of the kinase activation loop. Peptide binding induced an inhibitory interlobe conformer that was reversed by alterations in the activation loop. Structure class analysis revealed the subtle structural mechanisms for allosteric signaling between the peptide-binding site and activation loop. Biochemical data from isothermal calorimetry, fluorescence energy transfer, and enzyme inhibition demonstrated affinity differences among the three peptides that were consistent with structural observations.
Breuer,2012 (23140174) Breuer D, Kotelkin A, Ammosova T, Kumari N, Ivanov A, Ilatovskiy AV, Beullens M, Roane PR, Bollen M, Petukhov MG, Kashanchi F, Nekhai S "CDK2 regulates HIV-1 transcription by phosphorylation of CDK9 on serine 90." Retrovirology 2012 Dec 11
BACKGROUND: HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity. RESULTS: The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation. CONCLUSION: Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1 provirus.
Fry,2012 (23132929) Fry AM, O'Regan L, Sabir SR, Bayliss R "Cell cycle regulation by the NEK family of protein kinases." J Cell Sci 2012 Nov 20
Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.
Lau,2012 (23109716) Lau SY, Procko E, Gaudet R "Distinct properties of Ca2+-calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel." J Gen Physiol 2012 Oct 30
Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca(2+)-CaM) in complex with the TRPV1-CT segment, determined to 1.95-A resolution. The two lobes of Ca(2+)-CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca(2+)-CaM, respectively. This structure is similar to canonical Ca(2+)-CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca(2+)-CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca(2+)-CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca(2+)-CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.
Tian,2012 (23091007) Tian W, Li B, Warrington R, Tomchick DR, Yu H, Luo X "Structural analysis of human Cdc20 supports multisite degron recognition by APC/C." Proc Natl Acad Sci U S A 2012 Nov 07
The anaphase-promoting complex/cyclosome (APC/C) promotes anaphase onset and mitotic exit through ubiquitinating securin and cyclin B1. The mitotic APC/C activator, the cell division cycle 20 (Cdc20) protein, directly interacts with APC/C degrons--the destruction (D) and KEN boxes. APC/C(Cdc20) is the target of the spindle checkpoint. Checkpoint inhibition of APC/C(Cdc20) requires the binding of a BubR1 KEN box to Cdc20. How APC/C recognizes substrates is not understood. We report the crystal structures of human Cdc20 alone or bound to a BubR1 KEN box. Cdc20 has a disordered N-terminal region and a C-terminal WD40 beta propeller with a preformed KEN-box-binding site at its top face. We identify a second conserved surface at the side of the Cdc20 beta propeller as a D-box-binding site. The D box of securin, but not its KEN box, is critical for securin ubiquitination by APC/C(Cdc20). Although both motifs contribute to securin ubiquitination by APC/C(Cdh1), securin mutants lacking either motif are efficiently ubiquitinated. Furthermore, D-box peptides diminish the ubiquitination of KEN-box substrates by APC/C(Cdh1), suggesting possible competition between the two motifs. Our results indicate the lack of strong positive cooperativity between the two degrons of securin. We propose that low-cooperativity, multisite target recognition enables APC/C to robustly ubiquitinate diverse substrates and helps to drive cell cycle oscillations.
Panas,2012 (23087212) Panas MD, Varjak M, Lulla A, Eng KE, Merits A, Karlsson Hedestam GB, McInerney GM "Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection." Mol Biol Cell 2012 Dec 14
Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain-binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.
Tidow,2012 (23086147) Tidow H, Poulsen LR, Andreeva A, Knudsen M, Hein KL, Wiuf C, Palmgren MG, Nissen P "A bimodular mechanism of calcium control in eukaryotes." Nature 2012 Nov 15
Calcium ions (Ca(2+)) have an important role as secondary messengers in numerous signal transduction processes, and cells invest much energy in controlling and maintaining a steep gradient between intracellular ( approximately 0.1-micromolar) and extracellular ( approximately 2-millimolar) Ca(2+) concentrations. Calmodulin-stimulated calcium pumps, which include the plasma-membrane Ca(2+)-ATPases (PMCAs), are key regulators of intracellular Ca(2+) in eukaryotes. They contain a unique amino- or carboxy-terminal regulatory domain responsible for autoinhibition, and binding of calcium-loaded calmodulin to this domain releases autoinhibition and activates the pump. However, the structural basis for the activation mechanism is unknown and a key remaining question is how calmodulin-mediated PMCA regulation can cover both basal Ca(2+) levels in the nanomolar range as well as micromolar-range Ca(2+) transients generated by cell stimulation. Here we present an integrated study combining the determination of the high-resolution crystal structure of a PMCA regulatory-domain/calmodulin complex with in vivo characterization and biochemical, biophysical and bioinformatics data that provide mechanistic insights into a two-step PMCA activation mechanism mediated by calcium-loaded calmodulin. The structure shows the entire PMCA regulatory domain and reveals an unexpected 2:1 stoichiometry with two calcium-loaded calmodulin molecules binding to different sites on a long helix. A multifaceted characterization of the role of both sites leads to a general structural model for calmodulin-mediated regulation of PMCAs that allows stringent, highly responsive control of intracellular calcium in eukaryotes, making it possible to maintain a stable, basal level at a threshold Ca(2+) concentration, where steep activation occurs.
Cruciat,2013 (23085770) Cruciat CM, Niehrs C "Secreted and transmembrane wnt inhibitors and activators." Cold Spring Harb Perspect Biol 2013 Mar 04
Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand-receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-beta and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease.
Suijkerbuijk,2012 (23079597) Suijkerbuijk SJ, Vleugel M, Teixeira A, Kops GJ "Integration of kinase and phosphatase activities by BUBR1 ensures formation of stable kinetochore-microtubule attachments." Dev Cell 2012 Oct 19
Maintenance of chromosomal stability depends on error-free chromosome segregation. The pseudokinase BUBR1 is essential for this, because it is a core component of the mitotic checkpoint and is required for formation of stable kinetochore-microtubule attachments. We have identified a conserved and highly phosphorylated domain (KARD) in BUBR1 that is crucial for formation of kinetochore-microtubule attachments. Deletion of this domain or prevention of its phosphorylation abolishes formation of kinetochore microtubules, which can be reverted by inhibiting Aurora B activity. Phosphorylation of KARD by PLK1 promotes direct interaction of BUBR1 with the PP2A-B56alpha phosphatase that counters excessive Aurora B activity at kinetochores. As a result, removal of BUBR1 from mitotic cells or inhibition of PLK1 reduces PP2A-B56alpha kinetochore binding and elevates phosphorylation of Aurora B substrates on the outer kinetochore. We propose that PLK1 and BUBR1 cooperate to stabilize kinetochore-microtubule interactions by regulating PP2A-B56alpha-mediated dephosphorylation of Aurora B substrates at the kinetochore-microtubule interface.
Garai,2012 (23047924) Garai A, Zeke A, Gogl G, Toro I, Fordos F, Blankenburg H, Barkai T, Varga J, Alexa A, Emig D, Albrecht M, Remenyi A "Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove." Sci Signal 2012 Oct 10
Mitogen-activated protein kinases (MAPKs) have a docking groove that interacts with linear "docking" motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of 15 docking motifs from diverse MAPK partners to bind to c-Jun amino-terminal kinase 1 (JNK1), p38alpha, and extracellular signal-regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only those motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus differentiated between the topographically similar docking grooves of ERK and p38alpha. Crystal structures of four complexes of MAPKs with docking peptides, representing JNK-specific, ERK-specific, or ERK- and p38-selective binding modes, revealed that the regions located between consensus positions in the docking motifs showed conformational diversity. Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a nondiscriminatory fashion, the conformation of the intervening region between the anchor points mostly determined specificity. We designed peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of intervening regions located between anchor points. These results suggest a coherent structural model for MAPK docking specificity that reveals how short linear motifs binding to a common kinase docking groove can mediate diverse interaction patterns and contribute to correct MAPK partner selection in signaling networks.
Alemu,2012 (23043107) Alemu EA, Lamark T, Torgersen KM, Birgisdottir AB, Larsen KB, Jain A, Olsvik H, Overvatn A, Kirkin V, Johansen T "ATG8 family proteins act as scaffolds for assembly of the ULK complex: sequence requirements for LC3-interacting region (LIR) motifs." J Biol Chem 2012 Nov 19
Autophagy is a lysosome-dependent degradation system conserved among eukaryotes. The mammalian Atg1 homologues, Unc-51 like kinase (ULK) 1 and 2, are multifunctional proteins with roles in autophagy, neurite outgrowth, and vesicle transport. The mammalian ULK complex involved in autophagy consists of ULK1, ULK2, ATG13, FIP200, and ATG101. We have used pulldown and peptide array overlay assays to study interactions between the ULK complex and six different ATG8 family proteins. Strikingly, in addition to ULK1 and ULK2, ATG13 and FIP200 interacted with human ATG8 proteins, all with strong preference for the GABARAP subfamily. Similarly, yeast and Drosophila Atg1 interacted with their respective Atg8 proteins, demonstrating the evolutionary conservation of the interaction. Use of peptide arrays allowed precise mapping of the functional LIR motifs, and two-dimensional scans of the ULK1 and ATG13 LIR motifs revealed which substitutions that were tolerated. This information, combined with an analysis of known LIR motifs, provides us with a clearer picture of sequence requirements for LIR motifs. In addition to the known requirements of the aromatic and hydrophobic residues of the core motif, we found the interactions to depend strongly on acidic residues surrounding the central core LIR motifs. A preference for either a hydrophobic residue or an acidic residue following the aromatic residue in the LIR motif is also evident. Importantly, the LIR motif is required for starvation-induced association of ULK1 with autophagosomes. Our data suggest that ATG8 proteins act as scaffolds for assembly of the ULK complex at the phagophore.
Garay-Arroyo,2012 (23027524) Garay-Arroyo A, De La Paz Sanchez M, Garcia-Ponce B, Azpeitia E, Alvarez-Buylla ER "Hormone symphony during root growth and development." Dev Dyn 2012 Nov 21
Hormones regulate plant growth and development in response to external environmental stimuli via complex signal transduction pathways, which in turn form complex networks of interaction. Several classes of hormones have been reported, and their activity depends on their biosynthesis, transport, conjugation, accumulation in the vacuole, and degradation. However, the activity of a given hormone is also dependent on its interaction with other hormones. Indeed, there is a complex crosstalk between hormones that regulates their biosynthesis, transport, and/or signaling functionality, although some hormones have overlapping or opposite functions. The plant root is a particularly useful system in which to study the complex role of plant hormones in the plastic control of plant development. Physiological, cellular, and molecular genetic approaches have been used to study the role of plant hormones in root meristem homeostasis. In this review, we discuss recent findings on the synthesis, signaling, transport of hormones and role during root development and examine the role of hormone crosstalk in maintaining homeostasis in the apical root meristem.
von Muhlinen,2012 (23022382) von Muhlinen N, Akutsu M, Ravenhill BJ, Foeglein A, Bloor S, Rutherford TJ, Freund SM, Komander D, Randow F "LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy." Mol Cell 2012 Nov 12
Autophagy protects cellular homeostasis by capturing cytosolic components and invading pathogens for lysosomal degradation. Autophagy receptors target cargo to autophagy by binding ATG8 on autophagosomal membranes. The expansion of the ATG8 family in higher eukaryotes suggests that specific interactions with autophagy receptors facilitate differential cargo handling. However, selective interactors of ATG8 orthologs are unknown. Here we show that the selectivity of the autophagy receptor NDP52 for LC3C is crucial for innate immunity since cells lacking either protein cannot protect their cytoplasm against Salmonella. LC3C is required for antibacterial autophagy because in its absence the remaining ATG8 orthologs do not support efficient antibacterial autophagy. Structural analysis revealed that the selectivity of NDP52 for LC3C is conferred by a noncanonical LIR, in which lack of an aromatic residue is balanced by LC3C-specific interactions. Our report illustrates that specificity in the interaction between autophagy receptors and autophagy machinery is of functional importance to execute selective autophagy.
Kombrink,2012 (23011567) Kombrink E "Chemical and genetic exploration of jasmonate biosynthesis and signaling paths." Planta 2012 Oct 26
Jasmonates are lipid-derived compounds that act as signals in plant stress responses and developmental processes. Enzymes participating in biosynthesis of jasmonic acid (JA) and components of JA signaling have been extensively characterized by biochemical and molecular-genetic tools. Mutants have helped to define the pathway for synthesis of jasmonoyl-L-isoleucine (JA-Ile), the bioactive form of JA, and to identify the F-box protein COI1 as central regulatory unit. Details on the molecular mechanism of JA signaling were recently unraveled by the discovery of JAZ proteins that together with the adaptor protein NINJA and the general co-repressor TOPLESS form a transcriptional repressor complex. The current model of JA perception and signaling implies the SCF(COI1) complex operating as E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ proteins for degradation by the 26S proteasome pathway, thereby allowing MYC2 and other transcription factors to activate gene expression. Chemical strategies, as integral part of jasmonate research, have helped the establishment of structure-activity relationships and the discovery of (+)-7-iso-JA-L-Ile as the major bioactive form of the hormone. The transient nature of its accumulation highlights the need to understand catabolism and inactivation of JA-Ile and recent studies indicate that oxidation of JA-Ile by cytochrome P450 monooxygenase is the major mechanism for turning JA signaling off. Plants contain numerous JA metabolites, which may have pronounced and differential bioactivity. A major challenge in the field of plant lipid signaling is to identify the cognate receptors and modes of action of these bioactive jasmonates/oxylipins.
Izawa,2012 (23007648) Izawa D, Pines J "Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation." J Cell Biol 2012 Oct 02
The spindle assembly checkpoint (SAC) is essential to ensure proper chromosome segregation and thereby maintain genomic stability. The SAC monitors chromosome attachment, and any unattached chromosomes generate a "wait anaphase" signal that blocks chromosome segregation. The target of the SAC is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C) that triggers anaphase and mitotic exit by ubiquitylating securin and cyclin B1. The inhibitory complex formed by the SAC has recently been shown to inhibit Cdc20 by acting as a pseudosubstrate inhibitor, but in this paper, we show that Mad2 also inhibits Cdc20 by binding directly to a site required to bind the APC/C. Mad2 and the APC/C competed for Cdc20 in vitro, and a Cdc20 mutant that does not bind stably to Mad2 abrogated the SAC in vivo. Thus, we provide insights into how Cdc20 binds the APC/C and uncover a second mechanism by which the SAC inhibits the APC/C.
Hain,2012 (22982544) Hain AU, Weltzer RR, Hammond H, Jayabalasingham B, Dinglasan RR, Graham DR, Colquhoun DR, Coppens I, Bosch J "Structural characterization and inhibition of the Plasmodium Atg8-Atg3 interaction." J Struct Biol 2012 Nov 12
The autophagy-related proteins are thought to serve multiple functions in Plasmodium and are considered essential to parasite survival and development. We have studied two key interacting proteins, Atg8 and Atg3, of the autophagy pathway in Plasmodium falciparum. These proteins are vital for the formation and elongation of the autophagosome and essential to the process of macroautophagy. Autophagy may be required for conversion of the sporozoite into erythrocytic-infective merozoites and may be crucial for other functions during asexual blood stages. Here we describe the identification of an Atg8 family interacting motif (AIM) in Plasmodium Atg3, which binds Plasmodium Atg8. We determined the co-crystal structure of PfAtg8 with a short Atg3(1)(0)(3)(-)(1)(1)(0) peptide, corresponding to this motif, to 2.2 A resolution. Our in vitro interaction studies are in agreement with our X-ray crystal structure. Furthermore they suggest an important role for a unique Apicomplexan loop absent from human Atg8 homologues. Prevention of the protein-protein interaction of full length PfAtg8 with PfAtg3 was achieved at low micromolar concentrations with a small molecule, 1,2,3-trihydroxybenzene. Together our structural and interaction studies represent a starting point for future antimalarial drug discovery and design for this novel protein-protein interaction.
Zhang,2013 (22926519) Zhang J, Chen QM "Far upstream element binding protein 1: a commander of transcription, translation and beyond." Oncogene 2013 Jun 13
The far upstream binding protein 1 (FBP1) was first identified as a DNA-binding protein that regulates c-Myc gene transcription through binding to the far upstream element (FUSE) in the promoter region 1.5 kb upstream of the transcription start site. FBP1 collaborates with TFIIH and additional transcription factors for optimal transcription of the c-Myc gene. In recent years, mounting evidence suggests that FBP1 acts as an RNA-binding protein and regulates mRNA translation or stability of genes, such as GAP43, p27(Kip) and nucleophosmin. During retroviral infection, FBP1 binds to and mediates replication of RNA from Hepatitis C and Enterovirus 71. As a nuclear protein, FBP1 may translocate to the cytoplasm in apoptotic cells. The interaction of FBP1 with p38/JTV-1 results in FBP1 ubiquitination and degradation by the proteasomes. Transcriptional and post-transcriptional regulations by FBP1 contribute to cell proliferation, migration or cell death. FBP1 association with carcinogenesis has been reported in c-Myc dependent or independent manner. This review summarizes biochemical features of FBP1, its mechanism of action, FBP family members and the involvement of FBP1 in carcinogenesis.
Schmidt,2012 (22923767) Schmidt K, Xu Z, Mathews DH, Butler JS "Air proteins control differential TRAMP substrate specificity for nuclear RNA surveillance." RNA 2012 Sep 18
RNA surveillance systems function at critical steps during the formation and function of RNA molecules in all organisms. The RNA exosome plays a central role in RNA surveillance by processing and degrading RNA molecules in the nucleus and cytoplasm of eukaryotic cells. The exosome functions as a complex of proteins composed of a nine-member core and two ribonucleases. The identity of the molecular determinants of exosome RNA substrate specificity remains an important unsolved aspect of RNA surveillance. In the nucleus of Saccharomyces cerevisiae, TRAMP complexes recognize and polyadenylate RNAs, which enhances RNA degradation by the exosome and may contribute to its specificity. TRAMPs contain either of two putative RNA-binding factors called Air proteins. Previous studies suggested that these proteins function interchangeably in targeting the poly(A)-polymerase activity of TRAMPs to RNAs. Experiments reported here show that the Air proteins govern separable functions. Phenotypic analysis and RNA deep-sequencing results from air mutants reveal specific requirements for each Air protein in the regulation of the levels of noncoding and coding RNAs. Loss of these regulatory functions results in specific metabolic and plasmid inheritance defects. These findings reveal differential functions for Air proteins in RNA metabolism and indicate that they control the substrate specificity of the RNA exosome.
Avruch,2012 (22898666) Avruch J, Zhou D, Fitamant J, Bardeesy N, Mou F, Barrufet LR "Protein kinases of the Hippo pathway: regulation and substrates." Semin Cell Dev Biol 2012 Sep 26
The "Hippo" signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in Saccharomyces cerevisiae, e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologs Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin, e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP, promotes integrin clustering, actin remodeling and motility while restraining the proliferation of naive T cells. This review will summarize current knowledge of the structure and regulation of the kinases Hippo/Mst1&2, their noncatalytic binding partners, Salvador and the Rassf polypeptides, and their major substrates Warts/Lats1&2, Trc/ndr1&2, Mats/Mob1 and FOXO.
Cromer,2012 (22844260) Cromer L, Heyman J, Touati S, Harashima H, Araou E, Girard C, Horlow C, Wassmann K, Schnittger A, De Veylder L, Mercier R "OSD1 promotes meiotic progression via APC/C inhibition and forms a regulatory network with TDM and CYCA1;2/TAM." PLoS Genet 2012 Jul 30
Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor.
Takahashi,2012 (22837710) Takahashi T, Suzuki H, Inuzuka T, Shibata H, Maki M "Prediction of a New Ligand-Binding Site for Type 2 Motif based on the Crystal Structure of ALG-2 by Dry and Wet Approaches." Int J Mol Sci 2012 Jul 27
ALG-2 is a penta-EF-hand Ca(2+)-binding protein and interacts with a variety of intracellular proteins. Two types of ALG-2-binding motifs have been determined: type 1, PXYPXnYP (X, variable; n = 4), in ALIX and PLSCR3; type 2, PXPGF, in Sec31A and PLSCR3. The previously solved X-ray crystal structure of the complex between ALG-2 and an ALIX peptide containing type 1 motif showed that the peptide binds to Pocket 1 and Pocket 2. Co-crystallization of ALG-2 and type 2 motif-containing peptides has not been successful. To gain insights into the molecular basis of type 2 motif recognition, we searched for a new hydrophobic cavity by computational algorithms using MetaPocket 2.0 based on 3D structures of ALG-2. The predicted hydrophobic pocket designated Pocket 3 fits with N-acetyl-ProAlaProGlyPhe-amide, a virtual penta-peptide derived from one of the two types of ALG-2-binding sites in PLSCR3 (type 2 motif), using the molecular docking software AutoDock Vina. We investigated effects of amino acid substitutions of the predicted binding sites on binding abilities by pulldown assays using glutathione-S-transferase -fused ALG-2 of wild-type and mutant proteins and lysates of cells expressing green fluorescent protein -fused PLSCR3 of wild-type and mutants. Substitution of either L52 with Ala or F148 with Ser of ALG-2 caused loss of binding abilities to PLSCR3 lacking type 1 motif but retained those to PLSCR3 lacking type 2 motif, strongly supporting the hypothesis that Pocket 3 is the binding site for type 2 motif.
Fros,2012 (22837213) Fros JJ, Domeradzka NE, Baggen J, Geertsema C, Flipse J, Vlak JM, Pijlman GP "Chikungunya virus nsP3 blocks stress granule assembly by recruitment of G3BP into cytoplasmic foci." J Virol 2012 Sep 11
Chikungunya virus nonstructural protein nsP3 has an essential but unknown role in alphavirus replication and interacts with Ras-GAP SH3 domain-binding protein (G3BP). Here we describe the first known function of nsP3, to inhibit stress granule assembly by recruiting G3BP into cytoplasmic foci. A conserved SH3 domain-binding motif in nsP3 is essential for both nsP3-G3BP interactions and viral RNA replication. This study reveals a novel role for nsP3 as a regulator of the cellular stress response.
Doidge,2012 (22817755) Doidge R, Mittal S, Aslam A, Winkler GS "Deadenylation of cytoplasmic mRNA by the mammalian Ccr4-Not complex." Biochem Soc Trans 2012 Jul 23
The Ccr4-Not complex is one of the major deadenylase factors present in eukaryotic cells. This multi-subunit protein complex is composed of at least seven stably associated subunits in mammalian cells including two enzymatic deadenylase subunits: one DEDD (Asp-Glu-Asp-Asp)-type deadenylase (either CNOT7/human Caf1/Caf1a or CNOT8/human Pop2/Caf1b/Calif) and one EEP (endonuclease-exonuclease-phosphatase)-type enzyme (either CNOT6/human Ccr4/Ccr4a or CNOT6L/human Ccr4-like/Ccr4b). Here, the role of the human Ccr4-Not complex in cytoplasmic deadenylation of mRNA is discussed, including the mechanism of its recruitment to mRNA and the role of the BTG/Tob proteins.
Shindo,2012 (22814604) Shindo N, Kumada K, Hirota T "Separase sensor reveals dual roles for separase coordinating cohesin cleavage and cdk1 inhibition." Dev Cell 2012 Jul 20
Complete dissociation of sister chromatid cohesion and subsequent induction of poleward movement of disjoined sisters are two essential events underlying chromosome segregation; however, how cells coordinate these two processes is not well understood. Here, we developed a fluorescence-based sensor for the protease separase that mediates cohesin cleavage. We found that separase undergoes an abrupt activation shortly before anaphase onset in the vicinity of chromosomes. This activation profile of separase depends on the abilities of two of its binding proteins, securin and cyclin B1, to inhibit its protease activity and target it to chromosomes. Subsequent to its proteolytic activation, separase then binds to and inhibits a subset of cyclin B1-cdk1, which antagonizes cdk1-mediated phosphorylation on chromosomes and facilitates poleward movement of sisters in anaphase. Therefore, by consecutively acting as a protease and a cdk1 inhibitor, separase coordinates two key processes to achieve simultaneous and abrupt separation of sister chromatids.
Inuzuka,2012 (22770219) Inuzuka H, Gao D, Finley LW, Yang W, Wan L, Fukushima H, Chin YR, Zhai B, Shaik S, Lau AW, Wang Z, Gygi SP, Nakayama K, Teruya-Feldstein J, Toker A, Haigis MC, Pandolfi PP, Wei W "Acetylation-dependent regulation of Skp2 function." Cell 2012 Jul 09
Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.
Tian,2012 (22743616) Tian H, Zhang B, Di J, Jiang G, Chen F, Li H, Li L, Pei D, Zheng J "Keap1: one stone kills three birds Nrf2, IKKbeta and Bcl-2/Bcl-xL." Cancer Lett 2012 Aug 21
Oxidative stress, implicated in the etiology of cancer, results from an imbalance in the production of Reactive Oxygen Species (ROS) and cell's own antioxidant defenses. As a oxidative stress sensor, Keap1 functions as both an adaptor for Cul3Rbx1 E3 ligase complex mediated degradation of the transcription factor Nrf2, and a master regulator of cytoprotective gene expression. Although Nrf2 is a well known substrate for Keap1, the DGR domain of Keap1 has been reported also to bind other proteins directly or indirectly. IKKbeta as positive regulator of NF-kappaB is also destabilized by Keap1, which resulted in inhibiting NF-kappaB-derived tumor promotion. In addition, anti-apoptotic Bcl-2/Bcl-xL protein was identified as another substrate for the Keap1-Cul3-E3 ligase complex. Keap1 led to the repression and destabilization of Bcl-2, decreased Bcl-2:Bax heterodimers and facilitated cancer cells apoptosis. Given that Keap1 might function as a tumor suppressor protein to mitigate tumor progression, the different kinds of Keap1 somatic mutations were detected in numerous cancer cells. Therefore, it is important to understand the Keap1-involved signaling cascades. This review primarily focuses on the prevention of tumorigenesis role of Keap1 through negative regulation of three substrates Nrf2, IKKbeta and Bcl-2/Bcl-xL, with emphasis on the recent findings indicating the cancer guarder function of Keap1.
Labit,2012 (22713866) Labit H, Fujimitsu K, Bayin NS, Takaki T, Gannon J, Yamano H "Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C." EMBO J 2012 Aug 02
The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.
Holdsworth,2012 (22696217) Holdsworth G, Slocombe P, Doyle C, Sweeney B, Veverka V, Le Riche K, Franklin RJ, Compson J, Brookings D, Turner J, Kennedy J, Garlish R, Shi J, Newnham L, McMillan D, Muzylak M, Carr MD, Henry AJ, Ceska T, Robinson MK "Characterization of the interaction of sclerostin with the low density lipoprotein receptor-related protein (LRP) family of Wnt co-receptors." J Biol Chem 2012 Aug 06
LRP5 and LRP6 are proteins predicted to contain four six-bladed beta-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the beta-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another beta-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.
Srinivasan,2012 (22666276) Srinivasan M, Dunker AK "Proline rich motifs as drug targets in immune mediated disorders." Int J Pept 2012 Jun 05
The current version of the human immunome network consists of nearly 1400 interactions involving approximately 600 proteins. Intermolecular interactions mediated by proline-rich motifs (PRMs) are observed in many facets of the immune response. The proline-rich regions are known to preferentially adopt a polyproline type II helical conformation, an extended structure that facilitates transient intermolecular interactions such as signal transduction, antigen recognition, cell-cell communication and cytoskeletal organization. The propensity of both the side chain and the backbone carbonyls of the polyproline type II helix to participate in the interface interaction makes it an excellent recognition motif. An advantage of such distinct chemical features is that the interactions can be discriminatory even in the absence of high affinities. Indeed, the immune response is mediated by well-orchestrated low-affinity short-duration intermolecular interactions. The proline-rich regions are predominantly localized in the solvent-exposed regions such as the loops, intrinsically disordered regions, or between domains that constitute the intermolecular interface. Peptide mimics of the PRM have been suggested as potential antagonists of intermolecular interactions. In this paper, we discuss novel PRM-mediated interactions in the human immunome that potentially serve as attractive targets for immunomodulation and drug development for inflammatory and autoimmune pathologies.
Dharmarajan,2012 (22665483) Dharmarajan V, Lee JH, Patel A, Skalnik DG, Cosgrove MS "Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases." J Biol Chem 2012 Aug 13
Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are associated with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 associates with a subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoprecipitate WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we determined structures of WDR5 bound to six different naturally occurring Win motif sequences at resolutions ranging from 1.9 to 1.2 A. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.
Baietti,2012 (22660413) Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, Ivarsson Y, Depoortere F, Coomans C, Vermeiren E, Zimmermann P, David G "Syndecan-syntenin-ALIX regulates the biogenesis of exosomes." Nat Cell Biol 2012 Jul 03
The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan-syntenin-ALIX in membrane transport and signalling processes.
Xu,2012 (22649097) Xu C, Jin J, Bian C, Lam R, Tian R, Weist R, You L, Nie J, Bochkarev A, Tempel W, Tan CS, Wasney GA, Vedadi M, Gish GD, Arrowsmith CH, Pawson T, Yang XJ, Min J "Sequence-specific recognition of a PxLPxI/L motif by an ankyrin repeat tumbler lock." Sci Signal 2012 May 31
Ankyrin repeat family A protein 2 (ANKRA2) interacts with the plasma membrane receptor megalin and the class IIa histone deacetylases HDAC4 and HDAC5. We report that the ankyrin repeat domains of ANKRA2 and its close paralog regulatory factor X-associated ankyrin-containing protein (RFXANK) recognize a PxLPxI/L motif found in diverse binding proteins, including HDAC4, HDAC5, HDAC9, megalin, and regulatory factor X, 5 (RFX5). Crystal structures of the ankyrin repeat domain of ANKRA2 in complex with its binding peptides revealed that each of the middle three ankyrin repeats of ANKRA2 recognizes a residue from the PxLPxI/L motif in a tumbler-lock binding mode, with ANKRA2 acting as the lock and the linear binding motif serving as the key. Structural analysis showed that three disease-causing mutations in RFXANK affect residues that are critical for binding to RFX5. These results suggest a fundamental principle of longitudinal recognition of linear sequences by a repeat-type domain. In addition, phosphorylation of serine 350, a residue embedded within the PxLPxI/L motif of HDAC4, impaired the binding of ANKRA2 but generated a high-affinity docking site for 14-3-3 proteins, which may help sequester this HDAC in the cytoplasm. Thus, the binding preference of the PxLPxI/L motif is signal-dependent. Furthermore, proteome-wide screening suggested that a similar phosphorylation-dependent switch may operate in other pathways. Together, our findings uncover a previously uncharacterized sequence- and signal-dependent peptide recognition mode for a repeat-type protein domain.
Arboleda,2012 (22634751) Arboleda VA "Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome." Nat Genet 2012 Jun 28
IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.
Rose,2012 (22634725) Rose R, Rose M, Ottmann C "Identification and structural characterization of two 14-3-3 binding sites in the human peptidylarginine deiminase type VI." J Struct Biol 2012 Oct 08
The regulation and function of peptidylarginine deiminase isoform VI (PAD6), which is a highly abundant protein associated with the cytoplasmic lattices in mammalian oocytes, is poorly understood so far. It has been shown previously, that 14-3-3 proteins, a class of regulatory adapter proteins ubiquitous in eukaryotes, bind to PAD6 in vivo in a phosphorylation dependent manner. Here we identify possible 14-3-3 binding sites in human PAD6 by in silico methods, looking for conserved, surface exposed serine residues. Two of these sites were confirmed as 14-3-3 binding sites by fluorescence polarization competition and X-ray crystallography. We furthermore suggest a role of RSK-type kinases in the phosphorylation of one of these two binding sites and provide evidence in the form of in vitro kinase assays with p70S6 kinase and RSK1.
Schmidt,2012 (22624858) Schmidt F, Dietrich D, Eylenstein R, Groemping Y, Stehle T, Dodt G "The Role of Conserved PEX3 Regions in PEX19-Binding and Peroxisome Biogenesis." Traffic 2012 Aug 13
The human peroxins PEX3 and PEX19 are essential for peroxisome biogenesis. They mediate the import of membrane proteins as well as the de novo formation of peroxisomes. PEX19 binds newly synthesized peroxisomal membrane proteins post-translationally and directs them to peroxisomes by engaging PEX3, a protein anchored in the peroxisomal membrane. After protein insertion into the lipid bilayer, PEX19 is released back to the cytosol. Crystallographic analysis provided detailed insights into the PEX3-PEX19 interaction and identified three highly conserved regions, the PEX19-binding region, a hydrophobic groove and an acidic cluster, on the surface of PEX3. Here, we used site-directed mutagenesis and biochemical and functional assays to determine the role of these regions in PEX19-binding and peroxisome biogenesis. Mutations in the PEX19-binding region reduce the affinity for PEX19 and destabilize PEX3. Furthermore, we provide evidence for a crucial function of the PEX3-PEX19 complex during de novo formation of peroxisomes in peroxisome-deficient cells, pointing to a dual function of the PEX3-PEX19 interaction in peroxisome biogenesis. The maturation of preperoxisomes appears to require the hydrophobic groove near the base of PEX3, presumably by its involvement in peroxisomal membrane protein insertion, while the acidic cluster does not appear to be functionally relevant.
Finnen,2012 (22623775) Finnen RL, Pangka KR, Banfield BW "Herpes simplex virus 2 infection impacts stress granule accumulation." J Virol 2012 Jul 12
Interference with stress granule (SG) accumulation is gaining increased appreciation as a common strategy used by diverse viruses to facilitate their replication and to cope with translational arrest. Here, we examined the impact of infection by herpes simplex virus 2 (HSV-2) on SG accumulation by monitoring the localization of the SG components T cell internal antigen 1 (TIA-1), Ras-GTPase-activating SH3-domain-binding protein (G3BP), and poly(A)-binding protein (PABP). Our results indicate that SGs do not accumulate in HSV-2-infected cells and that HSV-2 can interfere with arsenite-induced SG accumulation early after infection. Surprisingly, SG accumulation was inhibited despite increased phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), implying that HSV-2 encodes previously unrecognized activities designed to maintain translation initiation downstream of eIF2alpha. SG accumulation was not inhibited in HSV-2-infected cells treated with pateamine A, an inducer that works independently of eIF2alpha phosphorylation. The SGs that accumulated following pateamine A treatment of infected cells contained G3BP and PABP but were largely devoid of TIA-1. We also identified novel nuclear structures containing TIA-1 that form late in infection. These structures contain the RNA binding protein 68-kDa Src-associated in mitosis (Sam68) and were noticeably absent in infected cells treated with inhibitors of viral DNA replication, suggesting that they arise as a result of late events in the virus replicative cycle.
Faesen,2012 (22616864) Faesen AC, Luna-Vargas MP, Sixma TK "The role of UBL domains in ubiquitin-specific proteases." Biochem Soc Trans 2012 May 23
Ubiquitin conjugation and deconjugation provides a powerful signalling system to change the fate of its target enzymes. Ubiquitination levels are organized through a balance between ubiquitinating E1, E2 and E3 enzymes and deubiquitination by DUBs (deubiquitinating enzymes). These enzymes are tightly regulated to control their activity. In the present article, we discuss the different ways in which DUBs of the USP (ubiquitin-specific protease) family are regulated by internal domains with a UBL (ubiquitin-like) fold. The UBL domain in USP14 is important for its localization at the proteasome, which enhances catalysis. In contrast, a UBL domain in USP4 binds to the catalytic domain and competes with ubiquitin binding. In this process, the UBL domain mimics ubiquitin and partially inhibits catalysis. In USP7, there are five consecutive UBL domains, of which the last two affect catalytic activity. Surprisingly, they do not act like ubiquitin and activate catalysis rather than inhibiting it. These C-terminal UBL domains promote a conformational change that allows ubiquitin binding and organizes the catalytic centre. Thus it seems that UBL domains have different functions in different USPs. Other proteins can modulate the roles of UBL domains in USP4 and USP7. On one hand, the inhibition of USP4 can be relieved when the UBL is sequestered by another USP. On the other, the activation of USP7 is increased, when the UBL-activated state is stabilized by allosteric binding of GMP synthetase. Altogether, UBL domains appear to be able to regulate catalytic activity in USPs, but they can use widely different mechanisms of action, in which they may, as in USP4, or may not, as in USP7, use the direct resemblance to ubiquitin.
Min,2012 (22608923) Min SH, Lau AW, Lee TH, Inuzuka H, Wei S, Huang P, Shaik S, Lee DY, Finn G, Balastik M, Chen CH, Luo M, Tron AE, Decaprio JA, Zhou XZ, Wei W, Lu KP "Negative regulation of the stability and tumor suppressor function of Fbw7 by the Pin1 prolyl isomerase." Mol Cell 2012 Jul 03
Fbw7 is the substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers; however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, overexpressing Pin1 reduces Fbw7 abundance and suppresses Fbw7's ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis, and Pin1 may be a promising drug target for anticancer therapy.
Gutierrez-Escribano,2012 (22589718) Gutierrez-Escribano P, Zeidler U, Suarez MB, Bachellier-Bassi S, Clemente-Blanco A, Bonhomme J, Vazquez de Aldana CR, d'Enfert C, Correa-Bordes J "The NDR/LATS kinase Cbk1 controls the activity of the transcriptional regulator Bcr1 during biofilm formation in Candida albicans." PLoS Pathog 2012 May 16
In nature, many microorganisms form specialized complex, multicellular, surface-attached communities called biofilms. These communities play critical roles in microbial pathogenesis. The fungal pathogen Candida albicans is associated with catheter-based infections due to its ability to establish biofilms. The transcription factor Bcr1 is a master regulator of C. albicans biofilm development, although the full extent of its regulation remains unknown. Here, we report that Bcr1 is a phosphoprotein that physically interacts with the NDR kinase Cbk1 and undergoes Cbk1-dependent phosphorylation. Mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to alanine markedly impaired Bcr1 function during biofilm formation and virulence in a mouse model of disseminated candidiasis. Cells lacking Cbk1, or any of its upstream activators, also had reduced biofilm development. Notably, mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to glutamate in cbk1Delta cells upregulated the transcription of Bcr1-dependent genes and partially rescued the biofilm defects of a cbk1Delta strain. Therefore, our data uncovered a novel role of the NDR/LATS kinase Cbk1 in the regulation of biofilm development through the control of Bcr1.
Gao,2012 (22586277) Gao Z, Poon HY, Li L, Li X, Palmesino E, Glubrecht DD, Colwill K, Dutta I, Kania A, Pawson T, Godbout R "Splice-mediated motif switching regulates disabled-1 phosphorylation and SH2 domain interactions." Mol Cell Biol 2012 Jun 29
Disabled-1 (Dab1) plays a key role in reelin-mediated neuronal migration during brain development. Tyrosine phosphorylation of Dab1 at two YQXI and two YXVP motifs recruits multiple SH2 domains, resulting in activation of a wide range of signaling cascades. However, the molecular mechanisms underlying the coordinated regulation of Dab1 downstream effectors remain poorly understood. Here, we show that alternative splicing results in inclusion of different combinations of YQXI and YXVP motifs in Dab1 isoforms during development. Dab1 variants with partial or complete loss of YQXI motifs are preferentially expressed at early developmental stages, whereas the commonly studied Dab1 is predominantly expressed at late developmental stages. Expression of Dab1 variants in 293T and Neuro2a cells reveals reduced levels or absence of tyrosine phosphorylation in variants that have lost one or both YQXI motifs. We further demonstrate that Dab1 variants differ in their abilities to activate Src and recruit distinct SH2 domains involved in specific downstream signaling pathways. We propose that coordinated expression of specific Dab1 isoforms in different populations of cells in the developing brain contributes to precise neuronal migration by modulating the activity of subsets of Dab1 downstream effectors.
Picard,2012 (22586270) Picard N, Caron V, Bilodeau S, Sanchez M, Mascle X, Aubry M, Tremblay A "Identification of estrogen receptor beta as a SUMO-1 target reveals a novel phosphorylated sumoylation motif and regulation by glycogen synthase kinase 3beta." Mol Cell Biol 2012 Jun 29
SUMO conjugation has emerged as a dynamic process in regulating protein function. Here we identify estrogen receptor beta (ERbeta) to be a new target of SUMO-1. ERbeta SUMO-1 modification occurs on a unique nonconsensus sumoylation motif which becomes fully competent upon phosphorylation of its contained serine residue, which provides the essential negative charge for sumoylation. This process is further regulated by phosphorylation of additional adjacent serine residues by glycogen synthase kinase 3beta (GSK3beta), which maximizes ERbeta sumoylation in response to hormone. SUMO-1 attachment prevents ERbeta degradation by competing with ubiquitin at the same acceptor site and dictates ERbeta transcriptional inhibition by altering estrogen-responsive target promoter occupancy and gene expression in breast cancer cells. These findings uncovered a novel phosphorylated sumoylation motif (pSuM), which consists of the sequence psiKXS (where psi represents a large hydrophobic residue) and which is connected to a GSK3-activated extension that functions as a SUMO enhancer. This extended pSuM offers a valuable signature to predict SUMO substrates under protein kinase regulation.
Zhang,2012 (22579256) Zhang M, Abrams C, Wang L, Gizzi A, He L, Lin R, Chen Y, Loll PJ, Pascal JM, Zhang JF "Structural basis for calmodulin as a dynamic calcium sensor." Structure 2012 May 14
Calmodulin is a prototypical and versatile Ca(2+) sensor with EF hands as its high-affinity Ca(2+) binding domains. Calmodulin is present in all eukaryotic cells, mediating Ca(2+)-dependent signaling. Upon binding Ca(2+), calmodulin changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on calmodulin, little is known on how target proteins regulate calmodulin's ability to bind Ca(2+). Here, we take advantage of two splice variants of SK2 channels, which are activated by Ca(2+)-bound calmodulin but show different sensitivity to Ca(2+) for their activation. Protein crystal structures and other experiments show that, depending on which SK2 splice variant it binds to, calmodulin adopts drastically different conformations with different affinities for Ca(2+) at its C-lobe. Such target protein-induced conformational changes make calmodulin a dynamic Ca(2+) sensor capable of responding to different Ca(2+) concentrations in cellular Ca(2+) signaling.
Matsuo,2012 (22542101) Matsuo K, Ohsumi K, Iwabuchi M, Kawamata T, Ono Y, Takahashi M "Kendrin is a novel substrate for separase involved in the licensing of centriole duplication." Curr Biol 2012 May 25
The centrosome, consisting of a pair of centrioles surrounded by pericentriolar material, directs the formation of bipolar spindles during mitosis. Aberrant centrosome number can promote chromosome instability, which is implicated in tumorigenesis. Thus, centrosome duplication needs to be tightly regulated to occur only once per cell cycle. Separase, a cysteine protease that triggers sister chromatid separation, is involved in centriole disengagement, which licenses centrosomes for the next round of duplication. However, at least two questions remain unsolved: what is the substrate relevant to the disengagement, and how does separase, activated at anaphase onset, act on the disengagement that occurs during late mitosis. Here, we show that kendrin, also named pericentrin, is cleaved by activated separase at a consensus site in vivo and in vitro, and this leads to the delayed release of kendrin from the centrosome later in mitosis. Furthermore, we demonstrate that expression of a noncleavable kendrin mutant suppresses centriole disengagement and subsequent centriole duplication. Based on these results, we propose that kendrin is a novel and crucial substrate for separase at the centrosome, protecting the engaged centrioles from premature disengagement and thereby blocking reduplication until the cell passes through mitosis.
Tasaki,2012 (22524314) Tasaki T, Sriram SM, Park KS, Kwon YT "The N-end rule pathway." Annu Rev Biochem 2012 Jun 05
The N-end rule pathway is a proteolytic system in which N-terminal residues of short-lived proteins are recognized by recognition components (N-recognins) as essential components of degrons, called N-degrons. Known N-recognins in eukaryotes mediate protein ubiquitylation and selective proteolysis by the 26S proteasome. Substrates of N-recognins can be generated when normally embedded destabilizing residues are exposed at the N terminus by proteolytic cleavage. N-degrons can also be generated through modifications of posttranslationally exposed pro-N-degrons of otherwise stable proteins; such modifications include oxidation, arginylation, leucylation, phenylalanylation, and acetylation. Although there are variations in components, degrons, and hierarchical structures, the proteolytic systems based on generation and recognition of N-degrons have been observed in all eukaryotes and prokaryotes examined thus far. The N-end rule pathway regulates homeostasis of various physiological processes, in part, through interaction with small molecules. Here, we review the biochemical mechanisms, structures, physiological functions, and small-molecule-mediated regulation of the N-end rule pathway.
Liu,2012 (22518098) Liu Z, Vogel HJ "Structural basis for the regulation of L-type voltage-gated calcium channels: interactions between the N-terminal cytoplasmic domain and Ca(2+)-calmodulin." Front Mol Neurosci 2012 Apr 20
It is well-known that the opening of L-type voltage-gated calcium channels can be regulated by calmodulin (CaM). One of the main regulatory mechanisms is calcium-dependent inactivation (CDI), where binding of apo-CaM to the cytoplasmic C-terminal domain of the channel can effectively sense an increase in the local calcium ion concentration. Calcium-bound CaM can bind to the IQ-motif region of the C-terminal region and block the calcium channel, thereby providing a negative feedback mechanism that prevents the rise of cellular calcium concentrations over physiological limits. Recently, an additional Ca(2+)/CaM-binding motif (NSCaTE, N-terminal spatial Ca(2+) transforming element) was identified in the amino terminal cytoplasmic region of Ca(v)1.2 and Ca(v)1.3. This motif exists only in Ca(v)1.2 and Ca(v)1.3 channels, and a pronounced N-lobe (Ca(2+)/CaM) CDI effect was found for Ca(v)1.3. To understand the molecular basis of this interaction, the complexes of Ca(2+)/CaM with the biosynthetically produced N-terminal region (residues 1-68) and NSCaTE peptide (residues 48-68) were investigated. We discovered that the NSCaTE motif in the N-terminal cytoplasmic region adopts an alpha-helical conformation, most likely due to its high alanine content. Additionally, the complex exhibits an unusual 1:2 protein:peptide stoichiometry when bound to Ca(2+)-CaM, and the N-lobe of CaM has a much stronger affinity for the peptide than the C-lobe. The complex structures of the isolated N- and C-lobe of Ca(2+)/CaM and the NSCaTE peptide were determined by nuclear magnetic resonance spectroscopy and data-driven protein-docking methods. Moreover, we also demonstrated that calcium binding protein 1, which competes with CaM for binding to the C-terminal cytoplasmic domain, binds only weakly to the NSCaTE region. The structures provide insights into the possible roles of this motif in the calcium regulatory network. Our study provides structural evidence for the CaM-bridge model proposed in previous studies.
Hanna,2012 (22505714) Hanna RA, Quinsay MN, Orogo AM, Giang K, Rikka S, Gustafsson AB "Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy." J Biol Chem 2012 Jun 04
Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. Bnip3 is an atypical BH3-only protein that is known to cause mitochondrial dysfunction and cell death. Interestingly, Bnip3 can also protect against cell death by inducing mitochondrial autophagy. The mechanism for this process, however, remains poorly understood. Bnip3 contains a C-terminal transmembrane domain that is essential for homodimerization and proapoptotic function. In this study, we show that homodimerization of Bnip3 is also a requirement for induction of autophagy. Several Bnip3 mutants that do not interfere with its mitochondrial localization but disrupt homodimerization failed to induce autophagy in cells. In addition, we discovered that endogenous Bnip3 is localized to both mitochondria and the endoplasmic reticulum (ER). To investigate the effects of Bnip3 at mitochondria or the ER on autophagy, Bnip3 was targeted specifically to each organelle by substituting the Bnip3 transmembrane domain with that of Acta or cytochrome b(5). We found that Bnip3 enhanced autophagy in cells from both sites. We also discovered that Bnip3 induced removal of both ER (ERphagy) and mitochondria (mitophagy) via autophagy. The clearance of these organelles was mediated in part via binding of Bnip3 to LC3 on the autophagosome. Although ablation of the Bnip3-LC3 interaction by mutating the LC3 binding site did not impair the prodeath activity of Bnip3, it significantly reduced both mitophagy and ERphagy. Our data indicate that Bnip3 regulates the apoptotic balance as an autophagy receptor that induces removal of both mitochondria and ER.
Thaler,2012 (22498450) Thaler JS, Humphrey PT, Whiteman NK "Evolution of jasmonate and salicylate signal crosstalk." Trends Plant Sci 2012 May 14
The evolution of land plants approximately 470 million years ago created a new adaptive zone for natural enemies (attackers) of plants. In response to attack, plants evolved highly effective, inducible defense systems. Two plant hormones modulating inducible defenses are salicylic acid (SA) and jasmonic acid (JA). Current thinking is that SA induces resistance against biotrophic pathogens and some phloem feeding insects and JA induces resistance against necrotrophic pathogens, some phloem feeding insects and chewing herbivores. Signaling crosstalk between SA and JA commonly manifests as a reciprocal antagonism and may be adaptive, but this remains speculative. We examine evidence for and against adaptive explanations for antagonistic crosstalk, trace its phylogenetic origins and provide a hypothesis-testing framework for future research on the adaptive significance of SA-JA crosstalk.
Nagae,2012 (22451694) Nagae M, Re S, Mihara E, Nogi T, Sugita Y, Takagi J "Crystal structure of alpha5beta1 integrin ectodomain: atomic details of the fibronectin receptor." J Cell Biol 2012 Apr 04
Integrin alpha5beta1 is a major cellular receptor for the extracellular matrix protein fibronectin and plays a fundamental role during mammalian development. A crystal structure of the alpha5beta1 integrin headpiece fragment bound by an allosteric inhibitory antibody was determined at a 2.9-A resolution both in the absence and presence of a ligand peptide containing the Arg-Gly-Asp (RGD) sequence. The antibody-bound beta1 chain accommodated the RGD ligand with very limited structural changes, which may represent the initial step of cell adhesion mediated by nonactivated integrins. Furthermore, a molecular dynamics simulation pointed to an important role for Ca(2+) in the conformational coupling between the ligand-binding site and the rest of the molecule. The RGD-binding pocket is situated at the center of a trenchlike exposed surface on the top face of alpha5beta1 devoid of glycosylation sites. The structure also enabled the precise prediction of the acceptor residue for the auxiliary synergy site of fibronectin on the alpha5 subunit, which was experimentally confirmed by mutagenesis and kinetic binding assays.
Suzuki,2012 (22448252) Suzuki A, Saba R, Miyoshi K, Morita Y, Saga Y "Interaction between NANOS2 and the CCR4-NOT deadenylation complex is essential for male germ cell development in mouse." PLoS One 2012 Mar 26
Nanos is one of the evolutionarily conserved proteins implicated in germ cell development and we have previously shown that it interacts with the CCR4-NOT deadenylation complex leading to the suppression of specific RNAs. However, the molecular mechanism and physiological significance of this interaction have remained elusive. In our present study, we identify CNOT1, a component of the CCR4-NOT deadenylation complex, as a direct factor mediating the interaction with NANOS2. We find that the first 10 amino acids (AAs) of NANOS2 are required for this binding. We further observe that a NANOS2 mutant lacking these first 10 AAs (NANOS2-DeltaN10) fails to rescue defects in the Nanos2-null mouse. Our current data thus indicate that the interaction with the CCR4-NOT deadenylation complex is essential for NANOS2 function. In addition, we further demonstrate that NANOS2-DeltaN10 can associate with specific mRNAs as well as wild-type NANOS2, suggesting the existence of other NANOS2-associated factor(s) that determine the specificity of RNA-binding independently of the CCR4-NOT deadenylation complex.
Osugi,2012 (22437941) Osugi K, Suzuki H, Nomura T, Ariumi Y, Shibata H, Maki M "Identification of the P-body component PATL1 as a novel ALG-2-interacting protein by in silico and far-Western screening of proline-rich proteins." J Biochem 2012 Jun 06
ALG-2 (also named PDCD6) is a 22-kDa Ca(2+)-binding protein that belongs to the penta-EF-hand family including calpain small subunit and interacts with various proteins such as ALIX and Sec31A at their specific sites containing an ALG-2-binding motif (ABM) present in their respective Pro-rich region (PRR). In this study, to search for novel ALG-2-interacting proteins, we first performed in silico screening of ABM-containing PRRs in a human protein database. After selecting 17 sequences, we expressed the PRR or full-length proteins fused with green fluorescent protein (GFP) in HEK293T cells and analysed their abilities to bind to ALG-2 by Far-Western blotting using biotinylated ALG-2 as a probe. As a result, we found 10 positive new ALG-2-binding candidates with different degrees of binding ability. For further investigation, we selected PATL1 (alternatively designated Pat1b), a component of the P-body, which is a cytoplasmic non-membranous granule composed of translation-inactive mRNAs and proteins involved in mRNA decay. Interactions between endogenous PATL1 and ALG-2 proteins were demonstrated by a co-immunoprecipitation assay using their specific antibodies. Furthermore, in immunofluorescence microscopic analyses, PATL1 as well as DCP1A, a well-known P-body marker, co-localized with a subset of ALG-2. This is the first report showing interaction of ALG-2 with a P-body component.
Chao,2012 (22437499) Chao WC, Kulkarni K, Zhang Z, Kong EH, Barford D "Structure of the mitotic checkpoint complex." Nature 2012 Apr 13
In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.
