PHOSPHORYLATION SITES IN LIGAND-INDUCED AND LIGAND-INDEPENDENT ACTIVATION OF THE PROGESTERONE-RECEPTOR

Steroid hormone receptors are phosphoproteins that undergo hyperphosphorylation upon binding of hormone. The mechanism and the role of this reaction remain poorly understood. Two-dimensional analysis of ligand-free progesterone receptor (PR) tryptic digests showed the existence of seven main phosphopeptides. Incubation of the cells with the progestin R5020 led to a global increase in the levels of PR phosphorylation. However, the same phosphopeptides were seen, and their levels of labeling relative to each other were unchanged. A similar result was observed after incubation of the cells with the antiprogestin RU486. The antiprogestin ZK98299 demonstrated only half of the activity of RU486 in terms of receptor hyperphosphorylation, but the same phosphopeptides, proportionally labeled to the same extent, were observed by chromatography electrophoresis. Ligand-induced DNA binding did not play a role in receptor hyperphosphorylation since the mutant Delta 547-592, which is devoid of the first zinc finger region, exhibited the same phosphopeptides, labeled to the same extent, as did wild-type receptor after incubation of cells with hormone. These results suggest that the same kinase(s) act in vivo on ligand-free and on agonist or antagonist-bound progesterone receptor. Binding of different ligands produces different conformational changes in the ligand binding domain of the receptor which enhance, to varying extents, affinity of the receptor for the kinase(s). The DNA binding region also plays a role in the interaction with the kinase(s), although binding to DNA per se is not necessary for the hyperphosphorylation of the receptor to take place. Similar phosphorylation patterns were induced by agonists and antagonists, suggesting that receptor hyperphosphorylation was not directly related to its transactivation properties. This conclusion was further supported in a cell-free transcription assay using purified receptor which had or had not undergone hyperphosphorylation in vivo. In conditions where their phosphorylation state was not changed during the incubation, the two receptor species produced the same enhancement of transcription. Receptor phosphorylation patterns were also shown to be unchanged during cAMP-induced, PR-mediated increase of transcription. Thus, both ligand-dependent and ligand-independent enhancement of PR biological activity is unrelated to a change in its phosphorylation state.