Progesterone receptor subunits are high-affinity substrates for phosphorylation by epidermal growth factor receptor.

Abstract

Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2 . Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80-or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/ min per mg of EGF receptor protein at 0°C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22°C.Tyrosine phosphorylation has become recognized as a protein modification reaction with potential roles in regulation of cell metabolism, reproduction, and differentiation. Viral transforming gene products and their normal cell homologues (1-4) and receptors for epidermal growth factor (EGF) (5), platelet-derived growth factor (6-8), and insulin (9-10) all catalyze transfer of phosphate from ATP to tyrosine residues of proteins. The best-characterized substrates for these kinases are the kinase proteins themselves; all undergo autophosphorylation. In addition, viral transformation of cells or binding of ligands to specific cell surface receptors stimulates tyrosine phosphorylation of cellular proteins (11-13). With the exception of vinculin (14) (20). To test for possible involvement of tyrosine phosphorylation in regulation of steroid receptor function, we examined purified hen oviduct progesterone receptor as a substrate for purified EGF receptor kinase. EXPERIMENTAL PROCEDURESPurification of Progesterone Receptor. The transformed progesterone receptor was purified from hen oviducts by a procedure similar to that of Chang et al. and B (105-kDa) subunits (kDa, kilodaltons). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/ PAGE) analysis of purified preparations showed minor impurities migrating at 280, 150, and 45 kDa on gels overloaded for receptor protein. In addition, there were faint bands immediately below the 80-and 105-kDa receptor bands that could arise from limited proteolysis; these increased when the serine protease inhibitor phenylmethylsulfonyl fluoride was omitted during receptor preparation. Receptor preparations were concentrated at the final step by precipitation with (NH4)2SO4 and dissolved in, and dialyzed against, 20 mM Hepes, pH 7.4. Photoaffinity labeling of the receptor subunits with 3H-labeled 17a, 21-dimethy...

Phosphorylation of Steroid Hormone Receptors*

“…As shown in our laboratory (unpublished results), protein kinase A also phosphorylates the glucocorticoid receptor in vitro. The oviduct progesterone receptor was found to be a good substrate for phosphorylation with purified epidermal growth factor receptor (86). This phosphorylation was exclusively at tyrosine residues, located in two major tryptic phosphopeptides.…”

Progress in understanding breast cancer: Epidemiological and biological interactions

“…Breast cancer cells have been reported to contain EGF receptors possibly in inverse proportion to estrogen receptors [153], although not all studies support this [154]. EGF receptors are capable of modifying progesterone receptor activity [155] and contain sequences closely related to those in the v-erb-B oncogene protein product [156] such that, once established, tumors might be able to sustain their own growth factor stimulus. The mechanism by which growth factors produced by the stoma might stimulate epithelial cell growth has been reviewed by Cuhna et al [157].…”

Nuclear estrogen receptor molecular heterogeneity in the mouse uterus.

“…Nonetheless, a paucity of data exists with regard to the actual regulation of receptor protein activity as directly induced by the hormone. Recent reports (10,11) have suggested the phosphorylation of tyrosines as a possible regulatory point; however, the relationship between this phenomenon and hormonal activation of receptor has not been established conclusively (12). Other reports indicate that, in the case of the estrogen receptor (ER), estradiol binding to the receptor directly influences receptor affinity for nonspecific nuclear binding sites (13), mechanistically theorized as a change in receptor conformation.…”