CELL CYCLE-DEPENDENT GLUCOCORTICOID RECEPTOR PHOSPHORYLATION AND ACTIVITY

Proliferating cells display striking cell cycle dependence in sensitiv ity to gene activation by glucocorticoids; they are sensitive in late gap 1/synthesis (G1/S) (late G1 and S phases) but resistant in gap 2/m itotic (G2/M), Here we describe large cell cycle-dependent variations in glucocorticoid receptor(GR) phosphorylation that accompany, and may account for, the changes in sensitivity, GRs are basally phosphorylat ed and undergo hyperphosphorylation after hormone-induced activation. Identified phosphorylated sites are all in the N-terminal domain. Seve ral iie in a region required for full transactivating activity and red uction of nonspecific binding to DNA. Most are in consensus sequences for cell cycle-associated kinases, suggesting that such kinases phosph orylate GRs, We now show with WCL2 cells (Chinese hamster ovary cells with overexpressed GRs) that: 1) glucocorticoid treatment fails to hyp erphosphorytate GRs in G2/M but doubles phosphorylation in S, more tha n seen with unsynchronized cells; and 2) basal GR phosphorylation is a lmost three times higher in G2/M than S, These results, along with ear lier observations, implicate GR phosphorylation with mechanisms of glu cocorticoid resistance in G2/M, Such mechanisms might underlie some fo rms of glucocorticoid resistance in inflammatory and lymphoproliferati ve diseases. HPLC phosphopeptide maps of GRs from S and G2/M reveal no significant qualitative differences in phosphorylated sites, consiste nt with a general increase during G2/M in negative charge of the N-ter minal domain. We also show that the previously described increase in G R hormone-binding capacity from G1 to S is accompanied by a parallel i ncrease in GR protein.