Phosphorylation of L-cell glucocorticoid receptors in immune complexes: evidence that the receptor is not a protein kinase.
Two phosphoproteins are absorbed to protein A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98K phosphoprotein that contains the steroid binding site, and the other is a 90K non-steroid-binding phosphoprotein that is associated with the molybdate-stabilized receptor [Housley, P. R., Sanchez, E. R., Westphal, H. M., Beato, M., & Pratt, W. B. (1985) J. Biol. Chem. 260, 13810-13817]. In this paper we have incubated L-cell cytosol with rabbit antiserum against the mouse glucocorticoid receptor and show that incubation of protein A-Sepharose-bound immune complexes with [gamma-32P]ATP and Mg2+ results in phosphorylation of the 98K steroid-binding protein but not of the 90K receptor-associated protein. Phosphorylation occurs regardless of whether the receptor is unoccupied or is present as the untransformed or transformed steroid-receptor complex. No phosphorylation occurs in the presence of Ca2+ instead of Mg2+. If protein A-Sepharose-bound immune complexes prepared with a monoclonal antibody against the receptor are incubated with [gamma-32P]ATP and Mg2+, neither protein is phosphorylated. If the protein A-Sepharose pellet is obtained from molybdate-stabilized cytosol that has been incubated both with monoclonal antibody to provide the 98K receptor and its 90K associated protein and with preimmune rabbit serum, which causes the nonspecific adsorption of an L-cell protein kinase, then incubation with [gamma-32P]ATP and Mg2+ causes receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D007739:L Cells / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007106:Immune Sera; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D000255:Adenosine Triphosphate; D011494:Protein Kinases