The long term goal of this project is to determine the role of phosphorylation in regulating activity of human sex steroid receptors particularly in breast cancer cells. Phosphorylation has been proposed to play a role in numerous aspects of receptor function, but little is actually known about receptor phosphorylation and its function. This application will focus on phosphorylation of human progesterone receptor (hPR). Since phosphorylations occur at different times in the receptor activation process, it is likely that phosphorylation plays multiple roles in regulating this receptor. Two approaches will be used to study the role of phosphorylation. The first will be to identify and mutate the phosphorylation sites in order to correlate phosphorylation of specific sites with receptor function. The second will examine the role of cellular kinases and phosphatases in the modulation of receptor activity in vivo to determine which pathways and sites are important for regulating receptor function.
The specific aims for this grant are: 1. To identify the constitutive and hormone dependent phosphorylation sites in human progesterone receptor. Sites will be identified by sequencing peptides prepared from [32P] labeled receptor isolated from T47D cells 2. To determine how the antagonists RU486 and ZK98299 alter receptor phosphorylation. RU486 causes hyperphosphorylation of receptor and ZK98299 treatment results in less phosphorylation than treatment with agonist. We will use peptide mapping to determine whether these compounds cause phosphorylation at different sites within the receptor or alter the phosphorylation (either up or down) of normal phosphorylation sites. 3. To examine the mechanisms by which altered cellular kinase and phosphatase activities modulate hPR function and the mechanism by which alteration in kinase activities converts RU486 from an antagonist to an agonist. The effect of modulators of kinases on receptor activation and on receptor phosphorylation will be examined to correlate changes in activity with specific phosphorylation sites and to examine the mechanisms by which other signal transduction pathways can interact with receptor mediated pathways to activate transcription. 4. To prepare mutations of the hPR phosphorylation sites and to begin to examine the role of these phosphorylations in receptor function. These studies will address the role of phosphorylation in normal receptor function and will examine the possibility that altered phosphorylation may result in altered levels of activity and function in cases where kinase levels are altered. These studies may have important implications in situations such as in cancers which display altered kinase activity in response to oncogene activation.
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