Phosphorylation of rat liver glucocorticoid receptor.
Rat liver glucocorticoid-receptor complex (GRc) was purified 2000-fold by a combination of methods including (NH4)2SO4-fractionation and phosphocellulose and DNA-cellulose chromatography. The purified glucocorticoid receptor preparation contained a major peptide of Mr = 90,000 and the GRc sedimented as 4 S in 5-20% sucrose gradients. An additional peptide of Mr = 45,000 (45K) was also observed. Some preparations yielded only the Mr = 90,000 (90K) peptide suggesting that the 45K peptide may be a proteolyzed portion of the 90K protein. The purified GRc was incubated with [gamma-32P]ATP in the presence of cAMP-dependent kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the above preparation revealed the presence of two 32P-containing bands with apparent Mr = 90,000 and 45,000. The 32P incorporation was dependent on the availability of divalent cation (Mg2+). GRc in cytosol labeled with [3H]dexamethasone mesylate and purified as above co-migrated with 32P-containing bands. GRc was also purified from cytosol obtained from livers of rats injected with [32P]orthophosphate. Both 32P and 3H bands were associated with 90K and 45K peptides. Our results indicate that rat liver glucocorticoid receptor is a phosphoprotein and that both the phosphorylated peptides 90K and 45K also contain the steroid and the DNA binding regions of the glucocorticoid receptor.
D000315:Adrenalectomy; D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D004247:DNA / Q000031:analogs & derivatives; D003907:Dexamethasone / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*
C033190:DNA-cellulose; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D003907:Dexamethasone; D002482:Cellulose; D004247:DNA