The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
SCF ubiquitin ligase binding Phosphodegrons
Functional site description:
Several phosphodegrons are required for cell state-dependent recognition of regulatory proteins by SCF complexes via repeat domains of associated F box proteins (FBPs) and their subsequent ubiquitin-mediated degradation. The SCF-FBW7 and the SCF-betaTrCP1 motifs, contain two phosphorylated residues, which are recognised via a WD40 domain. For example, the SCF-FBW7 degron TPxxS is found in cyclin E, which is required for the G1/S transition. The SCF-betaTrCP1 degron DSGxxS operates in a broader range of cell regulation. For example, NF-kappa-B inhibitors are phosphorylated and destroyed under immune stimulation while beta-catenin is degraded in the absence of Wnt signalling. Skp2, another FBP, recognises cell cycle regulators via its leucine-rich repeat. In case of the single-phosphorylated DEG_SCF_Skp2-Cks1_1 motif, Skp2 requires additional binding of Cks1 for recognition. So far, only a few cell cycle inhibitors, including p27Kip1 that is mainly involved in G1 arrest, have been found to carry this degron.
ELMs with same func. site: DEG_SCF_FBW7_1  DEG_SCF_FBW7_2  DEG_SCF_SKP2-CKS1_1  DEG_SCF_TRCP1_1 
ELM Description:
The Skp2 F box protein recognises substrates via its leucine-rich repeats (LRRs). However, the DEG_SCF_Skp2-Cks1_1 phosphodegron uniquely requires a pre-assembled target recognition site composed of Skp2 and Cks1. The motif is present in the Cip/Kip protein family members p27Kip1 and p57Kip2 and partially overlaps with the MOD_CDK_1 motif, which is required for phosphorylation of the threonine at the fifth position. The third and fifth motif residues are most relevant for the interaction. The fifth position always contains a phosphorylated threonine, which binds with high specificity into a positively charged pocket on Cks1. In p21Cip1, another Cip/Kip family protein, the threonine within a similar motif is substituted by a serine. However, there is no MOD_CDK_1 motif in p21Cip1. Furthermore, it is doubtful whether a phosphoserine fits into the binding pocket since it is likely that the additional methyl group of threonine interacts with Cks1 as well. Glutamate appears to be the third DEG_SCF_Skp2-Cks1_1 motif position and is highly conserved throughout the Cip/Kip protein family in Vertebrates. It forms hydrogen bonds with both Cks1 and Skp2, and therefore a tripartite interface is formed. Members of the retinoblastoma protein family, e.g. p130 and pRb, contain a similar DEG_SCF_Skp2-Cks1_1 motif, however, the conserved E is substituted by Y and no MOD_CDK_1 motif exists nearby. Computational structure mutations revealed that Y could also fit into the deep pocket formed by Cks1 and Skp2 but further experimental evidence is required to validate this hypothesis. The p130-specific YxSP motif is highly conserved in Vertebrates, though several Metazoans and plants show an F instead of a Y at the same position. The proline at the fourth position assists in binding by forming van der Waals interactions with a Cks1 tyrosine. The lysine at position 6 is part of the Cdk phosphorylation recognition site. This basic residue interacts with a negatively charged pocket of Cks1 considering the crystal structure 2AST.
Pattern: ..[DE].(T)P.K
Pattern Probability: 0.0000478
Present in taxon: Metazoa
Interaction Domain:
LRR_1 (PF00560) Leucine-rich repeat (Stochiometry: 2 : 1)
PDB Structure: 2AST
o See 3 Instances for DEG_SCF_SKP2-CKS1_1
o Abstract
Ubiquitin-mediated proteolysis has diverse regulatory functions in eukaryotic cells (Hershko,1998). The role of ubiquitylation in regulating cell state by targeting proteins for proteasomal destruction is a major field of research. The ubiquitylation of a specific protein is performed by an ubiquitin-protein ligase (also known as E3). Ubiquitin is covalently bound to an ubiquitin-activating enzyme (E1), which transfers the ubiquitin to an ubiquitin-conjugating enzyme (E2). The E2 binds to the E3 ligase, which specifically recognizes the target protein (Kamura,2003). There are two major types of E3 enzymes that ubiquitylate the substrate in different ways: HECT-type E3s first form an E3-ubiquitin thioester conjugate and then transfer the ubiquitin to the substrate. In contrast, RING-type E3s do not form this thioester bond but interact directly with E2 (reviewed in Pickart,2002). The SCF (Skp1-Cullin-F box) is a RING-type E3 consisting of four subunits. These are the scaffold protein Cul1, the RING-domain protein Rbx1/Roc1, the adaptor protein Skp1, and an F box protein that specifically recognises the substrate. F box proteins often contain WD40 beta propeller or leucine-rich repeat (LRR) modules, which interact with one or more short sequences, termed degrons, in their target substrates. Several F box proteins (FBPs), including Cdc4 and Grr1 in yeast, COI1 in plants and betaTrCP, Skp2 and Fbw7 (Fbxw7, hCdc4) in human, recognise phosphorylated degrons in their substrates.
