phosphorus-radioisotopes and Thymoma

Glucocorticoid receptors are basally phosphorylated in the absence of hormone and become hyperphosphorylated after hormone treatment of intact cells. To determine the sequence of changes which the receptor undergoes following hormone binding, we analyzed the kinetics of receptor phosphorylation in WEHI-7 mouse thymoma cells and in stably transfected Chinese hamster ovary cells that overexpress the mouse receptor. No major differences were found between these two cell types. Cells were preincubated with 32P(i) and [35S] methionine to label the receptors metabolically. The phosphate content of the receptor protein was determined from the ratio of 32P to 35S in radioactive gel slices after immunopurification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hormone-induced increases in phosphorylation were seen as early as 5 min after adding hormone and persisted for 20 h. Analysis of newly formed cytosolic and nuclear-bound activated (DNA-binding) receptors showed that activation precedes hyperphosphorylation. Nonactivated receptors, both unliganded and hormone-liganded, also became hyperphosphorylated but more slowly than activated receptors. The rate of receptor dephosphorylation, determined by chasing with unlabeled P(i), was much slower than the rate of phosphorylation or of hormone dissociation and appeared to be slightly increased by agonists and by the antagonist RU486 (which does not cause hyperphosphorylation). Mutant WEHI-7 cells lacking cAMP-dependent protein kinase activity gave basal and hormone-induced receptor phosphorylation indistinguishable from wild type cells. We conclude that (a) the substrate for hormone-dependent hyperphosphorylation is the activated hormone-receptor complex; (b) most hyperphosphorylated receptors are recycled and reutilized in hyperphosphorylated form; (c) control of receptor phosphorylation may not be cell-specific; (d) cAMP-dependent protein kinase is not involved directly or indirectly in phosphorylating major sites on the receptor in vivo.

Topics: Animals; Cell Nucleus; CHO Cells; Cricetinae; Cytosol; Kinetics; Methionine; Mice; Mifepristone; Mutation; Phosphates; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Receptors, Glucocorticoid; Sulfur Radioisotopes; Thymoma; Thymus Neoplasms; Time Factors; Transfection; Triamcinolone Acetonide; Tumor Cells, Cultured

We have used a monoclonal antibody to purify glucocorticoid-receptor complexes from WEHI-7 mouse thymoma cells. Molybdate-stabilized, nonactivated complexes were found to contain two distinct proteins which could be separated by polyacrylamide gel electrophoresis under denaturing and reducing conditions. One of the proteins, 100 kDa, was labeled when cytosol was incubated with the affinity ligand [3H]dexamethasone 21-mesylate. The second protein, 90 kDa, was not labeled. Several lines of evidence, including Western blot analysis of purified nonactivated complexes, indicate that only the 100-kDa protein is directly recognized by the antibody. The 90-kDa protein appears to be purified as a component of the nonactivated complex due to noncovalent association with the 100-kDa protein. Both the 100-kDa and 90-kDa components of the nonactivated complex become labeled with 35S when cells are grown in medium containing [35S]methionine. Using cells labeled in this manner, we have shown that activated (i.e. DNA-binding) cytosolic complexes, formed by warming either in intact cells or under cell-free conditions, contain only the 100-kDa protein. Complexes extracted from nuclei of warmed cells similarly contain only the 100-kDa protein. These results indicate that the 100-kDa and 90-kDa components of nonactivated complexes separate upon activation. Purification of nonactivated complexes from cells grown in medium containing [32P]orthophosphoric acid indicates that both the 100-kDa and 90-kDa components are phosphoproteins which can be labeled with 32P. Therefore, resolution of the two proteins will be essential in order to determine whether the receptor is dephosphorylated on activation.

Topics: Animals; Antibodies, Monoclonal; Cytosol; Mice; Molecular Weight; Molybdenum; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Receptors, Glucocorticoid; Thymoma