Differential recruitment of glucocorticoid receptor phospho-isoforms to glucocorticoid-induced genes

Abstract

The human glucocorticoid receptor (GR) is phosphorylated on its N-terminus at three major sites (S203, S211 and S226) within activation function 1 (AF1). Although GR has been shown to assemble at glucocorticoid responsive elements (GREs) in the presence of hormone, the timing and specificity of GR phospho-isoform recruitment to receptor target genes has not been established. Using chromatin immunoprecipitation (ChIP) and GR phosphorylation site-specific antibodies, we examined GR phospho-isoform recruitment to several glucocorticoid-induced genes including tyrosine aminotransferase (tat) and sulfonyltransferase-1A1 (sult) in rat hepatoma cells, and the glucocorticoid-induced leucine zipper (gilz) gene in human U2OS cells. GR P-S211 and GR P-S226 isoforms were efficiently recruited to the tat, sult and gilz GREs in a hormone-dependent manner. In contrast, the GR P-S203 isoform displayed no significant recruitment to any GREs of the genes analyzed, consistent with its lack of nuclear accumulation. Interestingly, the kinetics of GR P-S211 and GR P-S226 recruitment differed among genes. Our findings indicate that GR phospho-isoforms selectively occupy GR target genes, and suggests gene specific requirements for GR phosphorylation in receptor-dependent transcriptional activation.

Caveolin-1 Regulates Genomic Action of the Glucocorticoid Receptor in Neural Stem Cells

“…Therefore, the GC response of genes that require pSer211-GR to reach peak activation may be altered in Cav-1 KO NPSCs. For example, pSer211-GR is recruited to promoters of genes such as Gilz and Tat to activate their transcription (29). Since GR phosphorylated at S211 is associated with activated gene transcription, alterations in pSer211-GR are consistent with the attenuated activation of select GR target genes in Cav-1 KO NPSCs.…”

“…In fact, different phosphorylation patterns within the amino-terminal activation function-1 domain can dictate which target genes will be bound by GR (29). Both MAPKs, targets of rapid GR signaling and cyclin-dependent kinase 2 (CDK2), phosphorylate GR and influence its transcriptional activity (39).…”

“…Cav-1 did not act as a classical coregulator of GR on genomic targets but rather altered the phosphorylation of GR on at least one site, S211. Phosphorylation of S211 in U2OS cells influences the GC transcriptional program (29) and may likewise impact target genes regulated by GR in NPSCs. In fact, alterations of the GR transcriptome in NPSCs upon Cav-1 deletion define unique networks that potentially alter GC regulation of the cell cycle (see model in Fig.…”

“…IPVH00010; Millipore). Western blot analysis was performed using the p211-GR antibody (29) and an anti-rabbit horseradish peroxidase-conjugated secondary antibody (Bio-Rad, catalog no. 170-6515) using a chemiluminescence detection system (Advansta Western Bright ECL; catalog no.…”

Cellular Processing of the Glucocorticoid Receptor Gene and Protein: New Mechanisms for Generating Tissue-specific Actions of Glucocorticoids

“…Early studies showed that phosphorylation-deficient GR␣ mutants were compromised in their ability to activate reporter genes in a promoter-dependent fashion (79). Subsequently, it was reported that phosphorylation of Ser-211 correlated with the transcriptionally active form of GR␣, whereas phosphorylation of Ser-226 impaired its signaling capability (78,80,81). Phosphorylation of Ser-211 appears to be necessary for glucocorticoid-induced apoptosis of lymphoid cells, suggesting that a deficiency in this phosphorylation event may be a mechanism by which lymphocytes become resistant to glucocorticoids (82,83).…”