The estrogen proliferative signal, which plays important roles in reproductive tissue function and tumorigenesis, is tightly controlled by the counterbalancing influences of coactivators and corepressors. Our objectives are to determine the importance of 'phosphorylation codes' in maintaining the balance in protein levels between steroid receptor coactivator-3 (SRC-3) and the corepressors, Represser of Estrogen Action (REA) and prohibitin (PHB). Together, they provide a balance which is necessary for normal cellular responses to estrogen. We will determine the physiological consequences of perturbing this coregulator homeostasis on steroid-induced mammary ontogenesis and oncogenesis. For SRC-3, we have identified two distinct phosphorylation codes which exert opposing effects on its protein stability. While atypical protein kinase C zeta promotes SRC-3 stabilization, glucose synthase kinase 3 (GSK3) promotes SRC-3 degradation. Like SRC-3, we have evidence that REA and PHB also operate as phosphoproteins. Based on the foregoing, we hypothesize that permutations of phosphorylation sites on SRC-3, REA and PHB constitute a 'phosphorylation code', which not only codes for coactivator/corepressor activation states but defines the stability of these coregulators. We predict that the phosphorylation code is a pivotal post- translational mechanism that maintains a critical balance between the countervailing influences of SRC-3 (growth promotion) and the prohibitin family (growth suppression) which together ensures a normal proliferative response to estrogen. To advance this hypothesis, we will (1) demonstrate that phosphorylation status determines SRC-3 protein stability with consequences for steroid-dependent cell proliferation in vitro; (2) disclose the in vivo effects of perturbing SRC-3 phosphorylation status on steroid-induced epithelial proliferation; (3) define the regulatory events which control REGgamma mediated degradation of SRC-3; (4) determine the mechanism by which phosphorylation status coordinately controls the protein levels and repressive functions of REA and PHB in vitro; and (5) define the in vivo corepressor functions of REA and PHB in steroid-induced epithelial cell proliferation. Mass spectrometric analysis, transcript profiling and state-of-the-art mouse genetics will be employed to successfully execute our stated aims. Apart from addressing a new control paradigm for estrogen signaling in the epithelial cell, these studies will furnish a broader conceptual framework for understanding abnormal proliferative responses to steroid-exposure. Beyond providing important new information on coregulator control of estrogen action during breast development and cancer, we believe the results from these studies will aid in the design of novel diagnostic approaches and/or therapies for the future treatment and prevention of breast cancer, regulation of fertility and other reproductive diseases. ? ? ?
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