Biochemistry zyxwvut 1994,33, zyxwvu 13295- 13303 13295 Phosphorylation Sites in Ligand-Induced and Ligand-Independent Activation of the Progesterone Receptor: Anne Chauchereau, Karine Cohen-Solal, AndrC Jolivet, Alain Bailly, and Edwin Milgrom* Unit.4 de Recherches Hormones et Reproduction (INSERM U.135), Facult.4 de Mgdecine, Paris Sud, 94275 Le Kremlin, BicBtre Cedex, France Received July 27, 1994@ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ ABSTRACT: 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 A547-592, 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 zyx 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 pattems 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 pattems 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. The phosphorylation of transcription factors and activators has been extensively studied [review in Hunter and Karin (1992); Meek zyxwvutsrqp & Street, 19921. In many cases, phosphory- lation has been shown to modulate transcription factor activity, either by changing their subcellular localization (Kessler & Levy, 1991; Moll et al., 1991; Mosialos et al., 1991), by modifying their DNA binding properties (Luscher et al., 1990; Boyle et al., 1991a), or by regulating their transactivation efficiency (BinCtruy et al., 1991; Gonzalez et al., 1991; Smeal et al., 1991). Steroid hormone receptors are ligand-dependent transcription regulators. They have been shown to be phosphoproteins that undergo a hyper- phosphorylation event under the effect of the hormone (Logeat et al., 1985b; Pike & Sleator, 1985; Moudgil, 1990; Orti et al., 1992). With the exception of one series of results obtained with the estrogen receptor (Migliaccio et al., 1986), serines have been shown to be the major substrate of 'This work was supported by INSEFW, the Ligue Nationale FranGaise Contre le Cancer, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Mddicale, and the Facultd de MCdecine Paris Sud. * To whom correspondence should be addressed. Telephone: (1) 45 21 33 29. Fax: (1) 45 21 27 51. @ Abstract published in zyxwvutsrqpon Advance ACS Absrracrs, October 15, 1994. 0006-296019410433- 13295$04.50/0 phosphorylation. Studies have localized these modified residues to the N-terminal half of the progesterone (Sheridan et al., 1988; Sullivan et al., 1988; Chauchereau et al., 1991), estrogen (Le Goff et al., 1994), glucocorticoid (Hoeck & Groner, 1990; Bodwell et al., 1991), and androgen receptors (Kuiper et al., 1993). However phosphorylation sites outside this region have been described for the chicken progesterone receptor (Denner et al., 1990; Poletti & Weigel, 1993) and for the mouse (Lahooti et al., 1994) and human estrogen receptor (Ali et al., 1993). In all cases, multiple phospho- rylation sites have been reported. Based upon in vivo experiments (Takimoto et al., 1992) and in vitro transcription studies (Bagchi et al., 1991, 1992), it has been proposed that the hyperphosphorylated receptor is the biologically active species. Binding of the hormone to the receptor provoked receptor dissociation from a complex involving several heat shock proteins and the subsequent binding of receptor to DNA. At both of these steps, specific kinases were supposed to modify the receptor in several successive reactions. Furthermore, it appears that enhancement of transcription can occur through receptors and hormone responsive elements in the absence of ligand (Power et al., 1991a; Sartorius et al., 1993). This was achieved by treatment of cells with cyclic AMP or with zyxwvutsrq 0 1994 American Chemical Society