Hayashi,2012 (22433459) Hayashi K "The interaction and integration of auxin signaling components." Plant Cell Physiol 2012 Jun 06
IAA, a naturally occurring auxin, is a simple signaling molecule that regulates many diverse steps of plant development. Auxin essentially coordinates plant development through transcriptional regulation. Auxin binds to TIR1/AFB nuclear receptors, which are F-box subunits of the SCF ubiquitin ligase complex. The auxin signal is then modulated by the quantitative and qualitative responses of the Aux/IAA repressors and the auxin response factor (ARF) transcription factors. The specificity of the auxin-regulated gene expression profile is defined by several factors, such as the expression of these regulatory proteins, their post-transcriptional regulation, their stability and the affinity between these regulatory proteins. Auxin-binding protein 1 (ABP1) is a candidate protein for an auxin receptor that is implicated in non-transcriptional auxin signaling. ABP1 also affects TIR1/AFB-mediated auxin-responsive gene expression, implying that both the ABP1 and TIR1/AFB signaling machineries coordinately control auxin-mediated physiological events. Systematic approaches using the comprehensive mapping of the expression and interaction of signaling modules and computational modeling would be valuable for integrating our knowledge of auxin signals and responses.
Cunningham,2012 (22411985) Cunningham MR, McIntosh KA, Pediani JD, Robben J, Cooke AE, Nilsson M, Gould GW, Mundell S, Milligan G, Plevin R "Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4)." J Biol Chem 2012 Mar 13
Proteinase-activated receptors 4 (PAR(4)) is a Class A G-protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface in order for responsiveness to agonist to be retained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. In this study we found that PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit beta-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RxR) ER retention sequence located within intracellular loop-2 (R(183)AR), mutation of which ensured efficient membrane delivery of PAR(4). Surprisingly, we found that co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), through disruption of beta-COP1 binding and facilitating interaction with the chaperone protein 14-3-3 zeta. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signalling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.
van der Veen,2012 (22404627) van der Veen AG, Ploegh HL "Ubiquitin-like proteins." Annu Rev Biochem 2012 Jun 05
The eukaryotic ubiquitin family encompasses nearly 20 proteins that are involved in the posttranslational modification of various macromolecules. The ubiquitin-like proteins (UBLs) that are part of this family adopt the beta-grasp fold that is characteristic of its founding member ubiquitin (Ub). Although structurally related, UBLs regulate a strikingly diverse set of cellular processes, including nuclear transport, proteolysis, translation, autophagy, and antiviral pathways. New UBL substrates continue to be identified and further expand the functional diversity of UBL pathways in cellular homeostasis and physiology. Here, we review recent findings on such novel substrates, mechanisms, and functions of UBLs.
Kawano,2012 (22404616) Kawano T, Araseki M, Araki Y, Kinjo M, Yamamoto T, Suzuki T "A small peptide sequence is sufficient for initiating kinesin-1 activation through part of TPR region of KLC1." Traffic 2012 May 11
Kinesin-1 anterogradely transports vesicles containing cargo proteins when a protein-protein interaction activates it from an inhibited state. The C-terminal cytoplasmic region of kinesin-1 cargo protein Alcadeinalpha (Alcalpha) interacts with the KLC1 subunit's tetratricopeptide repeat (TPR) region, activating kinesin-1's association with vesicles and anterograde transport. We found that either of two 10-amino-acid WD motifs in Alcalpha cytoplasmic region was necessary and sufficient to initiate this activation. An artificial transmembrane protein containing either WD motif induced kinesin-1's vesicular association and anterograde transport in a KLC-dependent manner, even in the normally inhibiting presence of excess KLC1, thus allowing us to analyze the KLC1 TPR-WD functional interaction in detail in vivo. A part of TPR region was dispensable for the WD motifs' activation of kinesin-1 and transport, indicating that only part of the TPR structure is required for this function in vivo. For a different kinesin-1 cargo protein, JIP1, an 11-amino-acid C-terminal region was sufficient to recruit KLC1 to vesicles, but did not activate transport. These observations suggest that structurally different TPR-interacting peptides may have different effects on kinesin-1. This mechanism may partly explain how kinesin-1 can organize the transport of a wide variety of cargo molecules.
Schmidt,2012 (22361144) Schmidt O, Teis D "The ESCRT machinery." Curr Biol 2012 Feb 24
Heroes,2013 (22360570) Heroes E, Lesage B, Gornemann J, Beullens M, Van Meervelt L, Bollen M "The PP1 binding code: a molecular-lego strategy that governs specificity." FEBS J 2013 Jan 29
Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo.
Popovic,2012 (22354992) Popovic D, Akutsu M, Novak I, Harper JW, Behrends C, Dikic I "Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers." Mol Cell Biol 2012 Apr 11
Autophagy is an evolutionarily conserved degradation pathway characterized by dynamic rearrangement of membranes that sequester cytoplasm, protein aggregates, organelles, and pathogens for delivery to the vacuole and lysosome, respectively. The ability of autophagosomal membranes to act selectively toward specific cargo is dependent on the small ubiquitin-like modifier ATG8/LC3 and the LC3-interacting region (LIR) present in autophagy receptors. Here, we describe a comprehensive protein-protein interaction analysis of TBC (Tre2, Bub2, and Cdc16) domain-containing Rab GTPase-activating proteins (GAPs) as potential autophagy adaptors. We identified 14 TBC domain-containing Rab GAPs that bind directly to ATG8 modifiers and that colocalize with LC3-positive autophagy membranes in cells. Intriguingly, one of our screening hits, TBC1D5, contains two LIR motifs. The N-terminal LIR was critical for interaction with the retromer complex and transport of cargo. Direct binding of the retromer component VPS29 to TBC1D5 could be titrated out by LC3, indicating a molecular switch between endosomes and autophagy. Moreover, TBC1D5 could bridge the endosome and autophagosome via its C-terminal LIR motif. During starvation-induced autophagy, TBC1D5 was relocalized from endosomal localization to the LC3-positive autophagosomes. We propose that LC3-interacting Rab GAPs are implicated in the reprogramming of the endocytic trafficking events under starvation-induced autophagy.
Sczaniecka,2012 (22337874) Sczaniecka M, Gladstone K, Pettersson S, McLaren L, Huart AS, Wallace M "MDM2 protein-mediated ubiquitination of numb protein: identification of a second physiological substrate of MDM2 that employs a dual-site docking mechanism." J Biol Chem 2012 Apr 23
The E3 ubiquitin ligase, MDM2, uses a dual-site mechanism to ubiquitinate and degrade the tumor suppressor protein p53, involving interactions with the N-terminal hydrophobic pocket and the acidic domain of MDM2. The results presented here demonstrate that MDM2 also uses this same dual-site mechanism to bind to the cell fate determinant NUMB with both the N-terminal hydrophobic pocket and the acidic domain of MDM2 also involved in forming the interaction with NUMB. Furthermore, the acidic domain interactions are crucial for MDM2-mediated ubiquitination of NUMB. Contrary to p53, where two separate domains form the interface with MDM2, only one region within the phosphotyrosine binding domain of NUMB (amino acids 113-148) mediates binding to both these regions of MDM2. By binding to both domains on MDM2, NUMB disrupts the MDM2-p53 complex and MDM2-catalyzed ubiquitination of p53. Therefore, we have identified the mechanism NUMB uses to regulate the steady-state levels of the p53 in cells. By targeting the acidic domain of MDM2 using acid domain-binding ligands we can overcome MDM2-mediated ubiquitination and degradation of NUMB impacting on the stabilization of p53 in cells. Furthermore, delivery of MDM2 acid domain-binding ligands to cancer cells promotes p53-dependent growth arrest and the induction of apoptosis. This highlights the dual-site mechanism of MDM2 on another physiological substrate and identifies the acid domain as well as N terminus as a potential target for small molecules that inhibit MDM2.
Lee,2012 (22334659) Lee KY, Bang SW, Yoon SW, Lee SH, Yoon JB, Hwang DS "Phosphorylation of ORC2 protein dissociates origin recognition complex from chromatin and replication origins." J Biol Chem 2012 Apr 11
During the late M to the G(1) phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2-5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2-5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.
Ma,2012 (22331464) Ma J, Cai H, Wu T, Sobhian B, Huo Y, Alcivar A, Mehta M, Cheung KL, Ganesan S, Kong AN, Zhang DD, Xia B "PALB2 interacts with KEAP1 to promote NRF2 nuclear accumulation and function." Mol Cell Biol 2012 Mar 23
PALB2/FANCN is mutated in breast and pancreatic cancers and Fanconi anemia (FA). It controls the intranuclear localization, stability, and DNA repair function of BRCA2 and links BRCA1 and BRCA2 in DNA homologous recombination repair and breast cancer suppression. Here, we show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the master antioxidant transcription factor NRF2. PALB2 shares with NRF2 a highly conserved ETGE-type KEAP1 binding motif and can effectively compete with NRF2 for KEAP1 binding. PALB2 promotes NRF2 accumulation and function in the nucleus and lowers the cellular reactive oxygen species (ROS) level. In addition, PALB2 also regulates the rate of NRF2 export from the nucleus following induction. Our findings identify PALB2 as a regulator of cellular redox homeostasis and provide a new link between oxidative stress and the development of cancer and FA.
Shyu,2012 (22327740) Shyu C, Figueroa P, Depew CL, Cooke TF, Sheard LB, Moreno JE, Katsir L, Zheng N, Browse J, Howe GA "JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis." Plant Cell 2012 Mar 28
The lipid-derived hormone jasmonoyl-L-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxL-type EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif-containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors.
Kondo-Okamoto,2012 (22308029) Kondo-Okamoto N, Noda NN, Suzuki SW, Nakatogawa H, Takahashi I, Matsunami M, Hashimoto A, Inagaki F, Ohsumi Y, Okamoto K "Autophagy-related protein 32 acts as autophagic degron and directly initiates mitophagy." J Biol Chem 2012 Mar 26
Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.
Bhardwaj,2012 (22301153) Bhardwaj K, Liu P, Leibowitz JL, Kao CC "The coronavirus endoribonuclease Nsp15 interacts with retinoblastoma tumor suppressor protein." J Virol 2012 Mar 29
Coronaviruses encode an endoribonuclease, Nsp15, which has a poorly defined role in infection. Sequence analysis revealed a retinoblastoma protein-binding motif (LXCXE/D) in the majority of the Nsp15 of the severe acute respiratory syndrome coronavirus (SARS-CoV) and its orthologs in the alpha and beta coronaviruses. The endoribonuclease activity of the SARS-CoV Nsp15 (sNsp15) was stimulated by retinoblastoma protein (pRb) in vitro, and the two proteins can be coimmunoprecipitated from cellular extracts. Mutations in the pRb-binding motif rendered sNsp15 to be differentially modified by ubiquitin in cells, and cytotoxicity was observed upon its expression. Expression of the sNsp15 in cells resulted in an increased abundance of pRb in the cytoplasm, decreased overall levels of pRb, an increased proportion of cells in the S phase of the cell cycle, and an enhanced expression from a promoter normally repressed by pRb. The endoribonuclease activity of the mouse hepatitis virus (MHV) A59 Nsp15 was also increased by pRb in vitro, and an MHV with mutations in the LXCXE/D-motif, named vLC, exhibited a smaller plaque diameter and reduced the virus titer by approximately 1 log. Overexpression of pRb delayed the viral protein production by wild-type MHV but not by vLC. This study reveals that pRb and its interaction with Nsp15 can affect coronavirus infection and adds coronaviruses to a small but growing family of RNA viruses that encode a protein to interact with pRb.
Peti,2013 (22284538) Peti W, Nairn AC, Page R "Structural basis for protein phosphatase 1 regulation and specificity." FEBS J 2013 Jan 29
The ubiquitous serine/threonine protein phosphatase 1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. However, the free catalytic subunit of PP1, while an effective enzyme, lacks substrate specificity. Instead, it depends on a diverse set of regulatory proteins (>/= 200) to confer specificity towards distinct substrates. Here, we discuss recent advances in structural studies of PP1 holoenzyme complexes and summarize the new insights these studies have provided into the molecular basis of PP1 regulation and specificity.
Zhang,2012 (22266653) Zhang P, Lee H, Brunzelle JS, Couture JF "The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases." Nucleic Acids Res 2012 May 14
In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochemical and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative analysis of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations. Consistently, enzymatic assays reveal that WDR5 plays an important role in the optimal stimulation of MLL2-4, SET1A and SET1B methyltransferase activity by the RbBP5-ASH2L heterodimer. Overall, our findings illustrate the function of WDR5 in scaffolding the SET1 family of KMTs and further emphasize on the important role of WDR5 in regulating global histone H3 Lys-4 methylation.
Coon,2012 (22228094) Coon BG, Hernandez V, Madhivanan K, Mukherjee D, Hanna CB, Barinaga-Rementeria Ramirez I, Lowe M, Beales PL, Aguilar RC "The Lowe syndrome protein OCRL1 is involved in primary cilia assembly." Hum Mol Genet 2012 Mar 28
Lowe syndrome (LS) is a devastating, X-linked genetic disease characterized by the presence of congenital cataracts, profound learning disabilities and renal dysfunction. Unfortunately, children affected with LS often die early of health complications including renal failure. Although this syndrome was first described in the early 1950s and the affected gene, OCRL1, was identified more than 17 years ago, the mechanism by which Ocrl1 defects lead to LS's symptoms remains unknown. Here we show that LS display characteristics of a ciliopathy. Specifically, we found that patients' cells have defects in the assembly of primary cilia and this phenotype was reproduced in cell lines by knock-down of Ocrl1. Importantly, this defect could be rescued by re-introduction of WT Ocrl1 in both patient and Ocrl1 knock-down cells. In addition, a zebrafish animal model of LS exhibited cilia defects and multiple morphological and anatomical abnormalities typically seen in ciliopathies. Mechanistically, we show that Ocrl1 is involved in protein trafficking to the primary cilia in an Rab8-and IPIP27/Ses-dependent manner. Taking into consideration the relevance of the signaling pathways hosted by the primary cilium, our results suggest hitherto unrecognized mechanisms by which Ocrl1 deficiency may contribute to the phenotypic characteristics of LS. This conceptual change in our understanding of the disease etiology may provide an alternative avenue for the development of therapies.
Camp,2012 (22215675) Camp ND, James RG, Dawson DW, Yan F, Davison JM, Houck SA, Tang X, Zheng N, Major MB, Moon RT "Wilms tumor gene on X chromosome (WTX) inhibits degradation of NRF2 protein through competitive binding to KEAP1 protein." J Biol Chem 2012 Feb 27
WTX is a tumor suppressor protein that is lost or mutated in up to 30% of cases of Wilms tumor. Among its known functions, WTX interacts with the beta-transducin repeat containing family of ubiquitin ligase adaptors and promotes the ubiquitination and degradation of the transcription factor beta-catenin, a key control point in the WNT/beta-catenin signaling pathway. Here, we report that WTX interacts with a second ubiquitin ligase adaptor, KEAP1, which functions to regulate the ubiquitination of the transcription factor NRF2, a key control point in the antioxidant response. Surprisingly, we find that unlike its ability to promote the ubiquitination of beta-catenin, WTX inhibits the ubiquitination of NRF2. WTX and NRF2 compete for binding to KEAP1, and thus loss of WTX leads to rapid ubiquitination and degradation of NRF2 and a reduced response to cytotoxic insult. These results expand our understanding of the molecular mechanisms of WTX and reveal a novel regulatory mechanism governing the antioxidant response.
Gareau,2012 (22194619) Gareau JR, Reverter D, Lima CD "Determinants of small ubiquitin-like modifier 1 (SUMO1) protein specificity, E3 ligase, and SUMO-RanGAP1 binding activities of nucleoporin RanBP2." J Biol Chem 2012 Feb 15
The RanBP2 nucleoporin contains an internal repeat domain (IR1-M-IR2) that catalyzes E3 ligase activity and forms a stable complex with SUMO-modified RanGAP1 and UBC9 at the nuclear pore complex. RanBP2 exhibits specificity for SUMO1 as RanGAP1-SUMO1/UBC9 forms a more stable complex with RanBP2 compared with RanGAP1-SUMO2 that results in greater protection of RanGAP-SUMO1 from proteases. The IR1-M-IR2 SUMO E3 ligase activity also shows a similar preference for SUMO1. We utilized deletions and domain swap constructs in protease protection assays and automodification assays to define RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity. Our data suggest that elements in both IR1 and IR2 exhibit specificity for SUMO1. IR1 protects RanGAP1-SUMO1/UBC9 and functions as the primary E3 ligase of RanBP2, whereas IR2 retains the ability to interact with SUMO1 to promote SUMO1-specific E3 ligase activity. To determine the structural basis for SUMO1 specificity, a hybrid IR1 construct and IR1 were used to determine three new structures for complexes containing UBC9 with RanGAP1-SUMO1/2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2 and suggest that differences in SUMO specificity may be achieved through these subtle conformational differences.
Lara-Gonzalez,2012 (22193957) Lara-Gonzalez P, Scott MI, Diez M, Sen O, Taylor SS "BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner." J Cell Sci 2012 Jan 16
The spindle assembly checkpoint (SAC) is a signalling network that delays anaphase onset until all the chromosomes are attached to the mitotic spindle through their kinetochores. The downstream target of the spindle checkpoint is the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that targets several anaphase inhibitors for proteolysis, including securin and cyclin B1. In the presence of unattached kinetochores, the APC/C is inhibited by the mitotic checkpoint complex (MCC), a tetrameric complex composed of three SAC components, namely BubR1, Bub3 and Mad2, and the APC/C co-activator Cdc20. The molecular mechanisms underlying exactly how unattached kinetochores catalyse MCC formation and how the MCC then inhibits the APC/C remain obscure. Here, using RNAi complementation and in vitro ubiquitylation assays, we investigate the domains in BubR1 required for APC/C inhibition. We observe that kinetochore localisation of BubR1 is required for efficient MCC assembly and SAC response. Furthermore, in contrast to previous studies, we show that the N-terminal domain of BubR1 is the only domain involved in binding to Cdc20-Mad2 and the APC/C. Within this region, an N-terminal KEN box (KEN1) is essential for these interactions. By contrast, mutation of the second KEN box (KEN2) of BubR1 does not interfere with MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for inhibition of APC/C when activated by Cdc20 (APC/C(Cdc20)). Indeed, we show that this second KEN box promotes SAC function by blocking the recruitment of substrates to the APC/C. Thus, we propose a model in which the BubR1 KEN boxes play two very different roles, the first to promote MCC assembly and the second to block substrate recruitment to APC/C(Cdc20).
Rosa-Ferreira,2011 (22172677) Rosa-Ferreira C, Munro S "Arl8 and SKIP act together to link lysosomes to kinesin-1." Dev Cell 2011 Dec 16
Lysosomes move bidirectionally on microtubules, and this motility can be stimulated by overexpression of the small GTPase Arl8. By using affinity chromatography, we find that Arl8-GTP binds to the soluble protein SKIP (SifA and kinesin-interacting protein, aka PLEKHM2). SKIP was originally identified as a target of the Salmonella effector protein SifA and found to bind the light chain of kinesin-1 to activate the motor on the bacteria's replicative vacuole. We show that in uninfected cells both Arl8 and SKIP are required for lysosomes to distribute away from the microtubule-organizing center. We identify two kinesin light chain binding motifs in SKIP that are required for lysosomes to accumulate kinesin-1 and redistribute to the cell periphery. Thus, Arl8 binding to SKIP provides a link from lysosomal membranes to plus-end-directed motility. A splice variant of SKIP that lacks a light chain binding motif does not stimulate movement, suggesting fine-tuning by alternative splicing.
Ferrero,2011 (22163316) Ferrero M, Ferragud J, Orlando L, Valero L, Sanchez del Pino M, Farras R, Font de Mora J "Phosphorylation of AIB1 at mitosis is regulated by CDK1/CYCLIN B." PLoS One 2011 Dec 14
BACKGROUND: Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. METHODOLOGY/PRINCIPAL FINDINGS: Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. CONCLUSIONS: Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the transcriptional activity of AIB1.
Guettler,2011 (22153077) Guettler S, LaRose J, Petsalaki E, Gish G, Scotter A, Pawson T, Rottapel R, Sicheri F "Structural basis and sequence rules for substrate recognition by Tankyrase explain the basis for cherubism disease." Cell 2011 Dec 9
The poly(ADP-ribose)polymerases Tankyrase 1/2 (TNKS/TNKS2) catalyze the covalent linkage of ADP-ribose polymer chains onto target proteins, regulating their ubiquitylation, stability, and function. Dysregulation of substrate recognition by Tankyrases underlies the human disease cherubism. Tankyrases recruit specific motifs (often called RxxPDG "hexapeptides") in their substrates via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), each predicted to bind substrate. Here we report crystal structures of a representative ARC of TNKS2 bound to targeting peptides from six substrates. Using a solution-based peptide library screen, we derive a rule-based consensus for Tankyrase substrates common to four functionally conserved ARCs. This 8-residue consensus allows us to rationalize all known Tankyrase substrates and explains the basis for cherubism-causing mutations in the Tankyrase substrate 3BP2. Structural and sequence information allows us to also predict and validate other Tankyrase targets, including Disc1, Striatin, Fat4, RAD54, BCR, and MERIT40.
Molzan,2012 (22151054) Molzan M, Weyand M, Rose R, Ottmann C "Structural insights of the MLF1/14-3-3 interaction." FEBS J 2012 Jan 26
Myeloid leukaemia factor 1 (MLF1) binds to 14-3-3 adapter proteins by a sequence surrounding Ser34 with the functional consequences of this interaction largely unknown. We present here the high-resolution crystal structure of this binding motif [MLF1(29-42)pSer34] in complex with 14-3-3epsilon and analyse the interaction with isothermal titration calorimetry. Fragment-based ligand discovery employing crystals of the binary 14-3-3epsilon/MLF1(29-42)pSer34 complex was used to identify a molecule that binds to the interface rim of the two proteins, potentially representing the starting point for the development of a small molecule that stabilizes the MLF1/14-3-3 protein-protein interaction. Such a compound might be used as a chemical biology tool to further analyse the 14-3-3/MLF1 interaction without the use of genetic methods. Database Structural data are available in the Protein Data Bank under the accession number(s) 3UAL [14-3-3epsilon/MLF1(29-42)pSer34 complex] and 3UBW [14-3-3epsilon/MLF1(29-42)pSer34/3-pyrrolidinol complex] Structured digital abstract * 14-3-3 epsilon and MLF1 bind by x-ray crystallography (View interaction) * 14-3-3 epsilon and MLF1 bind by isothermal titration calorimetry (View Interaction: 1, 2).
Baek,2012 (22148351) Baek S, Kutchukian PS, Verdine GL, Huber R, Holak TA, Lee KW, Popowicz GM "Structure of the stapled p53 peptide bound to Mdm2." J Am Chem Soc 2012 Jan 12
Mdm2 is a major negative regulator of the tumor suppressor p53 protein, a protein that plays a crucial role in maintaining genome integrity. Inactivation of p53 is the most prevalent defect in human cancers. Inhibitors of the Mdm2-p53 interaction that restore the functional p53 constitute potential nongenotoxic anticancer agents with a novel mode of action. We present here a 2.0 A resolution structure of the Mdm2 protein with a bound stapled p53 peptide. Such peptides, which are conformationally and proteolytically stabilized with all-hydrocarbon staples, are an emerging class of biologics that are capable of disrupting protein-protein interactions and thus have broad therapeutic potential. The structure represents the first crystal structure of an i, i + 7 stapled peptide bound to its target and reveals that rather than acting solely as a passive conformational brace, a staple can intimately interact with the surface of a protein and augment the binding interface.
Namanja,2012 (22147707) Namanja AT, Li YJ, Su Y, Wong S, Lu J, Colson LT, Wu C, Li SS, Chen Y "Insights into high affinity small ubiquitin-like modifier (SUMO) recognition by SUMO-interacting motifs (SIMs) revealed by a combination of NMR and peptide array analysis." J Biol Chem 2012 Jan 30
The small ubiquitin-like modifiers (SUMOs) regulate many essential cellular functions. Only one type of SUMO-interacting motif (SIM) has been identified that can extend the beta-sheet of SUMO as either a parallel or an antiparallel strand. The molecular determinants of the bound orientation and paralogue specificity of a SIM are unclear. To address this question, we have conducted structural studies of SUMO1 in complex with a SUMO1-specific SIM that binds to SUMO1 with high affinity without post-translational modifications using nuclear magnetic resonance methods. In addition, the SIM sequence requirements have been investigated by peptide arrays in comparison with another high affinity SIM that binds in the opposing orientation. We found that antiparallel binding SIMs tolerate more diverse sequences, whereas the parallel binding SIMs prefer the more strict sequences consisting of (I/V)DLT that have a preference in high affinity SUMO2 and -3 binding. Comparison of two high affinity SUMO1-binding SIMs that bind in opposing orientations has revealed common SUMO1-specific interactions needed for high affinity binding. This study has significantly advanced our understanding of the molecular determinants underlining SUMO-SIM recognition.
Moschou,2012 (22121979) Moschou PN, Bozhkov PV "Separases: biochemistry and function." Physiol Plant 2012 Apr 16
Tight regulation of cell cycle is of critical importance for eukaryotic biology and is achieved through a combined action of a large number of highly specialized proteins. Separases are evolutionarily conserved caspase-like proteases playing a crucial role in cell cycle regulation, as they execute sister chromatid separation at metaphase to anaphase transition. In contrast to extensively studied yeast and metazoan separases, very little is known about the role of separases in plant biology. Here we describe the molecular mechanisms of separase-mediated chromatid segregation in yeast and metazoan models, discuss new emerging but less-understood functions of separases and highlight major gaps in our knowledge about plant separases.
Barford,2011 (22084387) Barford D "Structural insights into anaphase-promoting complex function and mechanism." Philos Trans R Soc Lond B Biol Sci 2011 Nov 15
The anaphase-promoting complex or cyclosome (APC/C) controls sister chromatid segregation and the exit from mitosis by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. This unusually large E3 RING-cullin ubiquitin ligase is assembled from 13 different proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D box and KEN box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. Recent structural and biochemical studies of the APC/C are beginning to reveal an understanding of the roles of individual APC/C subunits and co-activators and how they mutually interact to mediate APC/C functions. This review focuses on the findings showing how information on the structural organization of the APC/C provides insights into the role of co-activators and core APC/C subunits in mediating substrate recognition. Mechanisms of regulating and modulating substrate recognition are discussed in the context of controlling the binding of the co-activator to the APC/C, and the accessibility and conformation of the co-activator when bound to the APC/C.
Mizushima,2011 (22078875) Mizushima N, Komatsu M "Autophagy: renovation of cells and tissues." Cell 2011 Nov 14
Autophagy is the major intracellular degradation system by which cytoplasmic materials are delivered to and degraded in the lysosome. However, the purpose of autophagy is not the simple elimination of materials, but instead, autophagy serves as a dynamic recycling system that produces new building blocks and energy for cellular renovation and homeostasis. Here we provide a multidisciplinary review of our current understanding of autophagy's role in metabolic adaptation, intracellular quality control, and renovation during development and differentiation. We also explore how recent mouse models in combination with advances in human genetics are providing key insights into how the impairment or activation of autophagy contributes to pathogenesis of diverse diseases, from neurodegenerative diseases such as Parkinson disease to inflammatory disorders such as Crohn disease.
Bohdanowicz,2012 (22072788) Bohdanowicz M, Balkin DM, De Camilli P, Grinstein S "Recruitment of OCRL and Inpp5B to phagosomes by Rab5 and APPL1 depletes phosphoinositides and attenuates Akt signaling." Mol Biol Cell 2012 Jan 02
Sealing of phagosomes is accompanied by the disappearance of phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) from their cytoplasmic leaflet. Elimination of PtdIns(4,5)P(2), which is required for actin remodeling during phagosome formation, has been attributed to hydrolysis by phospholipase C and phosphorylation by phosphatidylinositol 3-kinase. We found that two inositol 5-phosphatases, OCRL and Inpp5B, become associated with nascent phagosomes. Both phosphatases, which are Rab5 effectors, associate with the adaptor protein APPL1, which is recruited to the phagosomes by active Rab5. Knockdown of APPL1 or inhibition of Rab5 impairs association of OCRL and Inpp5B with phagosomes and prolongs the presence of PtdIns(4,5)P(2) and actin on their membranes. Even though APPL1 can serve as an anchor for Akt, its depletion accentuated the activation of the kinase, likely by increasing the amount of PtdIns(4,5)P(2) available to generate phosphatidylinositol (3,4,5)-trisphosphate. Thus, inositol 5-phosphatases are important contributors to the phosphoinositide remodeling and signaling that are pivotal for phagocytosis.
Francis,2011 (22057126) Francis DM, Rozycki B, Koveal D, Hummer G, Page R, Peti W "Structural basis of p38alpha regulation by hematopoietic tyrosine phosphatase." Nat Chem Biol 2011 Nov 17
MAP kinases regulate essential cellular events, including cell growth, differentiation and inflammation. The solution structure of a complete MAPK-MAPK-regulatory protein complex, p38alpha-HePTP, was determined, enabling a comprehensive investigation of the molecular basis of specificity and fidelity in MAPK regulation. Structure determination was achieved by combining NMR spectroscopy and small-angle X-ray scattering data with a new ensemble calculation-refinement procedure. We identified 25 residues outside of the HePTP kinase interaction motif necessary for p38alpha recognition. The complex adopts an extended conformation in solution and rarely samples the conformation necessary for kinase deactivation. Complex formation also does not affect the N-terminal lobe, the activation loop of p38alpha or the catalytic domain of HePTP. Together, these results show how the downstream tyrosine phosphatase HePTP regulates p38alpha and provide for fundamentally new insights into MAPK regulation and specificity.
Moniz,2011 (22040655) Moniz L, Dutt P, Haider N, Stambolic V "Nek family of kinases in cell cycle, checkpoint control and cancer." Cell Div 2011 Nov 23
Early studies in lower Eukaryotes have defined a role for the members of the NimA related kinase (Nek) family of protein kinases in cell cycle control. Expansion of the Nek family throughout evolution has been accompanied by their broader involvement in checkpoint regulation and cilia biology. Moreover, mutations of Nek family members have been identified as drivers behind the development of ciliopathies and cancer. Recent advances in studying the physiological roles of Nek family members utilizing mouse genetics and RNAi-mediated knockdown are revealing intricate associations of Nek family members with fundamental biological processes. Here, we aim to provide a comprehensive account of our understanding of Nek kinase biology and their involvement in cell cycle, checkpoint control and cancer.
Collart,2011 (22027279) Collart MA, Panasenko OO "The Ccr4--not complex." Gene 2011 Dec 26
The Ccr4-Not complex is a unique, essential and conserved multi-subunit complex that acts at the level of many different cellular functions to regulate gene expression. Two enzymatic activities, namely ubiquitination and deadenylation, are provided by different subunits of the complex. However, studies over the last decade have demonstrated a tantalizing multi-functionality of this complex that extends well beyond its identified enzymatic activities. Most of our initial knowledge about the Ccr4-Not complex stemmed from studies in yeast, but an increasing number of reports on this complex in other species are emerging. In this review we will discuss the structure and composition of the complex, and describe the different cellular functions with which the Ccr4-Not complex has been connected in different organisms. Finally, based upon our current state of knowledge, we will propose a model to explain how one complex can provide such multi-functionality. This model suggests that the Ccr4-Not complex might function as a "chaperone platform".
Licausi,2011 (22020282) Licausi F, Kosmacz M, Weits DA, Giuntoli B, Giorgi FM, Voesenek LA, Perata P, van Dongen JT "Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization." Nature 2011 Nov 18
The majority of eukaryotic organisms rely on molecular oxygen for respiratory energy production. When the supply of oxygen is compromised, a variety of acclimation responses are activated to reduce the detrimental effects of energy depletion. Various oxygen-sensing mechanisms have been described that are thought to trigger these responses, but they each seem to be kingdom specific and no sensing mechanism has been identified in plants until now. Here we show that one branch of the ubiquitin-dependent N-end rule pathway for protein degradation, which is active in both mammals and plants, functions as an oxygen-sensing mechanism in Arabidopsis thaliana. We identified a conserved amino-terminal amino acid sequence of the ethylene response factor (ERF)-transcription factor RAP2.12 to be dedicated to an oxygen-dependent sequence of post-translational modifications, which ultimately lead to degradation of RAP2.12 under aerobic conditions. When the oxygen concentration is low-as during flooding-RAP2.12 is released from the plasma membrane and accumulates in the nucleus to activate gene expression for hypoxia acclimation. Our discovery of an oxygen-sensing mechanism opens up new possibilities for improving flooding tolerance in crops.
Gibbs,2011 (22020279) Gibbs DJ, Lee SC, Isa NM, Gramuglia S, Fukao T, Bassel GW, Correia CS, Corbineau F, Theodoulou FL, Bailey-Serres J, Holdsworth MJ "Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants." Nature 2011 Nov 18
Plants and animals are obligate aerobes, requiring oxygen for mitochondrial respiration and energy production. In plants, an unanticipated decline in oxygen availability (hypoxia), as caused by roots becoming waterlogged or foliage submergence, triggers changes in gene transcription and messenger RNA translation that promote anaerobic metabolism and thus sustain substrate-level ATP production. In contrast to animals, oxygen sensing has not been ascribed to a mechanism of gene regulation in response to oxygen deprivation in plants. Here we show that the N-end rule pathway of targeted proteolysis acts as a homeostatic sensor of severe low oxygen levels in Arabidopsis, through its regulation of key hypoxia-response transcription factors. We found that plants lacking components of the N-end rule pathway constitutively express core hypoxia-response genes and are more tolerant of hypoxic stress. We identify the hypoxia-associated ethylene response factor group VII transcription factors of Arabidopsis as substrates of this pathway. Regulation of these proteins by the N-end rule pathway occurs through a characteristic conserved motif at the amino terminus initiating with Met-Cys. Enhanced stability of one of these proteins, HRE2, under low oxygen conditions improves hypoxia survival and reveals a molecular mechanism for oxygen sensing in plants via the evolutionarily conserved N-end rule pathway. SUB1A-1, a major determinant of submergence tolerance in rice, was shown not to be a substrate for the N-end rule pathway despite containing the N-terminal motif, indicating that it is uncoupled from N-end rule pathway regulation, and that enhanced stability may relate to the superior tolerance of Sub1 rice varieties to multiple abiotic stresses.
Sriram,2011 (22016057) Sriram SM, Kim BY, Kwon YT "The N-end rule pathway: emerging functions and molecular principles of substrate recognition." Nat Rev Mol Cell Biol 2011 Oct 21
The N-end rule defines the protein-destabilizing activity of a given amino-terminal residue and its post-translational modification. Since its discovery 25 years ago, the pathway involved in the N-end rule has been thought to target only a limited set of specific substrates of the ubiquitin-proteasome system. Recent studies have provided insights into the components, substrates, functions and structural basis of substrate recognition. The N-end rule pathway is now emerging as a major cellular proteolytic system, in which the majority of proteins are born with or acquire specific N-terminal degradation determinants through protein-specific or global post-translational modifications.
Chen,2011 (22002310) Chen HZ, Li L, Wang WJ, Du XD, Wen Q, He JP, Zhao BX, Li GD, Zhou W, Xia Y, Yang QY, Hew CL, Liou YC, Wu Q "Prolyl isomerase Pin1 stabilizes and activates orphan nuclear receptor TR3 to promote mitogenesis." Oncogene 2011 Oct 17
Pin1 regulates a subset of phosphoproteins by isomerizing phospho-Ser/Thr-Pro motifs via a 'post-phosphorylation' mechanism. Here, we characterize TR3 as a novel Pin1 substrate, and the mitogenic function of TR3 depends on Pin1-induced isomerization. There are at least three phospho-Ser-Pro motifs on TR3 that bind to Pin1. The Ser95-Pro motif of TR3 is the key site through which Pin1 enhances TR3 stability by retarding its degradation. Pin1 can also catalyze TR3 through phospho-Ser431-Pro motif, which is phosphorylated by extracellular signal-regulated kinase 2 (ERK2), resulting in enhanced TR3 transactivation. Furthermore, Pin1 not only facilitates TR3 targeting to the promoter of cyclin D2, a novel downstream target of TR3, but also promotes TR3 to recruit p300, thereby inducing cell proliferation. Importantly, we found that Pin1 is indispensable for TR3 to promote tumor growth both in vitro and in vivo. Our study thus suggests that Pin1 has an important role in cell proliferation by isomerizing TR3.Oncogene advance online publication, 17 October 2011; doi:10.1038/onc.2011.463.
Ahn,2011 (22000856) Ahn VE, Chu ML, Choi HJ, Tran D, Abo A, Weis WI "Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6." Dev Cell 2011 Nov 14
LDL receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Wnt growth factors, and also bind Dkk proteins, secreted inhibitors of Wnt signaling. The LRP5/6 ectodomain contains four beta-propeller/EGF-like domain repeats. The first two repeats, LRP6(1-2), bind to several Wnt variants, whereas LRP6(3-4) binds other Wnts. We present the crystal structure of the Dkk1 C-terminal domain bound to LRP6(3-4), and show that the Dkk1 N-terminal domain binds to LRP6(1-2), demonstrating that a single Dkk1 molecule can bind to both portions of the LRP6 ectodomain and thereby inhibit different Wnts. Small-angle X-ray scattering analysis of LRP6(1-4) bound to a noninhibitory antibody fragment or to full-length Dkk1 shows that in both cases the ectodomain adopts a curved conformation that places the first three repeats at a similar height relative to the membrane. Thus, Wnts bound to either portion of the LRP6 ectodomain likely bear a similar spatial relationship to Frizzled coreceptors.
Tan,2011 (21998309) Tan BZ, Jiang F, Tan MY, Yu D, Huang H, Shen Y, Soong TW "Functional characterization of alternative splicing in the C terminus of L-type CaV1.3 channels." J Biol Chem 2011 Dec 05
Ca(V)1.3 channels are unique among the high voltage-activated Ca(2+) channel family because they activate at the most negative potentials and display very rapid calcium-dependent inactivation. Both properties are of crucial importance in neurons of the suprachiasmatic nucleus and substantia nigra, where the influx of Ca(2+) ions at subthreshold membrane voltages supports pacemaking function. Previously, alternative splicing in the Ca(V)1.3 C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), resulting in a pronounced activation at more negative voltages and faster inactivation in the latter. It was further shown that the C-terminal modulator in the Ca(V)1.3(42) isoforms modulates calmodulin binding to the IQ domain. Using splice variant-specific antibodies, we determined that protein localization of both splice variants in different brain regions were similar. Using the transcript-scanning method, we further identified alternative splicing at four loci in the C terminus of Ca(V)1.3 channels. Alternative splicing of exon 41 removes the IQ motif, resulting in a truncated Ca(V)1.3 protein with diminished inactivation. Splicing of exon 43 causes a frameshift and exhibits a robust inactivation of similar intensity to Ca(V)1.3(42A). Alternative splicing of exons 44 and 48 are in-frame, altering interaction of the distal modulator with the IQ domain and tapering inactivation slightly. Thus, alternative splicing in the C terminus of Ca(V)1.3 channels modulates its electrophysiological properties, which could in turn alter neuronal firing properties and functions.
Cheng,2011 (21984209) Cheng Z, Biechele T, Wei Z, Morrone S, Moon RT, Wang L, Xu W "Crystal structures of the extracellular domain of LRP6 and its complex with DKK1." Nat Struct Mol Biol 2011 Nov 07
Low-density-lipoprotein (LDL) receptor-related proteins 5 and 6 (LRP5/6) are Wnt co-receptors essential for Wnt/beta-catenin signaling. Dickkopf 1 (DKK1) inhibits Wnt signaling by interacting with the extracellular domains of LRP5/6 and is a drug target for multiple diseases. Here we present the crystal structures of a human LRP6-E3E4-DKK1 complex and the first and second halves of human LRP6's four propeller-epidermal growth factor (EGF) pairs (LRP6-E1E2 and LRP6-E3E4). Combined with EM analysis, these data demonstrate that LRP6-E1E2 and LRP6-E3E4 form two rigid structural blocks, with a short intervening hinge that restrains their relative orientation. The C-terminal domain of DKK1 (DKK1c) interacts with the top surface of the LRP6-E3 YWTD propeller and given their structural similarity, probably also that of the LRP6-E1 propeller, through conserved hydrophobic patches buttressed by a network of salt bridges and hydrogen bonds. Our work provides key insights for understanding LRP5/6 structure and the interaction of LRP5/6 with DKK, as well as for drug discovery.
Ito,2011 (21976065) Ito K, Takahashi A, Morita M, Suzuki T, Yamamoto T "The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability." Protein Cell 2011 Oct 06
The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation, although their precise roles remain to be established. In this study, we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells. Flow cytometric analysis revealed that the sub G(1) fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G1 fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits, suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity. Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits. Importantly, the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells. The formation of P-bodies, where mRNA decay is reported to take place, was largely suppressed in CNOT1-depleted cells. Therefore, CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex, and thus is critical in control of mRNA deadenylation and mRNA decay. We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4, which is associated with endoplasmic reticulum ER stress-induced apoptosis. Taken together, CNOT1 depletion structurally and functionally deteriorates the CCR4-NOTcomplex and induces stabilization of mRNAs, which results in the increment of translation causing ER stress-mediated apoptosis. We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.
Vicinanza,2011 (21971085) Vicinanza M, Di Campli A, Polishchuk E, Santoro M, Di Tullio G, Godi A, Levtchenko E, De Leo MG, Polishchuk R, Sandoval L, Marzolo MP, De Matteis MA "OCRL controls trafficking through early endosomes via PtdIns4,5P-dependent regulation of endosomal actin." EMBO J 2011 Dec 14
Mutations in the phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P(2)) 5-phosphatase OCRL cause Lowe syndrome, which is characterised by congenital cataracts, central hypotonia, and renal proximal tubular dysfunction. Previous studies have shown that OCRL interacts with components of the endosomal machinery; however, its role in endocytosis, and thus the pathogenic mechanisms of Lowe syndrome, have remained elusive. Here, we show that via its 5-phosphatase activity, OCRL controls early endosome (EE) function. OCRL depletion impairs the recycling of multiple classes of receptors, including megalin (which mediates protein reabsorption in the kidney) that are retained in engorged EEs. These trafficking defects are caused by ectopic accumulation of PtdIns4,5P(2) in EEs, which in turn induces an N-WASP-dependent increase in endosomal F-actin. Our data provide a molecular explanation for renal proximal tubular dysfunction in Lowe syndrome and highlight that tight control of PtdIns4,5P(2) and F-actin at the EEs is essential for exporting cargoes that transit this compartment.
Cong,2011 (21965678) Cong L, Pakala SB, Ohshiro K, Li DQ, Kumar R "SUMOylation and SUMO-interacting motif (SIM) of metastasis tumor antigen 1 (MTA1) synergistically regulate its transcriptional repressor function." J Biol Chem 2011 Dec 19
Metastasis tumor antigen 1 (MTA1), a component of the Mi-2.nucleosome remodeling and deacetylase complex, plays a crucial role in gene transcription, but the mechanism involved remains largely unknown. Here, we report that MTA1 is a substrate for small ubiquitin-related modifier 2/3 (SUMO2/3) in vivo. Protein inhibitor of activated STAT (PIAS) proteins enhance SUMOylation of MTA1 and may participate in paralog-selective SUMOylation, whereas sentrin/SUMO-specific protease 1 (SENP1) and 2 may act as deSUMOylation enzymes for MTA1. Moreover, MTA1 contains a functional SUMO-interacting motif (SIM) at its C terminus, and SIM is required for the efficient SUMOylation of MTA1. SUMO conjugation on Lys-509, which is located within the SUMO consensus site, together with SIM synergistically regulates the co-repressor activity of MTA1 on PS2 transcription, probably by recruiting HDAC2 onto the PS2 promoter. Interestingly, MTA1 may up-regulate the expression of SUMO2 via interaction with RNA polymerase II and SP1 at the SUMO2 promoter. These findings not only provide novel mechanistic insights into the regulation of the transcriptional repressor function of MTA1 by SUMOylation and SIM but also uncover a potential function of MTA1 in modulating the SUMOylation pathway.
Pauwels,2011 (21963667) Pauwels L, Goossens A "The JAZ proteins: a crucial interface in the jasmonate signaling cascade." Plant Cell 2011 Oct 28
Jasmonates are phytohormones that regulate many aspects of plant growth, development, and defense. Within the signaling cascades that are triggered by jasmonates, the JASMONATE-ZIM DOMAIN (JAZ) repressor proteins play a central role. The endogenous bioactive JA-Ile conjugate mediates the binding of JAZ proteins to the F-box protein CORONATINE INSENSITIVE1 (COI1), part of the Skp1/Cullin/F-box SCF(COI1) ubiquitin E3 ligase complex. Upon the subsequent destruction of the JAZ proteins by the 26S proteasome, multiple transcription factors are relieved from JAZ-mediated repression, allowing them to activate their respective downstream responses. However, many questions remain regarding the targets, specificity, function, and regulation of the different JAZ proteins. Here, we review recent studies on the model plant Arabidopsis thaliana that provided essential and novel insights. JAZ proteins have been demonstrated to interact with a broad array of transcription factors that each control specific downstream processes. Recruitment of the corepressor TOPLESS unveiled a mechanism for JAZ-mediated gene repression. Finally, the presence of JAZ proteins was also found to be regulated by alternative splicing and interactions with proteins from other hormonal signaling pathways. Overall, these contemporary findings underscore the value of protein-protein interaction studies to acquire fundamental insight into molecular signaling pathways.
Zhang,2012 (21952048) Zhang K, Rodriguez-Aznar E, Yabuta N, Owen RJ, Mingot JM, Nojima H, Nieto MA, Longmore GD "Lats2 kinase potentiates Snail1 activity by promoting nuclear retention upon phosphorylation." EMBO J 2012 Jan 04
Snail1 is a central regulator of epithelial cell adhesion and movement in epithelial-to-mesenchymal transitions (EMTs) during embryo development; a process reactivated during cancer metastasis. While induction of Snail1 transcription precedes EMT induction, post-translational regulation of Snail1 is also critical for determining Snail1's protein level, subcellular localization, and capacity to induce EMT. To identify novel post-translational regulators of Snail1, we developed a live cell, bioluminescence-based screen. From a human kinome RNAi screen, we have identified Lats2 kinase as a novel regulator of Snail1 protein level, subcellular localization, and thus, activity. We show that Lats2 interacts with Snail1 and directly phosphorylates Snail1 at residue T203. This occurs in the nucleus and serves to retain Snail1 in the nucleus thereby enhancing its stability. Lats2 was found to positively influence cellular EMT and tumour cell invasion, in a Snail1-dependent manner. Indeed during TGFbeta-induced EMT Lats2 is activated and Snail1 phosphorylated at T203. Analysis in mouse and zebrafish embryo development confirms that Lats2 acts as a positive modulator of Snail1 protein level and potentiates its in vivo EMT activity.
Bourhis,2011 (21944579) Bourhis E, Wang W, Tam C, Hwang J, Zhang Y, Spittler D, Huang OW, Gong Y, Estevez A, Zilberleyb I, Rouge L, Chiu C, Wu Y, Costa M, Hannoush RN, Franke Y, Cochran AG "Wnt antagonists bind through a short peptide to the first beta-propeller domain of LRP5/6." Structure 2011 Oct 17
The Wnt pathway inhibitors DKK1 and sclerostin (SOST) are important therapeutic targets in diseases involving bone loss or damage. It has been appreciated that Wnt coreceptors LRP5/6 are also important, as human missense mutations that result in bone overgrowth (bone mineral density, or BMD, mutations) cluster to the E1 propeller domain of LRP5. Here, we report a crystal structure of LRP6 E1 bound to an antibody, revealing that the E1 domain is a peptide recognition module. Remarkably, the consensus E1 binding sequence is a close match to a conserved tripeptide motif present in all Wnt inhibitors that bind LRP5/6. We show that this motif is important for DKK1 and SOST binding to LRP6 and for inhibitory function, providing a detailed structural explanation for the effect of the BMD mutations.
Scott,2011 (21940857) Scott DC, Monda JK, Bennett EJ, Harper JW, Schulman BA "N-terminal acetylation acts as an avidity enhancer within an interconnected multiprotein complex." Science 2011 Nov 04
Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.
Obsil,2011 (21920446) Obsil T, Obsilova V "Structural basis of 14-3-3 protein functions." Semin Cell Dev Biol 2011 Nov 02
The 14-3-3 proteins, a family of conserved regulatory molecules, participate in a wide range of cellular processes through binding interactions with hundreds of structurally and functionally diverse proteins. Several distinct mechanisms of the 14-3-3 protein function were described, including conformational modulation of the bound protein, masking of its sequence-specific or structural features, and scaffolding that facilitates interaction between two simultaneously bound proteins. Details of these functional modes, especially from the structural point of view, still remain mostly elusive. This review gives an overview of the current knowledge concerning the structure of 14-3-3 proteins and their complexes as well as the insights it provides into the mechanisms of their functions. We discuss structural basis of target recognition by 14-3-3 proteins, common structural features of their complexes and known mechanisms of 14-3-3 protein-dependent regulations.
Dodding,2011 (21915095) Dodding MP, Mitter R, Humphries AC, Way M "A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome." EMBO J 2011 Dec 22
Transport of cargoes by kinesin-1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit kinesin-1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define kinesin-1 binding. We now show that a bipartite tryptophan-based kinesin-1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for kinesin-1-dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan-based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases.
Davey,2011 (21909575) Davey NE, Van Roey K, Weatheritt RJ, Toedt G, Uyar B, Altenberg B, Budd A, Diella F, Dinkel H, Gibson TJ "Attributes of short linear motifs." Mol Biosyst 2011 Dec 02
Traditionally, protein-protein interactions were thought to be mediated by large, structured domains. However, it has become clear that the interactome comprises a wide range of binding interfaces with varying degrees of flexibility, ranging from rigid globular domains to disordered regions that natively lack structure. Enrichment for disorder in highly connected hub proteins and its correlation with organism complexity hint at the functional importance of disordered regions. Nevertheless, they have not yet been extensively characterised. Shifting the attention from globular domains to disordered regions of the proteome might bring us closer to elucidating the dense and complex connectivity of the interactome. An important class of disordered interfaces are the compact mono-partite, short linear motifs (SLiMs, or eukaryotic linear motifs (ELMs)). They are evolutionarily plastic and interact with relatively low affinity due to the limited number of residues that make direct contact with the binding partner. These features confer to SLiMs the ability to evolve convergently and mediate transient interactions, which is imperative to network evolution and to maintain robust cell signalling, respectively. The ability to discriminate biologically relevant SLiMs by means of different attributes will improve our understanding of the complexity of the interactome and aid development of bioinformatics tools for motif discovery. In this paper, the curated instances currently available in the Eukaryotic Linear Motif (ELM) database are analysed to provide a clear overview of the defining attributes of SLiMs. These analyses suggest that functional SLiMs have higher levels of conservation than their surrounding residues, frequently evolve convergently, preferentially occur in disordered regions and often form a secondary structure when bound to their interaction partner. These results advocate searching for small groupings of residues in disordered regions with higher relative conservation and a propensity to form the secondary structure. Finally, the most interesting conclusions are examined in regard to their functional consequences.
Chandran,2011 (21900244) Chandran S, Li H, Dong W, Krasinska K, Adams C, Alexandrova L, Chien A, Hallows KR, Bhalla V "Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) regulation by 14-3-3 protein binding at canonical serum and glucocorticoid kinase 1 (SGK1) phosphorylation sites." J Biol Chem 2011 Oct 24
Regulation of epithelial Na(+) channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed "minor," and one is termed "major," based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3epsilon mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.
Hamel,2011 (21873571) Hamel LP, Benchabane M, Nicole MC, Major IT, Morency MJ, Pelletier G, Beaudoin N, Sheen J, Seguin A "Stress-responsive mitogen-activated protein kinases interact with the EAR motif of a poplar zinc finger protein and mediate its degradation through the 26S proteasome." Plant Physiol 2011 Nov 04
Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors.
Safari,2011 (21873224) Safari F, Murata-Kamiya N, Saito Y, Hatakeyama M "Mammalian Pragmin regulates Src family kinases via the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif that is exploited by bacterial effectors." Proc Natl Acad Sci U S A 2011 Sep 07
Several pathogenic bacteria have adopted effector proteins that, upon delivery into mammalian cells, undergo tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) or EPIYA-like sequence motif by host kinases such as Src family kinases (SFKs). This EPIYA phosphorylation triggers complex formation of bacterial effectors with SH2 domain-containing proteins that results in perturbation of host cell signaling and subsequent pathogenesis. Although the presence of such an anomalous protein interaction suggests the existence of a mammalian EPIYA-containing protein whose function is mimicked or subverted by bacterial EPIYA effectors, no molecule that uses the EPIYA motif for biological function has so far been reported in mammals. Here we show that mammalian Pragmin/SgK223 undergoes tyrosine phosphorylation at the EPIYA motif by SFKs and thereby acquires the ability to interact with the SH2 domain of the C-terminal Src kinase (Csk), a negative regulator of SFKs. The Pragmin-Csk interaction prevents translocalization of Csk from the cytoplasm to the membrane and subsequent inactivation of membrane-associated SFKs. As a result, SFK activity is sustained in cells where Pragmin is phosphorylated at the EPIYA motif. Because EPIYA phosphorylation of Pragmin is mediated by SFKs, cytoplasmic sequestration of Csk by Pragmin establishes a positive feedback regulation of SFK activation. Remarkably, the Helicobacter pylori EPIYA effector CagA binds to the Csk SH2 domain in place of Pragmin and enforces membrane recruitment of Csk and subsequent inhibition of SFKs. This work identifies Pragmin as a mammalian EPIYA effector and suggests that bacterial EPIYA effectors target Pragmin to subvert SFKs for successful infection.
