Phosphorylation of Progesterone Receptor Serine 400 Mediates Ligand-Independent Transcriptional Activity in Response to Activation of Cyclin-Dependent Protein Kinase 2

Abstract

Human progesterone receptors (PR) are phosphorylated by cyclin-dependent protein kinase 2 (CDK2) at multiple sites, including Ser400. Herein, we have addressed the significance of phosphorylation of this residue. PR phospho-Ser400-specific antibodies revealed regulated phosphorylation of Ser400 in response to progestins and mitogens, and this correlated with increased CDK2 levels and activity. Expression of cyclin E elevated CDK2 activity and downregulated PR independently of ligand. Similarly, overexpression of activated mutant CDK2 increased PR transcriptional activity in the absence and presence of progestin. Mutation of PR Ser400 to alanine (S400A) blocked CDK2-induced PR activity in the absence, but not in the presence, of progestin. PR was unresponsive to activated CDK2 in breast cancer cells with elevated p27, and RNA interference knock-down of p27 partially restored CDK2-induced ligand-independent PR activation. Similarly, in p27^−/− mouse embryonic fibroblasts, elevated CDK2 activity increased wild-type (wt) but not S400A PR transcriptional activity in the absence of progestin. CDK2 induced nuclear localization of unliganded wt but not S400A PR; liganded S400A PR exhibited delayed nuclear accumulation. These studies demonstrate that CDK2 regulates PR in the absence of progestins via phosphorylation of Ser400, thus revealing a novel mechanism for upregulated PR transcriptional activity in human breast cancer cells expressing altered cell cycle regulatory molecules.

We thank Robert Sheaff (University of Minnesota) for numerous helpful comments and suggestions in addition to the p27 and activated CDK2 expression vectors, activated cyclin E/CDK2 complex, and the wt and p27^−/− MEFs and James DeGregori (University of Colorado Health Sciences Center) for cyclin E adenoviruses. We are grateful to Craig Jordan (Northwestern University) for PR-null T47D42W cells and to Kathryn B. Horwitz (University of Colorado Health Sciences Center) for T47Dco, T47D-YB, and YA breast cancer cell lines. We are grateful to Ming Qiu (University of Minnesota) and Jim McCabe and Jerry Sedgewick (University of Minnesota Bio-Imaging Processing Lab Core Facility) for excellent confocal microscopy technical assistance.

This work was supported by Department of Defense Breast Cancer Research Program DAMD17-02-1-0495 (to L. Pierson-Mullany) and NIH R01-DK053825 (to C. Lange).