Protein Kinase G Regulation of Granulosa Cell Viability

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DESCRIPTION (provided by applicant): Granulosa cell apoptosis is a major physiological event within the mammalian ovary that affects ovarian steroidogenesis, the number of ovarian follicles that ovulate and the rate at which follicles are depleted. Thus, understanding the mechanism that regulates granulosa cell apoptosis could provide insights into premature ovarian failure and certain types of "unexplained" infertility. In this grant application, we demonstrate that PKG [protein kinase G] is involved in maintaining granulosa cell viability. The mechanism through which PKG mediates its anti-apoptotic action is unknown and will be the focus of this grant application. A review of the literature indicates that there are at least 20 known PKG targets that could mediate its action. These potential PKG targets include such diverse proteins as ion channels, ion pumps, kinases, heat shock proteins, receptors and transcription factors. In our initial grant application, we proposed to determine which of these potential PKG targets are actually involved in regulating granulosa cell viability by utilizing a proteomic approach. We have now completed our initial proteomic screen. This screen revealed two potential proteins that could be involved in regulating granulosa cell apoptosis, 14-3-3 sigma and ATP synthase beta precursor. Other studies have shown that 14-3-3 sigma binds ATP synthase beta precursor and in mammalian cells, 14-3-3 directs proteins to the mitochondria. Once within the mitochondria, the presequence of ATP synthase beta is removed and the ATP synthase beta is activated. We propose that PKG dependent phosphorylation of both 14-3-3 sigma and ATP synthase beta precursor is essential for their interaction and the subsequent delivery of ATP synthase beta precursor to the mitochondria. The localization of ATP synthase beta to the mitochondria would allow for a continuous synthesis of ATP, which is required for cell viability. We now propose to test this hypothesis using biochemical, genetic, imaging and cell biological techniques.