Shpilka,2011 (21867568) Shpilka T, Weidberg H, Pietrokovski S, Elazar Z "Atg8: an autophagy-related ubiquitin-like protein family." Genome Biol 2011 Nov 04
Autophagy-related (Atg) proteins are eukaryotic factors participating in various stages of the autophagic process. Thus far 34 Atgs have been identified in yeast, including the key autophagic protein Atg8. The Atg8 gene family encodes ubiquitin-like proteins that share a similar structure consisting of two amino-terminal alpha helices and a ubiquitin-like core. Atg8 family members are expressed in various tissues, where they participate in multiple cellular processes, such as intracellular membrane trafficking and autophagy. Their role in autophagy has been intensively studied. Atg8 proteins undergo a unique ubiquitin-like conjugation to phosphatidylethanolamine on the autophagic membrane, a process essential for autophagosome formation. Whereas yeast has a single Atg8 gene, many other eukaryotes contain multiple Atg8 orthologs. Atg8 genes of multicellular animals can be divided, by sequence similarities, into three subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which are present in sponges, cnidarians (such as sea anemones, corals and hydras) and bilateral animals. Although genes from all three subfamilies are found in vertebrates, some invertebrate lineages have lost the genes from one or two subfamilies. The amino terminus of Atg8 proteins varies between the subfamilies and has a regulatory role in their various functions. Here we discuss the evolution of Atg8 proteins and summarize the current view of their function in intracellular trafficking and autophagy from a structural perspective.
Liou,2011 (21852138) Liou YC, Zhou XZ, Lu KP "Prolyl isomerase Pin1 as a molecular switch to determine the fate of phosphoproteins." Trends Biochem Sci 2011 Oct
Pin1 is a highly conserved enzyme that only isomerizes specific phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Such conformational changes represent a novel and tightly controlled signaling mechanism regulating a spectrum of protein activities in physiology and disease; often through phosphorylation-dependent, ubiquitin-mediated proteasomal degradation. In this review, we summarize recent advances in elucidating the role and regulation of Pin1 in controlling protein stability. We also propose a mechanism by which Pin1 functions as a molecular switch to control the fates of phosphoproteins. We finally stress the need to develop tools to visualize directly Pin1-catalyzed protein conformational changes as a way to determine their roles in the development and treatment of human diseases.
Wei,2011 (21832156) Wei R, Ngo B, Wu G, Lee WH "Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint." Mol Biol Cell 2011 Sep 30
The spindle assemble checkpoint (SAC) is critical for accurate chromosome segregation. Hec1 contributes to chromosome segregation in part by mediating SAC signaling and chromosome alignment. However, the molecular mechanism by which Hec1 modulates checkpoint signaling and alignment remains poorly understood. We found that Hec1 serine 165 (S165) is preferentially phosphorylated at kinetochores. Phosphorylated Hec1 serine 165 (pS165) specifically localized to kinetochores of misaligned chromosomes, showing a spatiotemporal distribution characteristic of SAC molecules. Expressing an RNA interference (RNAi)-resistant S165A mutant in Hec1-depleted cells permitted normal progression to metaphase, but accelerated the metaphase-to-anaphase transition. The S165A cells were defective in Mad1 and Mad2 localization to kinetochores, regardless of attachment status. These cells often entered anaphase with lagging chromosomes and elicited increased segregation errors and cell death. In contrast, expressing S165E mutant in Hec1-depleted cells triggered defective chromosome alignment and severe mitotic arrest associated with increased Mad1/Mad2 signals at prometaphase kinetochores. A small portion of S165E cells eventually bypassed the SAC but showed severe segregation errors. Nek2 is the primary kinase responsible for kinetochore pS165, while PP1 phosphatase may dephosphorylate pS165 during SAC silencing. Taken together, these results suggest that modifications of Hec1 S165 serve as an important mechanism in modulating SAC signaling and chromosome alignment.
Havens,2011 (21828267) Havens CG, Walter JC "Mechanism of CRL4(Cdt2), a PCNA-dependent E3 ubiquitin ligase." Genes Dev 2011 Aug 1
Eukaryotic cell cycle transitions are driven by E3 ubiquitin ligases that catalyze the ubiquitylation and destruction of specific protein targets. For example, the anaphase-promoting complex/cyclosome (APC/C) promotes the exit from mitosis via destruction of securin and mitotic cyclins, whereas CRL1(Skp2) allows entry into S phase by targeting the destruction of the cyclin-dependent kinase (CDK) inhibitor p27. Recently, an E3 ubiquitin ligase called CRL4(Cdt2) has been characterized, which couples proteolysis to DNA synthesis via an unusual mechanism that involves display of substrate degrons on the DNA polymerase processivity factor PCNA. Through its destruction of Cdt1, p21, and Set8, CRL4(Cdt2) has emerged as a master regulator that prevents rereplication in S phase. In addition, it also targets other factors such as E2F and DNA polymerase eta. In this review, we discuss our current understanding of the molecular mechanism of substrate recognition by CRL4(Cdt2) and how this E3 ligase helps to maintain genome integrity.
Chemes,2011 (21787785) Chemes LB, Sanchez IE, de Prat-Gay G "Kinetic recognition of the retinoblastoma tumor suppressor by a specific protein target." J Mol Biol 2011 Aug 25
The retinoblastoma tumor suppressor (Rb) plays a key role in cell cycle control and is linked to various types of human cancer. Rb binds to the LxCxE motif, present in a number of cellular and viral proteins such as AdE1A, SV40 large T-antigen and human papillomavirus (HPV) E7, all instrumental in revealing fundamental mechanisms of tumor suppression, cell cycle control and gene expression. A detailed kinetic study of RbAB binding to the HPV E7 oncoprotein shows that an LxCxE-containing E7 fragment binds through a fast two-state reaction strongly favored by electrostatic interactions. Conversely, full-length E7 binds through a multistep process involving a pre-equilibrium between E7 conformers, a fast electrostatically driven association step guided by the LxCxE motif and a slow conformational rearrangement. This kinetic complexity arises from the conformational plasticity and intrinsically disordered nature of E7 and from multiple interaction surfaces present in both proteins. Affinity differences between E7N domains from high- and low-risk types are explained by their dissociation rates. In fact, since Rb is at the center of a large protein interaction network, fast and tight recognition provides an advantage for disruption by the viral proteins, where the balance of physiological and pathological interactions is dictated by kinetic ligand competition. The localization of the LxCxE motif within an intrinsically disordered domain provides the fast, diffusion-controlled interaction that allows viral proteins to outcompete physiological targets. We describe the interaction mechanism of Rb with a protein ligand, at the same time an LxCxE-containing model target, and a paradigmatic intrinsically disordered viral oncoprotein.
Maki,2011 (21786200) Maki M, Suzuki H, Shibata H "Structure and function of ALG-2, a penta-EF-hand calcium-dependent adaptor protein." Sci China Life Sci 2011 Jul 25
ALG-2 (a gene product of PDCD6) is a 22-kD protein containing five serially repetitive EF-hand structures and belongs to the penta-EF-hand (PEF) family, including the subunits of typical calpains. ALG-2 is the most conserved protein among the PEF family members and its homologs are widely found in eukaryotes. X-ray crystal structures of various PEF proteins including ALG-2 have common features: presence of eight alpha-helices and dimer formation via paired EF5s that are positioned in anti-parallel orientation. ALG-2 forms a homodimer and a heterodimer with its closest paralog peflin. Like calmodulin, a well-known four-EF-hand protein, ALG-2 interacts with various proteins in a Ca(2+)-dependent fashion, but the binding motifs are completely different. With some exceptions, ALG-2-interacting proteins commonly contain Pro-rich regions, and ALG-2 recognizes at least two distinct Pro-containing motifs: PPYP(X)nYP (X, variable; n=4 in ALIX and PLSCR3) and PXPGF (represented by Sec31A). A shorter alternatively spliced isoform, lacking two residues and designated ALG-2(DeltaGF122), does not bind ALIX but maintains binding capacity to Sec31A. X-ray crystal structural analyses have revealed that binding of calcium ions induces the configuration of the side chain of R125 so that it opens Pocket 1, which accepts PPYP, but Pocket 1 remains closed in the case of ALG-2(DeltaGF122). ALG-2 dimer has two ligand-binding sites, each in a monomer molecule, and appears to function as a Ca(2+)-dependent adaptor protein to either stabilize a preformed complex or to bridge two proteins on scaffolds in systems of the endosomal sorting complex required for transport (ESCRT) and ER-to-Golgi transport.
Stafford,2011 (21763699) Stafford RL, Ear J, Knight MJ, Bowie JU "The molecular basis of the Caskin1 and Mint1 interaction with CASK." J Mol Biol 2011 Aug 19
Calcium/calmodulin-dependent serine protein kinase (CASK) is a conserved multi-domain scaffolding protein involved in brain development, synapse formation, and establishment of cell polarity. To accomplish these diverse functions, CASK participates in numerous protein-protein interactions. In particular, CASK forms competing CASK/Mint1/Velis and CASK/Caskin1/Velis tripartite complexes that physically associate with the cytoplasmic tail of neurexin, a transmembrane protein enriched at presynaptic sites. This study shows that a short linear EEIWVLRK peptide motif from Caskin1 is necessary and sufficient for binding CASK. We also identified the conserved binding site for the peptide on the CASK calmodulin kinase domain. A related EPIWVMRQ peptide from Mint1 was also discovered to be sufficient for binding. Searching all human proteins for the Mint1/Caskin1 consensus peptide ExIWVxR revealed that T-cell lymphoma invasion and metastasis 1 (TIAM1) contains a conserved EEVIWVRRE peptide that was also found to be sufficient for CASK binding in vitro. TIAM1 is well known for its role in tumor metastasis, but it also possesses overlapping cellular and neurological functions with CASK, suggesting a previously unknown cooperation between the two proteins. This new peptide interaction motif also explains how Caskin1 and Mint1 form competing complexes and suggests a new role for the cellular hub protein CASK.
Evans,2011 (21757287) Evans TI, Hell JW, Shea MA "Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2." Biophys Chem 2011 Nov
Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca(2+) channel (Ca(V)1.2) regulates Ca(2+) entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca(V)1.2 under low resting [Ca(2+)], but is poised to change conformation and position when intracellular [Ca(2+)] rises. CaM binding Ca(2+), and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A(1588), and C(1614) and the IQ motif studied as overlapping peptides IQ(1644) and IQ'(1650) as well as their effect on calcium binding. (Ca(2+))(4)-CaM bound to all four peptides very favorably (K(d)</=2 nM). Linkage analysis showed that IQ(1644-1670) bound with a K(d)~1 pM. In the pre-IQ region, (Ca(2+))(2)-N-domain bound preferentially to A(1588), while (Ca(2+))(2)-C-domain preferred C(1614). When bound to C(1614), calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.
Gaffarogullari,2011 (21740913) Gaffarogullari EC, Masterson LR, Metcalfe EE, Traaseth NJ, Balatri E, Musa MM, Mullen D, Distefano MD, Veglia G "A myristoyl/phosphoserine switch controls cAMP-dependent protein kinase association to membranes." J Mol Biol 2011 Aug 15
The cAMP-dependent protein kinase [protein kinase A (PKA)] mediates a myriad of cellular signaling events, and its activity is tightly regulated in both space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that, in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted byeither interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of the myristoylated C-subunit of PKA (PKA-C) steers the enzyme toward lipids independent of its regulatory subunit or an A-kinase anchoring protein, providing an additional mechanism to localize the enzyme near membrane-bound substrates.
Vernoux,2011 (21734647) Vernoux T, Brunoud G, Farcot E, Morin V, Van den Daele H, Legrand J, Oliva M, Das P, Larrieu A, Wells D, Guedon Y, Armitage L, Picard F, Guyomarc'h S, Cellier C, Parry G, Koumproglou R, Doonan JH, Estelle M, Godin C, Kepinski S, Bennett M, De Veylder L, Traas J "The auxin signalling network translates dynamic input into robust patterning at the shoot apex." Mol Syst Biol 2011 Jul 07
The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.
Barrera-Vilarmau,2011 (21731739) Barrera-Vilarmau S, Obregon P, de Alba E "Intrinsic order and disorder in the bcl-2 member harakiri: insights into its proapoptotic activity." PLoS One 2011 Jul 06
Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-x(L) and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual alpha-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-x(L) and Bcl-2. The structural data in water indicate that the alpha-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric alpha-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works.
Oliver,2011 (21726810) Oliver TG, Meylan E, Chang GP, Xue W, Burke JR, Humpton TJ, Hubbard D, Bhutkar A, Jacks T "Caspase-2-mediated cleavage of Mdm2 creates a p53-induced positive feedback loop." Mol Cell 2011 Jul 05
Caspase-2 is an evolutionarily conserved caspase, yet its biological function and cleavage targets are poorly understood. Caspase-2 is activated by the p53 target gene product PIDD (also known as LRDD) in a complex called the Caspase-2-PIDDosome. We show that PIDD expression promotes growth arrest and chemotherapy resistance by a mechanism that depends on Caspase-2 and wild-type p53. PIDD-induced Caspase-2 directly cleaves the E3 ubiquitin ligase Mdm2 at Asp 367, leading to loss of the C-terminal RING domain responsible for p53 ubiquitination. As a consequence, N-terminally truncated Mdm2 binds p53 and promotes its stability. Upon DNA damage, p53 induction of the Caspase-2-PIDDosome creates a positive feedback loop that inhibits Mdm2 and reinforces p53 stability and activity, contributing to cell survival and drug resistance. These data establish Mdm2 as a cleavage target of Caspase-2 and provide insight into a mechanism of Mdm2 inhibition that impacts p53 dynamics upon genotoxic stress.
Gaudioso,2011 (21726526) Gaudioso C, Carlier E, Youssouf F, Clare JJ, Debanne D, Alcaraz G "Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel." Biochem Biophys Res Commun 2011 Jul 29
Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca(++) depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca(++) could bind the Nav1.1 C-terminal region with micromolar affinity.
Caballe,2011 (21722282) Caballe A, Martin-Serrano J "ESCRT machinery and cytokinesis: the road to daughter cell separation." Traffic 2011 Sep 12
The endosomal sorting complex required for transport (ESCRT) machinery is a set of cellular protein complexes required for at least three topologically equivalent membrane scission events, namely multivesicular body (MVB) formation, retroviral particle release and midbody abscission during cytokinesis. Recently, several studies have explored the mechanism by which the core ESCRT-III subunits mediate membrane scission and might be differentially required according to the functions of the pathway. In this review, we discuss the links between the ESCRT machinery and cytokinesis, with special focus on abscission initiation and regulation.
Alexander,2011 (21712545) Alexander J, Lim D, Joughin BA, Hegemann B, Hutchins JR, Ehrenberger T, Ivins F, Sessa F, Hudecz O, Nigg EA, Fry AM, Musacchio A, Stukenberg PT, Mechtler K, Peters JM, Smerdon SJ, Yaffe MB "Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling." Sci Signal 2011 Jun 29
The timing and localization of events during mitosis are controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2 (never in mitosis kinase 2), Plk1 (Polo-like kinase 1), and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by positional scanning oriented peptide library screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.
Fitzgerald,2012 (21701498) Fitzgerald JC, Camprubi MD, Dunn L, Wu HC, Ip NY, Kruger R, Martins LM, Wood NW, Plun-Favreau H "Phosphorylation of HtrA2 by cyclin-dependent kinase-5 is important for mitochondrial function." Cell Death Differ 2012 Jan 10
The role of the serine protease HtrA2 in neuroprotection was initially identified by the demonstration of neurodegeneration in mice lacking HtrA2 expression or function, and the interesting finding that mutations adjacent to two putative phosphorylation sites (S142 and S400) have been found in Parkinson's disease patients. However, the mechanism of this neuroprotection and the signalling pathways associated with it remain mostly unknown. Here we report that cyclin-dependent kinase-5 (Cdk5), a kinase implicated in the pathogenesis of several neurodegenerative diseases, is responsible for phosphorylating HtrA2 at S400. HtrA2 and Cdk5 interact in human and mouse cell lines and brain, and Cdk5 phosphorylates S400 on HtrA2 in a p38-dependent manner. Phosphorylation of HtrA2 at S400 is involved in maintaining mitochondrial membrane potential under stress conditions and is important for mitochondrial function, conferring cells protection against cellular stress.
Bodmer,2011 (21689596) Bodmer D, Ascano M, Kuruvilla R "Isoform-specific dephosphorylation of dynamin1 by calcineurin couples neurotrophin receptor endocytosis to axonal growth." Neuron 2011 Jun 21
Endocytic events are critical for neuronal survival in response to target-derived neurotrophic cues, but whether local axon growth is mediated by endocytosis-dependent signaling mechanisms remains unclear. Here, we report that Nerve Growth Factor (NGF) promotes endocytosis of its TrkA receptors and axon growth by calcineurin-mediated dephosphorylation of the endocytic GTPase dynamin1. Conditional deletion of calcineurin in sympathetic neurons disrupts NGF-dependent innervation of peripheral target tissues. Calcineurin signaling is required locally in sympathetic axons to support NGF-mediated growth in a manner independent of transcription. We show that calcineurin associates with dynamin1 via a PxIxIT interaction motif found only in specific dynamin1 splice variants. PxIxIT-containing dynamin1 isoforms colocalize with surface TrkA receptors, and their phosphoregulation is selectively required for NGF-dependent TrkA internalization and axon growth in sympathetic neurons. Thus, NGF-dependent phosphoregulation of dynamin1 is a critical event coordinating neurotrophin receptor endocytosis and axonal growth.
Kevei,2011 (21687678) Kevei Z, Baloban M, Da Ines O, Tiricz H, Kroll A, Regulski K, Mergaert P, Kondorosi E "Conserved CDC20 cell cycle functions are carried out by two of the five isoforms in Arabidopsis thaliana." PLoS One 2011 Jun 20
BACKGROUND: The CDC20 and Cdh1/CCS52 proteins are substrate determinants and activators of the Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligase and as such they control the mitotic cell cycle by targeting the degradation of various cell cycle regulators. In yeasts and animals the main CDC20 function is the destruction of securin and mitotic cyclins. Plants have multiple CDC20 gene copies whose functions have not been explored yet. In Arabidopsis thaliana there are five CDC20 isoforms and here we aimed at defining their contribution to cell cycle regulation, substrate selectivity and plant development. METHODOLOGY/PRINCIPAL FINDINGS: Studying the gene structure and phylogeny of plant CDC20s, the expression of the five AtCDC20 gene copies and their interactions with the APC/C subunit APC10, the CCS52 proteins, components of the mitotic checkpoint complex (MCC) and mitotic cyclin substrates, conserved CDC20 functions could be assigned for AtCDC20.1 and AtCDC20.2. The other three intron-less genes were silent and specific for Arabidopsis. We show that AtCDC20.1 and AtCDC20.2 are components of the MCC and interact with mitotic cyclins with unexpected specificity. AtCDC20.1 and AtCDC20.2 are expressed in meristems, organ primordia and AtCDC20.1 also in pollen grains and developing seeds. Knocking down both genes simultaneously by RNAi resulted in severe delay in plant development and male sterility. In these lines, the meristem size was reduced while the cell size and ploidy levels were unaffected indicating that the lower cell number and likely slowdown of the cell cycle are the cause of reduced plant growth. CONCLUSIONS/SIGNIFICANCE: The intron-containing CDC20 gene copies provide conserved and redundant functions for cell cycle progression in plants and are required for meristem maintenance, plant growth and male gametophyte formation. The Arabidopsis-specific intron-less genes are possibly "retrogenes" and have hitherto undefined functions or are pseudogenes.
Aragon,2011 (21685363) Aragon E, Goerner N, Zaromytidou AI, Xi Q, Escobedo A, Massague J, Macias MJ "A Smad action turnover switch operated by WW domain readers of a phosphoserine code." Genes Dev 2011 Jun 20
When directed to the nucleus by TGF-beta or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-beta pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-beta signal delivery to turnover of the Smad signal transducers.
Padrick,2011 (21676863) Padrick SB, Doolittle LK, Brautigam CA, King DS, Rosen MK "Arp2/3 complex is bound and activated by two WASP proteins." Proc Natl Acad Sci U S A 2011 Aug 17
Actin related protein 2/actin related protein 3 (Arp2/3) complex nucleates new actin filaments in eukaryotic cells in response to signals from proteins in the Wiskott-Aldrich syndrome protein (WASP) family. The conserved VCA domain of WASP proteins activates Arp2/3 complex by inducing conformational changes and delivering the first actin monomer of the daughter filament. Previous models of activation have invoked a single VCA acting at a single site on Arp2/3 complex. Here we show that activation most likely involves engagement of two distinct sites on Arp2/3 complex by two VCA molecules, each delivering an actin monomer. One site is on Arp3 and the second is on ARPC1 and Arp2. The VCAs at these sites have distinct roles in activation. Our findings reconcile apparently conflicting literature on VCA activation of Arp2/3 complex and lead to a new model for this process.
Ti,2011 (21676862) Ti SC, Jurgenson CT, Nolen BJ, Pollard TD "Structural and biochemical characterization of two binding sites for nucleation-promoting factor WASp-VCA on Arp2/3 complex." Proc Natl Acad Sci U S A 2011 Aug 17
Actin-related protein (Arp) 2/3 complex mediates the formation of actin filament branches during endocytosis and at the leading edge of motile cells. The pathway of branch formation is ambiguous owing to uncertainty regarding the stoichiometry and location of VCA binding sites on Arp2/3 complex. Isothermal titration calorimetry showed that the CA motif from the C terminus of fission yeast WASP (Wsp1p) bound to fission yeast and bovine Arp2/3 complex with a stoichiometry of 2 to 1 and very different affinities for the two sites (K(d)s of 0.13 and 1.6 muM for fission yeast Arp2/3 complex). Equilibrium binding, kinetic, and cross-linking experiments showed that (i) CA at high-affinity site 1 inhibited Arp2/3 complex binding to actin filaments, (ii) low-affinity site 2 had a higher affinity for CA when Arp2/3 complex was bound to actin filaments, and (iii) Arp2/3 complex had a much higher affinity for free CA than VCA cross-linked to an actin monomer. Crystal structures showed the C terminus of CA bound to the low-affinity site 2 on Arp3 of bovine Arp2/3 complex. The C helix is likely to bind to the barbed end groove of Arp3 in a position for VCA to deliver the first actin subunit to the daughter filament.
Mouilleron,2011 (21673315) Mouilleron S, Langer CA, Guettler S, McDonald NQ, Treisman R "Structure of a pentavalent G-actin*MRTF-A complex reveals how G-actin controls nucleocytoplasmic shuttling of a transcriptional coactivator." Sci Signal 2011
Subcellular localization of the actin-binding transcriptional coactivator MRTF-A is controlled by its interaction with monomeric actin (G-actin). Signal-induced decreases in G-actin concentration reduce MRTF-A nuclear export, leading to its nuclear accumulation, whereas artificial increases in G-actin concentration in resting cells block MRTF-A nuclear import, retaining it in the cytoplasm. This regulation is dependent on three actin-binding RPEL motifs in the regulatory domain of MRTF-A. We describe the structures of pentavalent and trivalent G-actin*RPEL domain complexes. In the pentavalent complex, each RPEL motif and the two intervening spacer sequences bound an actin monomer, forming a compact assembly. In contrast, the trivalent complex lacked the C-terminal spacer- and RPEL-actins, both of which bound only weakly in the pentavalent complex. Cytoplasmic localization of MRTF-A in unstimulated fibroblasts also required binding of G-actin to the spacer sequences. The bipartite MRTF-A nuclear localization sequence was buried in the pentameric assembly, explaining how increases in G-actin concentration prevent nuclear import of MRTF-A. Analyses of the pentavalent and trivalent complexes show how actin loads onto the RPEL domain and reveal a molecular mechanism by which actin can control the activity of one of its binding partners.
Crawley,2011 (21671662) Crawley SW, Liburd J, Shaw K, Jung Y, Smith SP, Cote GP "Identification of calmodulin and MlcC as light chains for dictyostelium myosin-I isozymes." Biochemistry 2011 Aug 2
Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.
Greenbaum,2011 (21669984) Greenbaum MP, Iwamori T, Buchold GM, Matzuk MM "Germ cell intercellular bridges." Cold Spring Harb Perspect Biol 2011 Aug 02
Stable intercellular bridges are a conserved feature of gametogenesis in multicellular animals observed more than 100 years ago, but their function was unknown. Many of the components necessary for this structure have been identified through the study of cytokinesis in Drosophila; however, mammalian intercellular bridges have distinct properties from those of insects. Mammalian germ cell intercellular bridges are composed of general cytokinesis components with additional germ cell-specific factors including TEX14. TEX14 is an inactive kinase essential for the maintenance of stable intercellular bridges in gametes of both sexes but whose loss specifically impairs male meiosis. TEX14 acts to impede the terminal steps of abscission by competing for essential component CEP55, blocking its interaction in nongerm cells with ALIX and TSG101. Additionally, TEX14-interacting protein RBM44, whose localization in stabile intercellular bridges is limited to pachytene and secondary spermatocytes, may participate in processes such as RNA transport but is nonessential to the maintenance of intercellular bridge stability.
Pirruccello,2011 (21666675) Pirruccello M, Swan LE, Folta-Stogniew E, De Camilli P "Recognition of the F&H motif by the Lowe syndrome protein OCRL." Nat Struct Mol Biol 2011 Jul 06
Lowe syndrome and type 2 Dent disease are caused by defects in the inositol 5-phosphatase OCRL. Most missense mutations in the OCRL ASH-RhoGAP domain that are found in affected individuals abolish interactions with the endocytic adaptors APPL1 and Ses (both Ses1 and Ses2), which bind OCRL through a short phenylalanine and histidine (F&H) motif. Using X-ray crystallography, we have identified the F&H motif binding site on the RhoGAP domain of OCRL. Missense mutations associated with disease affected F&H binding indirectly by destabilizing the RhoGAP fold. By contrast, a disease-associated mutation that does not perturb F&H binding and ASH-RhoGAP stability disrupted the interaction of OCRL with Rab5. The F&H binding site of OCRL is conserved even in species that do not have an identified homolog for APPL or Ses. Our study predicts the existence of other OCRL binding partners and shows that the perturbation of OCRL interactions has a crucial role in disease.
Jia,2011 (21663793) Jia H, Wang X, Liu F, Guenther UP, Srinivasan S, Anderson JT, Jankowsky E "The RNA helicase Mtr4p modulates polyadenylation in the TRAMP complex." Cell 2011 Jun 13
Many steps in nuclear RNA processing, surveillance, and degradation require TRAMP, a complex containing the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for decay or trimming by the nuclear exosome. It has been unclear how polyadenylation by TRAMP differs from polyadenylation by conventional poly(A) polymerase, which produces poly(A) tails that stabilize RNAs. Using reconstituted S. cerevisiae TRAMP, we show that TRAMP inherently suppresses poly(A) addition after only 3-4 adenosines. This poly(A) tail length restriction is controlled by Mtr4p. The helicase detects the number of 3'-terminal adenosines and, over several adenylation steps, elicits precisely tuned adjustments of ATP affinities and rate constants for adenylation and TRAMP dissociation. Our data establish Mtr4p as a critical regulator of polyadenylation by TRAMP and reveal that an RNA helicase can control the activity of another enzyme in a highly complex fashion and in response to features in RNA.
Slupianek,2011 (21653319) Slupianek A, Dasgupta Y, Ren SY, Gurdek E, Donlin M, Nieborowska-Skorska M, Fleury F, Skorski T "Targeting RAD51 phosphotyrosine-315 to prevent unfaithful recombination repair in BCR-ABL1 leukemia." Blood 2011 Jul 29
Chronic myeloid leukemia chronic phase (CML-CP) CD34(+) cells contain numerous DNA double-strand breaks whose unfaithful repair may contribute to chromosomal instability and disease progression to blast phase (CML-BP). These phenomena are often associated with the appearance of imatinib-resistant BCR-ABL1 kinase mutants (eg, T315I) and overexpression of BCR-ABL1. Here we show that BCR-ABL1 (nonmutated and T315I mutant) promoted RAD51 recombinase-mediated unfaithful homeologous recombination repair (HomeoRR) in a dosage-dependent manner. BCR-ABL1 SH3 domain interacts with RAD51 proline-rich regions, resulting in direct phosphorylation of RAD51 on Y315 (pY315). RAD51(pY315) facilitates dissociation from the complex with BCR-ABL1 kinase, migrates to the nucleus, and enhances formation of the nuclear foci indicative of recombination sites. HomeoRR and RAD51 nuclear foci were strongly reduced by RAD51(Y315F) phosphorylation-less mutant. In addition, peptide aptamer mimicking RAD51(pY315) fragment, but not that with Y315F phosphorylation-less substitution, diminished RAD51 foci formation and inhibited HomeoRR in leukemia cells. In conclusion, we postulate that BCR-ABL1 kinase-mediated RAD51(pY315) promotes unfaithful HomeoRR in leukemia cells, which may contribute to accumulation of secondary chromosomal aberrations responsible for CML relapse and progression.
van der Vaart,2011 (21646404) van der Vaart B, Manatschal C, Grigoriev I, Olieric V, Gouveia SM, Bjelic S, Demmers J, Vorobjev I, Hoogenraad CC, Steinmetz MO, Akhmanova A "SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase." J Cell Biol 2011 Jun 13
The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network.
Goodarzi,2011 (21642969) Goodarzi AA, Kurka T, Jeggo PA "KAP-1 phosphorylation regulates CHD3 nucleosome remodeling during the DNA double-strand break response." Nat Struct Mol Biol 2011 Jul 06
KAP-1 poses a substantial barrier to DNA double-strand break (DSB) repair within heterochromatin that is alleviated by ATM-dependent KAP-1 phosphorylation (pKAP-1). Here we address the mechanistic consequences of pKAP-1 that promote heterochromatic DSB repair and chromatin relaxation. KAP-1 function involves autoSUMOylation and recruitment of nucleosome deacetylation, methylation and remodeling activities. Although heterochromatin acetylation or methylation changes were not detected, radiation-induced pKAP-1 dispersed the nucleosome remodeler CHD3 from DSBs and triggered concomitant chromatin relaxation; pKAP-1 loss reversed these effects. Depletion or inactivation of CHD3, or ablation of its interaction with KAP-1(SUMO1), bypassed pKAP-1's role in repair. Though KAP-1 SUMOylation was unaffected after irradiation, CHD3 dissociated from KAP-1(SUMO1) in a pKAP-1-dependent manner. We demonstrate that KAP-1(Ser824) phosphorylation generates a motif that directly perturbs interactions between CHD3's SUMO-interacting motif and SUMO1, dispersing CHD3 from heterochromatin DSBs and enabling repair.
Lobjois,2011 (21640712) Lobjois V, Froment C, Braud E, Grimal F, Burlet-Schiltz O, Ducommun B, Bouche JP "Study of the docking-dependent PLK1 phosphorylation of the CDC25B phosphatase." Biochem Biophys Res Commun 2011 Jun 27
CDC25 (A, B and C) phosphatases control cell cycle progression through the timely dephosphorylation and activation of cyclin-dependent kinases (CDK). At mitosis the CDC25B phosphatase activity is dependent on its phosphorylation by multiple kinases impinging on its localisation, stability and catalytic activity. Here we report that prior phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro, and we also show that phosphorylated S50 serves as a docking site for PLK1. Using a sophisticated strategy based on the sequential phosphorylation of CDC25B with (16)O and (18)O ATP prior to nanoLC-MS/MS analysis we identified 13 sites phosphorylated by PLK1. This study illustrates the complexity of the phosphorylation pattern and of the subsequent regulation of CDC25B activity.
Varshavsky,2011 (21633985) Varshavsky A "The N-end rule pathway and regulation by proteolysis." Protein Sci 2011 Jun 02
The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. The main determinant of an N-degron is a destabilizing N-terminal residue of a protein. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N-end rule and the Arg/N-end rule pathways. The Ac/N-end rule pathway targets proteins containing N(alpha) -terminally acetylated (Nt-acetylated) residues. The Arg/N-end rule pathway recognizes unacetylated N-terminal residues and involves N-terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt-acetylated. Thus most proteins harbor a specific degradation signal, termed (Ac) N-degron, from the moment of their birth. Specific N-end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N-degrons include methionine-aminopeptidases, caspases, calpains, Nt-acetylases, Nt-amidases, arginyl-transferases and leucyl-transferases. Regulated degradation of specific proteins by the N-end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights.
Pines,2011 (21633387) Pines J "Cubism and the cell cycle: the many faces of the APC/C." Nat Rev Mol Cell Biol 2011 Jun 23
One does not often look to analytic cubism for insights into the control of the cell cycle, but Pablo Picasso beautifully encapsulated the fundamentals when he said that "every act of creation is, first of all, an act of destruction". The rapid destruction of specific cell cycle regulators at just the right moment in the cell cycle ensures that daughter cells receive an equal and identical set of chromosomes from their mother and that DNA replication always follows mitosis. Remarkably, one protein complex is responsible for this surgical precision, the APC/C (anaphase-promoting complex, also known as the cyclosome). The APC/C is tightly regulated by its co-activators and by the spindle assembly checkpoint.
Rozenknop,2011 (21620860) Rozenknop A, Rogov VV, Rogova NY, Lohr F, Guntert P, Dikic I, Dotsch V "Characterization of the interaction of GABARAPL-1 with the LIR motif of NBR1." J Mol Biol 2011 Jun 24
Selective autophagy requires the specific segregation of targeted proteins into autophagosomes. The selectivity is mediated by autophagy receptors, such as p62 and NBR1, which can bind to autophagic effector proteins (Atg8 in yeast, MAP1LC3 protein family in mammals) anchored in the membrane of autophagosomes. Recognition of autophagy receptors by autophagy effectors takes place through an LC3 interaction region (LIR). The canonical LIR motif consists of a WXXL sequence, N-terminally preceded by negatively charged residues. The LIR motif of NBR1 presents differences to this classical LIR motif with a tyrosine residue and an isoleucine residue substituting the tryptophan residue and the leucine residue, respectively. We have determined the structure of the GABARAPL-1/NBR1-LIR complex and studied the influence of the different residues belonging to the LIR motif for the interaction with several mammalian autophagy modifiers (LC3B and GABARAPL-1). Our results indicate that the presence of a tryptophan residue in the LIR motif increases the binding affinity. Substitution by other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation. This indicates that different LIRs can interact with autophagy modifiers with unique binding properties.
Wild,2011 (21617041) Wild P, Farhan H, McEwan DG, Wagner S, Rogov VV, Brady NR, Richter B, Korac J, Waidmann O, Choudhary C, Dotsch V, Bumann D, Dikic I "Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth." Science 2011 Jul 08
Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitin-coated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin- or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.
Cornils,2011 (21593588) Cornils H, Kohler RS, Hergovich A, Hemmings BA "Downstream of human NDR kinases: impacting on c-myc and p21 protein stability to control cell cycle progression." Cell Cycle 2011 Jun 30
The mammalian genome encodes four members of the NDR/LATS kinase family: NDR1 (STK38), NDR2 (STK38L), LATS1 and LATS2, which are highly conserved from yeast to man. Members of the NDR/LATS kinase family have been implicated in a variety of biological processes ranging from cell division and morphology to apoptosis and tumor suppression. In mammals, LATS1/2 function as central parts of the HIPPO tumor suppressor pathway by restricting the activity of the YAP/TAZ proto-oncogenes. Recent evidence suggested that NDR1/2 are also part of an extended HIPPO tumor suppressor pathway. Apart from functions in apoptosis signaling and tumor suppression, NDR1/2 have been implicated in controlling centrosome duplication and mitotic chromosome alignment downstream of the HIPPO kinase homologs MST1 and MST2. Significantly, we also reported recently that NDR1/2 are controlling G 1/S transition downstream of a third MST family member MST3. Intriguingly, this newly described MST3-NDR1/2 axis promotes G 1 progression by stabilizing c-myc and preventing p21 accumulation, indicating a potential pro-tumorigenic role for NDR kinases. Here, we discuss these novel cell cycle functions of NDR kinases in a broader context and elaborate on possible explanations for the opposing functions of NDR kinases in normal and tumor biology.
Vitari,2011 (21572435) Vitari AC, Leong KG, Newton K, Yee C, O'Rourke K, Liu J, Phu L, Vij R, Ferrando R, Couto SS, Mohan S, Pandita A, Hongo JA, Arnott D, Wertz IE, Gao WQ, French DM, Dixit VM "COP1 is a tumour suppressor that causes degradation of ETS transcription factors." Nature 2011 Jun 16
The proto-oncogenes ETV1, ETV4 and ETV5 encode transcription factors in the E26 transformation-specific (ETS) family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COP1 (also known as RFWD2) as a tumour suppressor that negatively regulates ETV1, ETV4 and ETV5. ETV1, which is mutated in prostate cancer more often, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs and was 50-fold more stable than wild-type ETV1. Almost all patient translocations render ETV1 insensitive to COP1, implying that this confers a selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. Combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein, and elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a tumour suppressor whose downregulation promotes prostatic epithelial cell proliferation and tumorigenesis.
Lavoie,2011 (21565170) Lavoie G, St-Pierre Y "Phosphorylation of human DNMT1: implication of cyclin-dependent kinases." Biochem Biophys Res Commun 2011 Jun 06
DNA methylation plays a central role in the epigenetic regulation of gene expression during development and progression of cancer diseases. The inheritance of specific DNA methylation patterns are acquired in the early embryo and are specifically maintained after cellular replication via the DNA methyltransferase 1 (DNMT1). Recent studies have suggested that the enzymatic activity of DNMT1 is possibly modulated by phosphorylation of serine/threonine residues located in the N-terminal domain of the enzyme. In the present work, we report that cyclin-dependent kinases (CDKs) 1, 2 and 5 can phosphorylate Ser154 of human DNMT1 in vitro. Further evidence of phosphorylation of endogenous DNMT1 at position 154 by CDKs is also found in 293 cells treated with roscovitine, a specific inhibitor of CDK1, 2 and 5. To determine the importance of Ser154 phosphorylation, a mutant of DNMT1 encoding a single-point mutation at position 154 (S154A) was generated. This mutation induced a severe loss of enzymatic activity when compared to wild type DNMT1. Moreover, after treatment with 5-Aza-2'-Deoxycytidine (5-aza-dC), a faster decline in DNMT1 protein level was observed for HEK-293 cells expressing DNMT1(S154A) as compared to cells expressing wild type DNMT1. Our data suggest that phosphorylation of DNMT1 at Ser154 by CDKs is important for enzymatic activity and protein stability of DNMT1. Considering that tumour-associated cell cycle defects are often mediated by alterations in CDK activity, our results suggest that dysregulation of cell cycle via CDKs could induce abnormal phosphorylation of DNMT1 and lead to DNA hypermethylation often observed in cancer cells.
Keshwani,2011 (21561857) Keshwani MM, von Daake S, Newton AC, Harris TK, Taylor SS "Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1)." J Biol Chem 2011 Jun 27
p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.
ZeRuth,2011 (21543335) ZeRuth GT, Yang XP, Jetten AM "Modulation of the transactivation function and stability of Kruppel-like zinc finger protein Gli-similar 3 (Glis3) by Suppressor of Fused." J Biol Chem 2011 Jun 20
Glis3 is a member of the Glis subfamily of Kruppel-like zinc finger transcription factors. Recently, Glis3 has been linked to both type I and type II diabetes and shown to positively regulate insulin gene expression. In this study, we have identified a region within the N terminus of Glis3 that shares high levels of homology with the Cubitus interruptus (Ci)/Gli family of proteins. We demonstrated that Glis3 interacts with Suppressor of Fused (SUFU), which involves a VYGHF motif located within this conserved region. We further showed that SUFU is able to inhibit the activation of the insulin promoter by Glis3 but not the activation by a Glis3 mutant deficient in its ability to bind SUFU, suggesting that the inhibitory effect is dependent on the interaction between the two proteins. Exogenous SUFU did not affect the nuclear localization of Glis3; however, Glis3 promoted the nuclear accumulation of SUFU. Additionally, we demonstrated that SUFU stabilizes Glis3 in part by antagonizing the Glis3 association with a Cullin 3-based E3 ubiquitin ligase that promotes the ubiquitination and degradation of Glis3. This is the first reported instance of Glis3 interacting with SUFU and suggests a novel role for SUFU in the modulation of Glis3 signaling. Given the critical role of Glis3 in pancreatic beta-cell generation and maintenance, the elevated Glis3 expression in several cancers, and the established role of SUFU as a tumor suppressor, these data provide further insight into Glis3 regulation and its function in development and disease.
Voordeckers,2011 (21531713) Voordeckers K, Kimpe M, Haesendonckx S, Louwet W, Versele M, Thevelein JM "Yeast 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) Orthologs Pkh1-3 Differentially Regulate Phosphorylation of Protein Kinase A (PKA) and the Protein Kinase B (PKB)/S6K Ortholog Sch9." J Biol Chem 2011 Jun 24
Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh-dependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.
Schroder,2011 (21520322) Schroder T, Lilie H, Lange C "The myristoylation of guanylate cyclase-activating protein-2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca(2)(+)." Protein Sci 2011 Jun 20
Guanylate cyclase activating protein-2 (GCAP-2) is a Ca(2)(+)-binding protein of the neuronal calcium sensor (NCS) family. Ca(2)(+)-free GCAP-2 activates the retinal rod outer segment guanylate cyclases ROS-GC1 and 2. Native GCAP-2 is N-terminally myristoylated. Detailed structural information on the Ca(2)(+)-dependent conformational switch of GCAP-2 is missing so far, as no atomic resolution structures of the Ca(2)(+)-free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca(2)(+)-myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP-1, a Ca(2)(+)-independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non-myristoylated GCAP-2 in their Ca(2)(+)-bound and Ca(2)(+)-free forms, respectively, to gain information on the nature of the Ca(2)(+)-dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca(2)(+), the stability of the myristoylated and non-myristoylated forms was indistinguishable. Ca(2)(+) exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP-2. The stability data were corroborated by dye binding experiments performed to probe the solvent-accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent-exposed in Ca(2)(+)-free GCAP-2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca(2)(+)-bound state.
Arriagada,2011 (21490953) Arriagada G, Muntean LN, Goff SP "SUMO-interacting motifs of human TRIM5alpha are important for antiviral activity." PLoS Pathog 2011 Apr 14
Human TRIM5alpha potently restricts particular strains of murine leukemia viruses (the so-called N-tropic strains) but not others (the B- or NB-tropic strains) during early stages of infection. We show that overexpression of SUMO-1 in human 293T cells, but not in mouse MDTF cells, profoundly blocks N-MLV infection. This block is dependent on the tropism of the incoming virus, as neither B-, NB-, nor the mutant R110E of N-MLV CA (a B-tropic switch) are affected by SUMO-1 overexpression. The block occurred prior to reverse transcription and could be abrogated by large amounts of restricted virus. Knockdown of TRIM5alpha in 293T SUMO-1-overexpressing cells resulted in ablation of the SUMO-1 antiviral effects, and this loss of restriction could be restored by expression of a human TRIM5alpha shRNA-resistant plasmid. Amino acid sequence analysis of human TRIM5alpha revealed a consensus SUMO conjugation site at the N-terminus and three putative SUMO interacting motifs (SIMs) in the B30.2 domain. Mutations of the TRIM5alpha consensus SUMO conjugation site did not affect the antiviral activity of TRIM5alpha in any of the cell types tested. Mutation of the SIM consensus sequences, however, abolished TRIM5alpha antiviral activity against N-MLV. Mutation of lysines at a potential site of SUMOylation in the CA region of the Gag gene reduced the SUMO-1 block and the TRIM5alpha restriction of N-MLV. Our data suggest a novel aspect of TRIM5alpha-mediated restriction, in which the presence of intact SIMs in TRIM5alpha, and also the SUMO conjugation of CA, are required for restriction. We propose that at least a portion of the antiviral activity of TRIM5alpha is mediated through the binding of its SIMs to SUMO-conjugated CA.
Zhang,2011 (21478859) Zhang Y, Liu S, Mickanin C, Feng Y, Charlat O, Michaud GA, Schirle M, Shi X, Hild M, Bauer A, Myer VE, Finan PM, Porter JA, Huang SM, Cong F "RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling." Nat Cell Biol 2011 May
The Wnt/beta-catenin signalling pathway plays essential roles in embryonic development and adult tissue homeostasis, and deregulation of this pathway has been linked to cancer. Axin is a concentration-limiting component of the beta-catenin destruction complex, and its stability is regulated by tankyrase. However, the molecular mechanism by which tankyrase-dependent poly(ADP-ribosyl)ation (PARsylation) is coupled to ubiquitylation and degradation of axin remains undefined. Here, we identify RNF146, a RING-domain E3 ubiquitin ligase, as a positive regulator of Wnt signalling. RNF146 promotes Wnt signalling by mediating tankyrase-dependent degradation of axin. Mechanistically, RNF146 directly interacts with poly(ADP-ribose) through its WWE domain, and promotes degradation of PARsylated proteins. Using proteomics approaches, we have identified BLZF1 and CASC3 as further substrates targeted by tankyrase and RNF146 for degradation. Thus, identification of RNF146 as a PARsylation-directed E3 ligase establishes a molecular paradigm that links tankyrase-dependent PARsylation to ubiquitylation. RNF146-dependent protein degradation may emerge as a major mechanism by which tankyrase exerts its function.
Tong,2011 (21475249) Tong X, Gui H, Jin F, Heck BW, Lin P, Ma J, Fondell JD, Tsai CC "Ataxin-1 and Brother of ataxin-1 are components of the Notch signalling pathway." EMBO Rep 2011 Apr 28
Ataxin-1 (ATXN1), a causative factor for spinocerebellar ataxia type 1 (SCA1), and the related Brother of ATXN1 (BOAT1) are human proteins involved in transcriptional repression. So far, little is known about which transcriptional pathways mediate the effects of ATXN1 and BOAT1. From our analyses of the properties of BOAT1 in Drosophila and of both proteins in mammalian cells, we report here that BOAT1 and ATXN1 are components of the Notch signalling pathway. In Drosophila, BOAT1 compromises the activities of Notch. In mammalian cells, both ATXN1 and BOAT1 bind to the promoter region of Hey1 and inhibit the transcriptional output of Notch through direct interactions with CBF1, a transcription factor that is crucial for the Notch pathway. Our results suggest that, in addition to their involvement in SCA1, ATXN1 and BOAT1 might participate in several Notch-controlled developmental and pathological processes.
Chang,2011 (21474068) Chang CC, Naik MT, Huang YS, Jeng JC, Liao PH, Kuo HY, Ho CC, Hsieh YL, Lin CH, Huang NJ, Naik NM, Kung CC, Lin SY, Chen RH, Chang KS, Huang TH, Shih HM "Structural and functional roles of Daxx SIM phosphorylation in SUMO paralog-selective binding and apoptosis modulation." Mol Cell 2011 Apr 08
Small ubiquitin-like modifier (SUMO) conjugation and interaction are increasingly associated with various cellular processes. However, little is known about the cellular signaling mechanisms that regulate proteins for distinct SUMO paralog conjugation and interactions. Using the transcriptional coregulator Daxx as a model, we show that SUMO paralog-selective binding and conjugation are regulated by phosphorylation of the Daxx SUMO-interacting motif (SIM). NMR structural studies show that Daxx (732)E-I-I-V-L-S-D-S-D(740) is a bona fide SIM that binds to SUMO-1 in a parallel orientation. Daxx-SIM is phosphorylated by CK2 kinase at residues S737 and S739. Phosphorylation promotes Daxx-SIM binding affinity toward SUMO-1 over SUMO-2/3, causing Daxx preference for SUMO-1 conjugation and interaction with SUMO-1-modified factors. Furthermore, Daxx-SIM phosphorylation enhances Daxx to sensitize stress-induced cell apoptosis via antiapoptotic gene repression. Our findings provide structural insights into the Daxx-SIM:SUMO-1 complex, a model of SIM phosphorylation-enhanced SUMO paralog-selective modification and interaction, and phosphorylation-regulated Daxx function in apoptosis.
Nicholson,2011 (21468693) Nicholson B, Suresh Kumar KG "The multifaceted roles of USP7: new therapeutic opportunities." Cell Biochem Biophys 2011 May 16
The deubiquitylating enzyme USP7 (HAUSP) sits at a critical node regulating the activities of numerous proteins broadly characterized as tumor suppressors, DNA repair proteins, immune responders, viral proteins, and epigenetic modulators. Aberrant USP7 activity may promote oncogenesis and viral disease making it a compelling target for therapeutic intervention. Disclosed drug discovery programs have identified inhibitors of USP7 such as P005091 with cellular proof of concept and anti-proliferative activity in cancer models. Taken together, USP7 inhibitors hold promise as a new strategy for the treatment of disease.
Xu,2011 (21465563) Xu X, Ishima R, Ames JB "Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis." Proteins 2011 May 11
Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca(2+) -induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca(2+)-myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on (15) N-labeled recoverin to probe main chain conformational dynamics. (15) N NMR relaxation data suggest that Ca(2+)-free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca(2+) levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca(2+)-dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca(2+) saturated extruded state (R): T <--> I <--> R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (tau(ex) deltaomega < 1). The final step (I <--> R) is much slower (tau(ex) deltaomega > 1). The main chain structure of I is similar in part to the structure of half-saturated E85Q recoverin with a sequestered myristoyl group. We propose that millisecond dynamics during T <--> I may transiently increase the exposure of Ca(2+)-binding sites to initiate Ca(2+) binding that drives extrusion of the myristoyl group during I <--> R.
Ma,2011 (21464226) Ma C, Agrawal G, Subramani S "Peroxisome assembly: matrix and membrane protein biogenesis." J Cell Biol 2011 Apr 05
The biogenesis of peroxisomal matrix and membrane proteins is substantially different from the biogenesis of proteins of other subcellular compartments, such as mitochondria and chloroplasts, that are of endosymbiotic origin. Proteins are targeted to the peroxisome matrix through interactions between specific targeting sequences and receptor proteins, followed by protein translocation across the peroxisomal membrane. Recent advances have shed light on the nature of the peroxisomal translocon in matrix protein import and the molecular mechanisms of receptor recycling. Furthermore, the endoplasmic reticulum has been shown to play an important role in peroxisomal membrane protein biogenesis. Defining the molecular events in peroxisome assembly may enhance our understanding of the etiology of human peroxisome biogenesis disorders.
Burton,2011 (21460798) Burton JL, Xiong Y, Solomon MJ "Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1." EMBO J 2011 May 04
The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APC(Cdh1) inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APC(Cdh1) inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APC(Cdh1) whereas lysine removal from the APC substrate Hsl1 converted it into a potent APC(Cdh1) inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APC(Cdh1).
Wlotzka,2011 (21460797) Wlotzka W, Kudla G, Granneman S, Tollervey D "The nuclear RNA polymerase II surveillance system targets polymerase III transcripts." EMBO J 2011 May 04
A key question in nuclear RNA surveillance is how target RNAs are recognized. To address this, we identified in vivo binding sites for nuclear RNA surveillance factors, Nrd1, Nab3 and the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex poly(A) polymerase Trf4, by UV crosslinking. Hit clusters were reproducibly found over known binding sites on small nucleolar RNAs (snoRNAs), pre-mRNAs and cryptic, unstable non-protein-coding RNAs (ncRNAs) ('CUTs'), along with ~642 predicted long anti-sense ncRNAs (asRNAs), ~178 intergenic ncRNAs and, surprisingly, ~1384 mRNAs. Five putative asRNAs tested were confirmed to exist and were stabilized by loss of Nrd1, Nab3 or Trf4. Mapping of micro-deletions and substitutions allowed clear definition of preferred, in vivo Nab3 and Nrd1 binding sites. Nrd1 and Nab3 were believed to be Pol II specific but, unexpectedly, bound many oligoadenylated Pol III transcripts, predominately pre-tRNAs. Depletion of Nrd1 or Nab3 stabilized tested Pol III transcripts and their oligoadenylation was dependent on Nrd1-Nab3 and TRAMP. Surveillance targets were enriched for non-encoded A-rich tails. These were generally very short (1-5 nt), potentially explaining why adenylation destabilizes these RNAs while stabilizing mRNAs with long poly(A) tails.
Nakatsu,2011 (21454638) Nakatsu Y, Sakoda H, Kushiyama A, Zhang J, Ono H, Fujishiro M, Kikuchi T, Fukushima T, Yoneda M, Ohno H, Horike N, Kanna M, Tsuchiya Y, Kamata H, Nishimura F, Isobe T, Ogihara T, Katagiri H, Oka Y, Takahashi S, Kurihara H, Uchida T, Asano T "Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis." J Biol Chem 2011 Jun 10
Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.
