CONTROL BY NASAL PHOSPHORYLATION OF CELL CYCLE-DEPENDENT, HORMONE-INDUCED GLUCOCORTICOID RECEPTOR HYPERPHOSPHORYLATION

Mouse glucocorticoid receptors (GRs) are phosphorylated in the N-termi nal domain at serine/threonine residues, most lying in consensus seque nces for cell cycle-associated kinases, Glucocorticoid agonists, but n ot antagonists, induce hyperphosphorylation. Phosphorylation of GRs ov erexpressed in Chinese hamster ovary (CHO) cells is cell cycle-depende nt: basal phosphorylation in S phase is one third that in G2/M; glucoc orticoids induce hyperphosphorylation in S but not G2/M, paralleling t he reported sensitivity in S and resistance in G2/M of proliferating c ells to transcriptional activation by glucocorticoids, This parallel l ed us to investigate what controls hyperphosphorylation, We tested thr ee hypotheses: hyperphosphorylation is controlled by 1) negative charg e due to basal GR phosphorylation, being permitted in S by low charge and blocked in G2/M by high charge; 2) presence in S and absence in G2 /M of required kinases; 3) availability in S and lack in G2/M of unocc upied phosphorylatable sites, Our results are inconsistent with 2) and 3), but strongly support 1), GR mutants with alanines (A7GR) or gluta mates (E7GR) replacing all but one phosphorylated site were overexpres sed in CHO cells, Serine 122 remained intact to report GR phosphorylat ion. Consistent with hypothesis 1, with A7GRs hormone-induced hyperpho sphorylation occurred in both S and G2/M (thus revealing kinase activi ty for hyperphosphorylation of at least serine 122 in both phases), wh ereas with E7GRs it occurred in neither phase, We conclude that basal GR phosphorylation controls hormone-induced GR hyperphosphorylation by modulating negative charge in the N-terminal domain and could potenti ally control other cell cycle-dependent GR properties.