A biochemical analysis of the cell cycle specific degradation of the CDK inhibitor p27(kip1)

The activity of the cyclin dependent kinase 2 (CDK2) is required for S phase entry and progression. The mammalian CDK inhibitor p27 kip1 mediates cell cycle arrest by mainly inhibiting CDK2. In most cell types, as cells enter S phase p27kip1 protein stability reduces drastically. We used a cell free system to analyze the biochemical requirements for S phase specific p27kip1 degradation. We found that p27kip1 was degraded specifically in S phase cell extract and required the kinase activity of CDK2 and intact T187 on p27kip1 . We found that p27kip1 was ubiquitinated in an S phase specific manner, also requiring CDK2 activities and intact T187. The degradation of p27kip1 also required p27kip1 binding factor(s) other than cyclin-Cdk. The F box-protein skp2 has been recently reported to bind to phosphorylated p27kip1 and stimulate its ubiquitination and degradation. We found that our S phase extract required skp2 for its p27 kip1 ubiquitination and degradation activities. We found that adding cyclin E-cdk2 kinase and exogenous skp2/skp1 to a G1 cell extract did not induce p27kip1 degradation, whereas adding cyclinA-cdk2 did. We sought to understand the regulation of SCFskp2. We saw that skp2 was detected in a higher molecular weight complex in S phase extract and not in G1 phase extract. The presence of skp2 in the HMW complex was dependent on cyclin A-cdk2, independent of its kinase activity. We also found that adding cyclinA-cdk2 to a G1 extract increased cull binding to skp2/skp1, independent of cyclinA-cdk2 kinase activity. The data suggest that cyclin A-cdk2 complex might play a role in stabilizing the SCFskp2 complex. Furthermore, we found that adding catalytically inactive cyclin A-cdk2* to S extract suppressed p27kip1 degradation, even though it did not interfere with the endogenous kinase to phosphorylate p27kip1 on T187. We also found that adding inactive cyclin A-cdk2* did not interfere with the total cyclin A kinase activity, but it decreased the amount of active cyclin A-cdk2 found in the HMW complex. This suggests that cyclin A-cdk2* might have dominant negative effect by competing with active cyclin A-cdk2 for the SCF core, which suggests that SCFskp2 might require active cyclin A-cdk2 for its activity.