Calderwood,2011 (21440926) Calderwood MA, Lee S, Holthaus AM, Blacklow SC, Kieff E, Johannsen E "Epstein-Barr virus nuclear protein 3C binds to the N-terminal (NTD) and beta trefoil domains (BTD) of RBP/CSL; only the NTD interaction is essential for lymphoblastoid cell growth." Virology 2011 May 06
Association of EBV nuclear proteins EBNA2, EBNA3A and EBNA3C with RBP/CSL, is essential for lymphoblastoid cell line (LCL) proliferation. Conserved residues in the EBNA3 homology domain, required for RBP/CSL interaction, lack the WPhiP motif that mediates EBNA2 and Notch binding to the RBP/CSL beta-trefoil domain (BTD). We map RBP/CSL interacting residues within EBNA3A(aa128-204) and EBNA3C(aa211-233). The EBNA3A results are consistent with an earlier report (aa125-222), but the EBNA3C domain is unexpectedly small and includes a "WTP" sequence. This EBNA3C WTP motif confers RBP/CSL binding in vitro, in yeast, and in mammalian cells. Further, an EBNA3C WTP-->STP(W227S) mutation impaired BTD binding whereas EBNA3 homology domain mutations disrupted RBP/CSL N-terminal domain (NTD) binding. WTP was not essential for EBNA3C repression of EBNA2 in reporter assays or for maintenance of LCL growth. Our results indicate that EBNA3 proteins interact with multiple RBP/CSL domains, but only NTD interactions are required for LCL growth.
Feldkamp,2011 (21439835) Feldkamp MD, Yu L, Shea MA "Structural and energetic determinants of apo calmodulin binding to the IQ motif of the Na(V)1.2 voltage-dependent sodium channel." Structure 2011 May 11
The neuronal voltage-dependent sodium channel (Na(v)1.2), essential for generation and propagation of action potentials, is regulated by calmodulin (CaM) binding to the IQ motif in its alpha subunit. A peptide (Na(v)1.2(IQp), KRKQEEVSAIVIQRAYRRYLLKQKVKK) representing the IQ motif had higher affinity for apo CaM than (Ca(2+))(4)-CaM. Association was mediated solely by the C-domain of CaM. A solution structure (2KXW.pdb) of apo (13)C,(15)N-CaM C-domain bound to Na(v)1.2(IQp) was determined with NMR. The region of Na(v)1.2(IQp) bound to CaM was helical; R1902, an Na(v)1.2 residue implicated in familial autism, did not contact CaM. The apo C-domain of CaM in this complex shares features of the same domain bound to myosin V IQ motifs (2IX7) and bound to an SK channel peptide (1G4Y) that does not contain an IQ motif. Thermodynamic and structural studies of CaM-Na(v)1.2(IQp) interactions show that apo and (Ca(2+))(4)-CaM adopt distinct conformations that both permit tight association with Na(v)1.2(IQp) during gating.
Campbell,2011 (21421922) Campbell ID, Humphries MJ "Integrin structure, activation, and interactions." Cold Spring Harb Perspect Biol 2011 Mar
Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large "head" on two "legs," with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains. These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allow cells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure. This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor.
Luna-Vargas,2011 (21415856) Luna-Vargas MP, Faesen AC, van Dijk WJ, Rape M, Fish A, Sixma TK "Ubiquitin-specific protease 4 is inhibited by its ubiquitin-like domain." EMBO Rep 2011 Apr 01
USP4 is a member of the ubiquitin-specific protease (USP) family of deubiquitinating enzymes that has a role in spliceosome regulation. Here, we show that the crystal structure of the minimal catalytic domain of USP4 has the conserved USP-like fold with its typical ubiquitin-binding site. A ubiquitin-like (Ubl) domain inserted into the catalytic domain has autoregulatory function. This Ubl domain can bind to the catalytic domain and compete with the ubiquitin substrate, partially inhibiting USP4 activity against different substrates. Interestingly, other USPs, such as USP39, could relieve this inhibition.
Houben,2012 (21413015) Houben R, Adam C, Baeurle A, Hesbacher S, Grimm J, Angermeyer S, Henzel K, Hauser S, Elling R, Brocker EB, Gaubatz S, Becker JC, Schrama D "An intact retinoblastoma protein-binding site in Merkel cell polyomavirus large T antigen is required for promoting growth of Merkel cell carcinoma cells." Int J Cancer 2012 Jan 06
Merkel cell carcinoma (MCC) is a highly aggressive skin cancer that frequently harbours Merkel cell polyomavirus (MCV) DNA integrated in the genome of the tumor cells. In our study, we elaborate our recent finding that MCV-positive MCC cell lines require the expression of the viral T antigens (TA). Indeed, in a xeno-transplantation model, we prove that TA expression is essential also in an in vivo situation, as knock down of TA leads to tumor regression. Moreover, rescuing TA short hairpin RNA (shRNA)-treated MCV-positive MCC cells by ectopic expression of shRNA-insensitive TAs clearly demonstrates that the observed effect is caused by TA knockdown. Notably, introduction of a mutation in the LTA protein interfering with LTA binding to the retinoblastoma protein (RB) ablated this rescue. The importance of this interaction was further confirmed as LTA-specific knockdown leads to explicit cell growth inhibition. In summary, the presented data demonstrate that established MCV-positive MCC tumors critically depend on TA expression, in particular the LTA and RB interaction, for sustained tumor growth. Consequently, interference with LTA/RB interaction appears as promising strategy to treat MCC.
Ishihama,2011 (21386030) Ishihama N, Yamada R, Yoshioka M, Katou S, Yoshioka H "Phosphorylation of the Nicotiana benthamiana WRKY8 transcription factor by MAPK functions in the defense response." Plant Cell 2011 Apr 27
Mitogen-activated protein kinase (MAPK) cascades have pivotal roles in plant innate immunity. However, downstream signaling of plant defense-related MAPKs is not well understood. Here, we provide evidence that the Nicotiana benthamiana WRKY8 transcription factor is a physiological substrate of SIPK, NTF4, and WIPK. Clustered Pro-directed Ser residues (SP cluster), which are conserved in group I WRKY proteins, in the N-terminal region of WRKY8 were phosphorylated by these MAPKs in vitro. Antiphosphopeptide antibodies indicated that Ser residues in the SP cluster of WRKY8 are phosphorylated by SIPK, NTF4, and WIPK in vivo. The interaction of WRKY8 with MAPKs depended on its D domain, which is a MAPK-interacting motif, and this interaction was required for effective phosphorylation of WRKY8 in plants. Phosphorylation of WRKY8 increased its DNA binding activity to the cognate W-box sequence. The phospho-mimicking mutant of WRKY8 showed higher transactivation activity, and its ectopic expression induced defense-related genes, such as 3-hydroxy-3-methylglutaryl CoA reductase 2 and NADP-malic enzyme. By contrast, silencing of WRKY8 decreased the expression of defense-related genes and increased disease susceptibility to the pathogens Phytophthora infestans and Colletotrichum orbiculare. Thus, MAPK-mediated phosphorylation of WRKY8 has an important role in the defense response through activation of downstream genes.
Ho,2011 (21383157) Ho KC, Zhou Z, She YM, Chun A, Cyr TD, Yang X "Itch E3 ubiquitin ligase regulates large tumor suppressor 1 stability [corrected]." Proc Natl Acad Sci U S A 2011 Mar 23
The large tumor suppressor 1 (LATS1) is a serine/threonine kinase and tumor suppressor found down-regulated in a broad spectrum of human cancers. LATS1 is a central player of the emerging Hippo-LATS suppressor pathway, which plays important roles in cell proliferation, apoptosis, and stem cell differentiation. Despite the ample data supporting a role for LATS1 in tumor suppression, how LATS1 is regulated at the molecular level remains largely unknown. In this study, we have identified Itch, a HECT class E3 ubiquitin ligase, as a unique binding partner of LATS1. Itch can complex with LATS1 both in vitro and in vivo through the PPxY motifs of LATS1 and the WW domains of Itch. Significantly, we found that overexpression of Itch promoted LATS1 degradation by polyubiquitination through the 26S proteasome pathway. On the other hand, knockdown of endogenous Itch by shRNAs provoked stabilization of endogenous LATS1 proteins. Finally, through several functional assays, we also revealed that change of Itch abundance alone is sufficient for altering LATS1-mediated downstream signaling, negative regulation of cell proliferation, and induction of apoptosis. Taking these data together, our study identifies E3 ubiquitin ligase Itch as a unique negative regulator of LATS1 and presents a possibility of targeting LATS1/Itch interaction as a therapeutic strategy in cancer.
Itoh,2011 (21383079) Itoh T, Kanno E, Uemura T, Waguri S, Fukuda M "OATL1, a novel autophagosome-resident Rab33B-GAP, regulates autophagosomal maturation." J Cell Biol 2011 Mar 08
Macroautophagy is a bulk degradation system conserved in all eukaryotic cells. A ubiquitin-like protein, Atg8, and its homologues are essential for autophagosome formation and act as a landmark for selective autophagy of aggregated proteins and damaged organelles. In this study, we report evidence demonstrating that OATL1, a putative Rab guanosine triphosphatase-activating protein (GAP), is a novel binding partner of Atg8 homologues in mammalian cells. OATL1 is recruited to isolation membranes and autophagosomes through direct interaction with Atg8 homologues and is involved in the fusion between autophagosomes and lysosomes through its GAP activity. We further provide evidence that Rab33B, an Atg16L1-binding protein, is a target substrate of OATL1 and is involved in the fusion between autophagosomes and lysosomes, the same as OATL1. Because both its GAP activity and its Atg8 homologue-binding activity are required for OATL1 to function, we propose a model that OATL1 uses Atg8 homologues as a scaffold to exert its GAP activity and to regulate autophagosomal maturation.
Cargnello,2011 (21372320) Cargnello M, Roux PP "Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases." Microbiol Mol Biol Rev 2011 Mar 04
The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (alpha, beta, gamma, and delta), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.
Izawa,2011 (21336306) Izawa D, Pines J "How APC/C-Cdc20 changes its substrate specificity in mitosis." Nat Cell Biol 2011 Mar 02
Progress through mitosis requires that the right protein be degraded at the right time. One ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) targets most of the crucial mitotic regulators by changing its substrate specificity throughout mitosis. The spindle assembly checkpoint (SAC) acts on the APC/C co-activator, Cdc20 (cell division cycle 20), to block the degradation of metaphase substrates (for example, cyclin B1 and securin), but not others (for example, cyclin A). How this is achieved is unclear. Here we show that Cdc20 binds to different sites on the APC/C depending on the SAC. Cdc20 requires APC3 and APC8 to bind and activate the APC/C when the SAC is satisfied, but requires only APC8 to bind the APC/C when the SAC is active. Moreover, APC10 is crucial for the destruction of cyclin B1 and securin, but not cyclin A. We conclude that the SAC causes Cdc20 to bind to different sites on the APC/C and this alters APC/C substrate specificity.
Tang,2011 (21328310) Tang XN, Lo CW, Chuang YC, Chen CT, Sun YC, Hong YR, Yang CN "Prediction of the binding mode between GSK3beta and a peptide derived from GSKIP using molecular dynamics simulation." Biopolymers 2011 Apr 26
GSK3beta plays an important role in many physiological functions; dysregulated GSK3beta is involved in human diseases such as diabetes, cancer, and Alzheimer's disease. This study uses MD simulations to determine the interaction between GSK3beta and a peptide derived from GSKIP, a novel GSK3beta interacting protein. Results show that GSKIPtide is inlaid in a binding pocket consisting of an alpha-helix and an extended loop near the carboxy-terminal end. This binding pocket is hydrophobic, and is responsible for the protein-protein interaction of two other GSK3beta interacting proteins: FRAT and Axin. The GSKIPtide binding mode is closer to that of AxinGID (in the Axin-GSK3-interacting domain). The single-point mutations of V267G and Y288F in GSK3beta differentiate the binding modes between GSK3 and GSKIPtide, AxinGID, and FRATide. The V2677G mutation of GSK3beta reduces the GSKIPtide binding affinity by 70% and abolishes the binding affinity with AxinGID, but has no effect on FRATide. However, GSK3beta Y288F completely abolishes the FRATide binding without affecting GSKIPtide or AxinGID binding. An analysis of the GSK3beta-GSKIPtide complex structure and the X-ray crystal structures of GSK3beta-FRATide and GSK3beta-AxinGID complexes suggests that the hydroxyl group of Y288 is crucial to maintaining a hydrogen bond network in GSK3beta-FRATide. The hydrophobic side chain of V267 maintains the integrity of helix-helix ridge-groove hydrophobic interaction for GSK3beta-GSKIPtide and GSK3beta-AxinGID. This study simulates these two mutant systems to provide atomic-level evidence of the aforementioned experimental results and validate the wild-type complex structure prediction.
Hodeify,2011 (21325496) Hodeify R, Tarcsafalvi A, Megyesi J, Safirstein RL, Price PM "Cdk2-dependent phosphorylation of p21 regulates the role of Cdk2 in cisplatin cytotoxicity." Am J Physiol Renal Physiol 2011 May 05
Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21(WAF1/Cip1) was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21(S78D). Mutant p21(S78D) was an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatin-induced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.
Nishi,2011 (21296877) Nishi M, Akutsu H, Masui S, Kondo A, Nagashima Y, Kimura H, Perrem K, Shigeri Y, Toyoda M, Okayama A, Hirano H, Umezawa A, Yamamoto N, Lee SW, Ryo A "A distinct role for Pin1 in the induction and maintenance of pluripotency." J Biol Chem 2011 Apr 1
The prominent characteristics of pluripotent stem cells are their unique capacity to self-renew and pluripotency. Although pluripotent stem cell proliferation is maintained by specific intracellular phosphorylation signaling events, it has not been well characterized how the resulting phosphorylated proteins are subsequently regulated. We here report that the peptidylprolyl isomerase Pin1 is indispensable for the self-renewal and maintenance of pluripotent stem cells via the regulation of phosphorylated Oct4 and other substrates. Pin1 expression was found to be up-regulated upon the induction of induced pluripotent stem (iPS) cells, and the forced expression of Pin1 with defined reprogramming factors was observed to further enhance the frequency of iPS cell generation. The inhibition of Pin1 activity significantly suppressed colony formation and induced the aberrant differentiation of human iPS cells as well as murine ES cells. We further found that Pin1 interacts with the phosphorylated Ser(12)-Pro motif of Oct4 and that this in turn facilitates the stability and transcriptional activity functions of Oct4. Our current findings thus uncover an atypical role for Pin1 as a putative regulator of the induction and maintenance of pluripotency via the control of phosphorylation signaling. These data suggest that the manipulation of Pin1 function could be a potential strategy for the stable induction and proliferation of human iPS cells.
Corti,2011 (21282473) Corti A, Curnis F "Isoaspartate-dependent molecular switches for integrin-ligand recognition." J Cell Sci 2011 Feb 15
Integrins are cell-adhesion receptors that mediate cell-extracellular-matrix (ECM) and cell-cell interactions by recognizing specific ligands. Recent studies have shown that the formation of isoaspartyl residues (isoAsp) in integrin ligands by asparagine deamidation or aspartate isomerization could represent a mechanism for the regulation of integrin-ligand recognition. This spontaneous post-translational modification, which might occur in aged proteins of the ECM, changes the length of the peptide bond and, in the case of asparagine, also of the charge. Although these changes typically have negative effects on protein function, recent studies suggested that isoAsp formation at certain Asn-Gly-Arg (NGR) sites in ECM proteins have a gain-of-function effect, because the resulting isoAsp-Gly-Arg (isoDGR) sequence can mimic Arg-Gly-Asp (RGD), a well-known integrin-binding motif. Substantial experimental evidence suggests that the NGR-to-isoDGR transition can occur in vitro in natural proteins and in drugs containing this motif, thereby promoting integrin recognition and cell adhesion. In this Commentary, we review these studies and discuss the potential effects that isoAsp formation at NGR, DGR and RGD sites might have in the recognition of integrins by natural ligands and by drugs that contain these motifs, as well as their potential biological and pharmacological implications.
Jack,2011 (21281737) Jack BH, Pearson RC, Crossley M "C-terminal binding protein: A metabolic sensor implicated in regulating adipogenesis." Int J Biochem Cell Biol 2011 Mar 30
The development of mature adipocytes from preadipocyte precursor cells requires coordinated changes in gene expression. Management of these expression changes relies on the actions of both DNA-binding transcription factors and their coregulators. Recent studies have identified the corepressor C-terminal binding protein (CtBP) as a key transcriptional coregulator in adipose tissue. CtBP proteins work with several different partner proteins to regulate the development of both white and brown adipocytes. CtBP is of particular interest as it binds to NAD(+)/NADH and may respond to the metabolic state of the cell, thereby linking changes in nutrient levels to transcriptional outcomes.
Charbonnier,2011 (21238461) Charbonnier S, Nomine Y, Ramirez J, Luck K, Chapelle A, Stote RH, Trave G, Kieffer B, Atkinson RA "The structural and dynamic response of MAGI-1 PDZ1 with noncanonical domain boundaries to the binding of human papillomavirus E6." J Mol Biol 2011 Mar 11
PDZ domains are protein interaction domains that are found in cytoplasmic proteins involved in signaling pathways and subcellular transport. Their roles in the control of cell growth, cell polarity, and cell adhesion in response to cell contact render this family of proteins targets during the development of cancer. Targeting of these network hubs by the oncoprotein E6 of "high-risk" human papillomaviruses (HPVs) serves to effect the efficient disruption of cellular processes. Using NMR, we have solved the three-dimensional solution structure of an extended construct of the second PDZ domain of MAGI-1 (MAGI-1 PDZ1) alone and bound to a peptide derived from the C-terminus of HPV16 E6, and we have characterized the changes in backbone dynamics and hydrogen bonding that occur upon binding. The binding event induces quenching of high-frequency motions in the C-terminal tail of the PDZ domain, which contacts the peptide upstream of the canonical X-[T/S]-X-[L/V] binding motif. Mutations designed in the C-terminal flanking region of the PDZ domain resulted in a significant decrease in binding affinity for E6 peptides. This detailed analysis supports the notion of a global response of the PDZ domain to the binding event, with effects propagated to distal sites, and reveals unexpected roles for the sequences flanking the canonical PDZ domain boundaries.
Vaishnav,2011 (21237154) Vaishnav M, MacFarlane M, Dickens M "Disassembly of the JIP1/JNK molecular scaffold by caspase-3-mediated cleavage of JIP1 during apoptosis." Exp Cell Res 2011 Apr 15
We report here the cleavage of the c-Jun N-terminal Kinase (JNK) pathway scaffold protein, JNK Interacting Protein-1 (JIP1), by caspases during both Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and staurosporine-induced apoptosis in HeLa cells. During the initiation of apoptosis, maximal JNK activation is observed when JIP1 is intact, whereas cleavage of JIP1 correlates with JNK inactivation and progression of apoptosis. JIP1 is cleaved by caspase-3 at two sites, leading to disassembly of the JIP1/JNK complex. Inhibition of JIP1 cleavage by the caspase-3 inhibitor DEVD.fmk inhibits this disassembly, and is accompanied by sustained JNK activation. These data suggest that TRAIL and staurosporine induce JNK activation in a caspase-3-independent manner and that caspase-3-mediated JIP1 cleavage plays a role in JNK inactivation via scaffold disassembly during the execution phase of apoptosis. Caspase-mediated cleavage of JIP scaffold proteins may therefore represent an important mechanism for modulation of JNK signalling during apoptotic cell death.
Noakes,2011 (21233288) Noakes CJ, Lee G, Lowe M "The PH domain proteins IPIP27A and B link OCRL1 to receptor recycling in the endocytic pathway." Mol Biol Cell 2011 Mar 01
Mutation of the inositol polyphosphate 5-phosphatase OCRL1 results in two disorders in humans, namely Lowe syndrome (characterized by ocular, nervous system, and renal defects) and type 2 Dent disease (in which only the renal symptoms are evident). The disease mechanisms of these syndromes are poorly understood. Here we identify two novel OCRL1-binding proteins, termed inositol polyphosphate phosphatase interacting protein of 27 kDa (IPIP27)A and B (also known as Ses1 and 2), that also bind the related 5-phosphatase Inpp5b. The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1. IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN). The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane. Our results identify IPIP27A and B as key players in endocytic trafficking and strongly suggest that defects in this process are responsible for the pathology of Lowe syndrome and Dent disease.
Jorgensen,2011 (21220508) Jorgensen S, Eskildsen M, Fugger K, Hansen L, Larsen MS, Kousholt AN, Syljuasen RG, Trelle MB, Jensen ON, Helin K, Sorensen CS "SET8 is degraded via PCNA-coupled CRL4(CDT2) ubiquitylation in S phase and after UV irradiation." J Cell Biol 2011 Jan 10
The eukaryotic cell cycle is regulated by multiple ubiquitin-mediated events, such as the timely destruction of cyclins and replication licensing factors. The histone H4 methyltransferase SET8 (Pr-Set7) is required for chromosome compaction in mitosis and for maintenance of genome integrity. In this study, we show that SET8 is targeted for degradation during S phase by the CRL4(CDT2) ubiquitin ligase in a proliferating cell nuclear antigen (PCNA)-dependent manner. SET8 degradation requires a conserved degron responsible for its interaction with PCNA and recruitment to chromatin where ubiquitylation occurs. Efficient degradation of SET8 at the onset of S phase is required for the regulation of chromatin compaction status and cell cycle progression. Moreover, the turnover of SET8 is accelerated after ultraviolet irradiation dependent on the CRL4(CDT2) ubiquitin ligase and PCNA. Removal of SET8 supports the modulation of chromatin structure after DNA damage. These results demonstrate a novel regulatory mechanism, linking for the first time the ubiquitin-proteasome system with rapid degradation of a histone methyltransferase to control cell proliferation.
Dancheck,2011 (21218781) Dancheck B, Ragusa MJ, Allaire M, Nairn AC, Page R, Peti W "Molecular investigations of the structure and function of the protein phosphatase 1-spinophilin-inhibitor 2 heterotrimeric complex." Biochemistry 2011 Feb 15
Regulation of the major Ser/Thr phosphatase protein phosphatase 1 (PP1) is controlled by a diverse array of targeting and inhibitor proteins. Though many PP1 regulatory proteins share at least one PP1 binding motif, usually the RVxF motif, it was recently discovered that certain pairs of targeting and inhibitor proteins bind PP1 simultaneously to form PP1 heterotrimeric complexes. To date, structural information for these heterotrimeric complexes and, in turn, how they direct PP1 activity is entirely lacking. Using a combination of NMR spectroscopy, biochemistry, and small-angle X-ray scattering (SAXS), we show that major structural rearrangements in both spinophilin (targeting) and inhibitor 2 (I-2, inhibitor) are essential for the formation of the heterotrimeric PP1-spinophilin-I-2 (PSI) complex. The RVxF motif of I-2 is released from PP1 during the formation of PSI, making the less prevalent SILK motif of I-2 essential for complex stability. The release of the I-2 RVxF motif allows for enhanced flexibility of both I-2 and spinophilin in the heterotrimeric complex. In addition, we used inductively coupled plasma atomic emission spectroscopy to show that PP1 contains two metals in both heterodimeric complexes (PP1-spinophilin and PP1-I-2) and PSI, demonstrating that PSI retains the biochemical characteristics of the PP1-I-2 holoenzyme. Finally, we combined the NMR and biochemical data with SAXS and molecular dynamics simulations to generate a structural model of the full heterotrimeric PSI complex. Collectively, these data reveal the molecular events that enable PP1 heterotrimeric complexes to exploit both the targeting and inhibitory features of the PP1-regulatory proteins to form multifunctional PP1 holoenzymes.
Zhao,2011 (21205866) Zhao B, Li L, Lu Q, Wang LH, Liu CY, Lei Q, Guan KL "Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein." Genes Dev 2011 Jan 05
The Yes-associated protein (YAP) is a transcription coactivator that plays a crucial role in organ size control by promoting cell proliferation and inhibiting apoptosis. The Hippo tumor suppressor pathway inhibits YAP through phosphorylation-induced cytoplasmic retention and degradation. Here we report a novel mechanism of YAP regulation by angiomotin (AMOT) family proteins via a direct interaction. Knockdown of AMOT family protein AMOTL2 in polarized Madin-Darby canine kidney (MDCK) cells leads to YAP activation, as indicated by decreased YAP tight junction localization, attenuated YAP phosphorylation, accumulation of nuclear YAP, and induction of YAP target gene expression. Transcriptional coactivator with PDZ-binding motif (TAZ), the YAP paralog, is also regulated by AMOT in a similar fashion. Furthermore, AMOTL2 knockdown results in loss of cell contact inhibition in a manner dependent on the functions of YAP and TAZ. Our results indicate a potential tumor-suppressing role of AMOT family proteins as components of the Hippo pathway, and demonstrate a novel mechanism of YAP and TAZ inhibition by AMOT-mediated tight junction localization. These observations provide a potential link between the Hippo pathway and cell contact inhibition.
Bartlam,2011 (21203959) Bartlam M, Yamamoto T "The structural basis for deadenylation by the CCR4-NOT complex." Protein Cell 2011 Jan 04
The CCR4-NOT complex is a highly conserved, multifunctional machinery controlling mRNA metabolism. Its components have been implicated in several aspects of mRNA and protein expression, including transcription initiation, elongation, mRNA degradation, ubiquitination, and protein modification. In this review, we will focus on the role of the CCR4-NOT complex in mRNA degradation. The complex contains two types of deadenylase enzymes, one belonging to the DEDD-type family and one belonging to the EEP-type family, which shorten the poly(A) tails of mRNA. We will review the present state of structure-function analyses into the CCR4-NOT deadenylases and summarize current understanding of their roles in mRNA degradation. We will also review structural and functional work on the Tob/BTG family of proteins, which are known to interact with the CCR4-NOT complex and which have been reported to suppress deadenylase activity in vitro.
Petersen,2011 (21203436) Petersen K, Qiu JL, Lutje J, Fiil BK, Hansen S, Mundy J, Petersen M "Arabidopsis MKS1 is involved in basal immunity and requires an intact N-terminal domain for proper function." PLoS One 2011 Jan 04
BACKGROUND: Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. MAP kinase 4 (MPK4) functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn controls the production of anti-microbial phytoalexins. METHODOLOGY/PRINCIPAL FINDINGS: We investigate the role of MKS1 in basal resistance and the importance of its N- and C-terminal domains for MKS1 function. We used the information that mks1 loss-of-function partially suppresses the mpk4 loss-of-function phenotype, and that transgenic expression of functional MKS1 in mpk4/mks1 double mutants reverted the mpk4 dwarf phenotype. Transformation of mks1/mpk4 with mutant versions of MKS1 constructs showed that a single amino acid substitution in a putative MAP kinase docking domain, MKS1-L32A, or a truncated MKS1 version unable to interact with WRKY33, were deficient in reverting the double mutant to the mpk4 phenotype. These results demonstrate functional requirement in MKS1 for the interaction with MPK4 and WRKY33. In addition, nuclear localization of MKS1 was shown to depend on an intact N-terminal domain. Furthermore, loss-of-function mks1 mutants exhibited increased susceptibility to strains of Pseudomonas syringae and Hyaloperonospora arabidopsidis, indicating that MKS1 plays a role in basal defense responses. CONCLUSIONS: Taken together, our results indicate that MKS1 function and subcellular location requires an intact N-terminus important for both MPK4 and WRKY33 interactions.
Kubota,2011 (21199872) Kubota T, Matsuoka M, Xu S, Otsuki N, Takeda M, Kato A, Ozato K "PIASy inhibits virus-induced and interferon-stimulated transcription through distinct mechanisms." J Biol Chem 2011 Mar 10
The protein inhibitor of activated STAT (PIAS) family proteins regulates innate immune responses by controlling transcription induced by Toll-like receptor, RIG-I-like receptor signaling, and JAK/STAT pathways. Here, we show that PIASy negatively regulates type I interferon (IFN) transcription. Virus infection led to enhanced type I IFN induction in PIASy null cells, and conversely PIASy overexpression reduced IFN transcription. A mutation in the LXXLL motif of the SAP domain abolished inhibition of IFN-stimulated gene expression but did not affect virus or Toll-like receptor/RIG-I-like receptor-stimulated IFN transcription, indicating that PIASy employs distinct mechanisms to inhibit virus-induced and IFN-stimulated transcription. SUMO E3 activity was not required for PIASy inhibition of IFN transcription; however, PIASy relied on the SUMO modification mechanism to inhibit IFN transcription, because the activity of the SUMO-interacting motif was required for inhibition, and knockdown of SUMO E2 enzyme UBC9 decreased inhibitory activity of PIASy. Our results demonstrate that PIASy negatively regulates both IFN transcription and IFN-stimulated gene expression through multiple mechanisms utilizing the function of different domains.
Lai,2011 (21195170) Lai F, Zhou Y, Luo X, Fox J, King ML "Nanos1 functions as a translational repressor in the Xenopus germline." Mech Dev 2011 Feb 14
Nanos family members have been shown to act as translational repressors in the Drosophila and Caenorhabditis elegans germline, but direct evidence is missing for a similar function in vertebrates. Using a tethered function assay, we show that Xenopus Nanos1 is a translational repressor and that association with the RNA is required for this repression. We identified a 14 amino acid region within the N-terminal domain of Nanos1 that is conserved in organisms as diverse as sponge and Human. The region is found in all vertebrates but notably lacking in Drosophila and C. elegans. Deletion and substitution analysis revealed that this conserved region was required for Nanos1 repressive activity. Consistent with this observation, deletion of this region was sufficient to prevent abnormal development that results from ectopic expression of Nanos1 in oocytes. Although Nanos1 can repress capped and polyadenylated RNAs, Nanos1 mediated repression did not require the targeted RNA to have a cap or to be polyadenylated. These results suggest that Nanos1 is capable of repressing translation by several different mechanisms. We found that Nanos1, like Drosophila Nanos, associates with cyclin B1 RNA in vivo indicating that some Nanos targets may be evolutionarily conserved. Nanos1 protein was detected and thus available to repress mRNAs while PGCs were in the endoderm, but was not observed in PGCs after this stage.
Sung,2011 (21192925) Sung KS, Lee YA, Kim ET, Lee SR, Ahn JH, Choi CY "Role of the SUMO-interacting motif in HIPK2 targeting to the PML nuclear bodies and regulation of p53." Exp Cell Res 2011 Mar 14
Homeodomain-interacting protein kinase 2 (HIPK2) is a key regulator of various transcription factors including p53 and CtBP in the DNA damage signaling pathway. PML-nuclear body (NB) is required for HIPK2-mediated p53 phosphorylation at Ser46 and induction of apoptosis. Although PML-NB targeting of HIPK2 has been shown, much is not clear about the molecular mechanism of HIPK2 recruitment to PML-NBs. Here we show that HIPK2 colocalizes specifically with PML-I and PML-IV. Mutational analysis showed that HIPK2 recruitment to PML-IV-NBs is mediated by the SUMO-interaction motifs (SIMs) of both PML-IV and HIPK2. Wild-type HIPK2 associated with SUMO-conjugated PML-IV at a higher affinity than with un-conjugated PML-IV, while the association of a HIPK2 SIM mutant with SUMO-modified PML-IV was impaired. In colony formation assays, HIPK2 strongly suppressed cell proliferation, but HIPK2 SIM mutants did not. In addition, activation and phosphorylation of p53 at the Ser46 residue were impaired by HIPK2 SIM mutants. These results suggest that SIM-mediated HIPK2 targeting to PML-NBs is crucial for HIPK2-mediated p53 activation and induction of apoptosis.
Johansen,2011 (21189453) Johansen T, Lamark T "Selective autophagy mediated by autophagic adapter proteins." Autophagy 2011 May 02
Mounting evidence suggests that autophagy is a more selective process than originally anticipated. The discovery and characterization of autophagic adapters, like p62 and NBR1, has provided mechanistic insight into this process. p62 and NBR1 are both selectively degraded by autophagy and able to act as cargo receptors for degradation of ubiquitinated substrates. A direct interaction between these autophagic adapters and the autophagosomal marker protein LC3, mediated by a so-called LIR (LC3-interacting region) motif, their inherent ability to polymerize or aggregate as well as their ability to specifically recognize substrates are required for efficient selective autophagy. These three required features of autophagic cargo receptors are evolutionarily conserved and also employed in the yeast cytoplasm-to-vacuole targeting (Cvt) pathway and in the degradation of P granules in C. elegans. Here, we review the mechanistic basis of selective autophagy in mammalian cells discussing the degradation of misfolded proteins, p62 bodies, aggresomes, mitochondria and invading bacteria. The emerging picture of selective autophagy affecting the regulation of cell signaling with consequences for oxidative stress responses, tumorigenesis and innate immunity is also addressed.
Chan,2011 (21189416) Chan PM, Ng YW, Manser E "A robust protocol to map binding sites of the 14-3-3 interactome: Cdc25C requires phosphorylation of both S216 and S263 to bind 14-3-3." Mol Cell Proteomics 2011 Mar 02
Modern proteomic techniques have identified hundreds of proteins that bind 14-3-3s, the most widespread eukaryotic phosphoserine/threonine sensors, but accurate prediction of the target phospho-sites is difficult. Here we describe a systematic approach using synthetic peptides that tests large numbers of potential binding sites in parallel for human 14-3-3. By profiling the sequence requirements for three diverse 14-3-3 binding sites (from IRS-1, IRSp53 and GIT2), we have generated enhanced bioinformatics tools to score sites and allow more tractable testing by co-immunoprecipitation. This approach has allowed us to identify two additional sites other than Ser216 in the widely studied cell division cycle (Cdc) protein 25C, whose function depends on 14-3-3 binding. These Ser247 and Ser263 sites in human Cdc25C, which were not predicted by the existing Scansite search, are conserved across species and flank the nuclear localization region. Furthermore, we found strong interactions between 14-3-3 and peptides with the sequence Rxx[S/T]xR typical for PKC sites, and which is as abundant as the canonical Rxx[S/T]xP motif in the proteome. Two such sites are required for 14-3-3 binding in the polarity protein Numb. A recent survey of >200 reported sites identified only a handful containing this motif, suggesting that it is currently under-appreciated as a candidate binding site. This approach allows one to rapidly map 14-3-3 binding sites and has revealed alternate motifs.
Okamoto,2011 (21189250) Okamoto Y, Shikano S "Phosphorylation-dependent C-terminal binding of 14-3-3 proteins promotes cell surface expression of HIV co-receptor GPR15." J Biol Chem 2011 Mar 01
Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15(S359A) mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.
Panni,2011 (21182200) Panni S, Montecchi-Palazzi L, Kiemer L, Cabibbo A, Paoluzi S, Santonico E, Landgraf C, Volkmer-Engert R, Bachi A, Castagnoli L, Cesareni G "Combining peptide recognition specificity and context information for the prediction of the 14-3-3-mediated interactome in S. cerevisiae and H. sapiens." Proteomics 2011 Jan
Large-scale interaction studies contribute the largest fraction of protein interactions information in databases. However, co-purification of non-specific or indirect ligands, often results in data sets that are affected by a considerable number of false positives. For the fraction of interactions mediated by short linear peptides, we present here a combined experimental and computational strategy for ranking the reliability of the inferred partners. We apply this strategy to the family of 14-3-3 domains. We have first characterized the recognition specificity of this domain family, largely confirming the results of previous analyses, while revealing new features of the preferred sequence context of 14-3-3 phospho-peptide partners. Notably, a proline next to the carboxy side of the phospho-amino acid functions as a potent inhibitor of 14-3-3 binding. The position-specific information about residue preference was encoded in a scoring matrix and two regular expressions. The integration of these three features in a single predictive model outperforms publicly available prediction tools. Next we have combined, by a naive Bayesian approach, these "peptide features" with "protein features", such as protein co-expression and co-localization. Our approach provides an orthogonal reliability assessment and maps with high confidence the 14-3-3 peptide target on the partner proteins.
Cuchet,2010 (21172801) Cuchet D, Sykes A, Nicolas A, Orr A, Murray J, Sirma H, Heeren J, Bartelt A, Everett RD "PML isoforms I and II participate in PML-dependent restriction of HSV-1 replication." J Cell Sci 2010 Dec 28
Intrinsic antiviral resistance mediated by constitutively expressed cellular proteins is one arm of defence against virus infection. Promyelocytic leukaemia nuclear bodies (PML-NBs, also known as ND10) contribute to host restriction of herpes simplex virus type 1 (HSV-1) replication via mechanisms that are counteracted by viral regulatory protein ICP0. ND10 assembly is dependent on PML, which comprises several different isoforms, and depletion of all PML isoforms decreases cellular resistance to ICP0-null mutant HSV-1. We report that individual expression of PML isoforms I and II partially reverses the increase in ICP0-null mutant HSV-1 plaque formation that occurs in PML-depleted cells. This activity of PML isoform I is dependent on SUMO modification, its SUMO interaction motif (SIM), and each element of its TRIM domain. Detailed analysis revealed that the punctate foci formed by individual PML isoforms differ subtly from normal ND10 in terms of composition and/or Sp100 modification. Surprisingly, deletion of the SIM motif from PML isoform I resulted in increased colocalisation with other major ND10 components in cells lacking endogenous PML. Our observations suggest that complete functionality of PML is dependent on isoform-specific C-terminal sequences acting in concert.
Chagot,2011 (21167176) Chagot B, Chazin WJ "Solution NMR structure of Apo-calmodulin in complex with the IQ motif of human cardiac sodium channel NaV1.5." J Mol Biol 2011 Feb 11
The function of the human voltage-gated sodium channel Na(V)1.5 is regulated in part by intracellular calcium signals. The ubiquitous calcium sensor protein calmodulin (CaM) is an important part of the complex calcium-sensing apparatus in Na(V)1.5. CaM interacts with an IQ (isoleucine-glutamine) motif in the large intracellular C-terminal domain of the channel. Using co-expression and co-purification, we have been able to isolate a CaM-IQ motif complex and to determine its high-resolution structure in absence of calcium using multi-dimensional solution NMR. Under these conditions, the Na(V)1.5 IQ motif interacts with the C-terminal domain (C-lobe) of CaM, with the N-terminal domain remaining free in solution. The structure reveals that the C-lobe adopts a semi-open conformation with the IQ motif bound in a narrow hydrophobic groove. Sequence similarities between voltage-gated sodium channels and voltage-gated calcium channels suggest that the structure of the CaM-Na(V)1.5 IQ motif complex can serve as a general model for the interaction between CaM and ion channel IQ motifs under low-calcium conditions. The structure also provides insight into the biochemical basis for disease-associated mutations that map to the IQ motif in Na(V)1.5.
Shamas-Din,2011 (21146563) Shamas-Din A, Brahmbhatt H, Leber B, Andrews DW "BH3-only proteins: Orchestrators of apoptosis." Biochim Biophys Acta 2011 Mar 22
The BH3-only proteins of Bcl-2 family are essential initiators of apoptosis that propagate extrinsic and intrinsic cell death signals. The interaction of BH3-only proteins with other Bcl-2 family members is critical for understanding the core machinery that controls commitment to apoptosis by permeabilizing the mitochondrial outer membrane. BH3-only proteins promote apoptosis by both directly activating Bax and Bak and by suppressing the anti-apoptotic proteins at the mitochondria and the endoplasmic reticulum. To prevent constitutive cell death, BH3-only proteins are regulated by a variety of mechanisms including transcription and post-translational modifications that govern specific protein-protein interactions. Furthermore, BH3-only proteins also control the initiation of autophagy, another important pathway regulating cell survival and death. Emerging evidence indicates that the interaction of BH3-only proteins with membranes regulates binding to other Bcl-2 family members, thereby specifying function. Due to the important role of BH3-only proteins in the regulation of cell death, several promising BH3-mimetic drugs that are active in pre-clinical models are currently being tested as anti-cancer agents. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.
Davey,2011 (21146412) Davey NE, Trave G, Gibson TJ "How viruses hijack cell regulation." Trends Biochem Sci 2011 Mar 14
Viruses, as obligate intracellular parasites, are the pathogens that have the most intimate relationship with their host, and as such, their genomes have been shaped directly by interactions with the host proteome. Every step of the viral life cycle, from entry to budding, is orchestrated through interactions with cellular proteins. Accordingly, viruses will hijack and manipulate these proteins utilising any achievable mechanism. Yet, the extensive interactions of viral proteomes has yielded a conundrum: how do viruses commandeer so many diverse pathways and processes, given the obvious spatial constraints imposed by their compact genomes? One important approach is slowly being revealed, the extensive mimicry of host protein short linear motifs (SLiMs).
Michishita,2011 (21143559) Michishita M, Morimoto A, Ishii T, Komori H, Shiomi Y, Higuchi Y, Nishitani H "Positively charged residues located downstream of PIP box, together with TD amino acids within PIP box, are important for CRL4(Cdt2) -mediated proteolysis." Genes Cells 2011 Jan
PCNA links Cdt1 and p21 for proteolysis by Cul4-DDB1-Cdt2 (CRL4(Cdt2) ) in the S phase and after DNA damage in mammalian cells. However, other PCNA-interacting proteins, such as ligase I, are not targets of CRL4(Cdt2) . In this study, we created chimera constructs composed of Cdt1 and ligase I and examined how the proteolysis of PCNA-interacting proteins is regulated. Consistent with a recent report using the Xenopus egg system (Havens & Walter 2009), two amino acid elements are also required for degradation in HeLa cells: TD amino acid residues in the PIP box and the basic amino acid at +4 downstream of the PIP box. In addition, we demonstrate that a basic amino acid at +3 is also required for degradation and that an acidic amino acid residue following the basic amino acids abolishes the degradation. Electrostatic surface images suggest that the basic amino acid at +4 is involved in a contact with PCNA, while +3 position extending to opposite direction is important to create a positively charged surface. When all these required elements were introduced in ligase I peptide, the substituted form became degraded. Our results demonstrate that PCNA-dependent degron is strictly composed to avoid illegitimate destruction of PCNA-interacting proteins.
Tan,2011 (21118994) Tan GS, Magurno J, Cooper KF "Ama1p-activated anaphase-promoting complex regulates the destruction of Cdc20p during meiosis II." Mol Biol Cell 2011 Feb 01
The execution of meiotic divisions in Saccharomyces cerevisiae is regulated by anaphase-promoting complex/cyclosome (APC/C)-mediated protein degradation. During meiosis, the APC/C is activated by association with Cdc20p or the meiosis-specific activator Ama1p. We present evidence that, as cells exit from meiosis II, APC/C(Ama1) mediates Cdc20p destruction. APC/C(Ama1) recognizes two degrons on Cdc20p, the destruction box and destruction degron, with either domain being sufficient to mediate Cdc20p destruction. Cdc20p does not need to associate with the APC/C to bind Ama1p or be destroyed. Coimmunoprecipitation analyses showed that the diverged amino-terminal region of Ama1p recognizes both Cdc20p and Clb1p, a previously identified substrate of APC/C(Ama1). Domain swap experiments revealed that the C-terminal WD region of Cdh1p, when fused to the N-terminal region of Ama1p, could direct most of Ama1p functions, although at a reduced level. In addition, this fusion protein cannot complement the spore wall defect in ama1Delta strains, indicating that substrate specificity is also derived from the WD repeat domain. These findings provide a mechanism to temporally down-regulate APC/C(Cdc20) activity as the cells complete meiosis II and form spores.
da Fonseca,2011 (21107322) da Fonseca PC, Kong EH, Zhang Z, Schreiber A, Williams MA, Morris EP, Barford D "Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor." Nature 2011 Feb 10
The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box and KEN (Lys-Glu-Asn)-box. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/C(Cdh1)) bound to a D-box peptide at approximately 10 A resolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10--a core APC/C subunit previously implicated in substrate recognition--and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box-Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/C(Cdh1) complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.
Erdmann,2011 (21102557) Erdmann F, Schauble N, Lang S, Jung M, Honigmann A, Ahmad M, Dudek J, Benedix J, Harsman A, Kopp A, Helms V, Cavalie A, Wagner R, Zimmermann R "Interaction of calmodulin with Sec61alpha limits Ca2+ leakage from the endoplasmic reticulum." EMBO J 2011 Jan 5
In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61alpha that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.
Wacker,2011 (21102556) Wacker SA, Alvarado C, von Wichert G, Knippschild U, Wiedenmann J, Clauss K, Nienhaus GU, Hameister H, Baumann B, Borggrefe T, Knochel W, Oswald F "RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J." EMBO J 2011 Jan 05
The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.
Sato,2010 (21102411) Sato Y, Shibata H, Nakatsu T, Nakano H, Kashiwayama Y, Imanaka T, Kato H "Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p." EMBO J 2010 Dec 15
Peroxisomes require peroxin (Pex) proteins for their biogenesis. The interaction between Pex3p, which resides on the peroxisomal membrane, and Pex19p, which resides in the cytosol, is crucial for peroxisome formation and the post-translational targeting of peroxisomal membrane proteins (PMPs). It is not known how Pex3p promotes the specific interaction with Pex19p for the purpose of PMP translocation. Here, we present the three-dimensional structure of the complex between a cytosolic domain of Pex3p and the binding-region peptide of Pex19p. The overall shape of Pex3p is a prolate spheroid with a novel fold, the 'twisted six-helix bundle.' The Pex19p-binding site is at an apex of the Pex3p spheroid. A 16-residue region of the Pex19p peptide forms an alpha-helix and makes a contact with Pex3p; this helix is disordered in the unbound state. The Pex19p peptide contains a characteristic motif, consisting of the leucine triad (Leu18, Leu21, Leu22), and Phe29, which are critical for the Pex3p binding and peroxisome biogenesis.
Yang,2011 (21098120) Yang R, Gaidamakov SA, Xie J, Lee J, Martino L, Kozlov G, Crawford AK, Russo AN, Conte MR, Gehring K, Maraia RJ "La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability." Mol Cell Biol 2011 Feb
The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all approximately 150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.
Burkhard,2011 (21098038) Burkhard KA, Chen F, Shapiro P "Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity." J Biol Chem 2011 Jan 24
Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 muM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.
Zhao,2010 (21084559) Zhao B, Li L, Guan KL "Hippo signaling at a glance." J Cell Sci 2010 Nov 18
Im,2010 (21070952) Im YJ, Kuo L, Ren X, Burgos PV, Zhao XZ, Liu F, Burke TR Jr, Bonifacino JS, Freed EO, Hurley JH "Crystallographic and functional analysis of the ESCRT-I /HIV-1 Gag PTAP interaction." Structure 2010 Nov 12
Budding of HIV-1 requires the binding of the PTAP late domain of the Gag p6 protein to the UEV domain of the TSG101 subunit of ESCRT-I. The normal function of this motif in cells is in receptor downregulation. Here, we report the 1.4-1.6 A structures of the human TSG101 UEV domain alone and with wild-type and mutant HIV-1 PTAP and Hrs PSAP nonapeptides. The hydroxyl of the Thr or Ser residue in the P(S/T)AP motif hydrogen bonds with the main chain of Asn69. Mutation of the Asn to Pro, blocking the main-chain amide, abrogates PTAP motif binding in vitro and blocks budding of HIV-1 from cells. N69P and other PTAP binding-deficient alleles of TSG101 did not rescue HIV-1 budding. However, the mutant alleles did rescue downregulation of endogenous EGF receptor. This demonstrates that the PSAP motif is not rate determining in EGF receptor downregulation under normal conditions.
Ma,2010 (21070949) Ma W, Shang Y, Wei Z, Wen W, Wang W, Zhang M "Phosphorylation of DCC by ERK2 is facilitated by direct docking of the receptor P1 domain to the kinase." Structure 2010 Nov 12
Netrin receptor DCC plays critical roles in many cellular processes, including axonal outgrowth and migration, angiogenesis, and apoptosis, but the molecular basis of DCC-mediated signaling is largely unclear. ERK2, a member of the MAPK family, is one of the few proteins known to be involved in DCC-mediated signaling. Here, we report that ERK2 directly interacts with DCC, and the ERK2-binding region was mapped to the conserved intracellular P1 domain of the receptor. The structure of ERK2 in complex with the P1 domain of DCC reveals that DCC contains a MAPK docking motif. The docking of the P1 domain onto ERK2 physically positions several phosphorylation sites of DCC in the vicinity of the kinase active site. We further show that the docking interaction between the P1 domain and ERK2 is essential for the ERK2-mediated phosphorylation of DCC. We conclude that DCC signaling is directly coupled with MAPK signaling cascades.
Nagashima,2011 (21068219) Nagashima S, Takahashi M, Jirintai, Tanaka T, Yamada K, Nishizawa T, Okamoto H "A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells." J Gen Virol 2011 Feb
We have previously demonstrated that the release of hepatitis E virus (HEV) from infected cells depended on ORF3 protein, which harbours one or two PSAP motifs. To elucidate the PSAP motif(s) in the ORF3 protein during virion egress, five PSAP mutants derived from an infectious genotype 3 cDNA clone of pJE03-1760F/wt that can grow efficiently in PLC/PRF/5 cells were analysed. Four mutants, including mutLSAP, mutPSAL, mutLSAL (the substituted amino acids in the authentic PSAP motif are underlined) and mutPLAP/PSAP (the changed amino acid in the additional PSAP motif is underlined) generated progenies as efficiently as the wild-type virus. Conversely, the HEV RNA level in the culture supernatant of mutPLAP/LSAL RNA-transfected cells was significantly lower than in cells transfected with the wild-type RNA, similar to an ORF3-null mutant. Consistent with the ORF3-deficient mutant, the mutPLAP/LSAL mutant with no intact PSAP motifs banded at 1.26-1.27 g ml(-1) in sucrose, and was captured by anti-ORF2, but not by anti-ORF3, with or without prior treatment with detergent (0.1 % sodium deoxycholate). The absence of the ORF3 protein on the mutant particles in the culture supernatant was confirmed by Western blotting, despite the expression of ORF3 protein in the RNA-transfected cells, as detected by immunofluorescence and Western blotting. Therefore, at least one of the two intact PSAP motifs in the ORF3 protein is required for the formation of membrane-associated HEV particles possessing ORF3 proteins on their surface, thus suggesting that the PSAP motif plays a role as a functional domain for HEV budding.
Huntzinger,2010 (21063388) Huntzinger E, Braun JE, Heimstadt S, Zekri L, Izaurralde E "Two PABPC1-binding sites in GW182 proteins promote miRNA-mediated gene silencing." EMBO J 2010 Dec 15
miRNA-mediated gene silencing requires the GW182 proteins, which are characterized by an N-terminal domain that interacts with Argonaute proteins (AGOs), and a C-terminal silencing domain (SD). In Drosophila melanogaster (Dm) GW182 and a human (Hs) orthologue, TNRC6C, the SD was previously shown to interact with the cytoplasmic poly(A)-binding protein (PABPC1). Here, we show that two regions of GW182 proteins interact with PABPC1: the first contains a PABP-interacting motif 2 (PAM2; as shown before for TNRC6C) and the second contains the M2 and C-terminal sequences in the SD. The latter mediates indirect binding to the PABPC1 N-terminal domain. In D. melanogaster cells, the second binding site dominates; however, in HsTNRC6A-C the PAM2 motif is essential for binding to both Hs and DmPABPC1. Accordingly, a single amino acid substitution in the TNRC6A-C PAM2 motif abolishes the interaction with PABPC1. This mutation also impairs TNRC6s silencing activity. Our findings reveal that despite species-specific differences in the relative strength of the PABPC1-binding sites, the interaction between GW182 proteins and PABPC1 is critical for miRNA-mediated silencing in animal cells.
Ku,2011 (21060336) Ku B, Liang C, Jung JU, Oh BH "Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX." Cell Res 2011 Apr 07
Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic BCL-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague. Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, but was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-X(L), MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could be expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.
Kaustov,2011 (21047797) Kaustov L, Ouyang H, Amaya M, Lemak A, Nady N, Duan S, Wasney GA, Li Z, Vedadi M, Schapira M, Min J, Arrowsmith CH "Recognition and specificity determinants of the human cbx chromodomains." J Biol Chem 2011 Jan 03
The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1-8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1-8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity.
Aylon,2010 (21041410) Aylon Y, Ofir-Rosenfeld Y, Yabuta N, Lapi E, Nojima H, Lu X, Oren M "The Lats2 tumor suppressor augments p53-mediated apoptosis by promoting the nuclear proapoptotic function of ASPP1." Genes Dev 2010 Nov 02
Apoptosis is an important mechanism to eliminate potentially tumorigenic cells. The tumor suppressor p53 plays a pivotal role in this process. Many tumors harbor mutant p53, but others evade its tumor-suppressive effects by altering the expression of proteins that regulate the p53 pathway. ASPP1 (apoptosis-stimulating protein of p53-1) is a key mediator of the nuclear p53 apoptotic response. Under basal conditions, ASPP1 is cytoplasmic. We report that, in response to oncogenic stress, the tumor suppressor Lats2 (large tumor suppressor 2) phosphorylates ASPP1 and drives its translocation into the nucleus. Together, Lats2 and ASPP1 shunt p53 to proapoptotic promoters and promote the death of polyploid cells. These effects are overridden by the Yap1 (Yes-associated protein 1) oncoprotein, which disrupts Lats2-ASPP1 binding and antagonizes the tumor-suppressing function of the Lats2/ASPP1/p53 axis.
