Abstract
Human progesterone receptor (PR) is phosphorylated on multiple serine residues; three sites (Ser102, Ser294, and Ser345) are inducible by hormone agonist, while at least six others are basally phosphorylated and exhibit a general increase in response to hormone. In this study we have used high performance liquid chromatography phosphopeptide mapping and manual peptide sequencing to investigate how two different progestin antagonists, RU486 and ZK98299, affect site-specific phosphorylation of PR isolated from T47D breast cancer cells. As compared to the progestin agonist R5020, RU486 stimulated a similar increase in overall incorporation of [32P]phosphate per PR molecule (2.5-2.6-fold for PR-A and 2.1-fold for PR-B), and at the site-specific level, RU486 stimulated both the basal and inducible sites to the same extent as R5020. In contrast, ZK98299 produced only a minimal increase in overall phosphorylation (1.2-fold for PR-A and 1.1-fold for PR-B) which was due to a reduced stimulation of the basal sites and failure to induce any of the three hormone-dependent sites. No inappropriate phosphorylation sites were detected in response to either RU486 or ZK98299. In cotreatment studies, ZK98299 blocked the increase in overall phosphorylation of PR induced by R5020, demonstrating that the failure of this antagonist to stimulate specific phosphorylation sites is not due to an inefficient interaction with PR in the intact cell. These results indicate that the biological effects of RU486 are not mediated by an alternation in the phosphorylation state of PR, whereas failure to promote phosphorylation of certain sites may contribute to the antagonist action of ZK98299. Additionally these results support the concept of two mechanistic classes of anti-progestins that affect PR differently in vivo.
Highlights
All members of the steroid/thyroid hormone receptor family that have been analyzed so far are phosphoproteins, and as a general property they become hyperphosphorylated as a rapid response to binding hormone in the cell (1–7)
The Progestin Antagonists RU486 and ZK98299 Have Distinct Effects on Overall Phosphorylation of Human progesterone receptor (PR)—In previous time course studies we have shown that the progestin agonist R5020 stimulates a sequential increase in phosphorylation of human PR in T47D cells (12, 36)
In the present study we examined the effects of progestin antagonists on phosphorylation of PR by incubating T47D cells for a total of 6 h with [32P]orthophosphate and adding ligand for the last 2 h of labeling
Summary
Materials—[3H]R5020 (promegestone: [17␣-methyl-3H]17␣, 21-dimethyl-19-norpregna-4,9-diene-3, 20-one; 87 Ci/mmol), unlabeled R5020, carrier-free [32P]H3PO4 (8200 –9200 Ci/mmol), and EXPRE35S protein labeling mix (1,175 Ci/mmol) were obtained from DuPont NEN. For labeling with [35S]methionine, paired cultures of T47D cells were incubated for 48 h with minimal essential medium containing 5% charcoal-treated serum supplemented by the addition of 25 Ci/ml [35S]methionine. Protein A-Sepharose beads were washed with KPFM containing 0.3 M NaCl to remove nonspecifically bound protein, and immobilized receptors were eluted with 2% SDSsample buffer and electrophoresed on 7.0% discontinuous SDS-polyacrylamide gels as described previously (30, 36). Tryptic Digestion and HPLC Analysis of PR—Gel-purified receptors were extracted and digested with tosylphenylalanyl chloromethyl ketone-treated trypsin (20 g) as described previously (11, 12). This digestion protocol resulted in a highly reproducible phosphopeptide mapping profile, indicating digestion to completion, and a Ͼ70% recovery of 32P counts was obtained from gel pieces. The disk was washed five times with 1 ml of methanol before the cycle was started
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