Glucocorticoid receptor phosphorylation modulates transcription efficacy through GRIP-1 recruitment.
The role of GR phosphorylation in modulating GR-mediated transcription is not fully understood. Here we show that the hGR is rapidly phosphorylated at S211 and S226 in response to the synthetic agonist dexamethasone (dex) in COS-1 cells. Using a triple phosphorylation mutant hGR construct, we demonstrate that phosphorylation at one or more S residues (from S203, S211, and S226) is required for maximal hGR-mediated transcriptional activation on the MMTV promoter in response to dex in COS-1 cells, but that this effect is promoter selective. Phosphorylation at these residues does not affect unliganded or agonist-induced hGR degradation, suggesting that the mechanism whereby hGR phosphorylation at these residues regulates GR-mediated transactivation via a GRE does not involve changes in GR half-life. We have previously shown a direct correlation between efficacy for transactivation and interaction of the hGR with glucocorticoid receptor interacting protein-1 (GRIP-1). Here we show by pull-down assays in the absence and presence of glucocorticoid response elements (GREs) that phosphorylation of the hGR is required for GR-GRIP-1 interaction. Chromatin immunoprecipitation (ChIP) assays revealed that hGR phosphorylation at one or more S residues (from S226, S211, and S203) is required for the recruitment of GRIP-1 to the synthetic MMTV promoter as well as to the endogenous GRE-containing glucocorticoid-induced leucine zipper (GILZ) promoter in intact COS-1 cells, but not for nuclear localization. Our results support the conclusion that phosphorylation at S203, S211, and/or S226 of the hGR is required for a maximal transcriptional response via the synthetic MMTV and endogenous GILZ GREs in COS-1 cells, to enable recruitment of GRIP-1 to the hGR.
Avenant
Chanel
C
Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch 7700, South Africa.
Kotitschke
Andrea
A
Hapgood
Janet P
JP
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics / Q000378:metabolism; D051958:Nuclear Receptor Coactivator 2 / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics
C498001:NCOA2 protein, human; D051958:Nuclear Receptor Coactivator 2; D011965:Receptors, Glucocorticoid; D012694:Serine; D003907:Dexamethasone