Molecular Endocrinology, Vol 8, 1709-1713, Copyright © 1994 by Endocrine Society

ARTICLES

Cell cycle-dependent glucocorticoid receptor phosphorylation and activity

JM Hu, JE Bodwell and A Munck
Department of Physiology, Dartmouth Medical School Lebanon, New Hampshire 03756.

Proliferating cells display striking cell cycle dependence in sensitivity 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/mitotic (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 phosphorylated and undergo hyperphosphorylation after hormone- induced activation. Identified phosphorylated sites are all in the N- terminal domain. Several lie in a region required for full transactivating activity and reduction of nonspecific binding to DNA. Most are in consensus sequences for cell cycle-associated kinases, suggesting that such kinases phosphorylate GRs. We now show with WCL2 cells (Chinese hamster ovary cells with overexpressed GRs) that: 1) glucocorticoid treatment fails to hyperphosphorylate GRs in G2/M but doubles phosphorylation in S, more than seen with unsynchronized cells; and 2) basal GR phosphorylation is almost three times higher in G2/M than S. These results, along with earlier observations, implicate GR phosphorylation with mechanisms of glucocorticoid resistance in G2/M. Such mechanisms might underlie some forms of glucocorticoid resistance in inflammatory and lymphoproliferative diseases. HPLC phosphopeptide maps of GRs from S and G2/M reveal no significant qualitative differences in phosphorylated sites, consistent with a general increase during G2/M in negative charge of the N-terminal domain. We also show that the previously described increase in GR hormone-binding capacity from G1 to S is accompanied by a parallel increase in GR protein.

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