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 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.(ABSTRACT TRUNCATED AT 250 WORDS)