Cell cycle-dependent glucocorticoid receptor phosphorylation and activity.
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.
Hu
J M
JM
Department of Physiology, Dartmouth Medical School Lebanon, New Hampshire 03756.
Bodwell
J E
JE
Munck
A
A
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002453:Cell Cycle / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D016384:Consensus Sequence; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004351:Drug Resistance; D005938:Glucocorticoids / Q000494:pharmacology; D007700:Kinetics; D015735:Mifepristone / Q000494:pharmacology; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D016196:S Phase / Q000502:physiology
D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D015735:Mifepristone; D004247:DNA; D011494:Protein Kinases