p27^Kip1 and p21^Cip1 Are Not Required for the Formation of Active D Cyclin-cdk4 Complexes

Abstract

Our studies address questions pertaining to the regulation of D cyclin-cdk4 activity, and the following results were obtained. Conditions that increased the abundance of the D cyclins also increased the abundance of enzymatically active D cyclin-cdk4 complexes in mouse embryo fibroblasts (MEFs) lacking both p27^Kip1 and p21^Cip1 (p27/p21^−/−). Such conditions included ectopic expression of cyclin D1 and inhibition of D cyclin degradation by the proteasome inhibitor MG132. However, as determined by treatment of wild-type MEFs with MG132, maximal accumulation of D cyclin-cdk4 complexes required p27^Kip1 and p21^Cip1 and coincided with the formation of inactive D cyclin-cdk4-p27^Kip1 or -p21^Cip1 complexes. p27^Kip1 or p21^Cip1 also increased the abundance of D cyclin-cdk4 complexes and reduced amounts of cdk4 activity when ectopically expressed in p27/p21^−/− MEFs. Lastly, increases in the stability of the D cyclins accounted for their greater abundance in wild-type MEFs than in p27/p21^−/− MEFs. We conclude that (i) D cyclin-cdk4 complexes are formed and become active in the absence of p27^Kip1 and p21^Cip1 and (ii) p27^Kip1 and p21^Cip1 maximize the accumulation but inhibit the activity of D cyclin-cdk4 complexes. We suggest that D cyclin-cdk4 complexes are more stable when bound to p27^Kip1 or p21^Cip1 and that formation of ternary complexes also stabilizes the D cyclins.

ACKNOWLEDGMENTS

This work was supported by the Cortner-Couch Endowed Chair for Cancer Research and NIH Grant CA67360 to W.J.P.

We thank Nancy Olashaw for manuscript preparation and acknowledge the helpful service of the Molecular Imaging Core Laboratory at the Moffitt Cancer Center.