HORMONE-INDUCED HYPERPHOSPHORYLATION OF SPECIFIC PHOSPHORYLATED SITES IN THE MOUSE GLUCOCORTICOID RECEPTOR

The glucocorticoid receptor (GR) is phosphorylated in its basal state, and rapidly undergoes hormone-induced hyperphosphorylation after binding glucocorticoids. Previously, we have identified seven phosphorylated sites in the mouse GR. Most of the sites are located in the regions of the N-terminal domain that are necessary for maximum transcriptional activity and reduce nonspecific binding to DNA. Using WCL2 cells, which overexpress mouse GRs, we now quantitate hormone-induced hyperphosphorylation at each of these sites. Addition of triamcinolone acetonide to WCL2 cells results in significant hyperphosphorylation at the majority of the sites. The hyperphosphorylation ratio, i.e. the P-32 incorporation into GRs from hormone-treated cells divided by P-32 incorporation into GRs from untreated cells, was above 1.0 for all sites but serine 150 and threonine 159. Serine 220 displays marked hormone dependence, with a ratio of 3. For most sites the ratio was about 1.5. Hormone-induced hyperphosphorylation not only increases the charge at selected phosphorylated sites but also provides a substantial increase in the overall negative charge around the region of the N-terminal domain that is invoked in transactivation.