Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites

We have previously reported the identification of seven in vivo phosphoryla tion sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosph opeptides remained unidentified. In particular, we wished to determine whet her human PR contains a phosphorylation site in the hinge region, as do oth er steroid receptors including chicken PR, human androgen receptor, and mou se estrogen receptor. Previously, problematic trypsin cleavage sites hamper ed our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphoryla tion, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have i dentified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in b aculovirus-expressed human PR. Ser(676) is analogous to the hinge site iden tified in other steroid receptors, Additionally, precursor ion scans identi fied another phosphopeptide that contains Ser(130)-Pro(131), a likely candi date for phosphorylation. In vitro phosphorylation of PR with Cdk2 has reve aled five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213) , Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser (676), are authentic in, vivo sites. We confirmed the presence of the Cdk2- phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indica te that most, if not all, of the Ser-Pro motifs in human PR are sites for p hosphorylation. Taken together, these data indicate that the phosphorylatio n of PR is highly complex, with at least 14 phosphorylation sites.