Comprehensive Analysis of G1 Cyclin Docking Motif Sequences that Control CDK Regulatory Potency In Vivo

Abstract

Cyclin-dependent kinases (CDKs) control the ordered series of events during eukaryotic cell division. The stage at which individual CDK substrates are phosphorylated can be dictated by cyclin-specific docking motifs. In budding yeast, substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs and the conservation of this mechanism were unknown. Here we comprehensively analyzed LP motif requirements in vivo by combining a competitive growth assay with mutational scanning and deep sequencing. We quantified the impact of all single-residue replacements in five different LP motifs, using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results reveal the basis for variations in potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the potency of other candidate motifs. In one protein, Whi5, we found overlapping LP and phosphorylation motifs with partly redundant effects. In another protein, the CDK inhibitor Sic1, we found that its LP motif is inherently weak due to unfavorable residues at key positions, and this imposes a beneficial delay in its phosphorylation and degradation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in LP motif potency can tune the strength and timing of CDK regulation..

The Eukaryotic Linear Motif resource: 2022 release

“…Docking to G1-cyclins is mediated by motifs that target a surface distinct from the hp . The LP motif, DOC_CYCLIN_yCln2_LP_2, mediates binding to late G1-cyclins Cln1/2 and Ccn1 in yeasts ( 25–27 ) and is conserved through the fungal lineage ( 28 ). In mammals, docking of retinoblastoma-family proteins Rb, p107 and p130 to Cyclin D:Cdk4/6 complexes is mediated by a helical motif, DOC_CYCLIN_D_Helix_1, that cooperates with the RxL and LxCxE motifs to phosphorylate Rb-family proteins early in G1, a key step required for the G1 to S phase transition ( 29 ).…”

Bipartite binding of the N terminus of Skp2 to cyclin A

“…In the hypothetical antiparallel binding mode, these residues, which are embedded in the conserved and hydrophobic sequence 32 LLSGMGVSAL 41 , would be well placed to interact favorably with the hydrophobic properties of surface 1. Interestingly, a similar surface on the budding yeast cyclins Cln1 and Cln2 has been implicated in binding short linear motifs containing hydrophobic amino acids, particularly Leu and Pro (Figure 8B) (Bandyopadhyay et al, 2020;Bhaduri et al, 2015). Further work will be required to validate these inferences.…”

Docking to a Basic Helix Promotes Specific Phosphorylation by G1-Cdk1

“…A Set of G1 Cyclin Specific Cdk1 Targets Are Independent of the LP Docking While the Cln1,2-specific LP docking motif is important for phosphorylation of key G1 targets [11,13,14], our quantitative phosphorylation studies indicated that a fraction of known G1-specific Cdk1 targets does not use this docking interaction (Figure 1). We measured the cyclin specificity of these G1-Cdk1 targets using in vitro kinase assays with the major cyclin-Cdk1 complexes.…”

“…Secondly, in the case of several early CDK targets, the poor intrinsic activity of early complexes is compensated by phosphorylation mechanisms involving cyclin-specific substrate docking. For example, in budding yeast, the G1 cyclins Cln1,2 have been shown to dock a hydrophobic leucine-proline rich sequence designated as LP motif [11,13,14], the S-phase cyclin Clb5 uses at least two docking motifs: the classical R/KxL motif [15][16][17][18], and recently discovered NLxxxL motif [19], and the G2 cyclin Clb3 binds a motif PxxPxF [20]. Finally, despite the high intrinsic activity, the specificity of mitotic targets for M-CDK (Clb2,1-Cdk1) complex can be considerably improved using short linear motifs with minimal consensus LxF [21] In addition to the changes in CDK activity and specificity over the cell cycle, the counteracting phosphatases also have varying activity towards different CDK targets and this contributes to the timing of different phosphorylation and dephosphorylation switches [22][23][24][25].…”