NEW FUNCTIONAL ACTIVITIES FOR THE P21 FAMILY OF CDK INHIBITORS

The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrat e that the CDK inhibitors p21(CIP), p27(KIP), and p57(KIP2) all promot e the association of cdk4 with the D-type cyclins. This effect is spec ific and does not occur with other cdk inhibitors or cdk-binding prote ins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The pr omotion of assembly is not attributable to a simple cell cycle block a nd requires the function of both the cdk and cyclin-binding domains. K inetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase in K-a, respectively, mostly because of a decrease in K-off. At low concentrations, p21 promotes the assembly of active kinase comp lexes, whereas at higher concentrations, it inhibits activity. Moreove r, immunodepletion experiments demonstrate that most of the active cdk 4-associated kinase activity also associates with p21. To confirm thes e results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a G(0) arrest . In agreement with our other data, cyclin D1 and p21 bind concomitant ly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. Th is complex assembly occurs in parallel to an increase in cyclin D1-ass ociated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin D1 to the nucleus. We suggest that in addition to thei r roles as inhibitors, the p21 family of proteins, originally identifi ed as inhibitors, may also have roles as adaptor proteins that assembl e and program kinase complexes for specific functions.