P21 CONTAINS INDEPENDENT BINDING-SITES FOR CYCLIN AND CDK2 - BOTH SITES ARE REQUIRED TO INHIBIT CDK2 KINASE-ACTIVITY

Cyclin dependent kinases regulate the progression of eukaryotic cells through the cell cycle, p21(Cip1/Waf1/Sdi1) is an inhibitor of cdk-cyc lin kinase activity, and has been shown to form complexes with cdk-cyc lins and with PCNA, an accessory protein of DNA polymerase delta, The kinase inhibitory domain maps to the N-terminus (1-82) and contains th e cdk2 binding site (28-82), We have generated a panel of deletion mut ants of p21, A functional characterization of p21 mutants in the N-ter minal domain reveals that cyclins bind to this domain independently of cdk2, Correlating with these results we find that p21 can associate w ith cyclin-cdk kinases in two functionally distinct forms, one in whic h the kinase activity is inhibited and the other in which the kinase i s still active, The cdk2 and cyclin binding sites on p21 are both requ ired to inhibit kinase activity, The second type of interaction, in wh ich an active cyclin-cdk complex only interacts with p21 either via th e cyclin or the cdk2 binding site but not through both, does not lead to inhibition of cyclin kinase activity, These results thus provide a basis for understanding the mechanism by which p21, and perhaps other cdk-cyclin kinase inhibitory proteins, suppress kinase activity.