New functional activities for the p21 family of CDK inhibitors
J LaBaer, M D Garrett, L F Stevenson, J M Slingerland, C Sandhu, H S Chou, A Fattaey, E Harlow
Genes & Development
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 demonstrate that the CDK inhibitors pZl'^^", p27^^^, and p57^^^^ all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex
... eases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35-and 80-fold increase in K^, respectively, mostly because of a decrease in X^ff. At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a GQ arrest. In agreement with our other data, cyclin Dl and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin Dl complexes. This complex assembly occurs in parallel to an increase in cyclin Dl-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin Dl-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin Dl to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions. -Published by genesdev.cshlp.org Downloaded from Access the most recent version at doi: 11: 1997, Genes Dev.