ROLE OF SITE-SPECIFIC PHOSPHORYLATION IN THE ACTION OF GLUCOCORTICOID RECEPTOR

Raj Kumar

Department of Basic Sciences; Geisinger Commonwealth School of Medicine; Scranton, PA-18509, USA

Abstract

The N-terminal/AF1 activation function domain of the glucocorticoid receptor (GR) exists as an intrinsically disordered peptide (IDP), frequently found in many transcription factors. The IDPs are predicted to have much larger frequency of phosphorylation sites than ordered regions, suggesting an important role in their regulatory capacity. The GR is a phospho-protein and site-specific phosphorylation is known to be a critical step in the receptor activation and biological activity. In the human GR, all the major functionally important known phosphorylation sites (S203, S211, and S226) are located within the ID AF1. We propose that phosphorylation-induced disorder-order conformational transition in the AF1 domain may be a potential mechanism through which site-specific phosphorylation regulates GR functions. Therefore, in this study we examined the role of phosphorylation sites (S203/S211/S226) on the conformational transition of ID AF1 and subsequent AF1-mediated GR’s transcriptional activity. Our biophysical analyses showed that when compared to S203 or S226 residues, phosphorylation of S211 appears to contribute most to secondary structure formation in the AF1 protein. Further, CV-1 cells (lacking functional GR) constitutively expressing AF1 in a two domain GR fragment containing entire N-terminal and DNA-binding domains (GR500) or mutants (S203A/S211A/S226A) in various combinations were co-transfected with pGRE-SEAP plasmid to determine promoter-reporter activity. We found a differential effects on the promoter-reporter activities dependent upon various combinations of phosphorylation mutants. Together, these results demonstrate the role of different phosphorylation sites in the regulation of AF1-mediated GR actions.

Keywords: DNA, mechanism, phosphorylation, regulation, domain.