Identification of a Phosphorylation Site in the Hinge Region of the Human Progesterone Receptor and Additional Amino-terminal Phosphorylation Sites

Abstract

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. 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. progesterone receptor cyclin-dependent kinase-2 high performance liquid chromatography nitrilotriacetic acid ferric ion nitrilotriacetic acid resin phosphate-buffered saline Human progesterone receptor (PR),1 a ligand-activated transcription factor and member of the steroid receptor superfamily (1Evans R.M. 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Full Text Full Text PDF PubMed Scopus Google Scholar). different target unique of sites and these may have on receptor it is to which sites are by for studies to determine the on receptor We have previously identified seven phosphorylation sites in human PR from T47D of these sites is in the amino-terminal of the previously identified sites, and are unique to the Y. Beck C.A. Poletti A. Edwards D.P. N.L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). In of the PR in T47D that the PR is in the of at and the phosphorylation of these sites is of In addition, the phosphorylation of new phosphorylation sites, and in a for phosphorylation. These sites are to as the phosphorylation sites Y. Beck C.A. Poletti A. Edwards D.P. N.L. Mol. Endocrinol. 9: PubMed Google Scholar). human PR expressed in the phosphorylation as PR from T47D the is in C.A. Y. N.L. Edwards D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Additionally, we have that PR is a for several in We reported previously that in vivo phosphorylation sites, and are in vitro by the Y. Beck C.A. Poletti A. P. Edwards D.P. N.L. Mol. Endocrinol. 1997; PubMed Scopus Google and that is by Y. Beck C.A. Poletti A. Edwards D.P. N.L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). of the phosphorylation sites have been identified in PR, several peptide in the vivo PR have to be of the phosphorylation sites in steroid receptors and in the of the chicken PR of these motifs are O'Malley B.W. N.L. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, A. N.L. Mol. Endocrinol. 1993; 7: Google Scholar). Human PR contains of these several of which have been identified as phosphorylation sites Y. Beck C.A. Poletti A. Edwards D.P. N.L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, Y. Beck C.A. Poletti A. Edwards D.P. N.L. Mol. Endocrinol. 9: PubMed Google Scholar, Y. Beck C.A. Poletti A. P. Edwards D.P. N.L. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar). We in whether the Ser-Pro in the hinge region the and is as has been for chicken PR O'Malley B.W. N.L. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), mouse estrogen receptor H. J. Mol. Biol. PubMed Scopus Google Scholar), and human androgen receptors J.A. Mol. Endocrinol. 9: PubMed Google Scholar). by suggested that this site is not in human PR P.L. Evans R.M. Horwitz K.B. J. Biol. Chem. 1989; 264: 6520-6528Abstract Full Text PDF PubMed Google Scholar). and motifs are not by trypsin G. in and Scholar), and PR has an large of these of candidate Ser-Pro phosphorylation sites including Ser676 in the hinge region that are by these highly cleavage sites. these phosphopeptides be large and to by a Using trypsin by which has to we have the cleavage at these problematic sites, of new phosphopeptides in PR in vitro by Cdk2 and by mass spectrometry of phosphopeptides from PR expressed in we the identification of six candidate phosphorylation sites identified either in in vitro phosphorylation Cdk2 or by mass spectrometry of from PR expressed in These sites Ser676 in the hinge region, which was identified as vitro Cdk2 site and by mass additional sites, to PR-A and and two sites unique to the PR-B have been an additional site has been to a region amino acids and in the of the receptor unique to These studies that most, if not all, of the motifs in PR are and from and was from was from and from resin was from and and from was from and from J. trypsin was from and from and acid from and the was from from from and acids and from for the acid of with PR-B or PR-A and with was as previously P. 