Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

Linghu Nie,Carl G. Maki,Hongyan Zhu

doi:10.1074/jbc.m407352200

Abstract

p21 is a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors that includes p21, p27, and p57. Recent studies have suggested that Cdk2 activity may promote p21 degradation through a pathway similar to that for p27, although the mechanism by which this occurs has not been clarified. In the current report, co-expression with cyclin E and Cdk2 stabilized p21 in a manner that required the CDK-binding site of p21 and a cyclin-binding site (cy1) located in the p21 N terminus. Strikingly, however, a kinase-dead Cdk2 mutant stabilized p21 to a greater extent than did wild-type Cdk2, consistent with the notion that Cdk2 activity can destabilize p21. The ability of wild-type Cdk2 to destabilize p21 required a potential Cdk2 phosphorylation site in p21 at serine 130 and an intact cyclin-binding motif (cy2) in the p21 C terminus. Finally, p21 was phosphorylated by Cdk2 at Ser-130 in vitro, and this ability of Cdk2 to phosphorylate p21 was dependent, in large part, on the presence of cy2. These results support a model in which active Cdk2 destabilizes p21 via the cy2 cyclin-binding motif and p21 phosphorylation.

Highlights

P21 was first recognized as a protein that co-immunoprecipitated with mammalian cyclincyclin-dependent kinase (CDK)1 complexes [1]
P21 levels were increased, and the protein was stabilized in the presence of MG132 or when co-expressed with cyclin E and Cdk2 (t1⁄2 in each case Ͼ3 h)
The effect of cyclin and Cdk binding on p21 stability has not been fully clarified

Summary

Introduction

P21 ( called Waf or Cip1) was first recognized as a protein that co-immunoprecipitated with mammalian cyclincyclin-dependent kinase (CDK) complexes [1]. This finding provided an attractive model for the p53-dependent G1 phase cell cycle arrest observed in response to DNA damage According to this model, p53 activates p21 gene expression in response to DNA-damaging stress, and increased levels of p21 protein bind and inhibit G1 phase cyclin-CDK complexes, resulting in a G1 arrest. Active Cdk could enhance the in vitro ubiquitination of p21 mediated by a Skp2-containing SCF (Skp, Cullin, and F-box protein) complex [27] In contrast to these findings, cyclin D1 was reported to stabilize p21, perhaps by binding the cy site in the p21 C terminus and blocking p21 interaction with the C8 proteasome subunit [28]. The purpose of the current study was to examine the effect of cyclin E and Cdk on p21 protein stability

Objectives

Results

Conclusion