Phosphorylation of Glucocorticoid Receptor-associated and Free Forms of the -90-kDa Heat Shock Protein before and after Receptor Activation*

Several lines of evidence have suggested that glucocorticoid receptor function may be regulated by phosphorylation-dephosphorylation reactions, and it has been proposed that dephosphorylation accompanies activation to the DNA-binding form. The phosphate content of the -100-kDa steroid-binding protein has been determined directly and was found not to change during activation in intact cells (Mendel, D. B., Bodwell, J. E., and Munck, A. (1987) J. Biol. Chem. 262,56445648). We have now determined the effect of interaction with the receptor and of activation on the phosphate content of the -90-kDa heat shock protein (Hsp go), which is thought to be a non-steroid-binding subunit of nonactivated glucocorticoid receptors that dissociates on activation. Monoclonal antibodies AC88 and BuGR2 were used to purify free Hsp 90 and cytosolic nonactivated glucocorticoid-receptor complexes, respectively, from WEHI-7 cells grown in the presence of "Pi and ['"SI methionine. Cell-free activation of the nonactivated receptor-antibody complexes immobilized on protein A-Sepharose minicolumns allowed the recovery of the Hsp 90 dissociated from the complexes during activation. Proteins were separated by denaturing polyacrylamide gel electrophoresis, and the szP/s6S ratio, which was used as a measure of the phosphate content relative to protein, was determined for the free, receptor-associated, and dissociated forms of the Hsp 90, as well as for the -100-kDa steroid-binding protein of nonactivated and activated receptors. The three forms of the Hsp 90 had the same phosphate contents, as did the -100-kDa steroid-binding protein before and after activation. Based upon these results, we conclude that no net change in phosphorylation occurs when the Hsp 90 associates with the -100-kDa steroid-binding protein to form nonactivated receptors and that neither protein component of nonactivated complexes is dephosphorylated when they dissociate during thermal activation under cell-free conditions.