Boussetta,2010 (20956805) Boussetta T, Gougerot-Pocidalo MA, Hayem G, Ciappelloni S, Raad H, Arabi Derkawi R, Bournier O, Kroviarski Y, Zhou XZ, Malter JS, Lu PK, Bartegi A, Dang PM, El-Benna J "The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-alpha-induced priming of the NADPH oxidase in human neutrophils." Blood 2010 Dec 23
Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-alpha (TNF-alpha), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-alpha primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-alpha-induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-alpha. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-alpha-induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-alpha enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-alpha-induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy.
Chan,2011 (20945341) Chan SW, Lim CJ, Chen L, Chong YF, Huang C, Song H, Hong W "The Hippo pathway in biological control and cancer development." J Cell Physiol 2011 Jan 26
The Hippo pathway is an evolutionally conserved protein kinase cascade involved in regulating organ size in vivo and cell contact inhibition in vitro by governing cell proliferation and apoptosis. Deregulation of the Hippo pathway is linked to cancer development. Its first core kinase Warts was identified in Drosophila more than 15 years ago, but it gained much attention when other core components of the pathway were identified 8 years later. Major discoveries of the pathway were made during past several years. The core kinase components Hippo, Salvador, Warts, and Mats in the fly and Mst1/2, WW45, Lats1/2, and Mob1 in mammals phosphorylate and inactivate downstream transcriptional co-activators Yorkie in the fly, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in mammals, respectively. Phosphorylated Yorkie, YAP, and TAZ are sequestered in the cytoplasm by interaction with 14-3-3 proteins. Here we review recent progresses of this pathway by focusing on how these proteins communicate with each other and how loss of regulation results in cancers.
Jeshtadi,2010 (20943972) Jeshtadi A, Burgos P, Stubbs CD, Parker AW, King LA, Skinner MA, Botchway SW "Interaction of poxvirus intracellular mature virion proteins with the TPR domain of kinesin light chain in live infected cells revealed by two-photon-induced fluorescence resonance energy transfer fluorescence lifetime imaging microscopy." J Virol 2010 Nov 24
Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 +/- 0.1 ns to 2.1 +/- 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.
Matthess,2010 (20937773) Matthess Y, Raab M, Sanhaji M, Lavrik IN, Strebhardt K "Cdk1/cyclin B1 controls Fas-mediated apoptosis by regulating caspase-8 activity." Mol Cell Biol 2010 Nov 24
Caspase activation is a hallmark of apoptosis. However, the molecular mechanisms underlying the regulation of caspase-8 activation within the extrinsic death pathway are not well understood. In this study, we demonstrate that procaspase-8 is phosphorylated in mitotic cells by Cdk1/cyclin B1 on Ser-387, which is located at the N terminus of the catalytic subunit p10. This phosphorylation of procaspase-8 on Ser-387 occurs in cancer cell lines, as well as in primary breast tissues and lymphocytes. Furthermore, RNA interference-mediated silencing of cyclin B1 or treatment with the Cdk1 inhibitor RO-3306 enhances the Fas-mediated activation and processing of procaspase-8 in mitotic cells. A nonphosphorylatable procaspase-8 (S387A) facilitates Fas-induced apoptosis during mitosis. Our findings suggest that Cdk1/cyclin B1 activity shields human cells against extrinsic death stimuli and unravel the molecular details of the cross talk between cell cycle and extrinsic apoptotic pathways. Finally, this new mechanism may also contribute to tumorigenesis.
Visser,2010 (20935475) Visser S, Yang X "LATS tumor suppressor: a new governor of cellular homeostasis." Cell Cycle 2010 Oct 27
Accumulating evidence points to the LATS (Large Tumor Suppressor) family of human tumor suppressors (LATS1 and LATS2) as new resident governors of cellular homeostasis. Loss of function of either LATS1 or LATS2 leads to a variety of tumor types including soft tissue sarcomas, leukemia, as well as breast, prostate, lung and esophageal cancers. Due to their high degree of homology and functional overlap, LATS1 and LATS2 comprise a new tumor suppressor family. Classically identified within the Hippo-LATS signaling pathway, LATS also acts independently of this pathway, possessing multiple functions including regulation of cell proliferation, cell death and cell migration, as well as broad governing roles such as transcriptional regulation and maintenance of genetic stability. Activity of LATS is tightly controlled through various mechanisms including post-translational modifications, differential localization and expression. Although little is known about the specific underlying mechanisms of these activities, current data suggest that LATS signaling intersects with well-established tumor suppressive or oncogenic pathways including the p53, Ras or Akt networks. This review aims to identify what we know about the LATS tumor suppressor family, highlighting LATS1 and LATS2 redundancies and differences in terms of their structure, expression, regulation and functions, thereby establishing a novel tumor suppressor network.
Yang,2010 (20934435) Yang J, Phiel C "Functions of B56-containing PP2As in major developmental and cancer signaling pathways." Life Sci 2010 Nov 29
Members of the B'/B56/PR61 family regulatory subunits of PP2A determine the subcellular localization, substrate specificity, and catalytic activity of PP2A in a wide range of biological processes. Here, we summarize the structure and intracellular localization of B56-containing PP2As and review functions of B56-containing PP2As in several major developmental/cancer signaling pathways.
Centore,2010 (20932472) Centore RC, Havens CG, Manning AL, Li JM, Flynn RL, Tse A, Jin J, Dyson NJ, Walter JC, Zou L "CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase." Mol Cell 2010 Oct 8
The proper coordination between DNA replication and mitosis during cell-cycle progression is crucial for genomic stability. During G2 and mitosis, Set8 catalyzes monomethylation of histone H4 on lysine 20 (H4K20me1), which promotes chromatin compaction. Set8 levels decline in S phase, but why and how this occurs is unclear. Here, we show that Set8 is targeted for proteolysis in S phase and in response to DNA damage by the E3 ubiquitin ligase, CRL4(Cdt2). Set8 ubiquitylation occurs on chromatin and is coupled to DNA replication via a specific degron in Set8 that binds PCNA. Inactivation of CRL4(Cdt2) leads to Set8 stabilization and aberrant H4K20me1 accumulation in replicating cells. Transient S phase expression of a Set8 mutant lacking the degron promotes premature H4K20me1 accumulation and chromatin compaction, and triggers a checkpoint-mediated G2 arrest. Thus, CRL4(Cdt2)-dependent destruction of Set8 in S phase preserves genome stability by preventing aberrant chromatin compaction during DNA synthesis.
Sheard,2010 (20927106) Sheard LB, Tan X, Mao H, Withers J, Ben-Nissan G, Hinds TR, Kobayashi Y, Hsu FF, Sharon M, Browse J, He SY, Rizo J, Howe GA, Zheng N "Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor." Nature 2010 Nov 18
Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved alpha-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.
Sriram,2010 (20924402) Sriram SM, Kwon YT "The molecular principles of N-end rule recognition." Nat Struct Mol Biol 2010 Oct 06
The N-end rule pathway is a proteolytic system in which recognition components (N-recognins) recognize a set of N-terminal residues as part of degradation signals (N-degrons). Two studies in this issue report the structures of Ubr boxes, a substrate recognition domain of eukaryotic N-recognins. We discuss how eukaryotic and prokaryotic N-recognins use a similar molecular principle to recognize a different set of N-degrons.
Mitchell,2011 (20920535) Mitchell DJ, Butcher NJ, Minchin RF "Phosphorylation/dephosphorylation of human SULT4A1: role of Erk1 and PP2A." Biochim Biophys Acta 2011 Jan
SULT4A1 is a cytosolic sulfotransferase that shares little homology with other human sulfotransferases but is highly conserved between species. It is found in neurons located in several regions of the brain. Recently, the stability of SULT4A1 was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. Since Pin1 binds preferentially to phosphoproteins, these findings suggested that SULT4A1 is post-translationally modified. In this study, we show that the Thr(11) residue of SULT4A1, which is involved in Pin1 binding is phosphorylated. MEK inhibition was shown to abolish Pin1 mediated degradation of SULT4A1 while in vitro phosphorylation assays using alanine substitution mutants of SULT4A1 demonstrated phosphorylation of Thr(11) by ERK1. We also show that dephosphorylation was catalyzed by the protein phosphatase 2A. The PP2A regulatory subunit, Bbeta was identified from a yeast-2-hybrid screen of human brain cDNA as a SULT4A1 interacting protein. This was further confirmed by GST pull-downs and immunoprecipitation. Other members of the B subunit (alphadeltagamma) did not interact with SULT4A1. Taken together, these studies indicate that SULT4A1 stability is regulated by post-translational modification that involves the ERK pathway and PP2A. The phosphorylation of SULT4A1 allows interaction with Pin1, which then promotes degradation of the sulfotransferase.
Zhao,2010 (20890132) Zhao X, Jin S, Song Y, Zhan Q "Cdc2/cyclin B1 regulates centrosomal Nlp proteolysis and subcellular localization." Cancer Biol Ther 2010 Dec 29
The formation of proper mitotic spindles is required for appropriate chromosome segregation during cell division. Aberrant spindle formation often causes aneuploidy and results in tumorigenesis. However, the underlying mechanism of regulating spindle formation and chromosome separation remains to be further defined. Centrosomal Nlp (ninein-like protein) is a recently characterized BRCA1-regulated centrosomal protein and plays an important role in centrosome maturation and spindle formation. In this study, we show that Nlp can be phosphorylated by cell cycle protein kinase Cdc2/cyclin B1. The phosphorylation sites of Nlp are mapped at Ser185 and Ser589. Interestingly, the Cdc2/cyclin B1 phosphorylation site Ser185 of Nlp is required for its recognition by PLK1, which enable Nlp depart from centrosomes to allow the establishment of a mitotic scaffold at the onset of mitosis . PLK1 fails to dissociate the Nlp mutant lacking Ser185 from centrosome, suggesting that Cdc2/cyclin B1 might serve as a primary kinase of PLK1 in regulating Nlp subcellular localization. However, the phosphorylation at the site Ser589 by Cdc2/cyclin B1 plays an important role in Nlp protein stability probably due to its effect on protein degradation. Furthermore, we show that deregulated expression or subcellular localization of Nlp lead to multinuclei in cells, indicating that scheduled levels of Nlp and proper subcellular localization of Nlp are critical for successful completion of normal cell mitosis, These findings demonstrate that Cdc2/cyclin B1 is a key regulator in maintaining appropriate degradation and subcellular localization of Nlp, providing novel insights into understanding on the role of Cdc2/cyclin B1 in mitotic progression.
Bolanos-Garcia,2011 (20888775) Bolanos-Garcia VM, Blundell TL "BUB1 and BUBR1: multifaceted kinases of the cell cycle." Trends Biochem Sci 2011 Mar 14
The multidomain protein kinases BUB1 and BUBR1 (Mad3 in yeast, worms and plants) are central components of the mitotic checkpoint for spindle assembly (SAC). This evolutionarily conserved and essential self-monitoring system of the eukaryotic cell cycle ensures the high fidelity of chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bi-oriented on the mitotic spindle. Despite their amino acid sequence conservation and similar domain organization, BUB1 and BUBR1 perform different functions in the SAC. Recent structural information provides crucial molecular insights into the regulation and recognition of BUB1 and BUBR1, and a solid foundation to dissect the roles of these proteins in the control of chromosome segregation in normal and oncogenic cells.
Oka,2010 (20868367) Oka T, Remue E, Meerschaert K, Vanloo B, Boucherie C, Gfeller D, Bader GD, Sidhu SS, Vandekerckhove J, Gettemans J, Sudol M "Functional complexes between YAP2 and ZO-2 are PDZ domain-dependent, and regulate YAP2 nuclear localization and signalling." Biochem J 2010 Nov 26
The Hippo pathway regulates the size of organs by controlling two opposing processes: proliferation and apoptosis. YAP2 (Yes kinase-associated protein 2), one of the three isoforms of YAP, is a WW domain-containing transcriptional co-activator that acts as the effector of the Hippo pathway in mammalian cells. In addition to WW domains, YAP2 has a PDZ-binding motif at its C-terminus. We reported previously that this motif was necessary for YAP2 localization in the nucleus and for promoting cell detachment and apoptosis. In the present study, we show that the tight junction protein ZO (zonula occludens)-2 uses its first PDZ domain to form a complex with YAP2. The endogenous ZO-2 and YAP2 proteins co-localize in the nucleus. We also found that ZO-2 facilitates the nuclear localization and pro-apoptotic function of YAP2, and that this activity of ZO-2 is PDZ-domain-dependent. The present paper is the first report on a PDZ-based nuclear translocation mechanism. Moreover, since the Hippo pathway acts as a tumour suppressor pathway, the YAP2-ZO-2 complex could represent a target for cancer therapy.
Whisenant,2010 (20865152) Whisenant TC, Ho DT, Benz RW, Rogers JS, Kaake RM, Gordon EA, Huang L, Baldi P, Bardwell L "Computational prediction and experimental verification of new MAP kinase docking sites and substrates including Gli transcription factors." PLoS Comput Biol 2010 Sep 24
In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new 'D-site' class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates.
Heinzelmann,2010 (20861242) Heinzelmann K, Scholz BA, Nowak A, Fossum E, Kremmer E, Haas J, Frank R, Kempkes B "Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 4 (vIRF4/K10) is a novel interaction partner of CSL/CBF1, the major downstream effector of Notch signaling." J Virol 2010 Nov 04
In cells infected with the Kaposi's sarcoma-associated herpesvirus (KSHV), CSL/CBF1 signaling is essential for viral replication and promotes the survival of KSHV-infected cells. CSL/CBF1 is a DNA adaptor molecule which recruits coactivator and corepressor complexes to regulate viral and cellular gene transcription and which is a major downstream effector molecule of activated Notch. The interaction of KSHV RTA and LANA with CSL/CBF1 has been shown to balance the lytic and latent viral life cycle. Here we report that a third KSHV protein, viral interferon regulatory factor 4 (vIRF4/K10), but none of the three other KSHV-encoded vIRFs, interacts with CSL/CBF1. Two regions of vIRF4 with dissimilar affinities contribute to CSL/CBF1 binding. Similar to Notch, vIRF4 targets the hydrophobic pocket in the beta trefoil domain of CSL/CBF1 through a short peptide motif which closely resembles a motif found in Notch but does not strictly follow the PhiWPhiP consensus conserved in human and mouse Notch proteins. Our results suggest that vIRF4 might compete with Notch for CSL/CBF1 binding and signaling.
Matta-Camacho,2010 (20835242) Matta-Camacho E, Kozlov G, Li FF, Gehring K "Structural basis of substrate recognition and specificity in the N-end rule pathway." Nat Struct Mol Biol 2010 Oct 06
The N-end rule links the half-life of a protein to the identity of its N-terminal residue. Destabilizing N-terminal residues are recognized by E3 ubiquitin ligases, termed N-recognins. A conserved structural domain called the UBR box is responsible for their specificity. Here we report the crystal structures of the UBR boxes of the human N-recognins UBR1 and UBR2, alone and in complex with an N-end rule peptide, Arg-Ile-Phe-Ser. These structures show that the UBR box adopts a previously undescribed fold stabilized through the binding of three zinc ions to form a binding pocket for type 1 N-degrons. NMR experiments reveal a preference for N-terminal arginine. Peptide binding is abrogated by N-terminal acetylation of the peptide or loss of the positive charge of the N-terminal residue. These results rationalize and refine the empirical rules for the classification of type 1 N-degrons. We also confirm that a missense mutation in UBR1 that is responsible for Johanson-Blizzard syndrome leads to UBR box unfolding and loss of function.
Choi,2010 (20835240) Choi WS, Jeong BC, Joo YJ, Lee MR, Kim J, Eck MJ, Song HK "Structural basis for the recognition of N-end rule substrates by the UBR box of ubiquitin ligases." Nat Struct Mol Biol 2010 Oct 06
The N-end rule pathway is a regulated proteolytic system that targets proteins containing destabilizing N-terminal residues (N-degrons) for ubiquitination and proteasomal degradation in eukaryotes. The N-degrons of type 1 substrates contain an N-terminal basic residue that is recognized by the UBR box domain of the E3 ubiquitin ligase UBR1. We describe structures of the UBR box of Saccharomyces cerevisiae UBR1 alone and in complex with N-degron peptides, including that of the cohesin subunit Scc1, which is cleaved and targeted for degradation at the metaphase-anaphase transition. The structures reveal a previously unknown protein fold that is stabilized by a novel binuclear zinc center. N-terminal arginine, lysine or histidine side chains of the N-degron are coordinated in a multispecific binding pocket. Unexpectedly, the structures together with our in vitro biochemical and in vivo pulse-chase analyses reveal a previously unknown modulation of binding specificity by the residue at position 2 of the N-degron.
Shibata,2010 (20834162) Shibata H, Inuzuka T, Yoshida H, Sugiura H, Wada I, Maki M "The ALG-2 binding site in Sec31A influences the retention kinetics of Sec31A at the endoplasmic reticulum exit sites as revealed by live-cell time-lapse imaging." Biosci Biotechnol Biochem 2010 Sep 30
ALG-2, a member of the penta-EF-hand protein family, interacts Ca(2)+-dependently with a COPII component, Sec31A. In this study, we first established HeLa cells stably expressing green fluorescent protein-fused ALG-2 (GFP-ALG-2) and red fluorescent protein-fused Sec31A (Sec31A-RFP). After inducing Ca(2)+-mobilization, the cytoplasmic distribution of GFP-ALG-2 changed from a diffuse to a punctate pattern, which extensively overlapped with the Sec31A-RFP-positive structures, indicating that ALG-2 is recruited to the endoplasmic reticulum exit sites (ERES) in living cells. Next, overlay experiments with biotin-labeled ALG-2 were done to dissect the ALG-2 binding site (ABS). They revealed that a sequence comprising amino acid residues 839-851 in the Pro-rich region was necessary and sufficient for direct binding to ALG-2. Finally, fluorescence recovery after photobleaching analysis indicated that the ABS deletion reduced the high-affinity population of Sec31A to the ERES, suggesting that the ABS is one of the key determinants of the retention kinetics of Sec31A at ERES.
Schumacher,2010 (20823509) Schumacher B, Skwarczynska M, Rose R, Ottmann C "Structure of a 14-3-3sigma-YAP phosphopeptide complex at 1.15 A resolution." Acta Crystallogr Sect F Struct Biol Cryst Commun 2010 Sep 08
The 14-3-3 proteins are a class of eukaryotic acidic adapter proteins, with seven isoforms in humans. 14-3-3 proteins mediate their biological function by binding to target proteins and influencing their activity. They are involved in pivotal pathways in the cell such as signal transduction, gene expression, enzyme activation, cell division and apoptosis. The Yes-associated protein (YAP) is a WW-domain protein that exists in two transcript variants of 48 and 54 kDa in humans. By transducing signals from the cytoplasm to the nucleus, YAP is important for transcriptional regulation. In both variants, interaction with 14-3-3 proteins after phosphorylation of Ser127 is important for nucleocytoplasmic trafficking, via which the localization of YAP is controlled. In this study, 14-3-3sigma has been cloned, purified and crystallized in complex with a phosphopeptide from the YAP 14-3-3-binding domain, which led to a crystal that diffracted to 1.15 A resolution. The crystals belonged to space group C222(1), with unit-cell parameters a=82.3, b=112.1, c=62.9 A.
Liu,2010 (20818375) Liu H, Takeda S, Kumar R, Westergard TD, Brown EJ, Pandita TK, Cheng EH, Hsieh JJ "Phosphorylation of MLL by ATR is required for execution of mammalian S-phase checkpoint." Nature 2010 Sep 16
Cell cycle checkpoints are implemented to safeguard the genome, avoiding the accumulation of genetic errors. Checkpoint loss results in genomic instability and contributes to the evolution of cancer. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the role of an intact S-phase checkpoint in maintaining genome integrity. Although the basic framework of the S-phase checkpoint in multicellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome-11 band-q23 translocations disrupting the MLL gene lead to poor prognostic leukaemias. Here we assign MLL as a novel effector in the mammalian S-phase checkpoint network and identify checkpoint dysfunction as an underlying mechanism of MLL leukaemias. MLL is phosphorylated at serine 516 by ATR in response to genotoxic stress in the S phase, which disrupts its interaction with, and hence its degradation by, the SCF(Skp2) E3 ligase, leading to its accumulation. Stabilized MLL protein accumulates on chromatin, methylates histone H3 lysine 4 at late replication origins and inhibits the loading of CDC45 to delay DNA replication. Cells deficient in MLL showed radioresistant DNA synthesis and chromatid-type genomic abnormalities, indicative of S-phase checkpoint dysfunction. Reconstitution of Mll(-/-) (Mll also known as Mll1) mouse embryonic fibroblasts with wild-type but not S516A or DeltaSET mutant MLL rescues the S-phase checkpoint defects. Moreover, murine myeloid progenitor cells carrying an Mll-CBP knock-in allele that mimics human t(11;16) leukaemia show a severe radioresistant DNA synthesis phenotype. MLL fusions function as dominant negative mutants that abrogate the ATR-mediated phosphorylation/stabilization of wild-type MLL on damage to DNA, and thus compromise the S-phase checkpoint. Together, our results identify MLL as a key constituent of the mammalian DNA damage response pathway and show that deregulation of the S-phase checkpoint incurred by MLL translocations probably contributes to the pathogenesis of human MLL leukaemias.
Pawlowski,2010 (20818336) Pawlowski R, Rajakyla EK, Vartiainen MK, Treisman R "An actin-regulated importin alpha/beta-dependent extended bipartite NLS directs nuclear import of MRTF-A." EMBO J 2010 Oct 20
Myocardin-related transcription factors (MRTFs) are actin-regulated transcriptional coactivators, which bind G-actin through their N-terminal RPEL domains. In response to signal-induced actin polymerisation and concomitant G-actin depletion, MRTFs accumulate in the nucleus and activate target gene transcription through their partner protein SRF. Nuclear accumulation of MRTFs in response to signal is inhibited by increased G-actin level. Here, we study the mechanism by which MRTF-A enters the nucleus. We show that MRTF-A contains an unusually long bipartite nuclear localisation signal (NLS), comprising two basic elements separated by 30 residues, embedded within the RPEL domain. Using siRNA-mediated protein depletion in vivo, and nuclear import assays in vitro, we show that the MRTF-A extended bipartite NLS uses the importin (Imp)alpha/beta-dependent import pathway, and that import is inhibited by G-actin. Interaction of the NLS with the Impalpha-Impbeta heterodimer requires both NLS basic elements, and is dependent on the Impalpha major and minor binding pockets. Binding of the Impalpha-Impbeta heterodimer to the intact MRTF-A RPEL domain occurs competitively with G-actin. Thus, MRTF-A contains an actin-sensitive nuclear import signal.
Ma,2010 (20816748) Ma J, Martin JD, Xue Y, Lor LA, Kennedy-Wilson KM, Sinnamon RH, Ho TF, Zhang G, Schwartz B, Tummino PJ, Lai Z "C-terminal region of USP7/HAUSP is critical for deubiquitination activity and contains a second mdm2/p53 binding site." Arch Biochem Biophys 2010 Sep 29
USP7, also known as the hepes simplex virus associated ubiquitin-specific protease (HAUSP), deubiquitinates both mdm2 and p53, and plays an important role in regulating the level and activity of p53. Here, we report that deletion of the TRAF-like domain at the N-terminus of USP7, previously reported to contain the mdm2/p53 binding site, has no effect on USP7 mediated deubiquitination of Ub(n)-mdm2 and Ub(n)-p53. Amino acids 208-1102 were identified to be the minimal length of USP7 that retains proteolytic activity, similar to full length enzyme, towards not only a truncated model substrate Ub-AFC, but also Ub(n)-mdm2, Ub(n)-p53. In contrast, the catalytic domain of USP7 (amino acids 208-560) has 50-700 fold less proteolytic activity towards different substrates. Moreover, inhibition of the catalytic domain of USP7 by Ubal is also different from the full length or TRAF-like domain deleted proteins. Using glutathione pull-down methods, we demonstrate that the C-terminal domain of USP7 contains additional binding sites, a.a. 801-1050 and a.a. 880-1050 for mdm2 and p53, respectively. The additional USP7 binding site on mdm2 is mapped to be the C-terminal RING finger domain (a.a. 425-491). We propose that the C-terminal domain of USP7 is responsible for maintaining the active conformation for catalysis and inhibitor binding, and contains the prime side of the proteolytic active site.
Nair,2010 (20807815) Nair BC, Nair SS, Chakravarty D, Challa R, Manavathi B, Yew PR, Kumar R, Tekmal RR, Vadlamudi RK "Cyclin-dependent kinase-mediated phosphorylation plays a critical role in the oncogenic functions of PELP1." Cancer Res 2010 Sep 16
Estrogen receptor (ER) signaling plays an important role in breast cancer progression, and ER functions are influenced by coregulatory proteins. PELP1 (proline-, glutamic acid-, and leucine-rich protein 1) is a nuclear receptor coregulator that plays an important role in ER signaling. Its expression is deregulated in hormonal cancers. We identified PELP1 as a novel cyclin-dependent kinase (CDK) substrate. Using site-directed mutagenesis and in vitro kinase assays, we identified Ser(477) and Ser(991) of PELP1 as CDK phosphorylation sites. Using the PELP1 Ser(991) phospho-specific antibody, we show that PELP1 is hyperphosphorylated during cell cycle progression. Model cells stably expressing the PELP1 mutant that lack CDK sites had defects in estradiol (E2)-mediated cell cycle progression and significantly affected PELP1-mediated oncogenic functions in vivo. Mechanistic studies showed that PELP1 modulates transcription factor E2F1 transactivation functions, that PELP1 is recruited to pRb/E2F target genes, and that PELP1 facilitates ER signaling cross talk with cell cycle machinery. We conclude that PELP1 is a novel substrate of interphase CDKs and that its phosphorylation is important for the proper function of PELP1 in modulating hormone-driven cell cycle progression and also for optimal E2F transactivation function. Because the expression of both PELP1 and CDKs is deregulated in breast tumors, CDK-PELP1 interactions will have implications in breast cancer progression.
Astuti,2010 (20801879) Astuti P, Boutros R, Ducommun B, Gabrielli B "Mitotic phosphorylation of Cdc25B Ser321 disrupts 14-3-3 binding to the high affinity Ser323 site." J Biol Chem 2010 Nov 01
Cdc25B is a key regulator of entry into mitosis, and its activity and localization are regulated by binding of the 14-3-3 dimer. There are three 14-3-3 binding sites on Cdc25B, with Ser(323) being the highest affinity binding and is highly homologous to the Ser(216) 14-3-3 binding site on Cdc25C. Loss of 14-3-3 binding to Ser(323) increases cyclin/Cdk substrate access to the catalytic site, thereby increasing its activity. It also affects the localization of Cdc25B. Thus, phosphorylation and 14-3-3 binding to this site is essential for down-regulating Cdc25B activity, blocking its mitosis promoting function. The question of how this inhibitory signal is relieved to allow Cdc25B activation and entry into mitosis is yet to be resolved. Here, we show that Ser(323) phosphorylation is maintained into mitosis, but phosphorylation of Ser(321) disrupts 14-3-3 binding to Ser(323), mimicking the effect of inhibiting Ser(323) phosphorylation on both Cdc25B activity and localization. The unphosphorylated Ser(321) appears to have a role in stabilizing 14-3-3 binding to Ser(323), and loss of the Ser hydroxyl group appears to be sufficient to significantly reduce 14-3-3 binding. A consequence of loss of 14-3-3 binding is dephosphorylation of Ser(323). Ser(321) is phosphorylated in mitosis by Cdk1. The mitotic phosphorylation of Ser(321) acts to maintain full activation of Cdc25B by disrupting 14-3-3 binding to Ser(323) and enhancing the dephosphorylation of Ser(323) to block 14-3-3 binding to this site.
Magli,2010 (20801874) Magli A, Angelelli C, Ganassi M, Baruffaldi F, Matafora V, Battini R, Bachi A, Messina G, Rustighi A, Del Sal G, Ferrari S, Molinari S "Proline isomerase Pin1 represses terminal differentiation and myocyte enhancer factor 2C function in skeletal muscle cells." J Biol Chem 2010 Nov 5
Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser(98) and Ser(110), which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.
Matic,2010 (20797634) Matic I, Schimmel J, Hendriks IA, van Santen MA, van de Rijke F, van Dam H, Gnad F, Mann M, Vertegaal AC "Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif." Mol Cell 2010 Aug 27
Reversible protein modification by small ubiquitin-like modifiers (SUMOs) is critical for eukaryotic life. Mass spectrometry-based proteomics has proven effective at identifying hundreds of potential SUMO target proteins. However, direct identification of SUMO acceptor lysines in complex samples by mass spectrometry is still very challenging. We have developed a generic method for the identification of SUMO acceptor lysines in target proteins. We have identified 103 SUMO-2 acceptor lysines in endogenous target proteins. Of these acceptor lysines, 76 are situated in the SUMOylation consensus site [VILMFPC]KxE. Interestingly, eight sites fit the inverted SUMOylation consensus motif [ED]xK[VILFP]. In addition, we found direct mass spectrometric evidence for crosstalk between SUMOylation and phosphorylation with a preferred spacer between the SUMOylated lysine and the phosphorylated serine of four residues. In 16 proteins we identified a hydrophobic cluster SUMOylation motif (HCSM). SUMO conjugation of RanGAP1 and ZBTB1 via HCSMs is remarkably efficient.
Deshmukh,2010 (20739287) Deshmukh L, Gorbatyuk V, Vinogradova O "Integrin {beta}3 phosphorylation dictates its complex with the Shc phosphotyrosine-binding (PTB) domain." J Biol Chem 2010 Nov 5
Adaptor protein Shc plays a key role in mitogen-activated protein kinase (MAPK) signaling pathway, which can be mediated through a number of different receptors including integrins. By specifically recognizing the tyrosine-phosphorylated integrin beta(3), Shc has been shown to trigger integrin outside-in signaling, although the structural basis of this interaction remains nebulous. Here we present the detailed structural analysis of Shc phosphotyrosine-binding (PTB) domain in complex with the bi-phosphorylated beta(3)integrin cytoplasmic tail (CT). We show that this complex is primarily defined by the phosphorylation state of the integrin C-terminal Tyr(759), which fits neatly into the classical PTB pocket of Shc. In addition, we have identified a novel binding interface which concurrently accommodates phosphorylated Tyr(747) of the highly conserved NPXY motif of beta(3). The structure represents the first snapshot of an integrin cytoplasmic tail bound to a target for mediating the outside-in signaling. Detailed comparison with the known Shc PTB structure bound to a target TrkA peptide revealed some significant differences, which shed new light upon the PTB domain specificity.
Caride,2010 (20731332) Caride AJ, Bennett RD, Strehler EE "Kinetic analysis reveals differences in the binding mechanism of calmodulin and calmodulin-like protein to the IQ motifs of myosin-10." Biochemistry 2010 Sep 21
Myo10 is an unconventional myosin with important functions in filopodial motility, cell migration, and cell adhesion. The neck region of Myo10 contains three IQ motifs that bind calmodulin (CaM) or the tissue-restricted calmodulin-like protein (CLP) as light chains. However, little is known about the mechanism of light chain binding to the IQ motifs in Myo10. Binding of CaM and CLP to each IQ motif was assessed by nondenaturing gel electrophoresis and by stopped-flow experiments using fluorescence-labeled CaM and CLP. Although the binding kinetics are different in each case, there are similarities in the mechanism of binding of CaM and CLP to IQ1 and IQ2: for both IQ motifs Ca(2+) increased the binding affinity, mainly by increasing the rate of the forward steps. The general kinetic mechanism comprises a two-step process, which in some cases may involve the binding of a second IQ motif with lower affinity. For IQ3, however, the kinetics of CaM binding is very different from that of CLP. In both cases, binding in the absence of Ca(2+) is poor, and addition of Ca(2+) decreases the K(d) to below 10 nM. However, while the CaM binding kinetics are complex and best fitted by a multistep model, binding of CLP is fitted by a relatively simple two-step model. The results show that, in keeping with growing structural evidence, complexes between CaM or CaM-like myosin light chains and IQ motifs are highly diverse and depend on the specific sequence of the particular IQ motif as well as the light chain.
Odho,2010 (20716525) Odho Z, Southall SM, Wilson JR "Characterization of a novel WDR5-binding site that recruits RbBP5 through a conserved motif to enhance methylation of histone H3 lysine 4 by mixed lineage leukemia protein-1." J Biol Chem 2010 Oct 18
Histone modification is well established as a fundamental mechanism driving the regulation of transcription, replication, and DNA repair through the control of chromatin structure. Likewise, it is apparent that incorrect targeting of histone modifications contributes to misregulated gene expression and hence to developmental disorders and diseases of genomic instability such as cancer. The KMT2 family of SET domain methyltransferases, typified by mixed lineage leukemia protein-1 (MLL1), is responsible for histone H3 lysine 4 methylation, a marker of active genes. To ensure that this modification is correctly targeted, a multiprotein complex associates with the methyltransferase and directs activity. We have identified a novel interaction site on the core complex protein WD repeat protein-5 (WDR5), and we mapped the complementary site on its partner retinoblastoma-binding protein-5 (RbBP5). We have characterized this interaction by x-ray crystallography and show how it is fundamental to the assembly of the complex and to the regulation of methyltransferase activity. We show which region of RbBP5 contributes directly to mixed lineage leukemia activation, and we combine our structural and biochemical data to produce a model to show how WDR5 and RbBP5 act cooperatively to stimulate activity.
Sarkari,2010 (20713061) Sarkari F, La Delfa A, Arrowsmith CH, Frappier L, Sheng Y, Saridakis V "Further insight into substrate recognition by USP7: structural and biochemical analysis of the HdmX and Hdm2 interactions with USP7." J Mol Biol 2010 Oct 04
Ubiquitin-specific protease 7 (USP7) catalyzes the deubiquitination of several substrate proteins including p53 and Hdm2. We have previously shown that USP7, and more specifically its amino-terminal domain (USP7-NTD), interacts with distinct regions on p53 and Hdm2 containing P/AxxS motifs. The ability of USP7 to also deubiquitinate and control the turnover of HdmX was recently demonstrated. We utilized a combination of biochemistry and structural biology to identify which domain of USP7 interacts with HdmX as well as to identify regions of HdmX that interact with USP7. We showed that USP7-NTD recognized two of six P/AxxS motifs of HdmX ((8)AQCS(11) and (398)AHSS(401)). The crystal structure of the USP7-NTD:HdmX(AHSS) complex was determined providing the molecular basis of complex formation between USP7-NTD and the HdmX(AHSS) peptide. The HdmX peptide interacted within the same residues of USP7-NTD as previously demonstrated with p53, Hdm2, and EBNA1 peptides. We also identified an additional site on Hdm2 ((397)PSTS(400)) that interacts with USP7-NTD and determined the crystal structure of this complex. Finally, analysis of USP7-interacting peptides on filter arrays confirmed the importance of the serine residue at the fourth position for the USP7-NTD interaction and showed that phosphorylation of serines within the binding sequence prevents this interaction. These results lead to a better understanding of the mechanism of substrate recognition by USP7-NTD.
Cukier,2010 (20711187) Cukier CD, Hollingworth D, Martin SR, Kelly G, Diaz-Moreno I, Ramos A "Molecular basis of FIR-mediated c-myc transcriptional control." Nat Struct Mol Biol 2010 Sep 03
The far upstream element (FUSE) regulatory system promotes a peak in the concentration of c-Myc during cell cycle. First, the FBP transcriptional activator binds to the FUSE DNA element upstream of the c-myc promoter. Then, FBP recruits its specific repressor (FIR), which acts as an on/off transcriptional switch. Here we describe the molecular basis of FIR recruitment, showing that the tandem RNA recognition motifs of FIR provide a platform for independent FUSE DNA and FBP protein binding and explaining the structural basis of the reversibility of the FBP-FIR interaction. We also show that the physical coupling between FBP and FIR is modulated by a flexible linker positioned sequentially to the recruiting element. Our data explain how the FUSE system precisely regulates c-myc transcription and suggest that a small change in FBP-FIR affinity leads to a substantial effect on c-Myc concentration.
Hamill,2010 (20696927) Hamill S, Wolin SL, Reinisch KM "Structure and function of the polymerase core of TRAMP, a RNA surveillance complex." Proc Natl Acad Sci U S A 2010 Aug 25
The Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex recognizes aberrant RNAs in Saccharomyces cerevisiae and targets them for degradation. A TRAMP subcomplex consisting of a noncanonical poly(A) RNA polymerase in the Pol ss superfamily of nucleotidyl transferases, Trf4p, and a zinc knuckle protein, Air2p, mediates initial substrate recognition. Trf4p and related eukaryotic poly(A) and poly(U) polymerases differ from other characterized enzymes in the Pol ss superfamily both in sequence and in the lack of recognizable nucleic acid binding motifs. Here we report, at 2.7-A resolution, the structure of Trf4p in complex with a fragment of Air2p comprising two zinc knuckle motifs. Trf4p consists of a catalytic and central domain similar in fold to those of other noncanonical Pol beta RNA polymerases, and the two zinc knuckle motifs of Air2p interact with the Trf4p central domain. The interaction surface on Trf4p is highly conserved across eukaryotes, providing evidence that the Trf4p/Air2p complex is conserved in higher eukaryotes as well as in yeast and that the TRAMP complex may also function in RNA surveillance in higher eukaryotes. We show that Air2p, and in particular sequences encompassing a zinc knuckle motif near its N terminus, modulate Trf4p activity, and we present data supporting a role for this zinc knuckle in RNA binding. Finally, we show that the RNA 3' end plays a role in substrate recognition.
Hirschi,2010 (20694007) Hirschi A, Cecchini M, Steinhardt RC, Schamber MR, Dick FA, Rubin SM "An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein." Nat Struct Mol Biol 2010 Sep 03
The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking.
Inuzuka,2010 (20691033) Inuzuka T, Suzuki H, Kawasaki M, Shibata H, Wakatsuki S, Maki M "Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2DeltaGF122) and structural roles of F122 in target recognition." BMC Struct Biol 2010 Aug 25
BACKGROUND: ALG-2 (a gene product of PDCD6) belongs to the penta-EF-hand (PEF) protein family and Ca2+-dependently interacts with various intracellular proteins including mammalian Alix, an adaptor protein in the ESCRT system. Our previous X-ray crystal structural analyses revealed that binding of Ca2+ to EF3 enables the side chain of R125 to move enough to make a primary hydrophobic pocket (Pocket 1) accessible to a short fragment of Alix. The side chain of F122, facing a secondary hydrophobic pocket (Pocket 2), interacts with the Alix peptide. An alternatively spliced shorter isoform, designated ALG-2DeltaGF122, lacks Gly121Phe122 and does not bind Alix, but the structural basis of the incompetence has remained to be elucidated. RESULTS: We solved the X-ray crystal structure of the PEF domain of ALG-2DeltaGF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened alpha-helix 5 (alpha5) and changed the configuration of the R125 side chain so that it partially blocked Pocket 1. A wall created by the main chain of 121-GFG-123 and facing the two pockets was destroyed. Surprisingly, however, substitution of F122 with Ala or Gly, but not with Trp, increased the Alix-binding capacity in binding assays. The F122 substitutions exhibited different effects on binding of ALG-2 to other known interacting proteins, including TSG101 (Tumor susceptibility gene 101) and annexin A11. The X-ray crystal structure of the F122A mutant revealed that removal of the bulky F122 side chain not only created an additional open space in Pocket 2 but also abolished inter-helix interactions with W95 and V98 (present in alpha4) and that alpha5 inclined away from alpha4 to expand Pocket 2, suggesting acquirement of more appropriate positioning of the interacting residues to accept Alix. CONCLUSIONS: We found that the inability of the two-residue shorter ALG-2 isoform to bind Alix is not due to the absence of bulky side chain of F122 but due to deformation of a main-chain wall facing pockets 1 and 2. Moreover, a residue at the position of F122 contributes to target specificity and a smaller side chain is preferable for Alix binding but not favored to bind annexin A11.
Saxena,2010 (20682773) Saxena UH, Owens L, Graham JR, Cooper GM, Hansen U "Prolyl isomerase Pin1 regulates transcription factor LSF (TFCP2) by facilitating dephosphorylation at two serine-proline motifs." J Biol Chem 2010 Oct 8
Transcription factor LSF is essential for cell cycle progression, being required for activating expression of the thymidylate synthase (Tyms) gene at the G1/S transition. We previously established that phosphorylation of LSF in early G1 at Ser-291 and Ser-309 inhibits its transcriptional activity and that dephosphorylation later in G1 is required for its reactivation. Here we reveal the role of prolyl cis-trans isomerase Pin1 in activating LSF, by facilitating dephosphorylation at both Ser-291 and Ser-309. We demonstrate that Pin1 binds LSF both in vitro and in vivo. Using coimmunoprecipitation assays, we identify three SP/TP motifs in LSF (at residues Ser-291, Ser-309, and Thr-329) that are required and sufficient for association with Pin1. Co-expression of Pin1 enhances LSF transactivation potential in reporter assays. The Pin1-dependent enhancement of LSF activity requires residue Thr-329 in LSF, requires both the WW and PPiase domains of Pin1, and correlates with hypophosphorylation of LSF at Ser-291 and Ser-309. These findings support a model in which the binding of Pin1 at the Thr-329-Pro-330 motif in LSF permits isomerization by Pin1 of the peptide bonds at the nearby phosphorylated SP motifs (Ser-291 and Ser-309) to the trans configuration, thereby facilitating their dephosphorylation.
Molzan,2010 (20679480) Molzan M, Schumacher B, Ottmann C, Baljuls A, Polzien L, Weyand M, Thiel P, Rose R, Rose M, Kuhenne P, Kaiser M, Rapp UR, Kuhlmann J "Impaired binding of 14-3-3 to C-RAF in Noonan syndrome suggests new approaches in diseases with increased Ras signaling." Mol Cell Biol 2010 Sep 10
The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser(259), a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS(259) crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer(259)/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway.
Nakatsu,2010 (20675384) Nakatsu Y, Sakoda H, Kushiyama A, Ono H, Fujishiro M, Horike N, Yoneda M, Ohno H, Tsuchiya Y, Kamata H, Tahara H, Isobe T, Nishimura F, Katagiri H, Oka Y, Fukushima T, Takahashi S, Kurihara H, Uchida T, Asano T "Pin1 associates with and induces translocation of CRTC2 to the cytosol, thereby suppressing cAMP-responsive element transcriptional activity." J Biol Chem 2010 Oct 22
Pin1 is a unique regulator, which catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified CRTC2 as a Pin1-binding protein by overexpressing Pin1 with Myc and FLAG tags in mouse livers and subsequent purification of the complex containing Pin1. The association between Pin1 and CRTC2 was observed not only in overexpression experiments but also endogenously in the mouse liver. Interestingly, Ser(136) in the nuclear localization signal of CRTC2 was shown to be involved in the association with Pin1. Pin1 overexpression in HepG2 cells attenuated forskolin-induced nuclear localization of CRTC2 and cAMP-responsive element (CRE) transcriptional activity, whereas gene knockdown of Pin1 by siRNA enhanced both. Pin1 also associated with CRTC1, leading to their cytosol localization, essentially similar to the action of CRTC2. Furthermore, it was shown that CRTC2 associated with Pin1 did not bind to CREB. Taken together, these observations indicate the association of Pin1 with CRTC2 to decrease the nuclear CBP.CRTC.CREB complex. Indeed, adenoviral gene transfer of Pin1 into diabetic mice improved hyperglycemia in conjunction with normalizing phosphoenolpyruvate carboxykinase mRNA expression levels, which is regulated by CRE transcriptional activity. In conclusion, Pin1 regulates CRE transcriptional activity, by associating with CRTC1 or CRTC2.
Rellos,2010 (20668654) Rellos P, Pike AC, Niesen FH, Salah E, Lee WH, von Delft F, Knapp S "Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation." PLoS Biol 2010 Jul 29
Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.
Mitchell,2010 (20667914) Mitchell NC, Johanson TM, Cranna NJ, Er AL, Richardson HE, Hannan RD, Quinn LM "Hfp inhibits Drosophila myc transcription and cell growth in a TFIIH/Hay-dependent manner." Development 2010 Aug 11
An unresolved question regarding the RNA-recognition motif (RRM) protein Half pint (Hfp) has been whether its tumour suppressor behaviour occurs by a transcriptional mechanism or via effects on splicing. The data presented here demonstrate that Hfp achieves cell cycle inhibition via an essential role in the repression of Drosophila myc (dmyc) transcription. We demonstrate that regulation of dmyc requires interaction between the transcriptional repressor Hfp and the DNA helicase subunit of TFIIH, Haywire (Hay). In vivo studies show that Hfp binds to the dmyc promoter and that repression of dmyc transcription requires Hfp. In addition, loss of Hfp results in enhanced cell growth, which depends on the presence of dMyc. This is consistent with Hfp being essential for inhibition of dmyc transcription and cell growth. Further support for Hfp controlling dmyc transcriptionally comes from the demonstration that Hfp physically and genetically interacts with the XPB helicase component of the TFIIH transcription factor complex, Hay, which is required for normal levels of dmyc expression, cell growth and cell cycle progression. Together, these data demonstrate that Hfp is crucial for repression of dmyc, suggesting that a transcriptional, rather than splicing, mechanism underlies the regulation of dMyc and the tumour suppressor behaviour of Hfp.
Sharma,2010 (20634894) Sharma P, Ignatchenko V, Grace K, Ursprung C, Kislinger T, Gramolini AO "Endoplasmic reticulum protein targeting of phospholamban: a common role for an N-terminal di-arginine motif in ER retention?" PLoS One 2010 Jul 16
BACKGROUND: Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase, which transports Ca(2+) into the SR lumen, leading to muscle relaxation. A mutation of PLN in which one of the di-arginine residues at positions 13 and 14 was deleted led to a severe, early onset dilated cardiomyopathy. Here we were interested in determining the cellular mechanisms involved in this disease-causing mutation. METHODOLOGY/PRINCIPAL FINDING: Mutations deleting codons for either or both Arg13 or Arg14 resulted in the mislocalization of PLN from the ER. Our data show that PLN is recycled via the retrograde Golgi to ER membrane traffic pathway involving COP-I vesicles, since co-immunoprecipitation assays determined that COP I interactions are dependent on an intact di-arginine motif as PLN RDelta14 did not co-precipitate with COP I containing vesicles. Bioinformatic analysis determined that the di-arginine motif is present in the first 25 residues in a large number of all ER/SR Gene Ontology (GO) annotated proteins. Mutations in the di-arginine motif of the Sigma 1-type opioid receptor, the beta-subunit of the signal recognition particle receptor, and Sterol-O-acyltransferase, three proteins identified in our bioinformatic screen also caused mislocalization of these known ER-resident proteins. CONCLUSION: We conclude that PLN is enriched in the ER due to COP I-mediated transport that is dependent on its intact di-arginine motif and that the N-terminal di-arginine motif may act as a general ER retrieval sequence.
Graciet,2010 (20627801) Graciet E, Wellmer F "The plant N-end rule pathway: structure and functions." Trends Plant Sci 2010 Aug 09
The N-end rule pathway is a protein degradation pathway that relates the stability of a protein to the nature of its N-terminal amino acid residue. This pathway is part of the ubiquitin-proteasome system in eukaryotes and has been shown to be involved in a multitude of cellular and developmental processes in animals and fungi. However, in plants, its structure and functions have long been enigmatic. In this review, we discuss recent advances in the identification of the enzymatic components that mediate protein degradation through the N-end rule pathway in plants. We further describe the known functions of this pathway in the control of plant growth and development and outline open questions that will likely be the focus of future research.
Mehlitz,2010 (20624904) Mehlitz A, Banhart S, Maurer AP, Kaushansky A, Gordus AG, Zielecki J, Macbeath G, Meyer TF "Tarp regulates early Chlamydia-induced host cell survival through interactions with the human adaptor protein SHC1." J Cell Biol 2010 Jul 13
Many bacterial pathogens translocate effector proteins into host cells to manipulate host cell functions. Here, we used a protein microarray comprising virtually all human SRC homology 2 (SH2) and phosphotyrosine binding domains to comprehensively and quantitatively assess interactions between host cell proteins and the early phase Chlamydia trachomatis effector protein translocated actin-recruiting phosphoprotein (Tarp), which is rapidly tyrosine phosphorylated upon host cell entry. We discovered numerous novel interactions between human SH2 domains and phosphopeptides derived from Tarp. The adaptor protein SHC1 was among Tarp's strongest interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to tumor necrosis factor-induced apoptosis. Collectively, our findings reveal a critical role for SHC1 in early C. trachomatis-induced cell survival and suggest that Tarp functions as a multivalent phosphorylation-dependent signaling hub that is important during the early phase of chlamydial infection.
Moretto-Zita,2010 (20622153) Moretto-Zita M, Jin H, Shen Z, Zhao T, Briggs SP, Xu Y "Phosphorylation stabilizes Nanog by promoting its interaction with Pin1." Proc Natl Acad Sci U S A 2010 Jul 27
Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body, thus holding great promise as a renewable source of cells for human therapy. The mechanisms that maintain self-renewal of ESCs remain unclear. Here we show that Nanog, a transcription factor crucial for the self-renewal of ESCs, is phosphorylated at multiple Ser/Thr-Pro motifs. This phosphorylation promotes the interaction between Nanog and the prolyl isomerase Pin1, leading to Nanog stabilization by suppressing its ubiquitination. Inhibition of Pin1 activity or disruption of Pin1-Nanog interaction in ESCs suppresses their capability to self-renew and to form teratomas in immunodeficient mice. Therefore, in addition to the stringent transcriptional regulation of Nanog, the expression level of Nanog is also modulated by posttranslational mechanisms.
Yamaguchi,2010 (20615880) Yamaguchi M, Noda NN, Nakatogawa H, Kumeta H, Ohsumi Y, Inagaki F "Autophagy-related protein 8 (Atg8) family interacting motif in Atg3 mediates the Atg3-Atg8 interaction and is crucial for the cytoplasm-to-vacuole targeting pathway." J Biol Chem 2010 Sep 13
The autophagy-related protein 8 (Atg8) conjugation system is essential for the formation of double-membrane vesicles called autophagosomes during autophagy, a bulk degradation process conserved among most eukaryotes. It is also important in yeast for recognizing target vacuolar enzymes through the receptor protein Atg19 during the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective type of autophagy. Atg3 is an E2-like enzyme that conjugates Atg8 with phosphatidylethanolamine. Here, we show that Atg3 directly interacts with Atg8 through the WEDL sequence, which is distinct from canonical interaction between E2 and ubiquitin-like modifiers. Moreover, NMR experiments suggest that the mode of interaction between Atg8 and Atg3 is quite similar to that between Atg8/LC3 and the Atg8 family interacting motif (AIM) conserved in autophagic receptors, such as Atg19 and p62. Thus, the WEDL sequence in Atg3 is a canonical AIM. In vitro analyses showed that Atg3 AIM is crucial for the transfer of Atg8 from the Atg8 approximately Atg3 thioester intermediate to phosphatidylethanolamine but not for the formation of the intermediate. Intriguingly, in vivo experiments showed that it is necessary for the Cvt pathway but not for starvation-induced autophagy. Atg3 AIM attenuated the inhibitory effect of Atg19 on Atg8 lipidation in vitro, suggesting that Atg3 AIM may be important for the lipidation of Atg19-bound Atg8 during the Cvt pathway.
Kim,2010 (20606006) Kim DH, Budhavarapu VN, Herrera CR, Nam HW, Kim YS, Yew PR "The CRL4Cdt2 ubiquitin ligase mediates the proteolysis of cyclin-dependent kinase inhibitor Xic1 through a direct association with PCNA." Mol Cell Biol 2010 Sep
During DNA polymerase switching, the Xenopus laevis Cip/Kip-type cyclin-dependent kinase inhibitor Xic1 associates with trimeric proliferating cell nuclear antigen (PCNA) and is recruited to chromatin, where it is ubiquitinated and degraded. In this study, we show that the predominant E3 for Xic1 in the egg is the Cul4-DDB1-XCdt2 (Xenopus Cdt2) (CRL4(Cdt2)) ubiquitin ligase. The addition of full-length XCdt2 to the Xenopus extract promotes Xic1 turnover, while the N-terminal domain of XCdt2 (residues 1 to 400) cannot promote Xic1 turnover, despite its ability to bind both Xic1 and DDB1. Further analysis demonstrated that XCdt2 binds directly to PCNA through its C-terminal domain (residues 401 to 710), indicating that this interaction is important for promoting Xic1 turnover. We also identify the cis-acting sequences required for Xic1 binding to Cdt2. Xic1 binds to Cdt2 through two domains (residues 161 to 170 and 179 to 190) directly flanking the Xic1 PCNA binding domain (PIP box) but does not require PIP box sequences (residues 171 to 178). Similarly, human p21 binds to human Cdt2 through residues 156 to 161, adjacent to the p21 PIP box. In addition, we identify five lysine residues (K180, K182, K183, K188, and K193) immediately downstream of the Xic1 PIP box and within the second Cdt2 binding domain as critical sites for Xic1 ubiquitination. Our studies suggest a model in which both the CRL4(Cdt2) E3- and PIP box-containing substrates, like Xic1, are recruited to chromatin through independent direct associations with PCNA.
Kuang,2010 (20603330) Kuang Z, Lewis RS, Curtis JM, Zhan Y, Saunders BM, Babon JJ, Kolesnik TB, Low A, Masters SL, Willson TA, Kedzierski L, Yao S, Handman E, Norton RS, Nicholson SE "The SPRY domain-containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation." J Cell Biol 2010 Jul 13
Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain-containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB-iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections.