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Chem. 1994; Full Text PDF PubMed Google in of and at on a tryptic was in a and the phosphopeptides on a a in acid was for by an to by at a of phosphopeptide an as by for and in a determine the or of the acid in the was as previously S. PubMed Scopus Google the the tryptic phosphopeptides to and to of by the first amino acid of the which the first peptide with acid this and the amino acid from the of the the of was to additional of by the and was to the the was in and with of for at was with of in for at was by the in of of by the and of and for at the was in of and with of at for acid was as P. 1994; PubMed Scopus (125) Google Scholar). In phosphopeptide containing of acid in and in a was in and on a by for at in the first a the was the was and in the in at for acids the in amino acid by the with a of in amino acids by T47D at a of in in in to the was with for at was to the and the at to the with at a of was and the with and was to the and to for by and the was and to by at with and and with the in containing a of and C.A. N.L. Edwards D.P. Mol. Endocrinol. PubMed Scopus Google and by in a with to the to and at for was to the was in and with of in with of for at on a with of the PR L. N.L. Edwards D.P. Mol. Endocrinol. 2000; 14: PubMed Scopus Google was to the and at on a with and with was to the and at on a with with containing and with by in the by at to PR was from the by at in and on a PR was by PR was for in a PR was with of and for at additional of was and for an additional was in a and as Sci. 1997; PubMed Scopus (236) Google by a with of of of of of ferric in and of in and on the was with of of and of phosphopeptides with of resin to on a mass with a nanoelectrospray in an of and and in the nanoelectrospray for peptide identification ion precursor ion for with as the and a of ion the first as the mass and a of ion of the precursor to the site of phosphorylation with as the and of human PR or was by as and by and the PR was tryptic was on a that the phosphopeptide in the two in the phosphopeptide of PR-B and PR-A with the new to the previous Cdk2 with the we observed several phosphopeptide as as the previously identified and These new are to of the Cdk2 in the phosphorylation their not of these is in PR-B the new Cdk2 in with the previously of phosphopeptides from in vivo PR-B with we that the Cdk2 sites a of the sites in of the phosphopeptide from the in have not been Interestingly, the to a in the with with are in the in vivo that are previously unidentified authentic sites. demonstrated the cleavage is to cleavage of the of the be as an of the of phosphorylation of these sites. these new we a combination of and acid the data from the of the phosphopeptides in and in with an additional in has a in and in are in to of to incomplete and cleavage as as the of the are not an G. in and of indicate with acid of or are not in a new indicate with acid of or are not these with the and by on a peptide for and peptide the data and of and of the presence of be by the in the their not in a new of the presence of be by the in the their not of the presence of a phosphopeptide that not with with was in the of the not a Ser-Pro is for phosphorylation by acid was was the in this peptide the in PR to be in of a tryptic the peptide is in a the tryptic peptide not the Additionally, it is a peptide if from the revealed that incomplete tryptic of the peptide in a phosphopeptide with the peptide containing with by a to cleavage by trypsin G. in and Scholar). that this peptide is a incomplete tryptic phosphopeptide containing Thr430, additional with and These and with a with the peptide not the peptide of the incomplete tryptic phosphopeptide containing is with on the of these as of the Cdk2 phosphorylation sites. contains two phosphopeptides as by peptide that is the not the the two phosphopeptides be In this the containing the phosphopeptide with the with and the peptide with a that and two in PR a in by a the first candidate peptide is from the region unique to the candidate be the phosphopeptide was observed in PR-A and PR-B Additionally, the peptide is not a likely we have previously that this phosphopeptide of the candidate peptide containing that with the peptide be to cleavage to the of the motifs are at the of a peptide G. in and Scholar). of this peptide results in a phosphopeptide not the peptide be In the of the phosphopeptide with a of the region with to the to a the remained as These data the phosphopeptide from the the peptide containing to a peptide with a the two phosphopeptides in by Cdk2 in vitro to the containing Ser213 and observed in the PR-B a on by two of the candidate with are in the region of PR these, is a of the of the