The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
Cyclin N-terminal Domain Docking Motifs
Functional site description:
Cyclin-dependent kinases (Cdks) coordinate hundreds of molecular events during the cell cycle via Ser/Thr phosphorylation. With cell cycle progression, different cyclins bind to Cdks to control their function by providing docking sites for substrates and also by modulating Cdk active site specificity. Docking motifs control the timing of cell cycle events by enabling preferential interaction and phosphorylation of substrates by a specific cyclin/Cdk complex. Cyclins use the conserved hydrophobic pocket (hp) to bind docking motifs on partner proteins. In the budding yeast, the divergence of the hp has given rise to a family of related RxL-like docking motifs consisting of a hydrophobic core modulated by positively charged (RxLF, RxLxF) or hydrophobic (LxF, PxF, NLxxxL) residues. Cyclins may use additional surfaces to dock substrates, as with the mammalian Cyclin D-specific (DOC_CYCLIN_D_Helix_1) and the budding yeast Cln2-specific leucine- and proline-rich LP (DOC_CYCLIN_yCln2_LP_2) motifs.
ELMs with same func. site: DOC_CYCLIN_RxL_1  DOC_CYCLIN_yClb1_LxF_4  DOC_CYCLIN_yClb3_PxF_3  DOC_CYCLIN_yClb5_NLxxxL_5  DOC_CYCLIN_yCln2_LP_2 
ELM Description:
The G2-phase cyclin Clb3 found in a few budding yeast species is required for the proper timing of spindle assembly and this process depends on an intact hydrophobic pocket (hp). The expression level of Clb3 is lower than that of Clb2 and Clb5, suggesting that instead of contributing to the bulk Cdk activity, low levels of Clb3 can act by specific targeting of a few key substrates. A motif that specifically docks substrates to Clb3-Cdk1 was characterized in the Ypr174c, (Csa1) protein (Ord,2020; Koivomagi,2011). The phosphorylation of Ypr174c by Clb3-Cdk1 enhances its interaction with Polo-like kinase (PLK), driving its localization to spindle pole bodies (Ord,2020). The PxF motif is Clb3-specific, failing to enhance phosphorylation of model substrates by Clb4 or by other G1, S or M phase yeast cyclins (Ord,2020; Ord,2019). Nor does the motif bind to mammalian G2 cyclins A and B.
Targeted mutagenesis of Ypr174c identified a minimal motif that directs binding to the hp of Clb3 and consists of a PxF core with an additional Pro at the -5 position counting from the Phe (PxxPxF) (Ord,2020). Flanking residues spanning positions -6 to +3 further tune binding strength. Grafting of the Ypr174c PxF motif into a model substrate revealed that flanking residues P at the -6 position and K/R at the +3 position enhance substrate phosphorylation, and at least one of these flanking residues is found in functional motifs. The PxF motif was identified in other Clb3 substrates, Tgl5, Boi1, and Sen1 (Ord,2020). In a phospho-proteomics study using a yeast strain with Clb2 as the only cyclin driving S, G2 and M phases, a considerable decrease in phosphorylation of Clb3-specific targets Boi1 and Ypr174c was observed, showing the importance of cyclin specificity in the phosphorylation of these targets (Pirincci Ercan,2021). The substitution patterns found in the Clb3 hp (Q in the position corresponding to Cyclin A E220) explain the loss of specificity for the positively charged residue of the RxL motif.
Pattern: (PP..P.F)|(P..P.F..[KR])
Pattern Probability: 0.0000358
Present in taxons: Saccharomyces Saccharomycetaceae
Interaction Domain:
Cyclin_N (PF00134) Cyclin, N-terminal domain (Stochiometry: 1 : 1)
o See 4 Instances for DOC_CYCLIN_yClb3_PxF_3
o Abstract
Cyclin-dependent kinases (Cdks) are central regulatory enzymes of the eukaryotic cell cycle. The sequential attachment of different cyclins to Cdks represents the periodic driving force that ensures a controlled progression through the cell cycle. Although there can be functional overlap, the various cyclin/Cdk complexes are specialized for optimum performance of discrete tasks.

The cell cycle of the budding yeast Saccharomyces cerevisiae is remarkably simplified compared to that of mammalian cells and therefore it was the subject of many cell-cycle related studies and is currently better understood. Here a single Cdk, Cdk1, associates with different cyclins to mediate all major cell cycle transitions. Cyclins Cln1–3 are triggers for G1 and G1/S, while among B-type cyclins Clb5 and Clb6 drive S phase, Clb3 and Clb4 are specific for early mitotic events, and Clb1 and Clb2 complete the progression to mitosis. Detailed analyses of the budding yeast cell cycle provide important clues on the mechanisms that allow the fine-tuning of thresholds and the ordering of the switch points that drive cell cycle events. These mechanisms rely strongly on the linear encoding of SLiMs to direct cell cycle phosphorylation events (Ord,2019). Limited evidence suggests that these mechanisms have parallels in mammalian cyclin-Cdk regulation.

