The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
Cyclin D-specific Helical docking motif
Functional site description:
Progression through the cell cycle is characterized by a quantitative increase in Cdk activity, and the sequential phosphorylation of cell cycle targets. One of the first steps in retinoblastoma (Rb) protein inactivation is its phosphorylation by CyclinD/Cdk4-6 complexes during the G1 cell cycle phase. Cyclin D proteins harbour a specific docking site that binds to helical motifs present in the substrates. This motif acts in cooperation with RxL and LxCxE motifs to promote Rb protein family phosphorylation at the beginning of G1, a key step required for the subsequent hyperphosphorylation of Rb family proteins that leads to their inactivation and entry into S phase.
ELM Description:
Cyclin D proteins in mammals (Cyclins D1, D2 and D3) use a combination of three SLiMs to cooperatively target Rb family proteins Rb, p107 and p130, leading to their phosphorylation during the early G1 phase of the cell cycle (Rubin,2013). These motifs include the RxL motif of pocket proteins (DOC_CYCLIN_RxL_1), which docks to the hydrophobic pocket of Cyclin D (Topacio,2019), the LxCxE motif (LIG_Rb_LxCxE_1) in Cyclin D that docks to the LxCxE binding pocket in the Rb B-domain (Dowdy,1993; Lee,1998), and a third SLiM present in the disordered C-terminal domain of all Rb family proteins (DOC_CYCLIN_D_Helix_1) that is predicted from sequence to form a helical structure (Topacio,2019; Pan,2001). The motif is Cyclin D-specific, as it does not mediate docking to mammalian G1, S or M cyclins.

Mutational analysis and the sequence and structural preferences of the binding motif suggest that binding occurs at a surface on Cyclin D that is distinct from the hydrophobic pocket, although the lack of a structure prevents a more precise definition of the binding site. The functionality of the motif is supported by deletion studies, and in vitro phosphorylation assays with natural and model substrates. These show that the motif mediates docking of the Rb, p107 and p130 pocket proteins to all types of Cyclin D (1,2,3) and Cdk (4,6) complexes and that targeted removal of the motif leads to a 20-fold decrease in phosphorylation efficiency (Topacio,2019). The helical motif is broadly conserved across metazoan Rb family proteins, but has been lost in some insect species such as Drosophila melanogaster and is absent in Rb proteins from plants and green algae. A mutational scan together with the conservation pattern allow the definition of a regular expression (Topacio,2019). Consistent with the 3-turn alpha helical configuration, proline residues are disallowed, and conserved hydrophobic and positively charged positions alternate with a regular spacing of two to three residues.
Pattern: [FLV][^P][^P][KR]L[^P][^P][LMIV][^P][^P][^P]R
Pattern Probability: 0.0000066
Present in taxon: Metazoa
Interaction Domain:
Cyclin (IPR039361) Cyclins are eukaryotic proteins that play an active role in controlling nuclear cell division cycles, and regulate cyclin dependent kinases (CDKs) (Stochiometry: 1 : 1)
o See 3 Instances for DOC_CYCLIN_D_Helix_1
o Abstract
Cyclins are major regulators of cell cycle progression that act by regulating cyclin dependent kinase (Cdk) activity. Several cyclins are able to target specific substrates for phosphorylation by different cyclin/Cdk complexes, yet the molecular determinants underlying this specificity are still poorly understood in mammalian cyclins. Cyclins from budding yeast and animals harbour a highly conserved surface patch called the hydrophobic pocket (hp), which docks RxL sequences present in substrates and inhibitors (DOC_CYCLIN_RxL_1) (Lowe,2002; Cheng,2006). In budding yeast, the evolution of cyclin specificity led to the appearance of distinct sets of RxL-like motifs that target the hp and drive the phosphorylation of G2-specific targets by Clb3/4 (DOC_CYCLIN_yClb3_PxF_3), mitotic targets by Clb1/2 (DOC_CYCLIN_yClb1_LxF_4) (Ord,2019; Ord,2019) and S phase targets by Clb 5/6 (DOC_CYCLIN_yClb5_NLxxxL_5; Faustova,2021). The phosphorylation of G1 targets by Cln2 in yeast is mediated by a leucine- and proline-rich (LP) docking motif (DOC_CYCLIN_yCln2_LP_2) that resembles the core hydrophobic sequence of RxL-like motifs but whose binding site has not been conclusively determined (Koivomagi,2011; Bhaduri,2011). These motifs allow different yeast cyclins to specifically target cell cycle regulators at distinct cell cycle stages, and often the RxL-like motifs act cooperatively with additional docking sites (DOC_CKS1_1) to allow processive target phosphorylation (Koivomagi,2013). Independent evidence from mammalian Cyclin D regulation indicates that a novel helical motif present in metazoan Rb family proteins (DOC_CYCLIN_D_Helix_1) targets a separate and yet unidentified binding surface on Cyclin D proteins, cooperating with the RxL and LxCxE motifs to direct Rb protein family phosphorylation during the G1 phase of the cell cycle (Topacio,2019).

Cyclin D proteins bind specifically to cyclin-dependent kinases 4 and 6 (Cdk4/6) to phosphorylate substrates during the early G1 phase of the cell cycle. The retinoblastoma (Rb) family proteins Rb, p107 and p130, also called pocket proteins, are among the best-studied CyclinD-Cdk4/6 substrates (Giacinti,2006; Burkhart,2008). Pocket proteins bind to the E2F family of transcription factors, repressing their activity and acting as major controllers of the G1/S transition (van den Heuvel,2008). Pocket protein phosphorylation by CyclinD-Cdk4/6 and by CyclnE-Cdk2 in early and late G1 respectively leads to destabilization of the Rb-E2F repressive complex and to the transcription of E2F-regulated genes necessary for entry into S-phase (Dick,2013; Rubin,2013). The phosphorylation of Rb by CyclinD/Cdk4-6 is an important first step in driving the G1/S transition (Topacio,2019).

Deregulation of the CyclinD-Rb-E2F axis, and prominently Cyclin D1 and Rb deregulation are a hallmark of many types of human cancer (Musgrove,2011; Bosco,2007). Increased Cyclin D1 expression leads to a hyper activation of Cdk4/6 promoting Rb phosphorylation, entry into S-phase and the ensuing proliferation of tumor cells. Specific Cdk4/6 inhibitors such as Palbociclib are able to block deregulated proliferation, and combination therapies have emerged as a promising treatment for ER+ breast cancers (Johnson,2016). Because of this, SLiM-mediated interactions in the CyclinD-Rb-E2F axis also emerge as potential drug targets for cancer.
o 10 selected references:

o 9 GO-Terms:

o 3 Instances for DOC_CYCLIN_D_Helix_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
Q08999 RBL2
1125 1136 ENHSALLRRLQDVANDRGSH TP 3 Homo sapiens (Human)
P28749 RBL1
1054 1065 ENDDVLLKRLQDVVSERANH TP 3 Homo sapiens (Human)
P06400 RB1
897 908 GESKFQQKLAEMTSTRTRMQ TP 7 Homo sapiens (Human)
Please cite: ELM — the eukaryotic linear motif resource in 2020. (PMID:31680160)

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