IDENTIFICATION OF A CYCLIN-CDK2 RECOGNITION MOTIF PRESENT IN SUBSTRATES AND P21-LIKE CYCLIN-DEPENDENT KINASE INHIBITORS

Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A- cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphor ylation of the retinoblastoma protein by cyclin A- but not cyclin B-as sociated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 a nd E2F1 likewise prevented their phosphorylation by cyclin A-associate d kinases in vitro. The cdk inhibitor p21 was found to contain two fun ctional copies of this recognition motif, as determined by in vitro ki nase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate r ecognition motif. Furthermore, these data suggest that p21-like cdk in hibitors function, at least in part, by blocking the interaction of su bstrates with cyclin-cdk2 complexes.