Skp2 Contains a Novel Cyclin A Binding Domain That Directly Protects Cyclin A from Inhibition by p27Kip1

Skp2 is well known as the F-box protein of the SCFSkp2Roc1 complex targeting p27 for ubiquitylation. Skp2 also forms complexes with cyclin A, which is particularly abundant in cancer cells due to frequent Skp2 overexpres- sion, but the mechanism and significance of this interaction remain unknown. Here, we report that Skp2-cyclin A inter- action is mediated by novel interaction sequences on both Skp2 and cyclin A, distinguishing it from the well known RXL-hydrophobic patch interaction between cyclins and cyclin-binding proteins. Furthermore, a short peptide derived from the mapped cyclin A binding sequences of Skp2 can block Skp2-cyclin A interaction but not p27-cyclin A interaction, whereas a previously identified RXL peptide can block p27-cyclin A interaction but not Skp2-cyclin A interac- tion. Functionally, Skp2-cyclin A interaction is separable from Skp2 ability to mediate p27 ubiquitylation. Rather, Skp2-cyclin A interaction serves to directly protect cyclin A-Cdk2 from inhibition by p27 through competitive binding. Finally, we show that disruption of cyclin A binding with point mutations in the cyclin A binding domain of Skp2 com- promises the ability of overexpressed Skp2 to counter cell cycle arrest by a p53/p21-mediated cell cycle checkpoint without affecting its ability to cause degradation of cellular p27 and p21. These findings reveal a new functional mecha- nism of Skp2 and a new regulatory mechanism of cyclin A.