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Recruitment of Coactivator Glucocorticoid Receptor Interacting Protein 1 to an Estrogen Receptor Transcription Complex Is Regulated by the 3',5'-Cyclic Adenosine 5'-Monophosphate-Dependent Protein Kinase

Institute of Medicine, Section for Endocrinology, University of Bergen (I.S.F., T.H., M.H., J.V.S., E.A.L., G.M.) and The Hormone Laboratory, Haukeland University Hospital (I.S.F., E.A.L., G.M.), N-5021 Bergen, Norway

Address all correspondence and requests for reprints to: Gunnar Mellgren, M.D., Ph.D., The Hormone Laboratory, Institute of Medicine, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. E-mail: gunnar.mellgren{at}med.uib.no.

Steroid receptor coactivators (SRCs), such as glucocorticoid receptor interacting protein 1 (GRIP1) are recruited to the DNA-bound nuclear receptors (NRs) and are also shown to enhance the gene transactivation by other transcription factors. In contrast to the two other members of the SRC family, SRC-1 and SRC-3/amplified in breast cancer 1, SRC-2/GRIP1 is regulated by the cAMP-dependent protein kinase [protein kinase A (PKA)] that stimulates its ubiquitination and degradation. In this report we demonstrate that COS-1 and MCF-7 cells treated with cAMP-elevating agents and 8-para-chlorophenylthio-cAMP for short periods of time showed an increase in GRIP1 coactivator function, whereas prolonged stimulation of the cAMP/PKA pathway led to a decline in GRIP1-mediated activation and protein levels. Furthermore, MCF-7 breast cancer cells were subjected to chromatin immunoprecipitation assays after stimulation of the cAMP/PKA pathway. cAMP/PKA initiated a rapid recruitment of GRIP1 to the endogenous estrogen receptor (ER)- target pS2 gene promoter. In contrast to the estradiol-induced recruitment of GRIP1 to pS2, we observed an additional increase in GRIP1 recruitment on inhibition of the proteasome, suggesting that inhibition of GRIP1 degradation leads to accumulation at the pS2. Real-time PCR experiments confirmed that cAMP/PKA enhanced the expression of pS2. Moreover, confocal imaging of COS-1 cells transfected with yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ER revealed that PKA led to redistribution and colocalization of yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ER in subnuclear foci. In conclusion, these results suggest that activation of the cAMP/PKA pathway stimulates recruitment of GRIP1 to an ER-responsive gene promoter. The initial stimulation of GRIP1 coactivator function is followed by an increased turnover and subsequent degradation of GRIP1 protein.

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