Epidermal growth factor stimulates tyrosine phosphorylation of human glucocorticoid receptor in cultured cells

Human breast epithelial HBL100 cells, which bind both epidermal growth factor (EGF) and glucocorticoids, were labelled to steady state specific activity with 32Pi and the glucocorticoid receptor was immunoprecipitated from cell lysates with polyclonal antiserum GR884. Immunoprecipitated receptor was resolved by NaDodSO4-polyacrylamide gel electrophoresis and identified by autoradiography. Immunoprecipitated receptor also was characterized by western blot analysis and affinity labelling with [3H]dexamethasone-21-mesylate. Phosphoamino acid analysis of 32P-glucocorticoid receptor revealed 89% phosphoserine and 11% phosphotyrosine. Treatment of steady state 32Pi-labelled cells with EGF stimulated total and alkali-stable phosphorylation in the 97 kDa receptor band by about 35%. Prior incubation with dexamethasone inhibited EGF stimulated, alkali-stable phosphorylation of the 97 kDa glucocorticoid receptor band.