Abstract
Many steroid receptors, including chicken progesterone receptor, have been shown to be activated in the absence of their cognate ligands by modulators of kinases and phosphatases. To investigate the molecular mechanism of ligand-independent activation, chicken progesterone receptor mutants in which either one or all four of the previously identified phosphorylation sites have been changed to nonphosphorylatable alanine were analyzed for their ability to be activated by progesterone, 8-bromoadenosine 3':5'-cyclic monophosphate, or a dopamine agonist, SKF82958. Our current study shows that the receptor is differently phosphorylated in ligand-dependent and ligand-independent activation. The transcriptional activity of the receptor in response to 8-bromoadenosine 3':5'-cyclic monophosphate is affected by mutation of either Ser211 or Ser260. In addition, our data demonstrated that none of the four sites is absolutely required for the activation of the receptor by either 8-bromoadenosine 3':5'-cyclic monophosphate or the dopamine agonist. Treatment with 8-bromoadenosine 3':5'-cyclic monophosphate did not increase the overall level of receptor phosphorylation or cause phosphorylation of the receptor at alternate sites. These data raise the possibility that ligand-independent activation of the chicken progesterone receptor may be mediated through changes in the phosphorylation of coregulators or other protein factors interacting with the receptors.
Highlights
§ To whom correspondence should be addressed: Dept of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030
Our phosphopeptide mapping analyses showed that cPRA is only phosphorylated at Ser211 and Ser260 during the ligand-independent activation by 8-Br-cAMP
Cells were treated with 10 nM progesterone (Prog), 2 mM 8-bromo-cAMP, or vehicle for the indicated times, and cell extracts were prepared for chloramphenicol acetyltransferase (CAT) activity as described under “Experimental Procedures.” con, control
Summary
§ To whom correspondence should be addressed: Dept of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Since steroid receptors are phosphoproteins, it is possible that alteration of receptor phosphorylation in response to different treatments mediates the ligand-independent activation. To examine whether any of the known phosphorylation sites in cPR is involved in or mediates the ligand-independent activation of the receptor, the four phosphorylation sites were mutated either individually or simultaneously to nonphosphorylatable alanines, and the responses of these mutant receptors to 8-bromo-cAMP and a dopamine agonist were assayed and compared with that of the wild type receptor.
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