tryptic peptide containing that of the tryptic peptide with the to a we a the other peptide candidate not the Ser-Pro a in These results the site in as in the a large and data that the acid is in the with in contains a Ser-Pro with either or a in of the peptide observed by peptide the two with a in that and the Ser676 site is with these Ser676 is the site a Ser-Pro and the peptide containing contains a it is to the which is to be to cleavage G. in and Scholar). the and of the Ser676 tryptic peptide is with the of this peptide from the these we have identified Ser676 as the peptide in Interestingly, this site is analogous to the site identified in other steroid receptors. of the phosphorylation sites identified in steroid receptors are in Human PR contains of these of which six have been previously to be Y. Beck C.A. Poletti A. Edwards D.P. N.L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, Y. Beck C.A. Poletti A. Edwards D.P. N.L. Mol. Endocrinol. 9: PubMed Google Scholar, Y. Beck C.A. Poletti A. P. Edwards D.P. N.L. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar). of the sites that tryptic from to amino acids in for and from a Our previous studies that PR expressed in the phosphopeptide as PR from T47D C.A. Y. N.L. Edwards D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We of the of mass to for additional phosphorylation sites in baculovirus-expressed PR from of a tryptic of PR-B in precursor ion revealed a of the in ion was to phosphopeptides for of the by revealed a large phosphopeptide at ion precursor ion of revealed a to and mass a peptide of mass to amino acids which a phosphorylation. peptide contains including which in a Ser-Pro not of the in this region of PR have been previously identified as phosphorylation sites, this a with with containing previously identified sites, and In ferric ion resin was to the phosphopeptides from PR-B was with and and the phosphopeptides on the ferric ion resin as the of for the ion and the of the a for amino acids by cleavage with trypsin and containing a at amino acid the in the peptide and of the of the phosphopeptide at that it to by a trypsin and which contains at identified phosphopeptides in this the peptide at identified as the peptide containing the peptide at identified as the peptide containing and the phosphopeptide at as the phosphopeptide containing not of phosphopeptides by mass ion of the ion at with the of the and to the mass of the as of PR. ion of peptide of and is with as is the and this site as in vivo phosphorylation sites identified by mass spectrometry in a new determine whether of the sites by Cdk2 in vitro are in sites, we in vivo of PR from T47D in the and presence of PR was by by and as previously for the in vitro with from in vivo vitro by peptide of the to the of cleavage and from and the from additional peptides. of the from in vivo PR with the as the containing Ser213 by Cdk2 that a phosphopeptide with the on as the peptide determine whether phosphorylation of this site is or whether the site is phosphorylation was in the presence and of with that of the which has previously been to be a phosphorylation site and phosphorylation is with that the relative in of Ser213 phosphorylation as a of is to that of These data that Ser213 is in vivo in T47D and has the of a phosphorylation presence of unidentified in our phosphopeptide Y. Beck C.A. Poletti A. P. Edwards D.P. N.L. Mol. Endocrinol. 1997; PubMed Scopus Google and the to Ser676 Ser-Pro that is to the hinge site in chicken PR O'Malley B.W. N.L. J. Biol. Chem. 1990; Full Text PDF PubMed Google as a phosphorylation site to new to phosphorylation sites in PR. of PR revealed that the Ser-Pro that not been identified as phosphorylation sites are likely to in large by incomplete at trypsin cleavage sites. Using trypsin to as as other we have identified Ser676 as a phosphorylation site in PR by mass spectrometry and have that it is a for Using these we have identified five other candidate phosphorylation sites. We previously identified phosphorylation sites, and which by Cdk2 in vitro Y. Beck C.A. Poletti A. P. Edwards D.P. N.L. