HORMONE-INDUCED HYPERPHOSPHORYLATION OF SPECIFIC PHOSPHORYLATED SITESIN THE MOUSE GLUCOCORTICOID RECEPTOR

The glucocorticoid receptor (GR) is phosphorylated in its basal state, and rapidly undergoes hormone-induced hyperphosphorylation after bind ing glucocorticoids. Previously, we have identified seven phosphorylat ed sites in the mouse GR. Most of the sites are located in the regions of the N-terminal domain that are necessary for maximum transcription al activity and reduce nonspecific binding to DNA. Using WCL2 cells, w hich overexpress mouse GRs, we now quantitate hormone-induced hyperpho sphorylation at each of these sites. Addition of triamcinolone acetoni de to WCL2 cells results in significant hyperphosphorylation at the ma jority of the sites. The hyperphosphorylation ratio, i.e. the P-32 inc orporation into GRs from hormone-treated cells divided by P-32 incorpo ration into GRs from untreated cells, was above 1.0 for all sites but serine 150 and threonine 159. Serine 220 displays marked hormone depen dence, with a ratio of 3. For most sites the ratio was about 1.5. Horm one-induced hyperphosphorylation not only increases the charge at sele cted 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.