PROTEIN SYNTHESIS POST-TRANSLATION MODIFICATION AND DEGRADATION
Identification of a Phosphorylation Site in the Hinge Region of the Human Progesterone Receptor and Additional Amino-terminal Phosphorylation Sites*

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We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser20 and Ser676, in baculovirus-expressed human PR. Ser676 is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser130-Pro131, a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser25, Ser213, Thr430, Ser554, and Ser676. At least two of these, Ser213 and Ser676, are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser213 in PR from in vivolabeled T47D cells, indicating that this is an in vivosite. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.

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Published, JBC Papers in Press, December 7, 2000, DOI 10.1074/jbc.M009805200

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This work was supported in part by Public Health Service Grant R01 CA57539 (to N. L. W.) and the University of Colorado Cancer Center Core Grant P30 CA46934 (to D. P. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.