Deciphering the phosphorylation "code" of the glucocorticoid receptor in vivo

Abstract

The glucocorticoid receptor (GR) is phosphorylated at multiple serine residues in a hormone-dependent manner, yet progress on elucidating the function of GR phosphorylation has been hindered by the lack of a simple assay to detect receptor phosphorylation in vivo. We have produced antibodies that specifically recognize phosphorylation sites within human GR at Ser203 and Ser211. In the absence of hormone, the level of GR phosphorylation at Ser211 was low compared with phosphorylation at Ser203. Phosphorylation of both residues increased upon treatment with the GR agonist dexamethasone. Using a battery of agonists and antagonists, we found that the transcriptional activity of GR correlated with the amount of phosphorylation at Ser211, suggesting that Ser211 phosphorylation is a biomarker for activated GR in vivo. Mechanistically, the kinetics of Ser203 and Ser211 phosphorylation in response to hormone differed, with Ser211 displaying a more robust and sustained phosphorylation relative to Ser203. Analysis of GR immunoprecipitates with phospho-GR-specific antibodies indicated that the receptor was phosphorylated heterogeneously at Ser203 in the absence of hormone, whereas in the presence of hormone, a subpopulation of receptors was phosphorylated at both Ser203 and Ser211. Interestingly, biochemical fractionation studies following hormone treatment indicated that the Ser203-phosphorylated form of the receptor was predominantly cytoplasmic, whereas Ser211phosphorylated GR was found in the nucleus. Likewise, by immunofluorescence, Ser203-phosphorylated GR was located in the cytoplasm and perinuclear regions of the cell, but not in the nucleoplasm, whereas strong phospho-Ser211 staining was evident in the nucleoplasm of hormone-treated cells. Our results suggest that differentially phosphorylated receptor species are located in unique subcellular compartments, likely modulating distinct aspects of receptor function.