Bergamin,2010 (20603078) Bergamin E, Hallock PT, Burden SJ, Hubbard SR "The cytoplasmic adaptor protein Dok7 activates the receptor tyrosine kinase MuSK via dimerization." Mol Cell 2010 Jul 9
Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.
Park,2010 (20599701) Park SY, Kim SY, Kang KB, Kim IS "Adaptor protein GULP is involved in stabilin-1-mediated phagocytosis." Biochem Biophys Res Commun 2010 Jul 30
The clearance of apoptotic cells is critical during cellular homeostasis as well as inflammation resolution. Recently, we found that stabilin-1 is a phagocytic receptor that is involved in the clearance of apoptotic cells. However, the downstream signaling pathway of stabilin-1-mediated phagocytosis remains to be investigated. Here we identify that GULP is able to specifically interact with the NPxF/Y motif of stabilin-1 cytoplasmic region. The PTB domain of GULP is necessary for interaction with stabilin-1. GULP is enriched around PS-coated beads for phagocytosis and co-localized with stabilin-1. Downregulation of endogenous GULP expression decreased stabilin-1-mediated phagocytosis. Thus, these results indicate that GULP functions as an adaptor protein for stabilin-1-mediated phagocytosis.
Ruan,2010 (20595394) Ruan L, Osawa M, Hosoda N, Imai S, Machiyama A, Katada T, Hoshino S, Shimada I "Quantitative characterization of Tob interactions provides the thermodynamic basis for translation termination-coupled deadenylase regulation." J Biol Chem 2010 Sep 3
Translation termination-coupled deadenylation is the first and often the rate-limiting step of eukaryotic mRNA decay in which two deadenylases, Ccr4-Caf1 and Pan2, play key roles. One of the deadenylases, Caf1, associates with Tob, which recruits Caf1 to the poly(A) tail through interactions with a cytoplasmic poly(A)-binding protein 1 (PABPC1). We previously proposed that the competition between Tob and eRF3 (a translation termination factor that interacts with PABPC1) is responsible for the regulation of deadenylase activity. However, the molecular mechanism of the regulation should be addressed by investigating the binding affinity and the cellular levels of these proteins. In this work, we characterized the human Tob interactions with Caf1 and a C-terminal domain of PABPC1 (PABC). Nuclear magnetic resonance (NMR) and Western blot analyses revealed that Tob consists of a structured N-terminal BTG-Tob domain and an unstructured C-terminal region with two conserved PAM2 (PABPC1-interacting motif 2) motifs. The BTG-TOB domain associates with Caf1, whereas the C-terminal PAM2 motif binds to PABC, with a K(d) value of 20 microM. Furthermore, we demonstrated that the levels of eRF3 and Tob in HeLa cells are 4-5 microM and less than 0.2 microM, respectively. On the basis of these results, we propose a thermodynamic mechanism for the translation termination-coupled deadenylation mediated by the Tob-Caf1 complex.
Du,2010 (20584900) Du JX, McConnell BB, Yang VW "A small ubiquitin-related modifier-interacting motif functions as the transcriptional activation domain of Kruppel-like factor 4." J Biol Chem 2010 Aug 30
The zinc finger transcription factor, Kruppel-like factor 4 (KLF4), regulates numerous biological processes, including proliferation, differentiation, and embryonic stem cell self-renewal. Although the DNA sequence to which KLF4 binds is established, the mechanism by which KLF4 controls transcription is not well defined. Small ubiquitin-related modifier (SUMO) is an important regulator of transcription. Here we show that KLF4 is both SUMOylated at a single lysine residue and physically interacts with SUMO-1 in a region that matches an acidic and hydrophobic residue-rich SUMO-interacting motif (SIM) consensus. The SIM in KLF4 is required for transactivation of target promoters in a SUMO-1-dependent manner. Mutation of either the acidic or hydrophobic residues in the SIM significantly impairs the ability of KLF4 to interact with SUMO-1, activate transcription, and inhibit cell proliferation. Our study provides direct evidence that SIM in KLF4 functions as a transcriptional activation domain. A survey of transcription factor sequences reveals that established transactivation domains of many transcription factors contain sequences highly related to SIM. These results, therefore, illustrate a novel mechanism by which SUMO interaction modulates the activity of transcription factors.
Phelan,2010 (20581824) Phelan CA, Gampe RT Jr, Lambert MH, Parks DJ, Montana V, Bynum J, Broderick TM, Hu X, Williams SP, Nolte RT, Lazar MA "Structure of Rev-erbalpha bound to N-CoR reveals a unique mechanism of nuclear receptor-co-repressor interaction." Nat Struct Mol Biol 2010 Jul 06
Repression of gene transcription by the nuclear receptor Rev-erbalpha plays an integral role in the core molecular circadian clock. We report the crystal structure of a nuclear receptor-co-repressor (N-CoR) interaction domain 1 (ID1) peptide bound to truncated human Rev-erbalpha ligand-binding domain (LBD). The ID1 peptide forms an unprecedented antiparallel beta-sheet with Rev-erbalpha, as well as an alpha-helix similar to that seen in nuclear receptor ID2 crystal structures but out of register by four residues. Comparison with the structure of Rev-erbbeta bound to heme indicates that ID1 peptide and heme induce substantially different conformational changes in the LBD. Although heme is involved in Rev-erb repression, the structure suggests that Rev-erbalpha could also mediate repression via ID1 binding in the absence of heme. The previously uncharacterized secondary structure induced by ID1 peptide binding advances our understanding of nuclear receptor-co-repressor interactions.
Dejosez,2010 (20581084) Dejosez M, Levine SS, Frampton GM, Whyte WA, Stratton SA, Barton MC, Gunaratne PH, Young RA, Zwaka TP "Ronin/Hcf-1 binds to a hyperconserved enhancer element and regulates genes involved in the growth of embryonic stem cells." Genes Dev 2010 Jul 15
Self-renewing embryonic stem (ES) cells have an exceptional need for timely biomass production, yet the transcriptional control mechanisms responsible for meeting this requirement are largely unknown. We report here that Ronin (Thap11), which is essential for the self-renewal of ES cells, binds with its transcriptional coregulator, Hcf-1, to a highly conserved enhancer element that previously lacked a recognized binding factor. The subset of genes bound by Ronin/Hcf-1 function primarily in transcription initiation, mRNA splicing, and cell metabolism; genes involved in cell signaling and cell development are conspicuously underrepresented in this target gene repertoire. Although Ronin/Hcf-1 represses the expression of some target genes, its activity at promoter sites more often leads to the up-regulation of genes essential to protein biosynthesis and energy production. We propose that Ronin/Hcf-1 controls a genetic program that contributes to the unimpeded growth of ES cells.
Schaffler,2010 (20573744) Schaffler K, Schulz K, Hirmer A, Wiesner J, Grimm M, Sickmann A, Fischer U "A stimulatory role for the La-related protein 4B in translation." RNA 2010 Aug
La-related proteins (LARPs) belong to an evolutionarily conserved family of factors with predicted roles in RNA metabolism. Here, we have analyzed the cellular interactions and function of LARP4B, a thus far uncharacterized member of the LARP family. We show that LARP4B is a cytosolic protein that accumulates upon arsenite treatment in cellular stress granules. Biochemical experiments further uncovered an interaction of LARP4B with the cytosolic poly(A) binding protein 1 (PABPC1) and the receptor for activated C Kinase (RACK1), a component of the 40S ribosomal subunit. Under physiological conditions, LARP4B co-sedimented with polysomes in cellular extracts, suggesting a role in translation. In agreement with this notion, overexpression of LARP4B stimulated protein synthesis, whereas knockdown of the factor by RNA interference impaired translation of a large number of cellular mRNAs. In sum, we identified LARP4B as a stimulatory factor of translation. We speculate that LARP4B exerts its function by bridging mRNA factors of the 3' end with initiating ribosomes.
Weir,2010 (20566885) Weir JR, Bonneau F, Hentschel J, Conti E "Structural analysis reveals the characteristic features of Mtr4, a DExH helicase involved in nuclear RNA processing and surveillance." Proc Natl Acad Sci U S A 2010 Jul 09
Mtr4 is a conserved RNA helicase that functions together with the nuclear exosome. It participates in the processing of structured RNAs, including the maturation of 5.8S ribosomal RNA (rRNA). It also interacts with the polyadenylating Trf4-Air2 heterodimer to form the so-called TRAMP (Trf4-Air2-Mtr4 Polyadenylation) complex. TRAMP is involved in exosome-mediated degradation of aberrant RNAs in nuclear surveillance pathways. We report the 2.9-A resolution crystal structure of Saccharomyces cerevisiae Mtr4 in complex with ADP and RNA. The structure shows a central ATPase core similar to that of other DExH helicases. Inserted in the DExH core is a region characteristic of Mtr4 orthologues that folds into an elongated stalk connected to a beta-barrel domain. This domain shows unexpected similarity to the KOW domain of L24, a ribosomal protein that binds 23S rRNA. We find that indeed the KOW domain of Mtr4 is able to bind in vitro transcribed tRNA(iMet), suggesting it might assist in presenting RNA substrates to the helicase core. The interaction of Mtr4 with Trf4-Air2 is mediated not by the stalk/KOW insertion but by the DExH core. We find that in the context of the TRAMP complex, the DExH core functions independently in vitro as an RNA helicase and a protein-binding platform. Mtr4 has thus evolved specific structural and surface features to perform its multiple functions.
Filippakopoulos,2010 (20561531) Filippakopoulos P, Low A, Sharpe TD, Uppenberg J, Yao S, Kuang Z, Savitsky P, Lewis RS, Nicholson SE, Norton RS, Bullock AN "Structural basis for Par-4 recognition by the SPRY domain- and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4." J Mol Biol 2010 Aug 03
The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4((59-77)) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.
Seitsonen,2010 (20554778) Seitsonen J, Susi P, Heikkila O, Sinkovits RS, Laurinmaki P, Hyypia T, Butcher SJ "Interaction of alphaVbeta3 and alphaVbeta6 integrins with human parechovirus 1." J Virol 2010 Sep
Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to alpha(V) integrins. The interaction of HPEV1 with alpha(V) integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins alpha(V)beta(3) and alpha(V)beta(6) to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, alpha(V)beta(6) bound more efficiently than alpha(V)beta(3) to immobilized HPEV1. Moreover, soluble alpha(V)beta(6), but not alpha(V)beta(3), blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.
Schmidt,2010 (20554521) Schmidt F, Treiber N, Zocher G, Bjelic S, Steinmetz MO, Kalbacher H, Stehle T, Dodt G "Insights into peroxisome function from the structure of PEX3 in complex with a soluble fragment of PEX19." J Biol Chem 2010 Aug 09
The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.
Ducka,2010 (20538977) Ducka AM, Joel P, Popowicz GM, Trybus KM, Schleicher M, Noegel AA, Huber R, Holak TA, Sitar T "Structures of actin-bound Wiskott-Aldrich syndrome protein homology 2 (WH2) domains of Spire and the implication for filament nucleation." Proc Natl Acad Sci U S A 2010 Jun 29
Three classes of proteins are known to nucleate new filaments: the Arp2/3 complex, formins, and the third group of proteins that contain ca. 25 amino acid long actin-binding Wiskott-Aldrich syndrome protein homology 2 domains, called the WH2 repeats. Crystal structures of the complexes between the actin-binding WH2 repeats of the Spire protein and actin were determined for the Spire single WH2 domain D, the double (SpirCD), triple (SpirBCD), quadruple (SpirABCD) domains, and an artificial Spire WH2 construct comprising three identical D repeats (SpirDDD). SpirCD represents the minimal functional core of Spire that can nucleate actin filaments. Packing in the crystals of the actin complexes with SpirCD, SpirBCD, SpirABCD, and SpirDDD shows the presence of two types of assemblies, "side-to-side" and "straight-longitudinal," which can serve as actin filament nuclei. The principal feature of these structures is their loose, open conformations, in which the sides of actins that normally constitute the inner interface core of a filament are flipped inside out. These Spire structures are distant from those seen in the filamentous nuclei of Arp2/3, formins, and in the F-actin filament.
Padrick,2010 (20533885) Padrick SB, Rosen MK "Physical mechanisms of signal integration by WASP family proteins." Annu Rev Biochem 2010 Jun 10
The proteins of the Wiskott-Aldrich syndrome protein (WASP) family are activators of the ubiquitous actin nucleation factor, the Arp2/3 complex. WASP family proteins contain a C-terminal VCA domain that binds and activates the Arp2/3 complex in response to numerous inputs, including Rho family GTPases, phosphoinositide lipids, SH3 domain-containing proteins, kinases, and phosphatases. In the archetypal members of the family, WASP and N-WASP, these signals are integrated through two levels of regulation, an allosteric autoinhibitory interaction, in which the VCA is sequestered from the Arp2/3 complex, and dimerization/oligomerization, in which multi-VCA complexes are better activators of the Arp2/3 complex than monomers. Here, we review the structural, biochemical, and biophysical details of these mechanisms and illustrate how they work together to control WASP activity in response to multiple inputs. These regulatory principles, derived from studies of WASP and N-WASP, are likely to apply broadly across the family.
Harley,2010 (20526282) Harley ME, Allan LA, Sanderson HS, Clarke PR "Phosphorylation of Mcl-1 by CDK1-cyclin B1 initiates its Cdc20-dependent destruction during mitotic arrest." EMBO J 2010 Jul 21
The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti-apoptotic protein Mcl-1 is regulated during the cell cycle and peaks at mitosis. Mcl-1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin-dependent kinase 1 (CDK1)-cyclin B1 initiates degradation of Mcl-1 in cells arrested in mitosis by microtubule poisons. Mcl-1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl-1 during mitotic arrest by mutation of either Thr92 or a D-box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl-1 by CDK1-cyclin B1 and its APC/C(Cdc20)-mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.
Xiao,2010 (20521764) Xiao Q, Zhang F, Nacev BA, Liu JO, Pei D "Protein N-terminal processing: substrate specificity of Escherichia coli and human methionine aminopeptidases." Biochemistry 2010 Jun 29
Methionine aminopeptidase (MetAP) catalyzes the hydrolytic cleavage of the N-terminal methionine from newly synthesized polypeptides. The extent of removal of methionyl from a protein is dictated by its N-terminal peptide sequence. Earlier studies revealed that MetAPs require amino acids containing small side chains (e.g., Gly, Ala, Ser, Cys, Pro, Thr, and Val) as the P1' residue, but their specificity at positions P2' and beyond remains incompletely defined. In this work, the substrate specificities of Escherichia coli MetAP1, human MetAP1, and human MetAP2 were systematically profiled by screening against a combinatorial peptide library and kinetic analysis of individually synthesized peptide substrates. Our results show that although all three enzymes require small residues at the P1' position, they have differential tolerance for Val and Thr at this position. The catalytic activity of human MetAP2 toward Met-Val peptides is consistently 2 orders of magnitude higher than that of MetAP1, suggesting that MetAP2 is responsible for processing proteins containing N-terminal Met-Val and Met-Thr sequences in vivo. At positions P2'-P5', all three MetAPs have broad specificity but are poorly active toward peptides containing a proline at the P2' position. In addition, the human MetAPs disfavor acidic residues at the P2'-P5' positions. The specificity data have allowed us to formulate a simple set of rules that can reliably predict the N-terminal processing of E. coli and human proteins.
Kim,2010 (20519406) Kim ET, Kim YE, Huh YH, Ahn JH "Role of noncovalent SUMO binding by the human cytomegalovirus IE2 transactivator in lytic growth." J Virol 2010 Jul 19
The 86-kDa immediate-early 2 (IE2) protein of human cytomegalovirus (HCMV) is a promiscuous transactivator essential for viral gene expression. IE2 is covalently modified by SUMO at two lysine residues (K175 and K180) and also interacts noncovalently with SUMO. Although SUMOylation of IE2 has been shown to enhance its transactivation activity, the role of SUMO binding is not clear. Here we showed that SUMO binding by IE2 is necessary for its efficient transactivation function and for viral growth. IE2 bound physically to SUMO-1 through a SUMO-interacting motif (SIM). Mutations in SIM (mSIM) or in both SUMOylation sites and SIM (KR/mSIM), significantly reduced IE2 transactivation effects on viral early promoters. The replication of IE2 SIM mutant viruses (mSIM or KR/mSIM) was severely depressed in normal human fibroblasts. Analysis of viral growth curves revealed that the replication defect of the mSIM virus correlated with low-level accumulation of SUMO-modified IE2 and of viral early and late proteins. Importantly, both the formation of viral transcription domains and the association of IE2 with viral promoters in infected cells were significantly reduced in IE2 SIM mutant virus infection. Furthermore, IE2 was found to interact with the SUMO-modified form of TATA-binding protein (TBP)-associated factor 12 (TAF12), a component of the TFIID complex, in a SIM-dependent manner, and this interaction enhanced the transactivation activity of IE2. Our data demonstrate that the interaction of IE2 with SUMO-modified proteins plays an important role for the progression of the HCMV lytic cycle, and they suggest a novel viral mechanism utilizing the cellular SUMO system.
Suijkerbuijk,2010 (20516114) Suijkerbuijk SJ, van Osch MH, Bos FL, Hanks S, Rahman N, Kops GJ "Molecular causes for BUBR1 dysfunction in the human cancer predisposition syndrome mosaic variegated aneuploidy." Cancer Res 2010 Jun 16
Genetic mutations in the mitotic regulatory kinase BUBR1 are associated with the cancer-susceptible disorder mosaic variegated aneuploidy (MVA). In patients with biallelic mutations, a missense mutation pairs with a truncating mutation. Here, we show that cell lines derived from MVA patients with biallelic mutations have an impaired mitotic checkpoint, chromosome alignment defects, and low overall BUBR1 abundance. Ectopic expression of BUBR1 restored mitotic checkpoint activity, proving that BUBR1 dysfunction causes chromosome segregation errors in the patients. Combined analysis of patient cells and functional protein replacement shows that all MVA mutations fall in two distinct classes: those that impose specific defects in checkpoint activity or microtubule attachment and those that lower BUBR1 protein abundance. Low protein abundance is the direct result of the absence of transcripts from truncating mutants combined with high protein turnover of missense mutants. In this group of missense mutants, the amino acid change consistently occurs in or near the BUBR1 kinase domain. Our findings provide a molecular explanation for chromosomal instability in patients with biallelic genetic mutations in BUBR1.
Lara-Chacon,2010 (20512930) Lara-Chacon B, de Leon MB, Leocadio D, Gomez P, Fuentes-Mera L, Martinez-Vieyra I, Ortega A, Jans DA, Cisneros B "Characterization of an Importin alpha/beta-recognized nuclear localization signal in beta-dystroglycan." J Cell Biochem 2010 May 31
Beta-dystroglycan (beta-DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of beta-DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of beta-DG, characterizing a functional nuclear localization signal (NLS) in the beta-DG cytoplasmic domain, within amino acids 776-782. The NLS either alone or in the context of the whole beta-DG protein was able to target the heterologous GFP protein to the nucleus, with site-directed mutagenesis indicating that amino acids R(779) and K(780) are critical for NLS functionality. The nuclear transport molecules Importin (Imp)alpha and Impbeta bound with high affinity to the NLS of beta-DG and were found to be essential for NLS-dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of beta-DG may result in cytoplasmic retention, with Y(892) playing a key role. beta-DG thus follows a conventional Impalpha/beta-dependent nuclear import pathway, with important implications for its potential function in the nucleus.
Lee,2010 (20509869) Lee HJ, Zheng JJ "PDZ domains and their binding partners: structure, specificity, and modification." Cell Commun Signal 2010 Jun 25
PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes.
Calderon-Villalobos,2010 (20504967) Calderon-Villalobos LI, Tan X, Zheng N, Estelle M "Auxin perception--structural insights." Cold Spring Harb Perspect Biol 2010 Jul 02
The identity of the auxin receptor(s) and the mechanism of auxin perception has been a subject of intense interest since the discovery of auxin almost a century ago. The development of genetic approaches to the study of plant hormone signaling led to the discovery that auxin acts by promoting degradation of transcriptional repressors called Aux/IAA proteins. This process requires a ubiquitin protein ligase (E3) called SCF(TIR1) and related SCF complexes. Surprisingly, auxin works by directly binding to TIR1, the F-box protein subunit of this SCF. Structural studies demonstrate that auxin acts like a "molecular glue," to stabilize the interaction between TIR1 and the Aux/IAA substrate. These exciting results solve an old problem in plant biology and reveal new mechanisms for E3 regulation and hormone perception.
Dolnik,2010 (20504928) Dolnik O, Kolesnikova L, Stevermann L, Becker S "Tsg101 is recruited by a late domain of the nucleocapsid protein to support budding of Marburg virus-like particles." J Virol 2010 Aug
The nucleoprotein NP of Marburg virus (MARV) is the major component of the viral nucleocapsid, which also consists of the viral proteins VP35, L, and VP30, as well as the viral genome. During virus assembly at the plasma membrane, the nucleocapsids are enwrapped by the major matrix protein VP40 and the viral envelope, which contains the transmembrane glycoprotein GP. Upon recombinant expression, VP40 alone is able to induce the formation and release of virus-like particles (VLPs) that closely resemble the filamentous morphology of MARV particles. Release of these VP40-induced VLPs is partially dependent on the cellular ESCRT machinery, which interacts with a late-domain motif in VP40. Coexpression with NP significantly enhances the budding of VP40-induced VLPs by an unknown mechanism. In the present study we analyzed the impact of late domains present in NP on the release of VLPs. We observed that the ESCRT I protein Tsg101 was recruited by NP into NP-induced inclusions in the perinuclear region. In the presence of VP40, NP was then recruited to VP40-positive membrane clusters and, in turn, recruited Tsg101 via a C-terminal PSAP late-domain motif in NP. This PSAP motif also mediated a dramatically enhanced incorporation of Tsg101 into VLPs, and its deletion significantly diminished the positive effect of NP on the release of VLPs. Taken together, these data indicate that NP enhances budding of VLPs by recruiting Tsg101 to the VP40-positive budding site through a PSAP late-domain motif.
Zhang,2010 (20493962) Zhang X, Kondo M, Chen J, Miyoshi H, Suzuki H, Ohashi T, Shida H "Inhibitory effect of human TRIM5alpha on HIV-1 production." Microbes Infect 2010 Sep 06
Tripartite motif-containing 5 isoform-alpha (TRIM5alpha), a host restriction factor, blocks infection of some retroviruses at a post-entry, pre-integration stage in a species-specific manner. A recent report by Sakuma et al. describes a second antiretroviral activity of rhesus macaque TRIM5alpha, which blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. Here, we find that human TRIM5alpha limits HIV-1 production. Transient expression of TRIM5alpha decreased HIV-1 production, whereas knockdown of TRIM5alpha in human cells increased virion release. A single amino acid substitution (R437C) in the SPRY domain diminished the restriction effect. Moderate levels of human wild-type TRIM5alpha and a little amount of R437C mutant were incorporated into HIV-1 virions. The R437C mutant also lost restriction activity against N-tropic murine leukemia virus infection. However, the corresponding R to C mutation in rhesus macaque TRIM5alpha had no effect on the restriction ability. Our findings suggest human TRIM5alpha is an intrinsic immunity factor against HIV-1 infection. The importance of arginine at 437 aa in SPRY domain for the late restriction is species-specific.
Li,2010 (20489202) Li X, Zhang R, Zhang H, He Y, Ji W, Min W, Boggon TJ "Crystal structure of CCM3, a cerebral cavernous malformation protein critical for vascular integrity." J Biol Chem 2010 Jul 26
CCM3 mutations are associated with cerebral cavernous malformation (CCM), a disease affecting 0.1-0.5% of the human population. CCM3 (PDCD10, TFAR15) is thought to form a CCM complex with CCM1 and CCM2; however, the molecular basis for these interactions is not known. We have determined the 2.5 A crystal structure of CCM3. This structure shows an all alpha-helical protein containing two domains, an N-terminal dimerization domain with a fold not previously observed, and a C-terminal focal adhesion targeting (FAT)-homology domain. We show that CCM3 binds CCM2 via this FAT-homology domain and that mutation of a highly conserved FAK-like hydrophobic pocket (HP1) abrogates CCM3-CCM2 interaction. This CCM3 FAT-homology domain also interacts with paxillin LD motifs using the same surface, and partial CCM3 co-localization with paxillin in cells is lost on HP1 mutation. Disease-related CCM3 truncations affect the FAT-homology domain suggesting a role for the FAT-homology domain in the etiology of CCM.
Huang,2010 (20473298) Huang E, Qu D, Zhang Y, Venderova K, Haque ME, Rousseaux MW, Slack RS, Woulfe JM, Park DS "The role of Cdk5-mediated apurinic/apyrimidinic endonuclease 1 phosphorylation in neuronal death." Nat Cell Biol 2010 Jun 02
Accumulating evidence suggests that deregulated cyclin-dependent kinase 5 (Cdk5) plays a critical part in neuronal death. However, the pathogenic targets of Cdk5 are not fully defined. Here we demonstrate that the Cdk5 activator p35 interacts directly with apurinic/apyrimidinic endonuclease 1 (Ape1), a protein crucial for base excision repair (BER) following DNA damage. Cdk5 complexes phosphorylate Ape1 at Thr 232 and thereby reduces its apurinic/apyrimidinic (AP) endonuclease activity. Ape1 phosphorylation is dependent on Cdk5 in in vitro and in vivo. The reduced endonuclease activity of phosphorylated Ape1 results in accumulation of DNA damage and contributes to neuronal death. Overexpression of Ape1(WT) and Ape1(T232A), but not the phosphorylation mimic Ape1(T232E), protects neurons against MPP(+)/MPTP. Loss of Ape1 sensitizes neurons to death. Importantly, increased phosphorylated Ape1 was also observed in post-mortem brain tissue from patients with Parkinson's and Alzheimer's diseases, suggesting a potential link between Ape1 phosphorylation and the pathogenesis of neurodegenerative diseases.
Ilinskaya,2010 (20463077) Ilinskaya A, Heidecker G, Derse D "Opposing effects of a tyrosine-based sorting motif and a PDZ-binding motif regulate human T-lymphotropic virus type 1 envelope trafficking." J Virol 2010 Jul
Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein mediates binding of the virus to its receptor on the surface of target cells and subsequent fusion of virus and cell membranes. To better understand the mechanisms that control HTLV-1 Env trafficking and activity, we have examined two protein-protein interaction motifs in the cytoplasmic domain of Env. One is the sequence YSLI, which matches the consensus YXXPhi motifs that are known to interact with various adaptor protein complexes; the other is the sequence ESSL at the C terminus of Env, which matches the consensus PDZ-binding motif. We show here that mutations that destroy the YXXPhi motif increased Env expression on the cell surface and increased cell-cell fusion activity. In contrast, mutation of the PDZ-binding motif greatly diminished Env expression in cells, which could be restored to wild-type levels either by mutating the YXXPhi motif or by silencing AP2 and AP3, suggesting that interactions with PDZ proteins oppose an Env degradation pathway mediated by AP2 and AP3. Silencing of the PDZ protein hDlg1 did not affect Env expression, suggesting that hDlg1 is not a binding partner for Env. Substitution of the YSLI sequence in HTLV-1 Env with YXXPhi elements from other cell or virus membrane-spanning proteins resulted in alterations in Env accumulation in cells, incorporation into virions, and virion infectivity. Env variants containing YXXPhi motifs that are predicted to have high-affinity interaction with AP2 accumulated to lower steady-state levels. Interestingly, mutations that destroy the YXXPhi motif resulted in viruses that were not infectious by cell-free or cell-associated routes of infection. Unlike YXXPhi, the function of the PDZ-binding motif manifests itself only in the producer cells; AP2 silencing restored the incorporation of PDZ-deficient Env into virus-like particles (VLPs) and the infectivity of these VLPs to wild-type levels.
Jain,2010 (20452972) Jain A, Lamark T, Sjottem E, Larsen KB, Awuh JA, Overvatn A, McMahon M, Hayes JD, Johansen T "p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription." J Biol Chem 2010 Jul 12
The p62/SQSTM1 (sequestosome 1) protein, which acts as a cargo receptor for autophagic degradation of ubiquitinated targets, is up-regulated by various stressors. Induction of the p62 gene by oxidative stress is mediated by NF-E2-related factor 2 (NRF2) and, at the same time, p62 protein contributes to the activation of NRF2, but hitherto the mechanisms involved were not known. Herein, we have mapped an antioxidant response element (ARE) in the p62 promoter that is responsible for its induction by oxidative stress via NRF2. Chromatin immunoprecipitation and gel mobility-shift assays verified that NRF2 binds to this cis-element in vivo and in vitro. Also, p62 docks directly onto the Kelch-repeat domain of Kelch-like ECH-associated protein 1 (KEAP1), via a motif designated the KEAP1 interacting region (KIR), thereby blocking binding between KEAP1 and NRF2 that leads to ubiquitylation and degradation of the transcription factor. The KIR motif in p62 is located immediately C-terminal to the LC3-interacting region (LIR) and resembles the ETGE motif utilized by NRF2 for its interaction with KEAP1. KIR is required for p62 to stabilize NRF2, and inhibition of KEAP1 by p62 occurs from a cytoplasmic location within the cell. The LIR and KIR motifs cannot be engaged simultaneously by LC3 and KEAP1, but because p62 is polymeric the interaction between KEAP1 and p62 leads to accumulation of KEAP1 in p62 bodies, which is followed by autophagic degradation of KEAP1. Our data explain how p62 contributes to activation of NRF2 target genes in response to oxidative stress through creating a positive feedback loop.
Lu,2010 (20446724) Lu W, Liu CC, Thottassery JV, Bu G, Li Y "Mesd is a universal inhibitor of Wnt coreceptors LRP5 and LRP6 and blocks Wnt/beta-catenin signaling in cancer cells." Biochemistry 2010 Jun 01
Mesd is a specialized chaperone for low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6. In our previous studies, we found that Mesd binds to mature LRP6 on the cell surface and blocks the binding of Wnt antagonist Dickkopf-1 (Dkk1) to LRP6. Herein, we demonstrate that Mesd also binds to LRP5 with a high affinity and is a universal inhibitor of LRP5 and LRP6 ligands. Mesd not only blocks binding of Wnt antagonists Dkk1 and Sclerostin to LRP5 and LRP6 but also inhibits Wnt3A and Rspondin1-induced Wnt/beta-catenin signaling in LRP5- and LRP6-expressing cells. We also found that Mesd, Dkk1, and Sclerostin compete with one another for binding to LRP5 and LRP6 at the cell surface. More importantly, we demonstrated that Mesd is able to suppress LRP6 phosphorylation and Wnt/beta-catenin signaling in prostate cancer PC-3 cells and inhibits PC-3 cell proliferation. Our results indicate that recombinant Mesd protein is a useful tool for studying Wnt/beta-catenin signaling on the cell surface and has a potential therapeutic role in Wnt-dependent cancers.
Chen,2010 (20430886) Chen Z, Medina F, Liu MY, Thomas C, Sprang SR, Sternweis PC "Activated RhoA binds to the pleckstrin homology (PH) domain of PDZ-RhoGEF, a potential site for autoregulation." J Biol Chem 2010 Jul 2
Guanine nucleotide exchange factors (GEFs) catalyze exchange of GDP for GTP by stabilizing the nucleotide-free state of the small GTPases through their Dbl homology/pleckstrin homology (DH.PH) domains. Unconventionally, PDZ-RhoGEF (PRG), a member of the RGS-RhoGEFs, binds tightly to both nucleotide-free and activated RhoA (RhoA.GTP). We have characterized the interaction between PRG and activated RhoA and determined the structure of the PRG-DH.PH-RhoA.GTPgammaS (guanosine 5'-O-[gamma-thio]triphosphate) complex. The interface bears striking similarity to a GTPase-effector interface and involves the switch regions in RhoA and a hydrophobic patch in PRG-PH that is conserved among all Lbc RhoGEFs. The two surfaces that bind activated and nucleotide-free RhoA on PRG-DH.PH do not overlap, and a ternary complex of PRG-DH.PH bound to both forms of RhoA can be isolated by size-exclusion chromatography. This novel interaction between activated RhoA and PH could play a key role in regulation of RhoGEF activity in vivo.
Kumeta,2010 (20428927) Kumeta H, Watanabe M, Nakatogawa H, Yamaguchi M, Ogura K, Adachi W, Fujioka Y, Noda NN, Ohsumi Y, Inagaki F "The NMR structure of the autophagy-related protein Atg8." J Biomol NMR 2010 Jun 21
Mukherjee,2010 (20424264) Mukherjee K, Sharma M, Jahn R, Wahl MC, Sudhof TC "Evolution of CASK into a Mg2+-sensitive kinase." Sci Signal 2010 Apr 28
All known protein kinases, except CASK [calcium/calmodulin (CaM)-activated serine-threonine kinase], require magnesium ions (Mg(2+)) to stimulate the transfer of a phosphate from adenosine 5'-triphosphate (ATP) to a protein substrate. The CaMK (calcium/calmodulin-dependent kinase) domain of CASK shows activity in the absence of Mg(2+); indeed, it is inhibited by divalent ions including Mg(2+). Here, we converted the Mg(2+)-inhibited wild-type CASK kinase (CASK(WT)) into a Mg(2+)-stimulated kinase (CASK(4M)) by substituting four residues within the ATP-binding pocket. Crystal structures of CASK(4M) with and without bound nucleotide and Mn(2+), together with kinetic analyses, demonstrated that Mg(2+) accelerates catalysis of CASK(4M) by stabilizing the transition state, enhancing the leaving group properties of adenosine 5'-diphosphate, and indirectly shifting the position of the gamma-phosphate of ATP. Phylogenetic analysis revealed that the four residues conferring Mg(2+)-mediated stimulation were substituted from CASK during early animal evolution, converting a primordial, Mg(2+)-coordinating form of CASK into a Mg(2+)-inhibited kinase. This emergence of Mg(2+) sensitivity (inhibition by Mg(2+)) conferred regulation of CASK activity by divalent cations, in parallel with the evolution of the animal nervous systems.
Kozlov,2010 (20418951) Kozlov G, Gehring K "Molecular basis of eRF3 recognition by the MLLE domain of poly(A)-binding protein." PLoS One 2010
PABPC1 (cytosolic poly(A)-binding protein 1) is an RNA-binding protein that binds to the poly(A) tail of mRNAs to promote translation and mRNA turnover. In addition to RNA-binding domains, PABPC1 contains a unique protein-protein interaction domain, MLLE (also known as PABC) that binds regulatory proteins and translation factors that contain a conserved 12 amino acid peptide motif termed PAM2. Eukaryotic Release Factor 3 (eRF3/GSPT1) contains two overlapping PAM2 sequences, which are required for its activity. Here, we determined the crystal structures of the MLLE domain from PABPC1 in complex with the two PAM2 regions of eRF3. The structures reveal a mechanism of cooperativity between the two PAM2 sites that increases the binding affinity but prevents the binding of more than one molecule of eRF3 to PABPC1. Relative to previous structures, the high-resolution crystal structures force a re-evaluation of the PAM2 motif and improve our understanding of the molecular basis of MLLE peptide recognition.
Keller,2010 (20403971) Keller C, Woolcock K, Hess D, Buhler M "Proteomic and functional analysis of the noncanonical poly(A) polymerase Cid14." RNA 2010 May 24
The fission yeast Cid14 protein belongs to a family of noncanonical poly(A) polymerases which have been implicated in a broad range of biological functions. Here we describe an extensive Cid14 protein-protein interaction network and its biochemical dissection. Cid14 most stably interacts with the zinc-knuckle protein Air1 to form the Cid14-Air1 complex (CAC). Providing a link to ribosomal RNA processing, Cid14 sediments with 60S ribosomal subunits and copurifies with 60S assembly factors. In contrast, no physical link to chromatin has been identified, although gene expression profiling revealed that efficient silencing of a few heterochromatic genes depends on Cid14 and/or Air1.
Bollen,2010 (20399103) Bollen M, Peti W, Ragusa MJ, Beullens M "The extended PP1 toolkit: designed to create specificity." Trends Biochem Sci 2010 Aug 06
Protein Ser/Thr phosphatase-1 (PP1) catalyzes the majority of eukaryotic protein dephosphorylation reactions in a highly regulated and selective manner. Recent studies have identified an unusually diversified PP1 interactome with the properties of a regulatory toolkit. PP1-interacting proteins (PIPs) function as targeting subunits, substrates and/or inhibitors. As targeting subunits, PIPs contribute to substrate selection by bringing PP1 into the vicinity of specific substrates and by modulating substrate specificity via additional substrate docking sites or blocking substrate-binding channels. Many of the nearly 200 established mammalian PIPs are predicted to be intrinsically disordered, a property that facilitates their binding to a large surface area of PP1 via multiple docking motifs. These novel insights offer perspectives for the therapeutic targeting of PP1 by interfering with the binding of PIPs or substrates.
Goetz,2010 (20395968) Goetz SC, Anderson KV "The primary cilium: a signalling centre during vertebrate development." Nat Rev Genet 2010 Apr 16
The primary cilium has recently stepped into the spotlight, as a flood of data show that this organelle has crucial roles in vertebrate development and human genetic diseases. Cilia are required for the response to developmental signals, and evidence is accumulating that the primary cilium is specialized for hedgehog signal transduction. The formation of cilia, in turn, is regulated by other signalling pathways, possibly including the planar cell polarity pathway. The cilium therefore represents a nexus for signalling pathways during development. The connections between cilia and developmental signalling have begun to clarify the basis of human diseases associated with ciliary dysfunction.
Dilley,2010 (20392845) Dilley KA, Gregory D, Johnson MC, Vogt VM "An LYPSL late domain in the Gag protein contributes to the efficient release and replication of Rous sarcoma virus." J Virol 2010 Apr 14
The efficient release of newly assembled retrovirus particles from the plasma membrane requires the recruitment of a network of cellular proteins (ESCRT machinery) normally involved in the biogenesis of multivesicular bodies and cytokinesis. Retroviruses and other enveloped viruses recruit the ESCRT machinery through three classes of short amino acid consensus sequences termed late domains: PT/SAP, PPXY, and LYPXnL. The major late domain of Rous sarcoma virus (RSV) has been mapped to a PPPY motif in Gag that binds members of the Nedd4 family of ubiquitin ligases. RSV Gag also contains a second putative late domain motif, LYPSL, positioned five amino acids downstream of PPPY. LYPXnL motifs have been shown to support budding in other retroviruses by binding the ESCRT adaptor protein Alix. To investigate a possible role of the LYPSL motif in RSV budding, we constructed PPPY and LYPSL mutants in the context of an infectious virus, and then analyzed the budding rates, spreading profiles, and budding morphology. The data imply that the LYPSL motif acts as a secondary late domain, and that its role in budding is amplified in the absence of a fully functional PPPY motif. The LYPXL motif proved to be a stronger late domain when an aspartic acid was substituted for the native serine, recapitulating the properties of the LYPDL late domain of equine infectious anemia virus. Over-expression of human Alix in the absence of a fully functional PPPY late domain partially rescued both the viral budding rate and viral replication, supporting a model in which the RSV LYPSL motif mediates budding through an interaction with the ESCRT adaptor protein Alix.
Blomster,2010 (20388717) Blomster HA, Imanishi SY, Siimes J, Kastu J, Morrice NA, Eriksson JE, Sistonen L "In vivo identification of sumoylation sites by a signature tag and cysteine-targeted affinity purification." J Biol Chem 2010 Jun 15
Small ubiquitin-like modifier (SUMO) is conjugated to its substrates via an enzymatic cascade consisting of three enzymes, E1, E2, and E3. The active site of the E2 enzyme, Ubc9, recognizes the substrate through binding to a consensus tetrapeptide PsiKXE. However, recent proteomics studies suggested that a considerable part of sumoylation occurs on non-consensus sites. Current unbiased sumoylation site identification techniques typically require high stoichiometry in vitro sumoylation, mass spectrometry, and complex data analysis. To facilitate in vivo analysis, we have designed a mass spectrometric method based on an engineered human SUMO-1 construct that creates a signature tag on SUMO substrates. This construct enables affinity purification by covalent binding to cysteine residues in LysC/trypsin-cleaved peptides and site identification by diglycyl lysine tagging of sumoylation sites. As a proof of concept, site-specific and substrate-unbiased in vivo sumoylation analysis of HeLa cells was performed. We identified 14 sumoylation sites, including well known sites, such as Lys(524) of RanGAP1, and novel non-consensus sites. Only 3 of the 14 sites matched consensus sites, supporting the emerging view that non-consensus sumoylation is a common event in live cells. Six of the non-consensus sites had a nearby SUMO interaction motif (SIM), which emphasizes the role of SIM in non-consensus sumoylation. Nevertheless, the lack of nearby SIM residues among the remaining non-consensus sites indicates that there are also other specificity determinants of non-consensus sumoylation. The method we have developed proved to be a useful tool for sumoylation studies and will facilitate identification of novel SUMO substrates containing both consensus and non-consensus sites.
Humke,2010 (20360384) Humke EW, Dorn KV, Milenkovic L, Scott MP, Rohatgi R "The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins." Genes Dev 2010 Apr 02
The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. We show that the interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. We propose that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer.
Suryadinata,2010 (20337599) Suryadinata R, Sadowski M, Sarcevic B "Control of cell cycle progression by phosphorylation of cyclin-dependent kinase (CDK) substrates." Biosci Rep 2010 Mar 26
The eukaryotic cell cycle is a fundamental evolutionarily conserved process that regulates cell division from simple unicellular organisms, such as yeast, through to higher multicellular organisms, such as humans. The cell cycle comprises several phases, including the S-phase (DNA synthesis phase) and M-phase (mitotic phase). During S-phase, the genetic material is replicated, and is then segregated into two identical daughter cells following mitotic M-phase and cytokinesis. The S- and M-phases are separated by two gap phases (G1 and G2) that govern the readiness of cells to enter S- or M-phase. Genetic and biochemical studies demonstrate that cell division in eukaryotes is mediated by CDKs (cyclin-dependent kinases). Active CDKs comprise a protein kinase subunit whose catalytic activity is dependent on association with a regulatory cyclin subunit. Cell-cycle-stage-dependent accumulation and proteolytic degradation of different cyclin subunits regulates their association with CDKs to control different stages of cell division. CDKs promote cell cycle progression by phosphorylating critical downstream substrates to alter their activity. Here, we will review some of the well-characterized CDK substrates to provide mechanistic insights into how these kinases control different stages of cell division.
Ragusa,2010 (20305656) Ragusa MJ, Dancheck B, Critton DA, Nairn AC, Page R, Peti W "Spinophilin directs protein phosphatase 1 specificity by blocking substrate binding sites." Nat Struct Mol Biol 2010 Apr
The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets. PP1 associates with >or=200 regulatory proteins to form highly specific holoenzymes. These regulatory proteins target PP1 to its point of action within the cell and prime its enzymatic specificity for particular substrates. However, how they direct PP1's specificity is not understood. Here we show that spinophilin, a neuronal PP1 regulator, is entirely unstructured in its unbound form, and it binds PP1 through a folding-upon-binding mechanism in an elongated fashion, blocking one of PP1's three putative substrate binding sites without altering its active site. This mode of binding is sufficient for spinophilin to restrict PP1's activity toward a model substrate in vitro without affecting its ability to dephosphorylate its neuronal substrate, glutamate receptor 1 (GluR1). Thus, our work provides the molecular basis for the ability of spinophilin to dictate PP1 substrate specificity.
Cosgrove,2010 (20236310) Cosgrove MS, Patel A "Mixed lineage leukemia: a structure-function perspective of the MLL1 protein." FEBS J 2010 Apr 16
Several acute lymphoblastic and myelogenous leukemias are correlated with alterations in the human mixed lineage leukemia protein-1 (MLL1) gene. MLL1 is a member of the evolutionarily conserved SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for the regulation of distinct groups of developmentally regulated genes in metazoans. Despite the important biological role of SET1 family enzymes and their involvement in human leukemias, relatively little is understood about how these enzymes work. Here we review several recent structural and biochemical studies that are beginning to shed light on the molecular mechanisms for the regulation of H3K4 methylation by the human MLL1 enzyme.
Katis,2010 (20230747) Katis VL, Lipp JJ, Imre R, Bogdanova A, Okaz E, Habermann B, Mechtler K, Nasmyth K, Zachariae W "Rec8 phosphorylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates cohesin cleavage by separase during meiosis." Dev Cell 2010 Mar 16
During meiosis, two rounds of chromosome segregation after a single round of DNA replication produce haploid gametes from diploid precursors. At meiosis I, maternal and paternal kinetochores are pulled toward opposite poles, and chiasmata holding bivalent chromosomes together are resolved by cleavage of cohesin's alpha-kleisin subunit (Rec8) along chromosome arms. This creates dyad chromosomes containing a pair of chromatids joined solely by cohesin at centromeres that had resisted cleavage. The discovery that centromeric Rec8 is protected from separase during meiosis I by shugoshin/MEI-S332 proteins that bind PP2A phosphatase suggests that phosphorylation either of separase or cohesin may be necessary for Rec8 cleavage. We show here that multiple phosphorylation sites within Rec8 as well as two different kinases, casein kinase 1delta/epsilon (CK1delta/epsilon) and Dbf4-dependent Cdc7 kinase (DDK), are required for Rec8 cleavage and meiosis I nuclear division. Rec8 with phosphomimetic mutations is no longer protected from separase at centromeres and is cleaved even when the two kinases are inhibited. Our data suggest that PP2A protects centromeric cohesion by opposing CK1delta/epsilon- and DDK-dependent phosphorylation of Rec8.
Suzuki,2010 (20213681) Suzuki T, Moriya K, Nagatoshi K, Ota Y, Ezure T, Ando E, Tsunasawa S, Utsumi T "Strategy for comprehensive identification of human N-myristoylated proteins using an insect cell-free protein synthesis system." Proteomics 2010 May 03
To establish a strategy for the comprehensive identification of human N-myristoylated proteins, the susceptibility of human cDNA clones to protein N-myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell-free protein synthesis system. One-hundred-and-forty-one cDNA clones with N-terminal Met-Gly motifs were selected as potential candidates from approximately 2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N-myristoylation was evaluated using fusion proteins, in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N-myristoylated. The metabolic labeling experiments both in an insect cell-free protein synthesis system and in the transfected COS-1 cells using full-length cDNA revealed that 27 out of 29 proteins were in fact N-myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N-myristoylated proteins that have not been reported previously to be N-myristoylated, indicating that this strategy is useful for the comprehensive identification of human N-myristoylated proteins from human cDNA resources.
Mazars,2010 (20200153) Mazars R, Gonzalez-de-Peredo A, Cayrol C, Lavigne AC, Vogel JL, Ortega N, Lacroix C, Gautier V, Huet G, Ray A, Monsarrat B, Kristie TM, Girard JP "The THAP-zinc finger protein THAP1 associates with coactivator HCF-1 and O-GlcNAc transferase: a link between DYT6 and DYT3 dystonias." J Biol Chem 2010 Apr 30
THAP1 is a sequence-specific DNA binding factor that regulates cell proliferation through modulation of target genes such as the cell cycle-specific gene RRM1. Mutations in the THAP1 DNA binding domain, an atypical zinc finger (THAP-zf), have recently been found to cause DYT6 dystonia, a neurological disease characterized by twisting movements and abnormal postures. In this study, we report that THAP1 shares sequence characteristics, in vivo expression patterns and protein partners with THAP3, another THAP-zf protein. Proteomic analyses identified HCF-1, a potent transcriptional coactivator and cell cycle regulator, and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of O-GlcNAc, as major cellular partners of THAP3. THAP3 interacts with HCF-1 through a consensus HCF-1-binding motif (HBM), a motif that is also present in THAP1. Accordingly, THAP1 was found to bind HCF-1 in vitro and to associate with HCF-1 and OGT in vivo. THAP1 and THAP3 belong to a large family of HCF-1 binding factors since seven other members of the human THAP-zf protein family were identified, which harbor evolutionary conserved HBMs and bind to HCF-1. Chromatin immunoprecipitation (ChIP) assays and RNA interference experiments showed that endogenous THAP1 mediates the recruitment of HCF-1 to the RRM1 promoter during endothelial cell proliferation and that HCF-1 is essential for transcriptional activation of RRM1. Together, our findings suggest HCF-1 is an important cofactor for THAP1. Interestingly, our results also provide an unexpected link between DYT6 and DYT3 (X-linked dystonia-parkinsonism) dystonias because the gene encoding the THAP1/DYT6 protein partner OGT maps within the DYT3 critical region on Xq13.1.
Morgan,2010 (20195521) Morgan GW, Hollinshead M, Ferguson BJ, Murphy BJ, Carpentier DC, Smith GL "Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export." PLoS Pathog 2010 Mar 02
Vaccinia virus (VACV) uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC), a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs). Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD) motif, which is conserved in the kinesin-1-binding sequence (KBS) of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.
Kozlov,2010 (20181956) Kozlov G, Safaee N, Rosenauer A, Gehring K "Structural basis of binding of P-body-associated proteins GW182 and ataxin-2 by the Mlle domain of poly(A)-binding protein." J Biol Chem 2010 Apr 30
Poly(A)-binding protein (PABPC1) is involved in multiple aspects of mRNA processing and translation. It is a component of RNA stress granules and binds the RNA-induced silencing complex to promote degradation of silenced mRNAs. Here, we report the crystal structures of the C-terminal Mlle (or PABC) domain in complex with peptides from GW182 (TNRC6C) and Ataxin-2. The structures reveal overlapping binding sites but with unexpected diversity in the peptide conformation and residues involved in binding. The mutagenesis and binding studies show low to submicromolar binding affinity with overlapping but distinct specificity determinants. These results rationalize the role of the Mlle domain of PABPC1 in microRNA-mediated mRNA deadenylation and suggest a more general function in the assembly of cytoplasmic RNA granules.
Yang,2010 (20176810) Yang SH, Sharrocks AD "The SUMO E3 ligase activity of Pc2 is coordinated through a SUMO interaction motif." Mol Cell Biol 2010 Apr 08
Protein modification by SUMO conjugation has emerged to be an important regulatory event. Recently, the mechanisms through which SUMO elicits its effects on target proteins have been elucidated. One of these is the noncovalent association between SUMO and coregulatory proteins via SUMO interaction motifs (SIMs). We therefore searched for additional binding proteins to elucidate how SUMO acts as a signal to potentiate novel noncovalent interactions with SUMO-binding proteins. We identified an E3 ligase, Pc2, as a SUMO-binding protein with two functionally distinct SIMs. Here, we focus on the role of SIM2 and demonstrate that it is crucial for many of the documented Pc2 functions, which converge on determining its E3 ligase activity. One role of SUMO binding in this context is the subnuclear partitioning of the active form of Ubc9 (SUMO approximately Ubc9) by Pc2. The significance of the SIM2-dependent functions of Pc2 is demonstrated in the control of the precise expression of lineage-specific genes during embryonic stem cell differentiation.
Iwamori,2010 (20176808) Iwamori T, Iwamori N, Ma L, Edson MA, Greenbaum MP, Matzuk MM "TEX14 interacts with CEP55 to block cell abscission." Mol Cell Biol 2010 Apr 08
In somatic cells, abscission, the physical separation of daughter cells at the completion of cytokinesis, requires CEP55, ALIX, and TSG101. In contrast, cytokinesis is arrested prior to abscission in differentiating male germ cells that are interconnected by TEX14-positive intercellular bridges. We have previously shown that targeted deletion of TEX14 disrupts intercellular bridges in all germ cells and causes male sterility. Although these findings demonstrate that intercellular bridges are essential for spermatogenesis, it remains to be shown how TEX14 and other proteins come together to prevent abscission and form stable intercellular bridges. Using a biochemical enrichment of male germ cell intercellular bridges, we identified additional bridge proteins, including CEP55. Although CEP55 is highly expressed in testes at the RNA level, there is no report of the presence of CEP55 in germ cells. We show here that CEP55 becomes a stable component of the intercellular bridge and that an evolutionarily conserved GPPX3Y motif of TEX14 binds strongly to CEP55 to block similar GPPX3Y motifs of ALIX and TSG101 from interacting and localizing to the midbody. Thus, TEX14 prevents the completion of cytokinesis by altering the destiny of CEP55 from a nidus for abscission to an integral component of the intercellular bridge.
Podkowa,2010 (20176805) Podkowa M, Zhao X, Chow CW, Coffey ET, Davis RJ, Attisano L "Microtubule stabilization by bone morphogenetic protein receptor-mediated scaffolding of c-Jun N-terminal kinase promotes dendrite formation." Mol Cell Biol 2010 Apr 08
Neuronal outgrowth occurs via coordinated remodeling of the cytoskeleton involving both actin and microtubules. Microtubule stabilization drives the extending neurite, yet little is known of the molecular mechanisms whereby extracellular cues regulate microtubule dynamics. Bone morphogenetic proteins (BMPs) play an important role in neuronal differentiation and morphogenesis, and BMP7 in particular induces the formation of dendrites. Here, we show that BMP7 induces stabilization of microtubules in both a MAP2-dependent neuronal cell culture model and in dendrites of primary cortical neurons. BMP7 rapidly activates c-Jun N-terminal kinases (JNKs), known regulators of microtubule dynamics, and we show that JNKs associate with the carboxy terminus of the BMP receptor, BMPRII. Activation and binding of JNKs to BMPRII is required for BMP7-induced microtubule stabilization and for BMP7-mediated dendrite formation in primary cortical neurons. These data indicate that BMPRII acts as a scaffold to localize and coordinate cytoskeletal remodeling and thereby provides an efficient means for extracellular cues, such as BMPs, to control neuronal dendritogenesis.
