Protein Kinase G Regulation of Granulosa Cell Viability

描述（由申请人提供）：颗粒细胞凋亡是哺乳动物卵巢内的一个主要生理事件，它影响卵巢甾体生成、卵巢卵泡排卵数量和卵泡耗尽的速度。因此，了解调控颗粒细胞凋亡的机制可以为卵巢早衰和某些类型的“不明原因”不孕症提供见解。在这项拨款申请中，我们证明了PKG[蛋白激酶G]参与维持颗粒细胞的活力。PKG介导其抗凋亡作用的机制尚不清楚，这将是本次拨款申请的重点。文献综述表明，至少有20个已知的PKG靶点可以介导其作用。这些潜在的PKG靶点包括离子通道、离子泵、激酶、热休克蛋白、受体和转录因子等多种蛋白质。在我们最初的资助申请中，我们建议利用蛋白质组学方法确定哪些潜在的PKG靶点实际上参与调节颗粒细胞的活力。我们现在已经完成了最初的蛋白质组学筛选。通过筛选发现两种可能参与颗粒细胞凋亡调控的蛋白，14-3-3 sigma和ATP合成酶β前体。其他研究表明，14-3-3 sigma结合ATP合成酶β前体，在哺乳动物细胞中，14-3-3将蛋白质引导到线粒体。一旦进入线粒体，ATP合成酶的前体被移除，ATP合成酶被激活。我们认为14-3-3 sigma和ATP合成酶β前体的PKG依赖性磷酸化对于它们的相互作用和随后将ATP合成酶β前体递送到线粒体至关重要。ATP合成酶β在线粒体的定位将允许ATP的连续合成，这是细胞生存所必需的。我们现在建议使用生化、遗传、成像和细胞生物学技术来验证这一假设。

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.