Phosphorylated sites within the functional domains of the ∼ 100-kDa steroid-binding subunit of glucocorticoid receptors

The steroid-binding subunit of the glucocorticoid receptor is known to be a ∼ 100-kDa phosphoprotein composed of an immunogenic, DNA-binding, and steroid-binding domain. To identify the domains that contain these phosphorylated sites, the authors have analyzed the phosphate content of selected proteolytic fragments of the ∼ 100-kDa steroid-binding protein from nonactivated and activated receptors. The ∼ 100-kDa steroid-binding protein from WEHI-7 cells grown in the presence of [32P]orthophosphate was covalently labeled with [3H]dexamethasone 21-mesylate, purified with the BuGR2 monoclonal antibody, digested with chymotrypsin or trypsin, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chymotrypsin digestion of this protein yields a ∼ 45-kDa fragment containing both the steroid-binding and DNA-binding domains, which contained both 32P and 3H. The 32P/3H ratio of each fragment provides a measure of phosphate content per steroid-binding site and indicated that each fragment has approximately 30% of the phosphate content of the intact protein. This is sufficient to account for one of the three receptor phosphoryl groups. To determine more directly the phosphate content of the DNA-binding domain of the ∼ 100-kDa protein, they isolated a ∼ 16-kDa tryptic fragment from cytosol of WEHI-7 cells grown in the presence of [32P]orthophosphate and [35S]cysteine. Comparison of the 32P/35S ratio of this fragment to that of the intact protein indicated that it contains less than 0.2 mol of phosphoserine/mol of protein