PHOSPHORYLATION OF IMMUNOPURIFIED RAT-LIVER GLUCOCORTICOID RECEPTOR BY THE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE

We have examined phosphorylation of the rat liver glucocorticoid recep tor (GR) and GR-associated protein kinase (PK) activity in the immunop urified receptor preparations. Affinity labeling of hepatic cytosol wi th [H-3]dexamethasone U-mesylate showed a covalent association of the steroid with a 94 kDa protein. GR was immunopurified with antireceptor monoclonal antibody BuGR2 (Gametchu and Harrison, Endocrinology 114:2 74-279, 1984) to near homogeneity. A 23 degrees C incubation of the im munoprecipitated protein A-Sepharose adsorbed GR with [gamma-P-32]ATP, Mg2+ and the catalytic subunit of cAMP-dependent PK (cAMP-PK) from bo vine heart, led to an incorporation of radioactivity in the 94 kDa pro tein. Phosphorylation of GR was not evident in the absence of the adde d kinase. Of the radioinert nucleotides (ATP, GTP, UTP or CTP) tested, only ATP successfully competed with [gamma-P-32]ATP demonstrating a n ucleotide specific requirement for the phosphorylation of GR. Other di valent cations, such as Mn2+ or Ca2+, could not be substituted for Mg2 + during the phosphorylation reaction. Phosphorylation of GR was sensi tive to the presence of the protein kinase inhibitor, H-8, an isoquino line sulfonamide derivative. In addition, the incorporation of radioac tivity into GR was both time- and temperature-dependent. The phosphory lation of GR by cAMP-PK was independent of the presence of hsp-90 and transformation state of the receptor. The results of this study demons trate that GR is an effective substrate for action of cAMP-PK and that the immunopurified protein A-Sepharose adsorbed GR lacks intrinsic ki nase activity but can be conveniently used for the characterization of the phosphorylation reaction in the presence of an exogenous kinase.