Cyclins from yeasts and animals harbour a highly conserved surface patch called the hydrophobic pocket (hp) that recognizes docking motifs on partner proteins (DOC_CYCLIN_RxL_1; Loog,2005). The RxL docking motif mediates binding to the hp of a broad range of cyclins from budding yeast (Clb1-6) and mammalian cells (cyclins D/E/A/B). Studies in budding yeast have identified more specific motifs that target the hp. For example, G2 cyclin Clb3 recognizes substrates with the PxF motif (DOC_CYCLIN_yClb3_PxF_3; Ord,2020), and when Cdk is coupled to mitotic cyclins Clb1 or Clb2, the resulting M-Cdk complex recognizes the LxF motif (DOC_CYCLIN_yClb1_LxF_4; Ord,2019). Likewise, the NLxxxL motif is homologous to RxL, but has evolved exclusive specificity for S-phase cyclins Clb 5/6 (DOC_CYCLIN_yClb5_NLxxxL_5; Faustova,2021). Other cyclin-specific motifs include the leucine- and proline-rich LP docking motif (DOC_CYCLIN_yCln2_LP_2; Koivomagi,2011; Bhaduri,2011), which directs binding to late G1-cyclins Cln1/2. Specific docking motifs are also present in mammalian cyclins, as with the cyclin D-specific helical docking motif (DOC_CYCLIN_D_helix_1; Topacio,2019) that mediates binding of Rb proteins to Cyclin D to drive the G1/S transition. Cyclin docking motifs are not only employed by substrates, they are also frequently employed by regulators of cyclin/Cdk complexes, for example the mammalian p27Kip1 and p21Cip1 cyclin inhibitors (1JSU; 6P8E, 6P8H) which hide the site from substrates or the yeast Swe1 that keeps M-CDK in an inactive state during earlier phases of the cell cycle (Ord,2019).

The differences in specificity of hp-docking motifs are explained by changes in the residues that make up the 210-MRAILVDW-217 helix in the hydrophobic pocket (numbering according to human cyclin A2) (Ord,2019). The structures of several RxL motifs (p53, pRb, E2F, and p107) bound to cyclin A2 (1H24; 1H25; 1H26; 1H28) reveal that the central R/K residue of the RxL motif hydrogen bonds to E220 in cyclin A2, while two hydrophobic/aromatic positions bind to an apolar groove made up by M210, I213, L214 and W217 (Russo,1996; Lowe,2002). Residues surrounding the hp (E224 and R250) shape its charge specificity and determine a preference for basic or hydrophobic residues in the vicinity of the core motif. In the budding yeast, loss of the acidic E220 residue in G2- and M-phase cyclins weakens their preference for RxL sequences, favouring the emergence of related (PxF and LxF) motifs that preserve the hydrophobic mode of interaction (Bhaduri,2011; Ord,2019; Ord,2020). Similar changes in the hydrophobic pocket of mammalian cyclins make M-cyclin (Cyclin B) a poor binder of RxL motifs.

Early cyclin/Cdk complexes have low intrinsic activity toward the optimal substrate motif compared to the potent mitotic Cdks, still they need to initiate such important events as Start and S phase. The cyclin-specific docking sites described above are able to compensate for the gradually decreasing specificity of early cyclin/Cdk complexes (Loog,2005; Koivomagi,2011; Bhaduri,2011; Bhaduri,2015; Ord,2019; Ord,2019). Also, cyclins are not just activators of Cdk1 but are also modulators of the catalytic specificity of the kinase active site (Koivomagi,2011). Therefore, modulation of Cdk1 active-site substrate specificity combined with cyclin-specific docking enables regulated changes in Cdk1 specificity and provides a wide range of selective switch points that drive cell cycle transitions (Koivomagi,2011). Mammalian cyclins might use similar mechanisms to ensure specific substrate docking at different stages of the cell cycle, but hp-docking motifs different from the canonical RxL sequence remain to be elucidated.
o 12 selected references:

o 8 GO-Terms:

o 4 Instances for DOC_CYCLIN_yClb3_PxF_3
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
Q00416 SEN1
1034 1042 RVVHPPSAPAFHTKSRGLSN TP 2 Saccharomyces cerevisiae S288c
P38041 BOI1
410 418 SPVQPPQSPSFNNRYTNNNA TP 2 Saccharomyces cerevisiae S288c
Q12043 TGL5
665 673 SPLKVPSRPQFKKRSSYYNQ TP 2 Saccharomyces cerevisiae S288c
Q06616 YPR174C
190 198 SSLQPPKGPNFYAKYPKLPQ TP 4 Saccharomyces cerevisiae S288c
Please cite: ELM — the eukaryotic linear motif resource in 2020. (PMID:31680160)

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