Regulatory effects of glucocorticoid receptor phosphorylation

Abstract

The glucocorticoid receptor contains several phosphorylation sites that have been mapped to the N-terminal transactivation domain. We are investigating whether GR phosphorylation affects basic GR parameters such as ligand-binding affinity, nuclear translocation, and reporter-gene transactivation. Using U2-OS osteosarcoma cells overexpressing either wild type GR or phosphomutant GR, we have found that ligand-binding affinity is not significantly altered by GR's phosphorylation status. However, significant effects are observed in transactivation and translocation assays. At 1 nM dexamethasone treatment for 8 hours, reporter-gene induction was about 2 fold higher in the wild type GR cells compared to the phosphomutant GR cells. In addition, 1 hour treatment with 1 nM dexamethasone was sufficient to drive YFP-tagged GR into the nucleus while phosphorylation-deficient GR required 10 times more hormone to obtain the same affect. These data indicate that the phosphorylation status of GR can influence the subcellular distribution of GR, and the regulation of target genes. Therefore, the effects of GR on gene expression might be modulated by kinase pathways and provide an important regulatory mechanism.