Komatsu,2010 (20173742) Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y, Sou YS, Ueno I, Sakamoto A, Tong KI, Kim M, Nishito Y, Iemura S, Natsume T, Ueno T, Kominami E, Motohashi H, Tanaka K, Yamamoto M "The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1." Nat Cell Biol 2010 Mar 01
Impaired selective turnover of p62 by autophagy causes severe liver injury accompanied by the formation of p62-positive inclusions and upregulation of detoxifying enzymes. These phenotypes correspond closely to the pathological conditions seen in human liver diseases, including alcoholic hepatitis and hepatocellular carcinoma. However, the molecular mechanisms and pathophysiological processes in these events are still unknown. Here we report the identification of a novel regulatory mechanism by p62 of the transcription factor Nrf2, whose target genes include antioxidant proteins and detoxification enzymes. p62 interacts with the Nrf2-binding site on Keap1, a component of Cullin-3-type ubiquitin ligase for Nrf2. Thus, an overproduction of p62 or a deficiency in autophagy competes with the interaction between Nrf2 and Keap1, resulting in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Our findings indicate that the pathological process associated with p62 accumulation results in hyperactivation of Nrf2 and delineates unexpected roles of selective autophagy in controlling the transcription of cellular defence enzyme genes.
Fu,2010 (20173098) Fu X, Yucer N, Liu S, Li M, Yi P, Mu JJ, Yang T, Chu J, Jung SY, O'Malley BW, Gu W, Qin J, Wang Y "RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage." Proc Natl Acad Sci U S A 2010 Mar 10
In unstressed cells, the tumor suppressor p53 is maintained at low levels by ubiquitin-mediated proteolysis mainly through Mdm2. In response to DNA damage, p53 is stabilized and becomes activated to turn on transcriptional programs that are essential for cell cycle arrest and apoptosis. Activation of p53 leads to accumulation of Mdm2 protein, a direct transcriptional target of p53. It is not understood how p53 is protected from degradation when Mdm2 is up-regulated. Here we report that p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination. We found that an E3 ubiquitin ligase RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53 and is required to stabilize p53 in the late response to DNA damage. This process is regulated by the DNA damage checkpoint, because RFWD3 is phosphorylated by ATM/ATR kinases and the phosphorylation mutant fails to stimulate p53 ubiquitination. In vitro experiments suggest that RFWD3 is a p53 E3 ubiquitin ligase and that RFWD3-Mdm2 complex restricts the polyubiquitination of p53 by Mdm2. Our study identifies RFWD3 as a positive regulator of p53 stability when the G(1) cell cycle checkpoint is activated and provides an explanation for how p53 is protected from degradation in the presence of high levels of Mdm2.
Wade,2010 (20172729) Wade M, Wang YV, Wahl GM "The p53 orchestra: Mdm2 and Mdmx set the tone." Trends Cell Biol 2010 May 03
The activities of p53 cover diverse aspects of cell biology, including cell cycle control, apoptosis, metabolism, fertility, differentiation and cellular reprogramming. Although loss of p53 function engenders tumor susceptibility, hyperactivation of p53 is lethal. Therefore, p53 activity must be strictly regulated to maintain normal tissue homeostasis. Critical for the control of p53 function are its two main negative regulators: Mdm2 and Mdmx. Recent reports have provided insight into the complex mechanisms that regulate these two proteins and have revealed novel functions for each. Here, we review and evaluate models of Mdm2- and Mdmx-dependent regulation of p53 activity. Both Mdm2 and Mdmx receive input from numerous signaling pathways and interact with many proteins in addition to p53. Therefore, we also consider roles for Mdm2 and Mdmx in additional cancer-related networks, including Notch signaling and the epithelial-to-mesenchymal transition.
Gfeller,2010 (20159850) Gfeller A, Liechti R, Farmer EE "Arabidopsis jasmonate signaling pathway." Sci Signal 2010 Feb 17
Jasmonates control defense gene expression, growth, and fertility throughout the plant kingdom and have been studied extensively in Arabidopsis thaliana. The prohormone jasmonic acid (JA) is conjugated to amino acids such as isoleucine to form the active hormone jasmonoyl-isoleucine (JA-Ile). A series of breakthroughs has identified the SCF [SCF consists of four subunits: a cullin, SKP1 (S-phase kinase-associated protein 1), a RING finger protein (RBX1/HRT1/ROC1), and an F-box protein] CORONATINE INSENSITIVE1 (COI1) E3 ubiquitin ligase complex and the JASMONATE ZIM-DOMAIN (JAZ) proteins as central components in the perception of and transcriptional response to JA-Ile. JAZ proteins (most probably as dimers) bind transcription factors such as MYC2 before JA-Ile production. JA-Ile binds to COI1 to facilitate the formation of COI1-JAZ complexes, leading to ubiquitination and subsequent degradation of JAZ proteins. The degradation of JAZ proteins liberates transcription factors that function in the presence of the RNA polymerase II coregulatory complex Mediator to permit the expression of a number of jasmonate-regulated genes. Recent developments include the identification of COI1 as a receptor for jasmonates. Upstream of the signaling events, microRNA319 (miR319) negatively regulates the production of JA and JA-derived signals.
Meinecke,2010 (20154681) Meinecke M, Cizmowski C, Schliebs W, Kruger V, Beck S, Wagner R, Erdmann R "The peroxisomal importomer constitutes a large and highly dynamic pore." Nat Cell Biol 2010 Mar 01
The peroxisomal protein import machinery differs fundamentally from known translocons (endoplasmic reticulum, mitochondria, chloroplasts, bacteria) as it allows membrane passage of folded, even oligomerized proteins. However, the mechanistic principles of protein translocation across the peroxisomal membrane remain unknown. There are various models that consider membrane invagination events, vesicle fusion or the existence of large import pores. Current data show that a proteinaceous peroxisomal importomer enables docking of the cytosolic cargo-loaded receptors, cargo translocation and receptor recycling. Remarkably, the cycling import receptor Pex5p changes its topology from a soluble cytosolic form to an integral membrane-bound form. According to the transient pore hypothesis, the membrane-bound receptor is proposed to form the core component of the peroxisomal import pore. Here, we demonstrate that the membrane-associated import receptor Pex5p together with its docking partner Pex14p forms a gated ion-conducting channel which can be opened to a diameter of about 9 nm by the cytosolic receptor-cargo complex. The newly identified pore shows striking dynamics, as expected for an import machinery translocating proteins of variable sizes.
Lanyon-Hogg,2010 (20146669) Lanyon-Hogg T, Warriner SL, Baker A "Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes." Biol Cell 2010 Feb 11
Peroxisomes are a family of organelles which have many unusual features. They can arise de novo from the endoplasmic reticulum by a still poorly characterized process, yet possess a unique machinery for the import of their matrix proteins. As peroxisomes lack DNA, their function, which is highly variable and dependent on developmental and/or environmental conditions, is determined by the post-translational import of specific metabolic enzymes in folded or oligomeric states. The two classes of matrix targeting signals for peroxisomal proteins [PTS1 (peroxisomal targeting signal 1) and PTS2] are recognized by cytosolic receptors [PEX5 (peroxin 5) and PEX7 respectively] which escort their cargo proteins to, or possibly across, the peroxisome membrane. Although the membrane translocation mechanism remains unclear, it appears to be driven by thermodynamically favourable binding interactions. Recycling of the receptors from the peroxisome membrane requires ATP hydrolysis for two linked processes: ubiquitination of PEX5 (and the PEX7 co-receptors in yeast) and the function of two peroxisome-associated AAA (ATPase associated with various cellular activities) ATPases, which play a role in recycling or turnover of the ubiquitinated receptors. This review summarizes and integrates recent findings on peroxisome matrix protein import from yeast, plant and mammalian model systems, and discusses some of the gaps in our understanding of this remarkable protein transport system.
Yan,2010 (20142502) Yan J, Pan L, Chen X, Wu L, Zhang M "The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins." Proc Natl Acad Sci U S A 2010 Mar 2
The hereditary hearing-vision loss disease, Usher syndrome I (USH1), is caused by defects in several proteins that can interact with each other in vitro. Defects in USH1 proteins are thought to be responsible for the developmental and functional impairments of sensory cells in the retina and inner ear. Harmonin/USH1C and Sans/USH1G are two of the USH1 proteins that interact with each other. Harmonin also binds to other USH1 proteins such as cadherin 23 (CDH23) and protocadherin 15 (PCDH15). However, the molecular basis governing the harmonin and Sans interaction is largely unknown. Here, we report an unexpected assembly mode between harmonin and Sans. We demonstrate that the N-terminal domain and the first PDZ domain of harmonin are tethered by a small-domain C-terminal to PDZ1 to form a structural and functional supramodule responsible for binding to Sans. We discover that the SAM domain of Sans, specifically, binds to the PDZ domain of harmonin, revealing previously unknown interaction modes for both PDZ and SAM domains. We further show that the synergistic PDZ1/SAM and PDZ1/carboxyl PDZ binding-motif interactions, between harmonin and Sans, lock the two scaffold proteins into a highly stable complex. Mutations in harmonin and Sans found in USH1 patients are shown to destabilize the complex formation of the two proteins.
Johnson,2010 (20141511) Johnson C, Crowther S, Stafford MJ, Campbell DG, Toth R, MacKintosh C "Bioinformatic and experimental survey of 14-3-3-binding sites." Biochem J 2010 Mar 15
More than 200 phosphorylated 14-3-3-binding sites in the literature were analysed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate, although the +2 proline residue occurs in less than half, and LX(R/K)SX(pS/pT)XP is prominent in plant 14-3-3-binding sites. Proline at +1 is rarely reported, and such motifs did not stand up to experimental reanalysis of human Ndel1. Instead, we discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the DISC1 (disrupted-in-schizophrenia 1)-interacting region of Ndel1 that is implicated in cognitive disorders. These data conform with the general findings that there are different subtypes of 14-3-3-binding sites that overlap with the specificities of different basophilic AGC (protein kinase A/protein kinase G/protein kinase C family) and CaMK (Ca2+/calmodulin-dependent protein kinase) protein kinases, and a 14-3-3 dimer often engages with two tandem phosphorylated sites, which is a configuration with special signalling, mechanical and evolutionary properties. Thus 14-3-3 dimers can be digital logic gates that integrate more than one input to generate an action, and coincidence detectors when the two binding sites are phosphorylated by different protein kinases. Paired sites are generally located within disordered regions and/or straddle either side of functional domains, indicating how 14-3-3 dimers modulate the conformations and/or interactions of their targets. Finally, 14-3-3 proteins bind to members of several multi-protein families. Two 14-3-3-binding sites are conserved across the class IIa histone deacetylases, whereas other protein families display differential regulation by 14-3-3s. We speculate that 14-3-3 dimers may have contributed to the evolution of such families, tailoring regulatory inputs to different physiological demands.
Swan,2010 (20133602) Swan LE, Tomasini L, Pirruccello M, Lunardi J, De Camilli P "Two closely related endocytic proteins that share a common OCRL-binding motif with APPL1." Proc Natl Acad Sci U S A 2010 Feb 25
Mutations of the inositol 5' phosphatase oculocerebrorenal syndrome of Lowe (OCRL) give rise to the congenital X-linked disorders oculocerebrorenal syndrome of Lowe and Dent disease, two conditions giving rise to abnormal kidney proximal tubule reabsorption, and additional nervous system and ocular defects in the case of Lowe syndrome. Here, we identify two closely related endocytic proteins, Ses1 and Ses2, which interact with the ASH-RhoGAP-like (ASPM-SPD-2-Hydin homology and Rho-GTPase Activating Domain-like) domain of OCRL. The interaction is mediated by a short amino acid motif similar to that used by the rab-5 effector APPL1 (Adaptor Protein containing pleckstrin homology [PH] domain, PTB domain and Leucine zipper motif 1) APPL1 for OCRL binding. Ses binding is mutually exclusive with APPL1 binding, and is disrupted by the same missense mutations in the ASH-RhoGAP-like domain that also disrupt APPL1 binding. Like APPL1, Ses1 and -2 are localized on endosomes but reside on different endosomal subpopulations. These findings define a consensus motif (which we have called a phenylalanine and histidine [F&H] motif) for OCRL binding and are consistent with a scenario in which Lowe syndrome and Dent disease result from perturbations at multiple sites within the endocytic pathway.
Wang,2010 (20129060) Wang Y, Liu C, Yang D, Yu H, Liou YC "Pin1At encoding a peptidyl-prolyl cis/trans isomerase regulates flowering time in Arabidopsis." Mol Cell 2010 Jan 15
Floral transition in plants is regulated by an integrated network of flowering genetic pathways. We show that an Arabidopsis PIN1-type parvulin 1, Pin1At, controls floral transition by accelerating cis/trans isomerization of the phosphorylated Ser/Thr-Pro motifs in two MADS-domain transcription factors, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and AGAMOUS-LIKE 24 (AGL24). Pin1At regulates flowering, which is genetically mediated by AGL24 and SOC1. Pin1At interacts with the phosphorylated AGL24 and SOC1 in vitro and with AGL24 and SOC1 in vivo and accelerates the cis/trans conformational change of phosphorylated Ser/Thr-Pro motifs of AGL24 and SOC1. We further demonstrate that these Ser/Thr-Pro motifs are important for Pin1At function in promoting flowering through AGL24 and SOC1 and that the interaction between Pin1At and AGL24 mediates the AGL24 stability in the nucleus. Taken together, we propose that phosphorylation-dependent prolyl cis/trans isomerization of key transcription factors is an important flowering regulatory mechanism.
Paquette,2010 (20122400) Paquette N, Broemer M, Aggarwal K, Chen L, Husson M, Erturk-Hasdemir D, Reichhart JM, Meier P, Silverman N "Caspase-mediated cleavage, IAP binding, and ubiquitination: linking three mechanisms crucial for Drosophila NF-kappaB signaling." Mol Cell 2010 Jan 29
Innate immune responses are critical for the immediate protection against microbial infection. In Drosophila, infection leads to the rapid and robust production of antimicrobial peptides through two NF-kappaB signaling pathways-IMD and Toll. The IMD pathway is triggered by DAP-type peptidoglycan, common to most Gram-negative bacteria. Signaling downstream from the peptidoglycan receptors is thought to involve K63 ubiquitination and caspase-mediated cleavage, but the molecular mechanisms remain obscure. We now show that PGN stimulation causes caspase-mediated cleavage of the imd protein, exposing a highly conserved IAP-binding motif (IBM) at its neo-N terminus. A functional IBM is required for the association of cleaved IMD with the ubiquitin E3-ligase DIAP2. Through its association with DIAP2, IMD is rapidly conjugated with K63-linked polyubiquitin chains. These results mechanistically connect caspase-mediated cleavage and K63 ubiquitination in immune-induced NF-kappaB signaling.
Im,2010 (20117114) Im YJ, Kang GB, Lee JH, Park KR, Song HE, Kim E, Song WK, Park D, Eom SH "Structural basis for asymmetric association of the betaPIX coiled coil and shank PDZ." J Mol Biol 2010 Mar 26
betaPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of betaPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of betaPIX, which is important for its physiological functions. We have solved the crystal structure of the betaPIX CC-Shank PDZ complex and determined the stoichiometry of complex formation. The betaPIX CC forms a 76-A-long parallel CC trimer. Despite the fact that the betaPIX CC exposes three PDZ binding motifs in the C-termini, the betaPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive beta-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of betaPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the betaPIX trimer. The structure shows canonical class I PDZ binding with a beta-sheet interaction extending to position -6 of betaPIX. The betaB-betaC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the beta-sheet interaction and to accommodate the bulky side chain of Trp -5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of betaPIX CC and Shank PDZ.
de Diego,2010 (20103772) de Diego I, Kuper J, Bakalova N, Kursula P, Wilmanns M "Molecular basis of the death-associated protein kinase-calcium/calmodulin regulator complex." Sci Signal 2010 Jan 27
Death-associated protein kinase (DAPK) provides a model for calcium-bound calmodulin (CaM)-dependent protein kinases (CaMKs). Here, we report the crystal structure of the binary DAPK-CaM complex, using a construct that includes the DAPK catalytic domain and adjacent autoregulatory domain. When DAPK was in a complex with CaM, the DAPK autoregulatory domain formed a long seven-turn helix. This DAPK-CaM module interacted with the DAPK catalytic domain through two separate domain-domain interfaces, which involved the upper and the lower lobe of the catalytic domain. When bound to DAPK, CaM adopted an extended conformation, which was different from that in CaM-CaMK peptide complexes. Complementary biochemical analysis showed that the ability of DAPK to bind CaM correlated with its catalytic activity. Because many features of CaM binding are conserved in other CaMKs, our findings likely provide a generally applicable model for regulation of CaMK activity.
Marcello,2010 (20100836) Marcello E, Gardoni F, Di Luca M, Perez-Otano I "An arginine stretch limits ADAM10 exit from the endoplasmic reticulum." J Biol Chem 2010 Mar 29
A disintegrin and metalloproteinase 10 (ADAM10) is a type I transmembrane glycoprotein responsible for the ectodomain shedding of a number of proteins implicated in the pathogenesis of diseases ranging from cancer to Alzheimer Disease. ADAM10 is synthesized in an inactive form, which is proteolytically activated during its forward transport along the secretory pathway and at the plasma membrane. Therefore, modulation of its trafficking could provide a mechanism to finely tune its shedding activity. Here we report the identification of an endoplasmic reticulum (ER) retention motif within the ADAM10 intracellular C-terminal tail. Sequential deletion/mutagenesis analyses showed that an arginine-rich ((723)RRR) sequence was responsible for the retention of ADAM10 in the ER and its inefficient surface trafficking. Mutating the second arginine to alanine was sufficient to allow ER exit and surface expression in both heterologous cells and hippocampal neurons. As synapse-associated protein 97 (SAP97) binds ADAM10 at its cytoplasmic tail and facilitates forward ADAM10 trafficking in neurons, we tested whether SAP97 could modulate ER export. However, neither expression nor Ser-39 phosphorylation of SAP97 in heterologous cells or hippocampal neurons were sufficient to allow the ER exit of ADAM10, suggesting that other signaling pathways or alternative binding partners are responsible for ADAM10 ER exit. Together, these results identify a novel mechanism regulating the intracellular trafficking and membrane delivery of ADAM10.
Jinek,2010 (20098421) Jinek M, Fabian MR, Coyle SM, Sonenberg N, Doudna JA "Structural insights into the human GW182-PABC interaction in microRNA-mediated deadenylation." Nat Struct Mol Biol 2010 Feb 04
GW182-family proteins are essential for microRNA-mediated translational repression and deadenylation in animal cells. Here we show that a conserved motif in the human GW182 paralog TNRC6C interacts with the C-terminal domain of polyadenylate binding protein 1 (PABC) and present the crystal structure of the complex. Mutations at the complex interface impair mRNA deadenylation in mammalian cell extracts, suggesting that the GW182-PABC interaction contributes to microRNA-mediated gene silencing.
Kozlov,2010 (20096703) Kozlov G, Menade M, Rosenauer A, Nguyen L, Gehring K "Molecular determinants of PAM2 recognition by the MLLE domain of poly(A)-binding protein." J Mol Biol 2010 Mar 26
MLLE (previously known as PABC) is a peptide-binding domain that is found in poly(A)-binding protein (PABP) and EDD (E3 isolated by differential display), a HECT E3 ubiquitin ligase also known as HYD (hyperplastic discs tumor suppressor) or UBR5. The MLLE domain from PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12 amino acid peptide motif called PAM2 (for PABP-interacting motif 2). Here, we determined crystal structures of the MLLE domain from PABP alone and in complex with PAM2 peptides from PABP-interacting protein 2. The structures provide a detailed view of hydrophobic determinants of the MLLE binding coded by PAM2 positions 3, 5, 7, 10, and 12 and reveal novel intermolecular polar contacts. In particular, the side chain of the invariant MLLE residue K580 forms hydrogen bonds with the backbone of PAM2 residues 5 and 7. The structures also show that peptide residues outside of the conserved PAM2 motif contribute to binding. Altogether, the structures provide a significant advance in understanding the molecular basis for the binding of PABP by PAM2-containing proteins involved in translational control, mRNA deadenylation, and other cellular processes.
Gallardo,2010 (20091728) Gallardo R, Ivarsson Y, Schymkowitz J, Rousseau F, Zimmermann P "Structural diversity of PDZ-lipid interactions." Chembiochem 2010 Mar 03
PDZ domains are globular protein modules that are over-and-above appreciated for their interaction with short peptide motifs found in the cytosolic tail of membrane receptors, channels, and adhesion molecules. These domains predominate in scaffold molecules that control the assembly and the location of large signaling complexes. Studies have now emerged showing that PDZ domains can also interact with membrane lipids, and in particular with phosphoinositides. Phosphoinositides control various aspects of cell signaling, vesicular trafficking, and cytoskeleton remodeling. When investigated, lipid binding appears to be extremely relevant for PDZ protein functionality. Studies point to more than one mechanism for PDZ domains to associate with lipids. Few studies have been focused on the structural basis of PDZ-phosphoinositide interactions, and the biological consequences of such interactions. Using the current knowledge on syntenin-1, syntenin-2, PTP-Bas, PAR-3 and PICK1, we recapitulate our understanding of the structural and biochemical aspects of PDZ-lipid interactions and the consequences for peptide interactions.
Prehaud,2010 (20086240) Prehaud C, Wolff N, Terrien E, Lafage M, Megret F, Babault N, Cordier F, Tan GS, Maitrepierre E, Menager P, Chopy D, Hoos S, England P, Delepierre M, Schnell MJ, Buc H, Lafon M "Attenuation of rabies virulence: takeover by the cytoplasmic domain of its envelope protein." Sci Signal 2010
The capacity of a rabies virus to promote neuronal survival (a signature of virulence) or death (a marker of attenuation) depends on the cellular partners recruited by the PDZ-binding site (PDZ-BS) of its envelope glycoprotein (G). Neuronal survival requires the selective association of the PDZ-BS of G with the PDZ domains of two closely related serine-threonine kinases, MAST1 and MAST2. Here, we found that a single amino acid change in the PDZ-BS triggered the apoptotic death of infected neurons and enabled G to interact with additional PDZ partners, in particular the tyrosine phosphatase PTPN4. Knockdown of PTPN4 abrogated virus-mediated apoptosis. Thus, we propose that attenuation of rabies virus requires expansion of the set of host PDZ proteins with which G interacts, which interferes with the finely tuned homeostasis required for survival of the infected neuron.
Noda,2010 (20083108) Noda NN, Ohsumi Y, Inagaki F "Atg8-family interacting motif crucial for selective autophagy." FEBS Lett 2010 Mar 22
Autophagy is a bulk degradation system conserved among most eukaryotes. Recently, autophagy has been shown to mediate selective degradation of various targets such as aggregated proteins and damaged or superfluous organelles. Structural studies have uncovered the conserved specific interactions between autophagic receptors and Atg8-family proteins through WXXL-like sequences, which we term the Atg8-family interacting motif (AIM). AIM functions in various autophagic receptors such as Atg19 in the cytoplasm-to-vacuole targeting pathway, p62 and neighbor of BRCA1 gene 1 (NBR1) in autophagic degradation of protein aggregates, and Atg32 and Nix in mitophagy, and may link the target-receptor complex to autophagic membranes and/or their forming machineries.
Penela,2010 (20080565) Penela P, Rivas V, Salcedo A, Mayor F Jr "G protein-coupled receptor kinase 2 (GRK2) modulation and cell cycle progression." Proc Natl Acad Sci U S A 2010 Jan 19
Cell cycle progression requires changes in the activity or levels of a variety of key signaling proteins. G protein-coupled receptor kinase 2 (GRK2) plays a central role in G protein-coupled receptor regulation. Recent research is uncovering its involvement in additional cellular functions, but the potential role of GRK2 in the cell cycle has not been addressed. We report that GRK2 protein levels are transiently down-regulated during the G2/M transition by a mechanism involving CDK2-mediated phosphorylation of GRK2 at Serine670, which triggers binding to the prolyl-isomerase Pin1 and subsequent degradation. Prevention of GRK2 phosphorylation at S670 impedes normal GRK2 down-regulation and markedly delays cell cycle progression. Interestingly, we find that endogenous GRK2 down-regulation is prevented on activation of the G2/M checkpoint by doxorubicin and that stabilized GRK2 levels in such conditions inversely correlate with the p53 response and the induction of apoptosis, suggesting that GRK2 participates in the regulatory network controlling cell cycle arrest and survival in such conditions.
Huang,2010 (20067577) Huang TN, Chang HP, Hsueh YP "CASK phosphorylation by PKA regulates the protein-protein interactions of CASK and expression of the NMDAR2b gene." J Neurochem 2010 Jan 7
J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06569.x Abstract Calcium/calmodulin-dependent serine kinase (CASK), a causative gene in X-linked mental retardation, acts as a multi-domain scaffold protein and interacts with more than 20 cellular proteins in different subcellular regions of neurons. It is of interest, therefore, to explore whether post-translational modification regulates CASK's protein-protein interactions. Here, we provide evidence that CASK is phosphorylated by protein kinase A (PKA), identifying residue S562 in the PSD-95-Dlg-ZO-1 domain and residue T724 in the guanylate kinase domain as PKA sites by an in vitro PKA kinase reaction and site-directed mutagenesis. Although the role of S562 phosphorylation is not clear, T724 phosphorylation up-regulates the interaction between CASK and T-box transcription factor T-brain-1 (Tbr-1). NMDAR2b, a downstream target of the CASK-Tbr-1 complex, was then used to explore the significance of CASK phosphorylation by PKA. In cultured cortical neurons, the PKA pathway stimulates both the protein expression and the promoter activity of NMDAR2b. Deletion of the Tbr-1-binding sites greatly reduces the 3'-5'-cyclic AMP responsiveness of the NMDAR2b promoter, and the CASK T724A mutation does not promote the 3'-5'-cyclic AMP responsiveness of NMDAR2b. In conclusion, our data provide evidence that PKA phosphorylates CASK, regulates the nuclear function of CASK, and consequently modulates NMDAR2b expression.
Curnis,2010 (20064928) Curnis F, Cattaneo A, Longhi R, Sacchi A, Gasparri AM, Pastorino F, Di Matteo P, Traversari C, Bachi A, Ponzoni M, Rizzardi GP, Corti A "Critical role of flanking residues in NGR-to-isoDGR transition and CD13/integrin receptor switching." J Biol Chem 2010 Mar 19
Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of alphavbeta3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the alpha-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind alphavbeta3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind alphavbeta3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (alphavbeta5, alphavbeta6, alphavbeta8, and alpha5beta1), although with 10-100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas alpha-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.
Rodenhuis-Zybert,2010 (20062797) Rodenhuis-Zybert IA, van der Schaar HM, da Silva Voorham JM, van der Ende-Metselaar H, Lei HY, Wilschut J, Smit JM "Immature dengue virus: a veiled pathogen?" PLoS Pathog 2010 Jan
Cells infected with dengue virus release a high proportion of immature prM-containing virions. In accordance, substantial levels of prM antibodies are found in sera of infected humans. Furthermore, it has been recently described that the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection. This suggests that immature dengue virus may play a role in disease pathogenesis. Interestingly, however, numerous functional studies have revealed that immature particles lack the ability to infect cells. In this report, we show that fully immature dengue particles become highly infectious upon interaction with prM antibodies. We demonstrate that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor-expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Together, these data suggest that during a secondary infection or primary infection of infants born to dengue-immune mothers, immature particles have the potential to be highly infectious and hence may contribute to the development of severe disease.
Soria,2010 (20060369) Soria G, Gottifredi V "PCNA-coupled p21 degradation after DNA damage: The exception that confirms the rule?" DNA Repair (Amst) 2010 Apr 4
While many are the examples of DNA damaging treatments that induce p21 accumulation, the conception of p21 upregulation as the universal response to genotoxic stress has come to an end. Compelling evidences have demonstrated the existence of converging signals that negatively regulate p21 bellow basal levels when replication forks are blocked. Moreover, conclusive reports identified the E3-ligase CRL4(CDT2) (CUL4-DDB1-CDT2) as the enzymatic complex that promotes p21 proteolysis when treatments such as UV irradiation trigger replication fork stress. A pre-requisite for CRL4(CDT2)-driven proteolysis is the interaction of p21 with PCNA. Interestingly as well, CRL4(CDT2)-dependent proteolysis is not limited to p21 and affects other PCNA partners, including the specialized DNA polymerase eta (pol eta). These recent discoveries are particularly intriguing since the UV-induced degradation of p21 has been shown to be required for efficient pol eta recruitment to DNA lesions. Herein we review the findings that lead to the identification of the molecular mechanism that triggers damage-induced PCNA-coupled protein proteolysis. We propose a novel model in which CRL4(CDT2)-dependent protein degradation facilitates a sequential and dynamic exchange between PIP box bearing proteins at stall forks during Translesion DNA synthesis (TLS). Moreover, given the tight spatiotemporal control that CRL4(CDT2)-driven proteolysis is able to confer to PCNA-regulated processes, we discuss the impact that this degradation mechanism might have in other molecular switches associated with the repair of damaged DNA.
Davidson,2010 (20059949) Davidson G, Shen J, Huang YL, Su Y, Karaulanov E, Bartscherer K, Hassler C, Stannek P, Boutros M, Niehrs C "Cell cycle control of wnt receptor activation." Dev Cell 2010 Jan 11
Low-density lipoprotein receptor related proteins 5 and 6 (LRP5/6) are transmembrane receptors that initiate Wnt/beta-catenin signaling. Phosphorylation of PPPSP motifs in the LRP6 cytoplasmic domain is crucial for signal transduction. Using a kinome-wide RNAi screen, we show that PPPSP phosphorylation requires the Drosophila Cyclin-dependent kinase (CDK) L63. L63 and its vertebrate homolog PFTK are regulated by the membrane tethered G2/M Cyclin, Cyclin Y, which mediates binding to and phosphorylation of LRP6. As a consequence, LRP6 phosphorylation and Wnt/beta-catenin signaling are under cell cycle control and peak at G2/M phase; knockdown of the mitotic regulator CDC25/string, which results in G2/M arrest, enhances Wnt signaling in a Cyclin Y-dependent manner. In Xenopus embryos, Cyclin Y is required in vivo for LRP6 phosphorylation, maternal Wnt signaling, and Wnt-dependent anteroposterior embryonic patterning. G2/M priming of LRP6 by a Cyclin/CDK complex introduces an unexpected new layer of regulation of Wnt signaling.
Zhao,2010 (20048001) Zhao B, Li L, Tumaneng K, Wang CY, Guan KL "A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP)." Genes Dev 2010 Jan 05
The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon in a phosphodegron. The phosphorylated phosphodegron then recruits the SCF(beta-TRCP) E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. Our study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).
Tyler,2010 (20047332) Tyler RC, Peterson FC, Volkman BF "Distal interactions within the par3-VE-cadherin complex." Biochemistry 2010 Feb 9
par3 is a multiple-PDZ-containing scaffold protein that is central to the organization of an evolutionarily conserved cell polarity complex consisting of par3, par6, and aPKC. The ability of par3 PDZ domains to target various adhesion molecules and enzymes at the plasma membrane leads to the controlled localization of par6 and aPKC, which has firmly established its role in epithelial cell polarity. Of the numerous PDZ ligands associated with par3, interaction of its third PDZ domain with the class II ligand found within the C-terminal tail of vascular endothelial cadherin (VE-Cad) suggests a role in endothelial cell polarity as well, but the molecular details of the interaction are unknown. Previously determined structures of par3-PDZ3 bound to the class I ligand found within the C-terminal tail of the phosphoinositide phosphatase PTEN revealed two discrete binding sites: a canonical PDZ-ligand interaction site and a distal site involving charge-charge complements. Currently, it is unclear if par3-PDZ3 employs both canonical and distal binding modes in its association with VE-Cad or if these modes are unique to the PTEN interaction, suggesting a possible mechanism for ligand specificity within the polarity network. The structure of par3-PDZ3 bound to the C-terminal tail of VE-Cad presented in this work shows that both canonical and distal interactions are utilized in binding. Biophysical measurements using fluorescence polarization and two-dimensional NMR implicate the intermolecular charge pairing of aspartic acid 777 (VE-Cad) and arginine 609 (par3-PDZ3) as a crucial modulator of complex formation. Phosphorylation of VE-Cad at serine 776 increases its affinity for par3, demonstrating that post-translational modifications outside of the canonical carboxylate binding site can enhance PDZ-ligand interactions. Comparison of the VE-Cad and PTEN complexes highlights how the unique molecular architecture of par3-PDZ3 can accommodate both canonical and distal interaction modes that allow dual-class specificity for these two ligand types.
Errico,2010 (20045433) Errico A, Deshmukh K, Tanaka Y, Pozniakovsky A, Hunt T "Identification of substrates for cyclin dependent kinases." Adv Enzyme Regul 2010 May 03
Gan,2010 (20044140) Gan L, Li L "Interleukin-1 Receptor-Associated Kinase-1 (IRAK-1) functionally associates with PKCepsilon and VASP in the regulation of macrophage migration." Mol Immunol 2010 Mar
Macrophage migration is mediated by complex cellular signaling processes and cytoskeleton re-arrangement. In particular, recent advances indicate that the innate immunity signaling process plays a key role in the regulation of macrophage migration. In this report, we have provided evidence demonstrating the involvement of a key innate immunity signaling kinase, Interleukin-1 Receptor-Associated Kinase-1 (IRAK-1) as a critical modulator of macrophage migration. Macrophage migration induced by phorbol 12-myristate 13-acetate (PMA) is significantly attenuated in IRAK-1(-/-) macrophages as compared to wild type macrophages. Mechanistically, we demonstrated that IRAK-1 works downstream of PKCepsilon and upstream of VASP, a member of Ena/VASP family proteins. IRAK-1 forms a close complex with PKCepsilon as well as VASP, and participates in PMA-induced phosphorylation of VASP. Notably, IRAK-1 contains a novel EVH1 domain binding motif (L(167)WPPPP) within its N-terminus, which is responsible for its interaction with VASP. The mutant IRAK-1 (L167A/W168A) fails to associate with VASP. Our findings provide a novel facet regarding the molecular signaling process regulating macrophage migration.
Howng,2010 (20043192) Howng SL, Hwang CC, Hsu CY, Hsu MY, Teng CY, Chou CH, Lee MF, Wu CH, Chiou SJ, Lieu AS, Loh JK, Yang CN, Lin CS, Hong YR "Involvement of the residues of GSKIP, AxinGID, and FRATtide in their binding with GSK3beta to unravel a novel C-terminal scaffold-binding region." Mol Cell Biochem 2010 May 10
The specificity and regulation of GSK3beta are thought to involve in the docking interactions at core kinase domain because of the particular amino acid residues. Recent X-ray diffraction studies illuminated the relative binding residues on AxinGID and FRATtide for GSK3beta docking and appeared that GSK3beta Val267Gly (V267G) and Tyr288Phe (Y288F) could distinguish the direct interaction between AxinGID and FRATtide. In order to explore the mode that involved the binding of GSKIP to GSK3beta and compare it with that of AxinGID and FRATtide, we pinpointed the binding sites of GSKIP to GSK3beta through the single-point mutation of four corresponding sites within GSK3beta (residues 260-300) as scaffold-binding region I (designated SBR-I(260-300)). Our data showed that these three binding proteins shared similar binding sites on GSK3beta. We also found that the binding of GSK3beta V267G mutant to GSKIP and AxinGID, but not that of Y288F mutant (effect on FRATtide), was affected. Further, based on the simulation data, the electron-density map of GSKIPtide bore closer similarity to the map AxinGID than to that of FRATtide. Interestingly, many C-terminal helix region point-mutants of GSK3beta L359P, F362A, E366K, and L367P were able to eliminate the binding with FRATtide, but not AxinGID or GSKIP. In addition, CABYR exhibited a unique mode in binding to C-terminal helix region of GSK3beta. Taken together, our data revealed that in addition to the core kinase domain, SBR-I(260-300), another novel C-terminus helix region, designated SBR-II(339-383), also appeared to participate in the recognition and specificity of GSK3beta in binding to other specific proteins.
Fulcher,2010 (20040518) Fulcher AJ, Roth DM, Fatima S, Alvisi G, Jans DA "The BRCA-1 binding protein BRAP2 is a novel, negative regulator of nuclear import of viral proteins, dependent on phosphorylation flanking the nuclear localization signal." FASEB J 2010 Apr 30
This study describes for the first time the ability of the novel BRCA1-binding protein 2 (BRAP2) to inhibit the nuclear import of specific viral proteins dependent on phosphorylation. Ectopic expression of BRAP2 in transfected African green monkey kidney COS-7 cells was found to significantly reduce nuclear localization signal (NLS)-dependent nuclear accumulation of either simian virus SV40 large-tumor antigen (T-ag) or human cytomegalovirus DNA polymerase processivity factor ppUL44; this was also observed in HL-60 human promyelocytic leukemia cells on induction of BRAP2 expression by vitamin D3 treatment. BRAP2 inhibition of nuclear accumulation was dependent on phosphorylation sites flanking the respective NLSs, where substitution of the cyclin-dependent kinase site T124 of T-ag with Ala or Asp prevented or enhanced BRAP2 inhibition of nuclear import, respectively. Substitution of T427 within the NLS of ppUL44 gave similar results, whereas no effect of BRAP2 was observed on nuclear targeting of other viral proteins, such as herpes simplex virus-1 pUL30, which lacks a phosphorylation site near its NLS, and the human immunodeficiency virus-1 Tat protein. Pulldowns/AlphaScreen assays indicated direct, high-affinity binding of BRAP2(442-592) to T-ag(111-135), strictly dependent on negative charge at T124 and the NLS. All results are consistent with BRAP2 being a novel, phosphorylation-regulated negative regulator of nuclear import, with potential as an antiviral agent.
de Chiara,2009 (20037628) de Chiara C, Menon RP, Strom M, Gibson TJ, Pastore A "Phosphorylation of s776 and 14-3-3 binding modulate ataxin-1 interaction with splicing factors." PLoS One 2009
Ataxin-1 (Atx1), a member of the polyglutamine (polyQ) expanded protein family, is responsible for spinocerebellar ataxia type 1. Requirements for developing the disease are polyQ expansion, nuclear localization and phosphorylation of S776. Using a combination of bioinformatics, cell and structural biology approaches, we have identified a UHM ligand motif (ULM), present in proteins associated with splicing, in the C-terminus of Atx1 and shown that Atx1 interacts with and influences the function of the splicing factor U2AF65 via this motif. ULM comprises S776 of Atx1 and overlaps with a nuclear localization signal and a 14-3-3 binding motif. We demonstrate that phosphorylation of S776 provides the molecular switch which discriminates between 14-3-3 and components of the spliceosome. We also show that an S776D Atx1 mutant previously designed to mimic phosphorylation is unsuitable for this aim because of the different chemical properties of the two groups. Our results indicate that Atx1 is part of a complex network of interactions with splicing factors and suggest that development of the pathology is the consequence of a competition of aggregation with native interactions. Studies of the interactions formed by non-expanded Atx1 thus provide valuable hints for understanding both the function of the non-pathologic protein and the causes of the disease.
Liu,2010 (20034488) Liu Q, Hirohashi Y, Du X, Greene MI, Wang Q "Nek2 targets the mitotic checkpoint proteins Mad2 and Cdc20: a mechanism for aneuploidy in cancer." Exp Mol Pathol 2010 Apr 02
In mitosis, the duplicated chromosomes are separated and equally distributed to progeny cells under the guidance of the spindle, a dynamic microtubule network. Previous studies revealed a mitotic checkpoint that prevents segregation of the chromosomes until all of the chromosomes are properly attached to microtubules through the kinetochores. A variety of kinetochore-localized proteins, including Mad2 and Cdc20, have been implicated in controlling the mitotic checkpoint. Here we report that both Mad2 and Cdc20 can physically associate with Nek2, a serine/threonine kinase implicated in centrosome functions. We show that, similar to Nek2, the endogenous Cdc20 protein can be detected in the centrosome and the spindle poles. Both Cdc20 and Mad2 can be phosphorylated by Nek2. Moreover, our studies demonstrate that overexpression of Nek2 enhances the ability of Mad2 to induce a delay in mitosis. These observations indicate that Nek2 may act upon the Mad2-Cdc20 protein complex and play a critical role in regulating the mitotic checkpoint protein complex. We propose that overexpression of Nek2 may promote aneuploidy by disrupting the control of the mitotic checkpoint.
Johnson,2010 (20028974) Johnson SE, Ilagan MX, Kopan R, Barrick D "Thermodynamic analysis of the CSL x Notch interaction: distribution of binding energy of the Notch RAM region to the CSL beta-trefoil domain and the mode of competition with the viral transactivator EBNA2." J Biol Chem 2010 Feb 22
The Notch signaling pathway is a cell-cell communication network giving rise to cell differentiation during metazoan development. Activation of the pathway releases the intracellular portion of the Notch receptor to translocate to the nucleus, where it is able to interact with the effector transcription factor CSL, converting CSL from a transcriptional repressor to an activator. This conversion is dependent upon the high affinity binding of the RAM region of the Notch receptor to the beta-trefoil domain (BTD) of CSL. Here we probe the energetics of binding to BTD of each conserved residue of RAM through the use of isothermal titration calorimetry and single residue substitution. We find that although the highly conserved PhiW PhiP motif is the largest determinant of binding, energetically significant interactions are contributed by N-terminal residues, including a conserved Arg/Lys-rich region. Additionally, we present a thermodynamic analysis of the interaction between the Epstein-Barr virus protein EBNA2 with BTD and explore the extent to which the EBNA2- and RAM-binding sites on BTD are nonoverlapping, as proposed by Fuchs et al. (Fuchs, K. P., Bommer, G., Dumont, E., Christoph, B., Vidal, M., Kremmer, E., and Kempkes, B. (2001) Eur. J. Biochem. 268, 4639-4646). Combining these results with displacement isothermal titration calorimetry, we propose a mechanism by which the PhiW PhiP motif of RAM and EBNA2 compete with one another for binding at the hydrophobic pocket of BTD using overlapping but specific interactions that are unique to each BTD ligand.
Pearce,2010 (20027184) Pearce LR, Komander D, Alessi DR "The nuts and bolts of AGC protein kinases." Nat Rev Mol Cell Biol 2010 Jan
The AGC kinase subfamily of protein kinases contains 60 members, including PKA, PKG and PKC. The family comprises some intensely examined protein kinases (such as Akt, S6K, RSK, MSK, PDK1 and GRK) as well as many less well-studied enzymes (such as SGK, NDR, LATS, CRIK, SGK494, PRKX, PRKY and MAST). Research has shed new light onto the architecture and regulatory mechanisms of these kinases. In addition, AGC kinases mediate diverse and important cellular functions, and their mutation and/or dysregulation contributes to the pathogenesis of many human diseases, including cancer and diabetes.
Elowe,2009 (20016069) Elowe S, Dulla K, Uldschmid A, Li X, Dou Z, Nigg EA "Uncoupling of the spindle-checkpoint and chromosome-congression functions of BubR1." J Cell Sci 2009 Dec 17
The BubR1 checkpoint protein performs multiple functions in mitosis. We have carried out a functional analysis of conserved motifs of human BubR1 (also known as BUB1B) and demonstrate that spindle assembly checkpoint (SAC) and chromosome attachment functions can be uncoupled from each other. Mutation of five proline-directed serine phosphorylation sites, identified in vivo by mass spectrometry, essentially abolishes attachment of chromosomes to the spindle but has no effect on SAC functionality. By contrast, mutation of the two conserved KEN boxes required for SAC function does not impact chromosome congression. Interestingly, the contribution of the two KEN-box motifs is not equal. Cdc20 associates with the N-terminal but not C-terminal KEN box, and mutation of the N-terminal KEN motif results in more severe acceleration of mitotic timing. Moreover, the two KEN motifs are not sufficient for maximal binding of Cdc20 and APC/C, which also requires sequences in the BubR1 C-terminus. Finally, mutation of the GLEBS motif causes loss of Bub3 interaction and mislocalization of BubR1 from the kinetochore; concomitantly, BubR1 phosphorylation as well as SAC activity and chromosome congression are impaired, indicating that the GLEBS motif is strictly required for both major functions of human BubR1.
Sharma,2010 (20015412) Sharma A, Bruns K, Roder R, Henklein P, Votteler J, Wray V, Schubert U "Solution structure of the equine infectious anemia virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein." BMC Struct Biol 2010 Jan 08
BACKGROUND: The equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell. RESULTS: In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this alpha-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein. CONCLUSIONS: These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.
Novak,2009 (20010802) Novak I, Kirkin V, McEwan DG, Zhang J, Wild P, Rozenknop A, Rogov V, Lohr F, Popovic D, Occhipinti A, Reichert AS, Terzic J, Dotsch V, Ney PA, Dikic I "Nix is a selective autophagy receptor for mitochondrial clearance." EMBO Rep 2009 Dec 24
Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.
Rodriguez-Castaneda,2010 (20010694) Rodriguez-Castaneda F, Maestre-Martinez M, Coudevylle N, Dimova K, Junge H, Lipstein N, Lee D, Becker S, Brose N, Jahn O, Carlomagno T, Griesinger C "Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short-term synaptic plasticity." EMBO J 2010 Feb 03
Ca(2+) signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca(2+)-CaM binds a conserved region in the priming proteins Munc13-1 and ubMunc13-2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca(2+) signals. We solved the structure of Ca(2+)(4)-CaM in complex with the CaM-binding domain of Munc13-1, which features a novel 1-5-8-26 CaM-binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13-2 isoform. The N-module can be dissociated with EGTA to form the half-loaded Munc13/Ca(2+)(2)-CaM complex. The Ca(2+) regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca(2+)-CaM interactions, where the C-module provides a high-affinity interaction activated at nanomolar [Ca(2+)](i), whereas the N-module acts as a sensor at micromolar [Ca(2+)](i). This Ca(2+)/CaM-binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca(2+)-dependent modulation of short-term synaptic plasticity.
Roy,2009 (19996458) Roy J, Cyert MS "Cracking the phosphatase code: docking interactions determine substrate specificity." Sci Signal 2009 Dec 09
Phosphoserine- and phosphothreonine-directed phosphatases display remarkable substrate specificity, yet the sites that they dephosphorylate show little similarity in amino acid sequence. Studies reveal that docking interactions are key for the recognition of substrates and regulators by two conserved phosphatases, protein phosphatase 1 (PP1) and the Ca2+-calmodulin-dependent phosphatase calcineurin. In each case, a small degenerate sequence motif in the interacting protein directs low-affinity binding to a docking surface on the phosphatase that is distinct from the active site; several such interactions combine to confer overall binding specificity. Some docking surfaces are conserved, such as a hydrophobic groove on a face opposite the active site that serves as a major recognition surface for the "RVxF" motif of proteins that interact with PP1 and the "PxIxIT" motif of substrates of calcineurin. Secondary motifs combine with this primary targeting sequence to specify phosphatase binding. A comprehensive interactome for mammalian PP1 was described, analysis of which defines several PP1-binding motifs. Studies of "LxVP," a secondary calcineurin-binding sequence, establish that this motif is a conserved feature of calcineurin substrates and that the immunosuppressants FK506 and cyclosporin A inhibit the phosphatase by interfering with LxVP-mediated docking.
Fujimoto,2010 (19996102) Fujimoto Y, Shiraki T, Horiuchi Y, Waku T, Shigenaga A, Otaka A, Ikura T, Igarashi K, Aimoto S, Tate S, Morikawa K "Proline cis/trans-isomerase Pin1 regulates peroxisome proliferator-activated receptor gamma activity through the direct binding to the activation function-1 domain." J Biol Chem 2010 Jan 25
The important roles of a nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) are widely accepted in various biological processes as well as metabolic diseases. Despite the worldwide quest for pharmaceutical manipulation of PPARgamma activity through the ligand-binding domain, very little information about the activation mechanism of the N-terminal activation function-1 (AF-1) domain. Here, we demonstrate the molecular and structural basis of the phosphorylation-dependent regulation of PPARgamma activity by a peptidyl-prolyl isomerase, Pin1. Pin1 interacts with the phosphorylated AF-1 domain, thereby inhibiting the polyubiquitination of PPARgamma. The interaction and inhibition are dependent upon the WW domain of Pin1 but are independent of peptidyl-prolyl cis/trans-isomerase activity. Gene knockdown experiments revealed that Pin1 inhibits the PPARgamma-dependent gene expression in THP-1 macrophage-like cells. Thus, our results suggest that Pin1 regulates macrophage function through the direct binding to the phosphorylated AF-1 domain of PPARgamma.
Luo,2010 (19995909) Luo Z, Wijeweera A, Oh Y, Liou YC, Melamed P "Pin1 facilitates the phosphorylation-dependent ubiquitination of SF-1 to regulate gonadotropin beta-subunit gene transcription." Mol Cell Biol 2010 Feb
Pin1 is a peptidyl-prolyl cis-trans isomerase which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 knockout mice have marked abnormalities in their reproductive development and function. However, the molecular mechanisms underlying their reproductive defects are poorly understood. Herein, we demonstrate that Pin1 is required for both basal and GnRH-induced gonadotropin beta-subunit gene transcription, through interactions with the transcription factors SF-1, Pitx1, and Egr-1. Pin1 activates transcription of the gonadotropin beta-subunit genes synergistically with these transcription factors, either by modulating their stability or by increasing their protein-protein interactions. Notably, we provide evidence that Pin1 is required for the Ser203 phosphorylation-dependent ubiquitination of SF-1, which facilitates SF-1-Pitx1 interactions and therefore results in an enhancement of SF-1 transcriptional activity. Furthermore, we demonstrate that in gonadotrope cells, sufficient levels of activated Pin1 are maintained through transcriptional and posttranslational regulation by GnRH-induced signaling cascades. Our results suggest that Pin1 functions as a novel player in GnRH-induced signal pathways and is involved in gonadotropin beta-subunit gene transcription by modulating the activity of various specific transcription factors.
Boulaflous,2009 (19995436) Boulaflous A, Saint-Jore-Dupas C, Herranz-Gordo MC, Pagny-Salehabadi S, Plasson C, Garidou F, Kiefer-Meyer MC, Ritzenthaler C, Faye L, Gomord V "Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER." BMC Plant Biol 2009 Dec 30
BACKGROUND: In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails. RESULTS: Here, using Arabidopsis glucosidase I as a model, we have identified two types of signals sufficient for the location of a type II membrane protein in the ER. A first signal is located in the luminal domain, while a second signal corresponds to a short amino acid sequence located in the cytosolic tail of the membrane protein. The cytosolic tail contains at its N-terminal end four arginine residues constitutive of three di-arginine motifs (RR, RXR or RXXR) independently sufficient to confer ER localization. Interestingly, when only one di-arginine motif is present, fusion proteins are located both in the ER and in mobile punctate structures, distinct but close to Golgi bodies. Soluble and membrane ER protein markers are excluded from these punctate structures, which also do not colocalize with an ER-exit-site marker. It is hypothesized they correspond to sites involved in Golgi to ER retrotransport. CONCLUSION: Altogether, these results clearly show that cytosolic and luminal signals responsible for ER retention could coexist in a same type II membrane protein. These data also suggest that both retrieval and retention mechanisms govern protein residency in the ER membrane. We hypothesized that mobile punctate structures not yet described at the ER/Golgi interface and tentatively named GERES, could be involved in retrieval mechanisms from the Golgi to the ER.
Nesic,2010 (19966800) Nesic D, Miller MC, Quinkert ZT, Stein M, Chait BT, Stebbins CE "Helicobacter pylori CagA inhibits PAR1-MARK family kinases by mimicking host substrates." Nat Struct Mol Biol 2010 Jan 06
The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.
Jansen,2010 (19962308) Jansen JM, Wanless AG, Seidel CW, Weiss EL "Cbk1 regulation of the RNA-binding protein Ssd1 integrates cell fate with translational control." Curr Biol 2010 Jan 12
Spatial control of gene expression, at the level of both transcription and translation, is critical for cellular differentiation [1-4]. In budding yeast, the conserved Ndr/warts kinase Cbk1 localizes to the new daughter cell, where it acts as a cell fate determinant. Cbk1 both induces a daughter-specific transcriptional program and promotes morphogenesis in a less well-defined role [5-8]. Cbk1 is essential in cells expressing functional Ssd1, an RNA-binding protein of unknown function [9-11]. We show here that Cbk1 inhibits Ssd1 in vivo. Loss of this regulation dramatically slows bud expansion, leading to highly aberrant cell wall organization at the site of cell growth. Ssd1 associates with specific mRNAs, a significant number of which encode cell wall remodeling proteins. Translation of these messages is rapidly and specifically suppressed when Cbk1 is inhibited; this suppression requires Ssd1. Transcription of several of these Ssd1-associated mRNAs is also regulated by Cbk1, indicating that the kinase controls both the transcription and translation of daughter-specific mRNAs. This work suggests a novel system by which cells coordinate localized expression of genes involved in processes critical for cell growth and division.
