Regulation of Cyclin A-Cdk2 by SCF Component Skp1 and F-Box Protein Skp2

Abstract

Cyclin A-Cdk2 complexes bind to Skp1 and Skp2 during S phase, but the function of Skp1 and Skp2 is unclear. Skp1, together with F-box proteins like Skp2, are part of ubiquitin-ligase E3 complexes that target many cell cycle regulators for ubiquitination-mediated proteolysis. In this study, we investigated the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2. We found that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 in vitro, both by direct inhibition of cyclin A-Cdk2 and by inhibition of the activation of Cdk2 by cyclin-dependent kinase (CDK)-activating kinase phosphorylation. Only the kinase activity of Cdk2, not of that of Cdc2 or Cdk5, is reduced by Skp2. Skp2 is phosphorylated by cyclin A-Cdk2 on residue Ser76, but nonphosphorylatable mutants of Skp2 can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1. The F box of Skp2 is required for binding to Skp1, and both the N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2. Furthermore, Skp2 and the CDK inhibitor p21^Cip1/WAF1 bind to cyclin A-Cdk2 in a mutually exclusive manner. Overexpression of Skp2, but not Skp1, in mammalian cells causes a G₁/S cell cycle arrest.

ACKNOWLEDGMENTS

We are very grateful for David Beach, Hermann Bujard, Emma Lees, Julian Gannon, Tim Hunt, Tony Hunter, Kun Ping Lu, Gertrud Orend, Hideo Toyoshima, and Masakane Yamashita for reagents. We thank Tim Hunt, Tony Hunter, and members of the Poon lab for help and discussions. We also thank Frances Chan for help with the FACS analysis.

This work was supported in part by Research Grants Council grant DAG96/97-SC26 and British Council/Research Grants Council grant JRS96/31 to R.Y.C.P. C.H.Y. is a Sir Edward Youde Memorial Fellow. R.Y.C.P. is a member of the Biotechnology Research Institute.