Differential Regulation of Cyclin-Dependent Kinase 4 (CDK4) and CDK6, Evidence that CDK4 Might Not Be Activated by CDK7, and Design of a CDK6 Activating Mutation

Abstract

The homologous cyclin-dependent kinases (CDK) CDK4 and CDK6 integrate mitogenic and oncogenic signaling cascades with the cell cycle. Their activation requires binding to a D-type cyclin and then T-loop phosphorylation at T172 and T177 (respectively) by the only CDK-activating kinase identified in animal cells, cyclin H-CDK7. At odds with the existing data showing the constitutive activity of CDK7, we have recently identified the T172 phosphorylation of cyclin D-bound CDK4 as a crucial cell cycle regulatory target. Here we show that T172 phosphorylation of CDK4 is conditioned by its unique proline 173 residue. In contrast to CDK4, CDK6 does not contain such a proline and, unexpectedly, remained poorly phosphorylated and active in a variety of cells. Mutations of proline 173 did not adversely affect CDK4 activation by CDK7, but in cells they abolished CDK4 T172 phosphorylation and activity. Conversely, substituting a proline for the corresponding residue of CDK6 enforced its complete, apparently cyclin-independent T177 phosphorylation and dramatically increased its activity. These results lead us to propose that CDK4 might not be phosphorylated by CDK7 in intact cells but is more likely phosphorylated by another, presumably proline-directed kinase(s). Moreover, they provide a new model of a potentially oncogenic activating mutation of a CDK.

ACKNOWLEDGMENTS

We thank Audrey Delacroix for participation in initial experiments, Katia Coulonval for advice on CAK assays, and Jacques Dumont for his continued interest, helpful discussions, and critical reading of the manuscript. The phospho-specific CDK4 (T172) antibody was a kind gift of Cell Signaling Technology Inc. (Beverly, MA). We thank J. Bartek and J. Lukas (Danish Cancer Society) for kindly providing several plasmids.

This study was supported by grants from the Belgian Federation against Cancer, the Communauté française de Belgique-Actions de Recherches Concertées, the Belgian Fund for Scientific Medical Research (FRSM), the National Fund for Scientific Research (FRS-FNRS, Belgium) and Télévie. X.B. is a fellow of the Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture (FRIA). L.B., S.P., and P.P.R. are a Scientific Research Worker, Postdoctoral Researcher, and Senior Research Associate of the FRS-FNRS, respectively.

We have no conflict of interest to disclose.