Zhang,2009 (19955409) Zhang Q, Shi Q, Chen Y, Yue T, Li S, Wang B, Jiang J "Multiple Ser/Thr-rich degrons mediate the degradation of Ci/Gli by the Cul3-HIB/SPOP E3 ubiquitin ligase." Proc Natl Acad Sci U S A 2009 Dec 18
The Cul3-based E3 ubiquitin ligases regulate many cellular processes using a large family of BTB domain-containing proteins as their target recognition components, but how they recognize targets remains unknown. Here we identify and characterize degrons that mediate the degradation of the Hedgehog pathway transcription factor cubitus interruptus (Ci)/Gli by Cul3-Hedghog-induced MATH and BTB domain-containing protein (HIB)/SPOP. Ci uses multiple Ser/Thr (S/T)-rich motifs that bind HIB cooperatively to mediate its degradation. We provide evidence that both HIB and Ci form dimers/oligomers and engage in multivalent interactions, which underlies the in vivo cooperativity among individual HIB-binding sites. We find that similar S/T-rich motifs are present in Gli proteins as well as in numerous HIB-interacting proteins and mediate Gli degradation by SPOP. Our results provide a mechanistic insight into how HIB/SPOP recognizes its substrates and have important implications for the genome-wide prediction of substrates for Cul3-based E3 ligases.
Delaval,2010 (19951897) Delaval B, Doxsey SJ "Pericentrin in cellular function and disease." J Cell Biol 2010 Jan 26
Pericentrin is an integral component of the centrosome that serves as a multifunctional scaffold for anchoring numerous proteins and protein complexes. Through these interactions, pericentrin contributes to a diversity of fundamental cellular processes. Recent studies link pericentrin to a growing list of human disorders. Studies on pericentrin at the cellular, molecular, and, more recently, organismal level, provide a platform for generating models to elucidate the etiology of these disorders. Although the complexity of phenotypes associated with pericentrin-mediated disorders is somewhat daunting, insights into the cellular basis of disease are beginning to come into focus. In this review, we focus on human conditions associated with loss or elevation of pericentrin and propose cellular and molecular models that might explain them.
Gulino,2010 (19944684) Gulino A, Di Marcotullio L, Screpanti I "The multiple functions of Numb." Exp Cell Res 2010 Mar 08
Numb is an evolutionary conserved protein that plays critical roles in cell fate determination. Mammalian Numb displays a higher degree of structural complexity compared to the Drosophila homolog based on the number of encoding genes (Numb and Numb-like) and of alternative spliced isoforms. Accordingly, Numb proteins display a complex pattern of functions such as the control of asymmetric cell division and cell fate choice, endocytosis, cell adhesion, cell migration, ubiquitination of specific substrates and a number of signaling pathways (i.e. Notch, Hedgehog, p53). Recent findings indicate that, besides controlling such physiologic developmental processes, subversion of the above Numb-dependent events plays a critical role in disease (e.g. cancer). We will review here the multiple functions of mNumb and their underlying molecular mechanisms in development and disease.
Brown,2009 (19935675) Brown CJ, Lain S, Verma CS, Fersht AR, Lane DP "Awakening guardian angels: drugging the p53 pathway." Nat Rev Cancer 2009 Nov 25
Currently, around 11 million people are living with a tumour that contains an inactivating mutation of TP53 (the human gene that encodes p53) and another 11 million have tumours in which the p53 pathway is partially abrogated through the inactivation of other signalling or effector components. The p53 pathway is therefore a prime target for new cancer drug development, and several original approaches to drug discovery that could have wide applications to drug development are being used. In one approach, molecules that activate p53 by blocking protein-protein interactions with MDM2 are in early clinical development. Remarkable progress has also been made in the development of p53-binding molecules that can rescue the function of certain p53 mutants. Finally, cell-based assays are being used to discover compounds that exploit the p53 pathway by either seeking targets and compounds that show synthetic lethality with TP53 mutations or by looking for non-genotoxic activators of the p53 response.
Schoch,2010 (19913032) Schoch GA, D'Arcy B, Stihle M, Burger D, Bar D, Benz J, Thoma R, Ruf A "Molecular switch in the glucocorticoid receptor: active and passive antagonist conformations." J Mol Biol 2010 Jan 28
Mifepristone is known to induce mixed passive antagonist, active antagonist, and agonist effects via the glucocorticoid receptor (GR) pathway. Part of the antagonist effects of mifepristone are due to the repression of gene transcription mediated by the nuclear receptor corepressor (NCoR). Here, we report the crystal structure of a ternary complex of the GR ligand binding domain (GR-LBD) with mifepristone and a receptor-interacting motif of NCoR. The structures of three different conformations of the GR-LBD mifepristone complex show in the oxosteroid hormone receptor family how helix 12 modulates LBD corepressor and coactivator binding. Differences in NCoR binding and in helix 12 conformation reveal how the 11beta substituent in mifepristone triggers the helix 12 molecular switch to reshape the coactivator site into the corepressor site. Two observed conformations exemplify the active antagonist state of GR with NCoR bound. In another conformation, helix 12 completely blocks the coregulator binding site and explains the passive antagonistic effect of mifepristone on GR.
Malumbres,2009 (19884882) Malumbres M, Harlow E, Hunt T, Hunter T, Lahti JM, Manning G, Morgan DO, Tsai LH, Wolgemuth DJ "Cyclin-dependent kinases: a family portrait." Nat Cell Biol 2009 Nov 03
Pincetic,2009 (19865606) Pincetic A, Leis J "The Mechanism of Budding of Retroviruses From Cell Membranes." Adv Virol 2009 Jan 1
Retroviruses have evolved a mechanism for the release of particles from the cell membrane that appropriates cellular protein complexes, referred to as ESCRT-I, -II, -III, normally involved in the biogenesis of multivesicular bodies. Three different classes of late assembly (L) domains encoded in Gag, with core sequences of PPXY, PTAP, and YPXL, recruit different components of the ESCRT machinery to form a budding complex for virus release. Here, we highlight recent progress in identifying the role of different ESCRT complexes in facilitating budding, ubiquitination, and membrane targeting of avian sarcoma and leukosis virus (ASLV) and human immunodeficiency virus, type 1 (HIV-1). These findings show that retroviruses adopt parallel budding pathways by recruiting different host factors from common cellular machinery for particle release.
Aoyama,2009 (19861499) Aoyama T, Hata S, Nakao T, Tanigawa Y, Oka C, Kawaichi M "Cayman ataxia protein caytaxin is transported by kinesin along neurites through binding to kinesin light chains." J Cell Sci 2009 Nov 13
Deficiency of caytaxin results in hereditary ataxia or dystonia in humans, mice and rats. Our yeast two-hybrid screen identified kinesin light chains (KLCs) as caytaxin-binding proteins. The tetratricopeptide-repeat region of KLC1 recognizes the ELEWED sequence (amino acids 115-120) of caytaxin. This motif is conserved among BNIP-2 family members and other KLC-interacting kinesin cargo proteins such as calsyntenins. Caytaxin associates with kinesin heavy chains (KHCs) indirectly by binding to KLCs, suggesting that caytaxin binds to the tetrameric kinesin molecule. In cultured hippocampal neurons, we found that caytaxin is distributed in both axons and dendrites in punctate patterns, and it colocalizes with microtubules and KHC. GFP-caytaxin expressed in hippocampal neurons is transported at a speed ( approximately 1 mum/second) compatible with kinesin movement. Inhibition of kinesin-1 by dominant-negative KHC decreases the accumulation of caytaxin in the growth cone. Caytaxin puncta do not coincide with vesicles containing known kinesin cargos such as APP or JIP-1. A part of caytaxin, however, colocalizes with mitochondria and suppression of caytaxin expression by RNAi redistributes mitochondria away from the distal ends of neurites. These data indicate that caytaxin binds to kinesin-1 and functions as an adaptor that mediates intracellular transport of specific cargos, one of which is the mitochondrion.
Chen,2009 (19851314) Chen HZ, Tsai SY, Leone G "Emerging roles of E2Fs in cancer: an exit from cell cycle control." Nat Rev Cancer 2009 Nov
Mutations of the retinoblastoma tumour suppressor gene (RB1) or components regulating the RB pathway have been identified in almost every human malignancy. The E2F transcription factors function in cell cycle control and are intimately regulated by RB. Studies of model organisms have revealed conserved functions for E2Fs during development, suggesting that the cancer-related proliferative roles of E2F family members represent a recent evolutionary adaptation. However, given that some human tumours have concurrent RB1 inactivation and E2F amplification and overexpression, we propose that there are alternative tumour-promoting activities for the E2F family, which are independent of cell cycle regulation.
Hsueh,2009 (19847910) Hsueh YP "Calcium/calmodulin-dependent serine protein kinase and mental retardation." Ann Neurol 2009 Oct 27
Calcium/calmodulin-dependent serine protein kinase (CASK) belongs to the membrane-associated guanylate kinase protein family. The members of this protein family function as multiple domain adaptor proteins originally identified at cell junctions and synapses. Insertional mutations or targeted disruption of the CASK gene in mice results in neonatal lethality, indicating an important role for CASK in development. Recently, several reports have also indicated that mutations in the human CASK gene result in X-linked malformations of the brain and mental retardation. At the molecular level, many studies indicate that CASK is critical for synapse formation at both presynaptic and postsynaptic junctions, and in the regulation of gene expression. The known molecular functions of CASK explain, at least partially, mental retardation and brain developmental defects in patients. In this review, recent findings about CASK are summarized and discussed.
Sundstrom,2009 (19820703) Sundstrom JF, Vaculova A, Smertenko AP, Savenkov EI, Golovko A, Minina E, Tiwari BS, Rodriguez-Nieto S, Zamyatnin AA Jr, Valineva T, Saarikettu J, Frilander MJ, Suarez MF, Zavialov A, Stahl U, Hussey PJ, Silvennoinen O, Sundberg E, Zhivotovsky B, Bozhkov PV "Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome." Nat Cell Biol 2009 Nov
Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.
Lee,2009 (19818716) Lee DF, Kuo HP, Liu M, Chou CK, Xia W, Du Y, Shen J, Chen CT, Huo L, Hsu MC, Li CW, Ding Q, Liao TL, Lai CC, Lin AC, Chang YH, Tsai SF, Li LY, Hung MC "KEAP1 E3 ligase-mediated downregulation of NF-kappaB signaling by targeting IKKbeta." Mol Cell 2009 Oct 12
IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.
Zhuang,2009 (19818708) Zhuang M, Calabrese MF, Liu J, Waddell MB, Nourse A, Hammel M, Miller DJ, Walden H, Duda DM, Seyedin SN, Hoggard T, Harper JW, White KP, Schulman BA "Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases." Mol Cell 2009 Oct 12
In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated protein-interaction domain such as MATH recruits substrates for ubiquitination. Here, we present biochemical and structural analyses of the MATH-BTB protein, SPOP. We define a SPOP-binding consensus (SBC) and determine structures revealing recognition of SBCs from the phosphatase Puc, the transcriptional regulator Ci, and the chromatin component MacroH2A. We identify a dimeric SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB domains, which we call "3-box" due to its facilitating Cul3 binding and its resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility between the substrate-binding MATH and Cul3-binding BTB/3-box domains potentially allows a SPOP dimer to engage multiple SBCs found within a single substrate, such as Puc. These studies provide a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3 and how their dimerization and conformational variability may facilitate avid interactions with diverse substrates.
Machida,2009 (19815555) Machida YJ, Machida Y, Vashisht AA, Wohlschlegel JA, Dutta A "The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1." J Biol Chem 2009 Dec 4
The deubiquitinating enzyme BRCA1-associated protein 1 (BAP1) possesses growth inhibitory activity and functions as a tumor suppressor. In this study we report that BAP1 also plays positive roles in cell proliferation. BAP1 depletion by RNAi inhibits cell proliferation as does overexpression of a dominant negative mutant of BAP1. Mass spectrometry analyses of copurified proteins revealed that BAP1 is associated with factors involved in chromatin modulation and transcriptional regulation. We show that the interaction with host cell factor-1 (HCF-1), a cell-cycle regulator composed of HCF-1N and HCF-1C, is critical for the BAP1-mediated growth regulation. We found that HCF-1N is modified with Lys-48-linked polyubiquitin chains on its Kelch domain. The HCF-1 binding motif of BAP1 is required for interaction with HCF-1N and mediates deubiquitination of HCF-1N by BAP1. The importance of the BAP1-HCF-1 interaction is underscored by the fact that growth suppression by the dominant negative BAP1 mutant is entirely dependent on the HCF-1 binding motif. These results suggest that BAP1 regulates cell proliferation by deubiquitinating HCF-1.
Urata,2010 (19812267) Urata S, Yasuda J "Regulation of Marburg virus (MARV) budding by Nedd4.1: a different WW domain of Nedd4.1 is critical for binding to MARV and Ebola virus VP40." J Gen Virol 2010 Jan
The VP40 matrix protein of Marburg virus (MARV) has been shown to be the driving force behind MARV budding, a process in which the PPPY L-domain motif of VP40 plays a critical role. Here, we report that Vps4B and Nedd4.1 play critical roles in MARV VP40-mediated budding. We showed that unidentified activities of the Nedd4.1 HECT domain, along with its E3 ubiquitin ligase activity, may be required for MARV budding. Moreover, we showed that the first WW domain of Nedd4.1, WW1, is critical for binding to MARV VP40, indicating that MARV VP40 and Ebola virus VP40 are recognized by a different WW domain of Nedd4.1. This is the first report showing that the viral L-domains containing PPxY have specificities for binding to WW domains. Our findings provide new insights into MARV budding, which may contribute to the development of novel anti-MARV therapeutic strategies.
Berndt,2009 (19812159) Berndt A, Hofmann-Winkler H, Tavalai N, Hahn G, Stamminger T "Importance of covalent and noncovalent SUMO interactions with the major human cytomegalovirus transactivator IE2p86 for viral infection." J Virol 2009 Nov 20
The major transactivator protein IE2p86 of human cytomegalovirus (HCMV) has previously been shown to undergo posttranslational modification by the covalent attachment of SUMO proteins, termed SUMOylation, which occurs at two lysine residues located at amino acid positions 175 and 180. Mutation of the acceptor lysines resulted in the abrogation of IE2p86 SUMOylation in mammalian cells and a strong reduction of IE2p86-mediated transactivation. In this paper, we identify an additional SUMO interaction motif (SIM) within IE2p86, which mediates noncovalent binding to SUMO, as shown by yeast two-hybrid analyses. Transient-expression experiments revealed that an IE2p86 SIM mutant exhibited significantly reduced SUMOylation, strongly suggesting that noncovalent SUMO interactions affect the efficacy of covalent SUMO coupling. In order to define the relevance of IE2p86 SUMO interactions for viral replication, recombinant viruses originating from two different HCMV strains (AD169 and VR1814) were generated. Analysis of viruses expressing SUMOylation-negative IE2p86 revealed strongly impaired replication due to reduced viral DNA and protein accumulation, as well as diminished initiation of immediate-early gene expression. The additional introduction of the SIM mutation into the viral genome did not further compromise viral replication but resulted in altered expression of viral proteins at late times postinfection. In summary, this paper clearly shows that IE2p86 SUMOylation is necessary for efficient replication of the HCMV laboratory strain AD169 and the clinical isolate VR1814 and thus for the in vivo function of this viral transcription factor.
Yeap,2009 (19811652) Yeap LS, Hayashi K, Surani MA "ERG-associated protein with SET domain (ESET)-Oct4 interaction regulates pluripotency and represses the trophectoderm lineage." Epigenetics Chromatin 2009 Oct 20
BACKGROUND: Pluripotency, the capacity for indefinite self-renewal and differentiation into diverse cell types is a unique state exhibited by embryonic stem (ES) cells. Transcriptional regulators, such as Oct4, are critical for pluripotency, but the role of epigenetic modifiers remains to be fully elucidated. RESULTS: Here, we show that ERG-associated protein with SET domain (ESET), a histone methyltransferase enzyme, maintains pluripotency through repression of Cdx2, a key trophectoderm determinant, by histone H3 lysine 9 trimethylation (H3K9me3) of the promoter region. Notably, this repression is mediated through the synergistic function of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4. ESET localises to the promyelocytic leukaemia (PML) nuclear bodies and is SUMOylated in ES cells. Interaction of ESET with Oct4 depends on a SUMO-interacting motif (SIM) in Oct4, which is critical for the repression of Cdx2. CONCLUSION: Loss of ESET or Oct4 results in strikingly similar phenotypes both in ES cells with their differentiation into trophectoderm cells, and in early embryos where there is a failure of development of the pluripotent inner cell mass (ICM) of blastocysts. We propose that SUMOylated ESET-Oct4 complex is critical for both the initiation and maintenance of pluripotency through repression of differentiation, particularly of the trophectoderm lineage by epigenetic silencing of Cdx2.
Terry,2009 (19801417) Terry LJ, Wente SR "Flexible gates: dynamic topologies and functions for FG nucleoporins in nucleocytoplasmic transport." Eukaryot Cell 2009 Dec
The nuclear envelope is a physical barrier between the nucleus and cytoplasm and, as such, separates the mechanisms of transcription from translation. This compartmentalization of eukaryotic cells allows spatial regulation of gene expression; however, it also necessitates a mechanism for transport between the nucleus and cytoplasm. Macromolecular trafficking of protein and RNA occurs exclusively through nuclear pore complexes (NPCs), specialized channels spanning the nuclear envelope. A novel family of NPC proteins, the FG-nucleoporins (FG-Nups), coordinates and potentially regulates NPC translocation. The extensive repeats of phenylalanine-glycine (FG) in each FG-Nup directly bind to shuttling transport receptors moving through the NPC. In addition, FG-Nups are essential components of the nuclear permeability barrier. In this review, we discuss the structural features, cellular functions, and evolutionary conservation of the FG-Nups.
Ciccia,2009 (19793862) Ciccia A, Bredemeyer AL, Sowa ME, Terret ME, Jallepalli PV, Harper JW, Elledge SJ "The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart." Genes Dev 2009 Oct 16
The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.
Oya,2009 (19776015) Oya H, Yokoyama A, Yamaoka I, Fujiki R, Yonezawa M, Youn MY, Takada I, Kato S, Kitagawa H "Phosphorylation of Williams syndrome transcription factor by MAPK induces a switching between two distinct chromatin remodeling complexes." J Biol Chem 2009 Nov 20
Changes in the environment of a cell precipitate extracellular signals and sequential cascades of protein modification and elicit nuclear transcriptional responses. However, the functional links between intracellular signaling-dependent gene regulation and epigenetic regulation by chromatin-modifying proteins within the nucleus are largely unknown. Here, we describe novel epigenetic regulation by MAPK cascades that modulate formation of an ATP-dependent chromatin remodeling complex, WINAC (WSTF Including Nucleosome Assembly Complex), an SWI/SNF-type complex containing Williams syndrome transcription factor (WSTF). WSTF, a specific component of two chromatin remodeling complexes (SWI/SNF-type WINAC and ISWI-type WICH), was phosphorylated by the stimulation of MAPK cascades in vitro and in vivo. Ser-158 residue in the WAC (WSTF/Acf1/cbpq46) domain, located close to the N terminus of WSTF, was identified as a major phosphorylation target. Using biochemical analysis of a WSTF mutant (WSTF-S158A) stably expressing cell line, the phosphorylation of this residue (Ser-158) was found to be essential for maintaining the association between WSTF and core BAF complex components, thereby maintaining the ATPase activity of WINAC. WINAC-dependent transcriptional regulation of vitamin D receptor was consequently impaired by this WSTF mutation, but the recovery from DNA damage mediated by WICH was not impaired. Our results suggest that WSTF serves as a nuclear sensor of the extracellular signals to fine-tune the chromatin remodeling activity of WINAC. WINAC mediates a previously unknown MAPK-dependent step in epigenetic regulation, and this MAPK-dependent switching mechanism between the two functionally distinct WSTF-containing complexes might underlie the diverse functions of WSTF in various nuclear events.
Nechama,2009 (19770516) Nechama M, Uchida T, Mor Yosef-Levi I, Silver J, Naveh-Many T "The peptidyl-prolyl isomerase Pin1 determines parathyroid hormone mRNA levels and stability in rat models of secondary hyperparathyroidism." J Clin Invest 2009 Oct
Secondary hyperparathyroidism is a major complication of chronic kidney disease (CKD). In experimental models of secondary hyperparathyroidism induced by hypocalcemia or CKD, parathyroid hormone (PTH) mRNA levels increase due to increased PTH mRNA stability. K-homology splicing regulator protein (KSRP) decreases the stability of PTH mRNA upon binding a cis-acting element in the PTH mRNA 3' UTR region. As the peptidyl-prolyl isomerase (PPIase) Pin1 has recently been shown to regulate the turnover of multiple cytokine mRNAs, we investigated the role of Pin1 in regulating PTH mRNA stability in rat parathyroids and transfected cells. The data generated were consistent with Pin1 being a PTH mRNA destabilizing protein. Initial analysis indicated that Pin1 activity was decreased in parathyroid protein extracts from both hypocalcemic and CKD rats and that pharmacologic inhibition of Pin1 increased PTH mRNA levels posttranscriptionally in rat parathyroid and in transfected cells. Pin1 mediated its effects via interaction with KSRP, which led to KSRP dephosphorylation and activation. In the rat parathyroid, Pin1 inhibition decreased KSRP-PTH mRNA interactions, increasing PTH mRNA levels. Furthermore, Pin1-/- mice displayed increased serum PTH and PTH mRNA levels, suggesting that Pin1 determines basal PTH expression in vivo. These results demonstrate that Pin1 is a key mediator of PTH mRNA stability and indicate a role for Pin1 in the pathogenesis of secondary hyperparathyroidism in individuals with CKD.
Yu,2009 (19766626) Yu B, Shao Y, Zhang C, Chen Y, Zhong Q, Zhang J, Yang H, Zhang W, Wan J "BS69 undergoes SUMO modification and plays an inhibitory role in muscle and neuronal differentiation." Exp Cell Res 2009 Nov 25
BS69, an adenovirus E1A binding protein, has been described as a co-repressor in association with various transcription factors. But its characteristics and exact biological functions remain largely unknown at present. Now we intensively investigated the localization of BS69 and its various truncated derivatives and found that: (a) BS69 forms oligomer through its C-terminus and (b) both PHD and MYND domain are important for the localization of BS69. Furthermore, we provided evidence showing that BS69 interacts with PIAS1 (a well-characterized SUMO E3 enzyme) and Ubc9 (the only SUMO E2 enzyme so far identified) through its distinct regions. And PIAS1 significantly increases the SUMO modification of BS69. More importantly, in terms of the biological function of BS69, we found that BS69 plays an inhibitory role in the muscle and neuronal differentiation process. By taking advantage of several PHD and MYND domain mutants of BS69, we found that the PHD domain plays indispensable roles in the localization, sumoylation and function of BS69. Thus, our work contributed to the more intensive understanding of BS69.
Huang,2009 (19759537) Huang SM, Mishina YM, Liu S, Cheung A, Stegmeier F, Michaud GA, Charlat O, Wiellette E, Zhang Y, Wiessner S, Hild M, Shi X, Wilson CJ, Mickanin C, Myer V, Fazal A, Tomlinson R, Serluca F, Shao W, Cheng H, Shultz M, Rau C, Schirle M, Schlegl J, Ghidelli S, Fawell S, Lu C, Curtis D, Kirschner MW, Lengauer C, Finan PM, Tallarico JA, Bouwmeester T, Porter JA, Bauer A, Cong F "Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling." Nature 2009 Oct 1
The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.
Humphries,2009 (19738201) Humphries JD, Byron A, Bass MD, Craig SE, Pinney JW, Knight D, Humphries MJ "Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6." Sci Signal 2009 Sep 09
The binding of integrin adhesion receptors to their extracellular matrix ligands controls cell morphology, movement, survival, and differentiation in various developmental, homeostatic, and disease processes. Here, we report a methodology to isolate complexes associated with integrin adhesion receptors, which, like other receptor-associated signaling complexes, have been refractory to proteomic analysis. Quantitative, comparative analyses of the proteomes of two receptor-ligand pairs, alpha(4)beta(1)-vascular cell adhesion molecule-1 and alpha(5)beta(1)-fibronectin, defined both core and receptor-specific components. Regulator of chromosome condensation-2 (RCC2) was detected in the alpha(5)beta(1)-fibronectin signaling network at an intersection between the Rac1 and adenosine 5'-diphosphate ribosylation factor 6 (Arf6) subnetworks. RCC2 knockdown enhanced fibronectin-induced activation of both Rac1 and Arf6 and accelerated cell spreading, suggesting that RCC2 limits the signaling required for membrane protrusion and delivery. Dysregulation of Rac1 and Arf6 function by RCC2 knockdown also abolished persistent migration along fibronectin fibers, indicating a functional role for RCC2 in directional cell movement. This proteomics workflow now opens the way to further dissection and systems-level analyses of adhesion signaling.
Xia,2009 (19737939) Xia C, Misra I, Iyanagi T, Kim JJ "Regulation of interdomain interactions by calmodulin in inducible nitric-oxide synthase." J Biol Chem 2009 Oct 26
Nitric-oxide synthases (NOSs) catalyze the conversion of l-arginine to nitric oxide and citrulline. There are three NOS isozymes, each with a different physiological role: neuronal NOS, endothelial NOS, and inducible NOS (iNOS). NOSs consist of an N-terminal oxygenase domain and a C-terminal reductase domain, linked by a calmodulin (CaM)-binding region. CaM is required for NO production, but unlike other NOS isozymes, iNOS binds CaM independently of the exogenous Ca(2+) concentration. We have co-expressed CaM and the FMN domain of human iNOS, which includes the CaM-binding region. The Ca(2+)-bound protein complex (CaCaMxFMN) forms an air-stable semiquinone when reduced with NADPH and reduces cytochrome c when reconstituted with the iNOS FAD/NADPH domain. We have solved the crystal structure of the CaCaMxFMN complex in four different conformations, each with a different relative orientation, between the FMN domain and the bound CaM. The CaM-binding region together with bound CaM forms a hinge, pivots on the conserved Arg(536), and regulates electron transfer from FAD to FMN and from FMN to heme by adjusting the relative orientation and distance among the three cofactors. In addition, the relative orientations of the N- and C-terminal lobes of CaM are also different among the four conformations, suggesting that the flexibility between the two halves of CaM also contributes to the fine tuning of the orientation/distance between the redox centers. The data demonstrate a possible mode for precise control of electron transfer by altering the distance and orientation of redox centers in a protein displaying domain movement.
Dettori,2009 (19723632) Dettori R, Sonzogni S, Meyer L, Lopez-Garcia LA, Morrice NA, Zeuzem S, Engel M, Piiper A, Neimanis S, Frodin M, Biondi RM "Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)." J Biol Chem 2009 Oct 30
The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.
Kleino,2009 (19718658) Kleino I, Ortiz RM, Yritys M, Huovila AP, Saksela K "Alternative splicing of ADAM15 regulates its interactions with cellular SH3 proteins." J Cell Biochem 2009 Oct 26
A Disintegrin And Metalloprotease (ADAM15) is a member of the adamalysin protein family and has been associated with cancer, possibly via its role in ectodomain shedding of cadherins. Alternative mRNA splicing generates several ADAM15 isoforms containing different combinations of putative Src homology-3 (SH3) domain binding sites in their cytosolic tails. Here we present a comprehensive characterization of SH3 binding potential of different ADAM15 isoforms. Alternative use of ADAM15 exons was found to profoundly influence selection of SH3-containing cellular partner proteins, including the avid interactions with nephrocystin and sorting nexin-33 (SNX33 a.k.a. SNX30). Specifically, strong co-precipitation of nephrocystin from cell lysates was specific to ADAM15 isoforms i4, i5, and i6. These isoforms contain one or both of the two almost identical proline-rich regions encoded by exons 20 and 21, wherein the residues RxLPxxP were found to be indispensable for nephrocystin SH3 binding. Similarly, robust cellular association with SNX33 was observed only for ADAM15 isoforms containing the most carboxyterminal proline cluster lacking in isoforms i1 and i3. Thus, alternative mRNA splicing provides a versatile mechanism for regulation of intracellular protein interactions and thereby likely the cellular functions of ADAM15, which could explain the association with cancer of some but not all ADAM15 isoforms.
Fonseca,2009 (19716757) Fonseca S, Chico JM, Solano R "The jasmonate pathway: the ligand, the receptor and the core signalling module." Curr Opin Plant Biol 2009 Oct 05
Jasmonates regulate specific developmental processes and plant adaptation to environment by controlling responses to external biotic or abiotic stimuli. The core events of jasmonate signalling are now defined. After hormone perception by SCF(COI1), JAZ (JAsmonate ZIM domain) repressors are targeted for proteasome degradation, releasing MYC2 and de-repressing transcriptional activation. JAZs are homomeric and heteromeric proteins and have been instrumental in recent advances in the field, such as the identification of COI1 as a critical component of the jasmonate receptor and the discovery of the bioactive jasmonate in Arabidopsis, (+)-7-iso-JA-Ile. Small changes in jasmonate structure result in hormone inactivation and might be the key to switching-off signalling for specific responses to stimulus and for long-distance signalling events.
Fabian,2009 (19716330) Fabian MR, Mathonnet G, Sundermeier T, Mathys H, Zipprich JT, Svitkin YV, Rivas F, Jinek M, Wohlschlegel J, Doudna JA, Chen CY, Shyu AB, Yates JR 3rd, Hannon GJ, Filipowicz W, Duchaine TF, Sonenberg N "Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation." Mol Cell 2009 Sep 28
MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.
Schmidt,2009 (19706427) Schmidt MR, Maritzen T, Kukhtina V, Higman VA, Doglio L, Barak NN, Strauss H, Oschkinat H, Dotti CG, Haucke V "Regulation of endosomal membrane traffic by a Gadkin/AP-1/kinesin KIF5 complex." Proc Natl Acad Sci U S A 2009 Oct 06
Endosomes and endosomal vesicles (EVs) rapidly move along cytoskeletal filaments allowing them to exchange proteins and lipids between different endosomal compartments, lysosomes, the trans-Golgi network (TGN), and the plasma membrane. The precise mechanisms that connect membrane traffic between the TGN and perinuclear endosomal compartments with motor-protein driven transport have largely remained elusive. Here we show that Gadkin (also termed gamma-BAR), a peripheral membrane protein localized to the TGN and to TGN-derived EVs, directly associates with the clathrin adaptor AP-1 and with the motor protein kinesin KIF5, thereby potentially regulating EV dynamics. Gadkin overexpression induced the dispersion of transferrin (Tf)- and Rab4-positive EVs to the cell periphery, whereas KIF5B-depleted cells displayed a perinuclear concentration. Functional experiments suggest that the role of Gadkin as a regulator of endosomal membrane traffic critically depends on complex formation with both AP-1 and KIF5. Our data thus provide a direct molecular link between TGN-derived EVs and the microtubule-based cytoskeleton.
Beamish,2009 (19703905) Beamish H, de Boer L, Giles N, Stevens F, Oakes V, Gabrielli B "Cyclin A/cdk2 regulates adenomatous polyposis coli-dependent mitotic spindle anchoring." J Biol Chem 2009 Oct 12
Mutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-beta-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis.
Barczyk,2009 (19693543) Barczyk M, Carracedo S, Gullberg D "Integrins." Cell Tissue Res 2009 Nov 30
Integrins are cell adhesion receptors that are evolutionary old and that play important roles during developmental and pathological processes. The integrin family is composed of 24 alphabeta heterodimeric members that mediate the attachment of cells to the extracellular matrix (ECM) but that also take part in specialized cell-cell interactions. Only a subset of integrins (8 out of 24) recognizes the RGD sequence in the native ligands. In some ECM molecules, such as collagen and certain laminin isoforms, the RGD sequences are exposed upon denaturation or proteolytic cleavage, allowing cells to bind these ligands by using RGD-binding receptors. Proteolytic cleavage of ECM proteins might also generate fragments with novel biological activity such as endostatin, tumstatin, and endorepellin. Nine integrin chains contain an alphaI domain, including the collagen-binding integrins alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1. The collagen-binding integrins recognize the triple-helical GFOGER sequence in the major collagens, but their ability to recognize these sequences in vivo is dependent on the fibrillar status and accessibility of the interactive domains in the fibrillar collagens. The current review summarizes some basic facts about the integrin family including a historical perspective, their structure, and their ligand-binding properties.
Cordeddu,2009 (19684605) Cordeddu V, Di Schiavi E, Pennacchio LA, Ma'ayan A, Sarkozy A, Fodale V, Cecchetti S, Cardinale A, Martin J, Schackwitz W, Lipzen A, Zampino G, Mazzanti L, Digilio MC, Martinelli S, Flex E, Lepri F, Bartholdi D, Kutsche K, Ferrero GB, Anichini C, Selicorni A, Rossi C, Tenconi R, Zenker M, Merlo D, Dallapiccola B, Iyengar R, Bazzicalupo P, Gelb BD, Tartaglia M "Mutation of SHOC2 promotes aberrant protein N-myristoylation and causes Noonan-like syndrome with loose anagen hair." Nat Genet 2009 Aug 27
N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat-containing protein that positively modulates RAS-MAPK signal flow, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721) shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2(S2G) in vitro enhanced MAPK activation in a cell type-specific fashion. Induction of SHOC2(S2G) in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.
Kristie,2010 (19682612) Kristie TM, Liang Y, Vogel JL "Control of alpha-herpesvirus IE gene expression by HCF-1 coupled chromatin modification activities." Biochim Biophys Acta 2010 Mar-Apr
The immediate early genes of the alpha-herpesviruses HSV and VZV are transcriptionally regulated by viral and cellular factors in a complex combinatorial manner. Despite this complexity and the apparent redundancy of activators, the expression of the viral IE genes is critically dependent upon the cellular transcriptional coactivator HCF-1. Although the role of HCF-1 had remained elusive, recent studies have demonstrated that the protein is a component of multiple chromatin modification complexes including the Set1/MLL1 histone H3K4 methyltransferases. Studies using model viral promoter-reporter systems as well as analyses of components recruited to the viral genome during the initiation of infection have elucidated the significance of HCF-1 chromatin modification complexes in contributing to the final state of modified histones assembled on the viral IE promoters. Strikingly, the absence of HCF-1 results in the accumulation of nucleosomes bearing repressive marks on the viral IE promoters and silencing of viral gene expression.
Watanabe,2009 (19679663) Watanabe M, Wake H, Moorhouse AJ, Nabekura J "Clustering of neuronal K+-Cl- cotransporters in lipid rafts by tyrosine phosphorylation." J Biol Chem 2009 Oct 05
The neuronal K(+)-Cl(-) cotransporter (KCC2) is a membrane transport protein that extrudes Cl(-) from neurons and helps maintain low intracellular [Cl(-)] and hyperpolarizing GABAergic synaptic potentials. Depolarizing gamma-aminobutyric acid (GABA) responses in neonatal neurons and following various forms of neuronal injury are associated with reduced levels of KCC2 expression. Despite the importance for plasticity of inhibitory transmission, less is known about cellular mechanisms involved in more dynamic changes in KCC2 function. In this study, we investigated the role of tyrosine phosphorylation in KCC2 localization and function in hippocampal neurons and in cultured GT1-7 cells. Mutation to the putative tyrosine phosphorylation site within the long intracellular carboxyl terminus of KCC2(Y1087D) or application of the tyrosine kinase inhibitor genistein shifted the GABA reversal potential (E(GABA)) to more depolarized values, indicating reduced KCC2 function. This was associated with a change in the expression pattern of KCC2 from a punctate distribution to a more uniform distribution, suggesting that functional tyrosine-phosphorylated KCC2 forms clusters in restricted membrane domains. Sodium vanadate, a tyrosine phosphatase inhibitor, increased the proportion of KCC2 associated with lipid rafts membrane domains. Loss of tyrosine phosphorylation also reduced oligomerization of KCC2. A loss of the punctuate distribution and oligomerization of KCC2 and a more depolarized E(GABA) were seen when the 28-amino-acid carboxyl terminus of KCC2 was deleted. These results indicate that direct tyrosine phosphorylation of KCC2 results in membrane clusters and functional transport activity, suggesting a mechanism by which intracellular Cl(-) concentrations and GABA responses can be rapidly modulated.
Kostelecky,2009 (19662078) Kostelecky B, Saurin AT, Purkiss A, Parker PJ, McDonald NQ "Recognition of an intra-chain tandem 14-3-3 binding site within PKCepsilon." EMBO Rep 2009 Sep 01
The phosphoserine/threonine binding protein 14-3-3 stimulates the catalytic activity of protein kinase C-epsilon (PKCepsilon) by engaging two tandem phosphoserine-containing motifs located between the PKCepsilon regulatory and catalytic domains (V3 region). Interaction between 14-3-3 and this region of PKCepsilon is essential for the completion of cytokinesis. Here, we report the crystal structure of 14-3-3zeta bound to a synthetic diphosphorylated PKCepsilon V3 region revealing how a consensus 14-3-3 site and a divergent 14-3-3 site cooperate to bind to 14-3-3 and so activate PKCepsilon. Thermodynamic data show a markedly enhanced binding affinity for two-site phosphopeptides over single-site 14-3-3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14-3-3 ligand. This dual-site intra-chain recognition has implications for other 14-3-3 targets, which seem to have only a single 14-3-3 motif, as other lower affinity and cryptic 14-3-3 gatekeeper sites might exist.
El Firar,2009 (19661287) El Firar A, Voisin T, Rouyer-Fessard C, Ostuni MA, Couvineau A, Laburthe M "Discovery of a functional immunoreceptor tyrosine-based switch motif in a 7-transmembrane-spanning receptor: role in the orexin receptor OX1R-driven apoptosis." FASEB J 2009 Dec 01
The orexin neuropeptides promote robust apoptosis in cancer cells. We have recently shown that the 7-transmembrane-spanning orexin receptor OX1R mediates apoptosis through an original mechanism. OX1R is equipped with a tyrosine-based inhibitory motif ITIM, which is tyrosine-phosphorylated on receptor activation, allowing the recruitment and activation of the tyrosine phosphatase SHP-2, leading to apoptosis. We show here that another motif, immunoreceptor tyrosine-based switch motif (ITSM), is present in OX1R and is mandatory for OX1R-mediated apoptosis. This conclusion is based on the following observations: 1) a canonical ITSM sequence is present in the first intracellular loop of OX1R; 2) mutation of Y(83) to F within ITSM abolished OX1R-mediated apoptosis but did not alter orexin-induced inositol phosphate formation or calcium transient via coupling of OX1R to G(q) protein; 3) mutation of Y(83) to F further abolished orexin-induced tyrosine phosphorylation in ITSM and subsequent recruitment of SHP-2 by the receptor. Finally, we developed a structural model of OX1R showing that the spatial localization of phosphotyrosines in ITSM and ITIM in OX1R is compatible with their interaction with the two SH2 domains of SHP-2. These data represent the first evidence for a functional role of an ITSM in a 7-transmembrane-spanning receptor.
Hughes,2009 (19656877) Hughes M, Gretton S, Shelton H, Brown DD, McCormick CJ, Angus AG, Patel AH, Griffin S, Harris M "A conserved proline between domains II and III of hepatitis C virus NS5A influences both RNA replication and virus assembly." J Virol 2009 Oct
We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.
Nelsen,2009 (19651771) Nelsen SM, Christian JL "Site-specific cleavage of BMP4 by furin, PC6, and PC7." J Biol Chem 2009 Oct 05
Bone morphogenetic proteins (BMPs) require proteolytic activation by members of the proprotein convertase (PC) family. Pro-BMP4 is initially cleaved at a site adjacent to the mature ligand domain (S1) and then at an upstream site (S2) within the prodomain. Cleavage at the S2 site, which appears to occur in a tissue-specific fashion, regulates the activity and signaling range of mature BMP4. To test the hypothesis that tissue-specific cleavage of pro-BMP4 is regulated by differential expression of a site-specific protease, we identified the PCs that cleave each site in vivo. In Xenopus oocytes, furin and PC6 function redundantly to cleave both the S1 and S2 sites of pro-BMP4, as evidenced by the results of antisense-mediated gene knockdown and the use of the furin- and PC6-selective inhibitor alpha(1)-PDX. By contrast, alpha(1)-PDX blocked cleavage of the S2 but not the S1 site of pro-BMP4 in embryos, suggesting the existence of a developmentally regulated S1 site-specific convertase. This protease is likely to be PC7 based on knowledge of its required substrate cleavage motif and resistance to alpha(1)-PDX. Consistent with this prediction, an alpha(1)-PDX variant engineered to target PC7, in addition to furin and PC6, completely inhibited cleavage of BMP4 in oocytes and embryos. Further studies showed that pc7 transcripts are expressed and polyadenylated, and that the PC7 precursor protein undergoes efficient autocatalytic activation in both oocytes and embryos. These results suggest that PC7, or a convertase with similar substrate specificity, functions to selectively cleave the S1 site of pro-BMP4 in a developmentally regulated fashion.
Lomonosova,2009 (19641503) Lomonosova E, Chinnadurai G "BH3-only proteins in apoptosis and beyond: an overview." Oncogene 2009 Jul 30
BH3-only BCL-2 family proteins are effectors of canonical mitochondrial apoptosis. They discharge their pro-apoptotic functions through BH1-3 pro-apoptotic proteins such as BAX and BAK, while their activity is suppressed by BH1-4 anti-apoptotic BCL-2 family members. The precise mechanism by which BH3-only proteins mediate apoptosis remains unresolved. The existing data are consistent with three mutually non-exclusive models (1) displacement of BH1-3 proteins from complexes with BH1-4 proteins; (2) direct interaction with and conformational activation of BH1-3 proteins; and (3) membrane insertion and membrane remodeling. The BH3-only proteins appear to play critical roles in restraining cancer and inflammatory diseases such as rheumatoid arthritis. Molecules that mimic the effect of BH3-only proteins are being used in treatments against these diseases. The cell death activity of a subclass of BH3-only members (BNIP3 and BNIP3L) is linked to cardiomyocyte loss during heart failure. In addition to their established role in apoptosis, several BH3-only members also regulate diverse cellular functions in cell-cycle regulation, DNA repair and metabolism. Several members are implicated in the induction of autophagy and autophagic cell death, possibly through unleashing of the BH3-only autophagic effector Beclin 1 from complexes with BCL-2/BCL-xL. The Chapters included in the current Oncogene Review issues provide in-depth discussions on various aspects of major BH3-only proteins.
van der Horst,2009 (19638580) van der Horst A, Khanna KK "The peptidyl-prolyl isomerase Pin1 regulates cytokinesis through Cep55." Cancer Res 2009 Aug 15
Failure of cytokinesis results in tetraploidy and can increase the genomic instability frequently observed in cancer. The peptidyl-prolyl isomerase Pin1, which is deregulated in many tumors, regulates several processes, including cell cycle progression. Here, we show a novel role for Pin1 in cytokinesis. Pin1 knockout mouse embryonic fibroblasts show a cytokinesis delay, and depletion of Pin1 from HeLa cells also causes a cytokinesis defect. Furthermore, we provide evidence that Pin1 localizes to the midbody ring and regulates the final stages of cytokinesis by binding to centrosome protein 55 kDa (Cep55), an essential component of this ring. This interaction induces Polo-like kinase 1-mediated phosphorylation of Cep55, which is critical for the function of Cep55 during cytokinesis. Importantly, Pin1 knockdown does not enhance the cytokinesis defect in Cep55-depleted cells, indicating that Pin1 and Cep55 act in the same pathway. These data are the first evidence that Pin1 regulates cytokinesis and may provide a mechanistic explanation as to how pathologic levels of Pin1 can stimulate tumorigenesis.
Yan,2009 (19635485) Yan X, Zhou H, Zhang J, Shi C, Xie X, Wu Y, Tian C, Shen Y, Long J "Molecular mechanism of inward rectifier potassium channel 2.3 regulation by tax-interacting protein-1." J Mol Biol 2009 Oct 2
Inwardly rectifying potassium channel 2.3 (Kir2.3) is specifically targeted on the basolateral membranes of epithelial and neuronal cells, and it thus plays an important role in maintaining potassium homeostasis. Tax-interacting protein-1 (TIP-1), an atypical PDZ-domain-containing protein, binds to Kir2.3 with a high affinity, causing the intracellular accumulation of Kir2.3 in cultured epithelial cells. However, the molecular basis of the TIP-1/Kir2.3 interaction is still poorly understood. Here, we present the crystal structure of TIP-1 in complex with the C-terminal Kir2.3-peptide (residues 436-445) to reveal the molecular details of the interaction between them. Moreover, isothermal titration calorimetry experiments show that the C-terminal Kir2.3-peptide binds much more strongly to TIP-1 than to mammalian Lin-7, indicating that TIP-1 can compete with mammalian Lin-7 to uncouple Kir2.3 from its basolateral membrane anchoring complex. We further show that the phosphorylation/dephosphorylation of Ser443 within the C-terminal Kir2.3 PDZ-binding motif RRESAI dynamically regulates the Kir2.3/TIP-1 association in heterologous HEK293T cells. These data suggest that TIP-1 may act as an important regulator for the endocytic pathway of Kir2.3.
Honnappa,2009 (19632184) Honnappa S, Gouveia SM, Weisbrich A, Damberger FF, Bhavesh NS, Jawhari H, Grigoriev I, van Rijssel FJ, Buey RM, Lawera A, Jelesarov I, Winkler FK, Wuthrich K, Akhmanova A, Steinmetz MO "An EB1-binding motif acts as a microtubule tip localization signal." Cell 2009 Jul 23
Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.
Messner,2009 (19622798) Messner S, Schuermann D, Altmeyer M, Kassner I, Schmidt D, Schar P, Muller S, Hottiger MO "Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function." FASEB J 2009 Oct 30
Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein and functions as a molecular stress sensor. At the cellular level, PARP1 has been implicated in a wide range of processes, such as maintenance of genome stability, cell death, and transcription. PARP1 functions as a transcriptional coactivator of nuclear factor kappaB (NF-kappaB) and hypoxia inducible factor 1 (HIF1). In proteomic studies, PARP1 was found to be modified by small ubiquitin-like modifiers (SUMOs). Here, we characterize PARP1 as a substrate for modification by SUMO1 and SUMO3, both in vitro and in vivo. PARP1 is sumoylated at the single lysine residue K486 within its automodification domain. Interestingly, modification of PARP1 with SUMO does not affect its ADP-ribosylation activity but completely abrogates p300-mediated acetylation of PARP1, revealing an intriguing crosstalk of sumoylation and acetylation on PARP1. Genetic complementation of PARP1-depleted cells with wild-type and sumoylation-deficient PARP1 revealed that SUMO modification of PARP1 restrains its transcriptional coactivator function and subsequently reduces gene expression of distinct PARP1-regulated target genes.
MacDonald,2009 (19619488) MacDonald BT, Tamai K, He X "Wnt/beta-catenin signaling: components, mechanisms, and diseases." Dev Cell 2009 Jul 21
Signaling by the Wnt family of secreted glycolipoproteins via the transcriptional coactivator beta-catenin controls embryonic development and adult homeostasis. Here we review recent progress in this so-called canonical Wnt signaling pathway. We discuss Wnt ligands, agonists, and antagonists, and their interactions with Wnt receptors. We also dissect critical events that regulate beta-catenin stability, from Wnt receptors to the cytoplasmic beta-catenin destruction complex, and nuclear machinery that mediates beta-catenin-dependent transcription. Finally, we highlight some key aspects of Wnt/beta-catenin signaling in human diseases including congenital malformations, cancer, and osteoporosis, and discuss potential therapeutic implications.
Theisgen,2010 (19616509) Theisgen S, Scheidt HA, Magalhaes A, Bonagamba TJ, Huster D "A solid-state NMR study of the structure and dynamics of the myristoylated N-terminus of the guanylate cyclase-activating protein-2." Biochim Biophys Acta 2010 Jan 27
Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca(2+) sensor protein. It plays a central role in shaping the photoreceptor light response and in light adaptation through the Ca(2+)-dependent regulation of the transmembrane retinal guanylate cyclase (GC). GCAP-2 is N-terminally myristoylated and the full activation of the GC requires this lipid modification. The structural and functional role of the N-terminus and particularly of the myristoyl moiety is currently not well understood. In particular, detailed structural information on the myristoylated N-terminus in the presence of membranes was not available. Therefore, we studied the structure and dynamics of a 19 amino acid peptide representing the myristoylated N-terminus of GCAP-2 bound to lipid membranes by solid-state NMR. (13)C isotropic chemical shifts revealed a random coiled secondary structure of the peptide. Peptide segments up to Ala(9) interact with the membrane surface. Order parameters for Calpha and side chain carbons obtained from DIPSHIFT experiments are relatively low, suggesting high mobility of the membrane-associated peptide. Static (2)H solid-state NMR measurements show that the myristoyl moiety is fully incorporated into the lipid membrane. The parameters of the myristoyl moiety and the DMPC host membrane are quite similar. Furthermore, dynamic parameters (obtained from (2)H NMR relaxation rates) of the peptide's myristic acid chain are also comparable to those of the lipid chains of the host matrix. Therefore, the myristoyl moiety of the N-terminal peptide of GCAP-2 fills a similar conformational space as the surrounding phospholipid chains.
Oliver,2009 (19609323) Oliver AW, Swift S, Lord CJ, Ashworth A, Pearl LH "Structural basis for recruitment of BRCA2 by PALB2." EMBO Rep 2009 Sep 01
The breast cancer 2, early onset protein (BRCA2) is central to the repair of DNA damage by homologous recombination. BRCA2 recruits the recombinase RAD51 to sites of damage, regulates its assembly into nucleoprotein filaments and thereby promotes homologous recombination. Localization of BRCA2 to nuclear foci requires its association with the partner and localizer of BRCA2 (PALB2), mutations in which are associated with cancer predisposition, as well as subtype N of Fanconi anaemia. We have determined the structure of the PALB2 carboxy-terminal beta-propeller domain in complex with a BRCA2 peptide. The structure shows the molecular determinants of this important protein-protein interaction and explains the effects of both cancer-associated truncating mutants in PALB2 and missense mutations in the amino-terminal region of BRCA2.
Mohd-Ismail,2009 (19605477) Mohd-Ismail NK, Deng L, Sukumaran SK, Yu VC, Hotta H, Tan YJ "The hepatitis C virus core protein contains a BH3 domain that regulates apoptosis through specific interaction with human Mcl-1." J Virol 2009 Sep 09
The hepatitis C virus (HCV) core protein is known to modulate apoptosis and contribute to viral replication and pathogenesis. In this study, we have identified a Bcl-2 homology 3 (BH3) domain in the core protein that is essential for its proapoptotic property. Coimmunoprecipitation experiments showed that the core protein interacts specifically with the human myeloid cell factor 1 (Mcl-1), a prosurvival member of the Bcl-2 family, but not with other prosurvival members (Bcl-X(L) and Bcl-w). Moreover, the overexpression of Mcl-1 protects against core-induced apoptosis. By using peptide mimetics, core was found to release cytochrome c from isolated mitochondria when complemented with Bad. Thus, core is a bona fide BH3-only protein having properties similar to those of Noxa, a BH3-only member of the Bcl-2 family that binds preferentially to Mcl-1. There are three critical hydrophobic residues in the BH3 domain of the core protein, and they are essential for the proapoptotic property of the core protein. Furthermore, the genotype 1b core protein is more effective than the genotype 2a core protein in inducing apoptosis due to a single-amino-acid difference at one of these hydrophobic residues (residue 119). Replacing this residue in the J6/JFH-1 infectious clone (genotype 2a) with the corresponding amino acid in the genotype 1b core protein produced a mutant virus, J6/JFH-1(V119L), which induced significantly higher levels of apoptosis in the infected cells than the parental J6/JFH-1 virus. Furthermore, the core protein of J6/JFH-1(V119L), but not that of J6/JFH-1, interacted with Mcl-1 in virus-infected cells. Taken together, the core protein is a novel BH3-only viral homologue that contributes to the induction of apoptosis during HCV infection.
Tamayev,2009 (19602287) Tamayev R, Zhou D, D'Adamio L "The interactome of the amyloid beta precursor protein family members is shaped by phosphorylation of their intracellular domains." Mol Neurodegener 2009 Aug 11
ABSTRACT: BACKGROUND: Brain tissue from patients with Alzheimer's disease has shown an increase of phosphorylation of Tyr-682, located on the conserved Y682ENPTY motif, and Thr-668 residues, both in the intracellular domain (AID) of amyloid beta precursor protein (APP), although the role of these two residues is not yet known. RESULTS: Here, we report that the phosphorylation status of Tyr-682, and in some cases Thr-668, shapes the APP interactome. It creates a docking site for SH2-domain containing proteins, such as ShcA, ShcB, ShcC, Grb7, Grb2, as well as adapter proteins, such as Crk and Nck, that regulate important biological processes, cytosolic tyrosine kinases, such as Abl, Lyn and Src, which regulate signal transduction pathways, and enzymes that control phosphatidylinositols levels and signaling, such as PLC-gamma. At the same time, it either reduces (like for JIP1, NUMB, NUMBL and ARH) or abolishes (like for Fe65, Fe65L1 and Fe65L2) binding of other APP interactors. Phosphorylation of Thr-668, unlike Tyr-682, does not seem