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar). Using the trypsin that and at a we have in the and have identified five additional Cdk2 sites in these Our studies the in phosphorylation sites in large by incomplete of the trypsin we in our previous studies with the new trypsin that was a in the phosphopeptide by the new to be with previously motifs be relative to other trypsin cleavage sites G. in and Scholar). that we are cleavage is in of the peptide with the be of the of phosphorylation for by these cleavage sites. to our to of these new in of T47D receptor. in large from with a as a as phosphorylation by other in vivo the of the of the new in to and our previous of from PR from T47D that is at least additional phosphopeptide in this region that has not been the of mass spectrometry to from PR expressed in and in of T47D cells, we have identified two new phosphorylation sites and and two phosphopeptides peptide containing and a peptide that with the Cdk2-phosphorylated mass have to additional phosphorylation sites in PR as as to previously identified sites. mass have in the by our studies, we PR by of and by to the and for we detect phosphopeptides in a ion precursor ion we that of of was to for phosphopeptides for by mass chromatography to the this was in phosphopeptides of we not detect the large phosphopeptides from incomplete with trypsin in the studies from our have that in of PR from T47D and are and aberrant phosphorylation was observed in C.A. Y. N.L. Edwards D.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we that the new phosphorylation sites identified by mass spectrometry are in receptor expressed in T47D of these that it be to detect the sites in T47D PR either by or by mass spectrometry of have been to the and sites, and we have to phosphorylation of PR in T47D L. N.L. Edwards D.P. Mol. Endocrinol. 2000; 14: PubMed Scopus Google Scholar). in the of of these new phosphorylation sites, generation of phosphorylation be in their phosphorylation in vivo and their regulation by and the sites previously our has identified a of sites by Cdk2 in five of these have been confirmed in vivo and of the Ser-Pro motifs in PR are for Cdk2 phosphorylation, the by In we observed that Cdk2 Ser25, not the that phosphorylation sites are the tryptic peptide and of the sites by Cdk2 in Ser25, Ser213, Thr430, and Ser554, are the Interestingly, and the is amino acids of the of these sites to the their ability to or be by or Cdk2 Ser676, was identified of the in the hinge region the and Ser676 is analogous to vivo phosphorylation sites identified in chicken PR O'Malley B.W. N.L. J. Biol. Chem. 1990; Full Text PDF PubMed Google and in human androgen receptor J.A. Mol. Endocrinol. 9: PubMed Google Scholar). Ser676 was not as a phosphorylation site in a of PR P.L. Evans R.M. Horwitz K.B. J. Biol. Chem. 1989; 264: 6520-6528Abstract Full Text PDF PubMed Google Scholar). this peptide have been for by by and have the other to sites. In androgen receptor and chicken PR, of the to in transactivation J.A. Mol. Endocrinol. 9: PubMed Google for chicken PR, the was observed at S. N.L. Mol. Endocrinol. 1994; PubMed Google Scholar). Interestingly, this site in human PR has been and the of an was in activity from to the and that this is an site in human PR G.S. L. Horwitz K.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We have confirmed by mass spectrometry that Ser676 is an in vivo phosphorylation to to the it is to that and the phosphorylation of Ser676. role of Cdk2 phosphorylation in receptor has been examined for several steroid receptors. In the of the human estrogen receptor, overexpression of a of estrogen transactivation Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). two and have been by in vitro phosphorylation and studies to be in this J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). receptor has been to be a for Cdk2 two and M.D. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). of receptor transactivation in that transactivation is by of the M.D. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). Cdk2 and are in it is to that the phosphorylation of mouse receptor is A. Mol. Endocrinol. 1994; Google Scholar). of this to a role for in steroid receptor by phosphorylation. In the of PR, the large of Cdk2 phosphorylation sites regulation of PR the In six new phosphorylation sites have been identified in PR of these are in vitro by Ser676, as as two phosphorylation sites, Ser20 and a peptide containing the site identified as in sites by mass phosphopeptide containing the identified Cdk2 site Ser213 of a phosphorylation site in of these additional phosphorylation sites in PR that we have to the of PR phosphorylation. We E. for with PR and of phosphopeptides for mass spectrometry, G. for the and and the of for of PR in