Phosphorylation of human progesterone receptors at serine-294 by mitogen-activated protein kinase signals their degradation by the 26S proteasome

Abstract

Ligand-dependent down-regulation that leads to rapid and extensive loss of protein is characteristic of several nuclear steroid receptors, including human progesterone receptors (PRs). In breast cancer cells, >95% of PRs are degraded 6 h after the start of progestin treatment. The mechanism for down-regulation is unknown. We examined the role of PR phosphorylation by mitogenactivated protein kinases (MAPKs) in this process. Lactacystin and calpain inhibitor I, specific inhibitors of the 26S proteasome, blocked progestin-induced down-regulation, and ubiquitinated conjugates of PR accumulated in cells. Ligand-dependent PR degradation was also blocked by specific inhibition of p42 and p44 MAPKs. To define the targets of phosphorylation by this kinase, two serine͞proline MAPK consensus sites on PR were mutated. We demonstrate that mutation of PR serine-294 to alanine (S294A) specifically and completely prevents ligand-dependent receptor down-regulation. We also find that rapid, ligand-independent degradation of immature PR intermediates occurs by a proteasome-mediated pathway. These results demonstrate that PR destruction, by either of two alternate routes, is mediated by the 26S proteasome. Specifically, down-regulation of mature PRs occurs by a mechanism in which ligand binding activates PR phosphorylation by MAPKs at a unique serine residue, which then targets the receptors for degradation.

Linkage of progestin and epidermal growth factor signaling: Phosphorylation of progesterone receptors mediates transcriptional hypersensitivity and increased ligand-independent breast cancer cell growth

“…Both agents are known to activate MAPKs within 5-15 minutes (25,26). However, EGF is a stronger activator of MAPK relative to progestins at this time point, and liganded PRs are relatively transient, most likely due to the rapid down-regulation of liganded and Ser294 phosphorylated species (14). We therefore compared the kinetics of PR Ser294 phosphorylation in response to treatment of T47D-YB breast cancer cells with either EGF or R5020 for 5-60 min (Fig.…”

“…PR Ser294 is a hormone-inducible MAPK consensus site in the PR N-terminus that may serve as a direct "sensor" for MAPK-dependent input to PR transcriptional activity (6,(13)(14)(15)(16)19,32). Therefore, we included our well-characterized S294A PR mutant receptor in the above experiments (Fig.…”

“…3). S294A PR-B contains a single point mutation at Ser294 to Ala that renders it resistant to progestin-induced downregulation in multiple cell lines (14). S294A PR-B is transcriptionally impaired when stably expressed in T47D or HeLa cells (16).…”

“…Additionally, EGF pretreatment of cells expressing wt PR-B resulted in the rapid downregulation of liganded PR in the presence of 0.10 to 10nM R5020 (lanes [10][11][12]. PR transcriptional activity is inversely related to PR stability (16,19); transcriptionally active PR turnover very rapidly and these events appear to be coupled via the phosphorylation of PR Ser294 (14)(15)(16)19). Consistent with these results, the ability of S294A PR-B to upshift in the presence of R5020 was unaltered by EGF pretreatment, and S294A PR were resistant to ligand-induced downregulation relative to wt PR-B (compare lanes 10-12 for each receptor).…”

“…kinase cascades) by peptide growth factors or liganded steroid hormone receptors stationed at the cell membrane is the direct phosphorylation of nuclear steroid receptors. We have previously defined functional roles for phosphorylation of human progesterone receptor (PR) Ser294 by mitogen-activated protein kinase (14)(15)(16) and PR Ser400 by cell-cycle-dependent protein kinase two (17). Phosphorylation of these sites is regulated in response to progestins or growth factor mitogens, including EGF and heregulin, and can potentiate ligand-dependent PR transcriptional activity, as well as induce ligand-independent PR actions (17,18).…”