BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

Abstract

BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G₁ and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G₁/S-specific increase in BRCA1 phosphorylation.

ACKNOWLEDGMENTS

We thank Tim Mayall for providing recombinant p16 and p21, Nik Somia for providing pCL-MFG-MCS and for excellent suggestions, Jill Meisenhelder for providing 9E10 antibody and for technical advice, Mirta Grifman and Matthew Weitzman for the pRK5-cyclin A expression construct, Lamya Shihabuddin for technical advice, and Brian Spain, Chris Larson, Tal Kafri, and other members of the Verma laboratory for valuable discussions. We thank Jean E. Rivier for his interest in our work.

H.R. is supported by consecutive funds from the Schweizerische Nationalfonds für wissenschaftliche Forschung, grant 823A-046698, and the California Breast Cancer Research Program of the University of California, grant 4FB-0102. W.J. is supported by a postdoctoral fellowship from the American Cancer Society. I.M.V., T.H., and A.G.C. are supported by grants from the National Institutes of Health. I.M.V. is an American Cancer Society Professor of Molecular Biology, and he is also supported by the Elsa Pardee Foundation. T.H. is a Frank and Else Schilling American Cancer Society Research Professor.