Structural and biochemical analysis of three FBPs, betaTrCP, FBW7 and Skp2, improved our understanding of the nature of their interactions with specific substrates. In a first step, single or double phosphorylation of their targets by kinases is required and serves as the ubiquitylation signal. In case of betaTrCP and FBW7 it has been revealed that short phosphodegrons containing two phosphorylated residues bind simultaneously to two phosphate-binding sites on the WD40 domain of the FBPs (Wu,2003; Hao,2007). On the other hand, Skp2 primarily binds substrates via its LRRs. However, in addition to Skp2, the DEG_SCF_Skp2-Cks1_1 motif requires interaction with the accessory Cks1 protein (cyclin-dependent kinases regulatory subunit 1) for its recognition. The Skp2-Cks1 degrons contain only one phosphorylated residue, which binds to Cks1 (Hao,2005). Well characterised phosphodegrons recognised by the ubiquitin ligase system are those of cyclins and cyclin-dependent kinase (Cdk) inhibitors. In yeast, the Cln-cdc28 kinase phosphorylates TPxxS motifs on Sic1 (an inhibitor of the cyclin B regulated kinase) thereby targeting it for degradation and enabling entry into S phase. The FBP Cdc4 recognizes the phosphorylated Sic1. The human ortholog of Cdc4, FBW7, interacts with phosphodegrons of cyclin E, c-Myc and c-Jun (DEG_SCF_FBW7_1; DEG_SCF_FBW7_2). The DEG_SCF-TrCP1_1 destruction motif may play a role in the G2/M checkpoint (e.g. by degrading claspin), but most substrates are regulators of cell state rather than purely responsible for cell cycle. DSGxxS phosphodegrons are found in beta-catenin, several IkB paralogues, Bora as well as the cytosolic side of Prolactin receptor and various other transmembrane receptors.
Two members of the Cdk inhibitor protein (Cip/Kip) family, p27Kip1 and p57Kip2, have been shown to contain a functional DEG_SCF_Skp2-Cks1_1 motif, which is recognized by Skp2-Cks1. Skp2 probably recognises other ubiquitylation targets via its LRRs and without supplementary binding of Cks1, e.g. Tob1 and Cdt1 (Zhang,2006). However, recognition of the DEG_SCF_Skp2-Cks1_1 motif requires binding of Cks1 to Skp2. Cks proteins are known as mutation suppressors and regulators of Cdks (Harper,2001) but only Cks1 seems to be able to bind Skp2 and play a role in Cdk inhibitor degradation. p27Kip1 and p57Kip2 (Hao,2005) play a role in maintenance of G1 phase and their degradation occurs during G1/S phase transition. Both proteins specifically inhibit Cdk2-cyclin A or E. In turn, these Cdk-cyclin complexes phosphorylate these Cip/Kip proteins at the motif site as soon as their levels exceed Cdk inhibitor levels in late G1 phase, thereby inducing motif recognition by the Skp2-Cks1 complex and subsequent ubiquitylation and degradation. After mediating relief of CDK inhibition, Cks1 will further regulate CDK activity by acting as a phospho-dependent docking site for cyclin-CDK substrates (DOC_CKS1_1), thereby increasing the specificity and efficiency of substrate phosphorylation by CDK (Koivomagi,2013). The DEG_SCF_Skp2-Cks1_1 motif binds with low affinity to its recognition site since it consists of few amino acids. However, the effective affinity is enhanced by a Cdk-cyclin complex that binds with high affinity across both the substrate targeted for ubiquitylation and the E3 ligase, thereby tightening the motif interaction (Xu,2007). Since Cip/Kip proteins are involved in cell cycle inhibition, they are altered in a broad variety of tumours, e.g. breast and bladder cancers. These alterations mainly comprise reduced expression or increased degradation as well as loss of heterozygosity through locus mutations (Tedesco,2002; Nakayama,1999). Some viruses have been found to use DEG_SCF_TRCP1_1 motifs for perturbation of cellular regulation. A TPxxS-like motif is present in SV40 Large T (ELMI001402) while DSGxxS pseudosubstrate motifs are found in the HIV VPU (ELMI001315) and EBV LMP1 (ELMI001315) proteins and are thought to mop up any available betaTrCP.
o 3 selected references:

o 8 GO-Terms:

o 3 Instances for DEG_SCF_SKP2-CKS1_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P49919 Cdkn1c
338 345 SPNVAPGVGAVEQTPRKRLR TP 4 Mus musculus (House mouse)
P49918 CDKN1C
306 313 SPSAAPGVGSVEQTPRKRLR TP 4 Homo sapiens (Human)
P46527 CDKN1B
183 190 GSPNAGSVEQTPKKPGLRRR TP 3 Homo sapiens (Human)
Please cite: ELM 2016-data update and new functionality of the eukaryotic linear motif resource. (PMID:26615199)

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