The long term goal of this project is to determine the physiological roles for phosphorylation of steroid receptors. As a model, our studies focus on the human progesterone receptor (hPR). In the previous grant period, eight phosphorylation sites were identified in hPR. This information will be utilized to determine the functions of the individual sites.
The specific aims for this grant period are 1. To mutate each hPR phosphorylation site and to determine the effects of these changes in vivo on protein expression, phosphorylation of other sites (hierarchal phosphorylation) and transcriptional activity. We will determine whether the phosphorylation of hPR is hierarchical and with transcriptional activation assays as an endpoint will determine which sites affect overall activity in vivo. 2. To determine mechanistically how phosphorylation sites affect specific factions of PR. Based on the results in Aim #1 selected analyses of specific receptor function (interaction with heat shock proteins, receptor dimerization, DNA binding and interaction with other factors) will be performed to determine the specific roles for each phosphorylation site. 3. To determine whether phosphorylation contributes to functional differences between the A and B forms of progesterone receptor. The finding that PR-B is a strong activator and PR-A can act as a repressor suggests very different functions for these proteins. That there are phosphorylations unique to PR-B suggests that phosphorylation plays a role in these unique functions. 4. To identify kinases that phosphorylate purified hPR in vitro on authentic phosphorylation sites and to use in vitro phosphorylated receptor to study the role of phosphorylation in receptor function. Purified receptor phosphorylated on specific sites or subsets of sites will be used to quantify the effects of these phosphorylations on binding to DNA using fluorescence anisotropy (FAS). That antagonists can act as agonists when kinase activity is altered has important implications in determining the mechanism by which hormone resistance occurs in breast cancer. Phosphorylated receptor will be used in a differential screen to identify proteins that interact specifically with the PR-B specific region of the receptor and are potential mediators of the PR-B specific antagonist/agonist switch in response to activators of Protein Kinase A.
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