AN OMICS APPROACH TO INDENTIFY PKA TARGETS IN GRANULOSA CELLS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，但不一定是研究者所在的机构。 女性的生育力需要卵母细胞、支持颗粒细胞（GC）和构成卵泡的卵泡膜细胞的受控成熟。 卵泡生长是一个动态的过程，需要精细的调控。卵泡在窦前阶段被抑制，直到它们被垂体激素卵泡刺激素（FSH）刺激。 响应FSH，GC产生类固醇和蛋白质激素以及生长因子，调节下丘脑/垂体轴和子宫容受性，并促进卵母细胞成熟和卵泡发育为排卵前表型。 所有记录的对FSH的反应都是通过cAMP及其主要的细胞内靶点cAMP依赖性蛋白激酶（PKA）介导的。 GC提供了一个最好的例子，细胞模型的反应是由PKA编排。 PKA通过磷酸化直接调节转录的底物或通过调节靶点调节转录的途径来实现这种整合功能。 在PKA激活的许多途径中，磷脂酰肌醇-3激酶（PI-3 K）途径被认为是GC存活、增殖和分化的基础。我们已经证明PKA磷酸化一种未鉴定的底物，该底物促进胰岛素受体底物-1的酪氨酸磷酸化，从而指导PI-3 K通路的激活。 方法：本申请的目的是利用磷酸化蛋白质组学方法鉴定GC中的PKA底物。 我们将获得一个纯化的制备大鼠GCs，将它们放置在培养中，预处理细胞没有和选择性PKA抑制剂肽，肉豆蔻酰化的PKI，然后处理GCs没有和FSH为15分钟。预期结果：结果应该阐明蛋白质的磷酸化刺激FSH和抑制PKI。 我们将通过标准细胞生物学和生物化学方法确认结果。 重要性：由于PKA是大多数细胞功能的基础，这些研究的结果应该提供更好的理解PKA如何整合细胞信号通路。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Fertility in females requires controlled maturation of the oocyte, supporting granulosa cells (GCs), and thecal cells that comprise the ovarian follicle. Follicle growth is a dynamic process that demands exquisite regulation. Follicles are restrained at the preantral stage until they are stimulated by the pituitary hormone follicle stimulating hormone (FSH). In response to FSH GCs produce steroid and protein hormones and growth factors that regulate the hypothalamic/pituitary axis and uterine receptivity, and promote oocyte maturation and development of the follicle to a preovulatory phenotype. All of the documented responses to FSH are mediated via cAMP and its predominate intracellular target, cAMP-dependent protein kinase (PKA). GCs offer one of the best examples of a cellular model whose responses are orchestrated by PKA. PKA accomplishes this integrating function by phosphorylating substrates that directly regulate transcription or by regulating pathways whose targets regulate transcription. Of the many pathways that PKA activates, the phosphatidylinositol-3 kinase (PI-3K) pathway is recognized to be fundamental to GC survival, proliferation, and differentiation. We have shown that PKA phosphorylates an unidentified substrate that promotes the tyrosine phosphorylation of insulin receptor substrate-1, thereby directing activation of the PI-3K pathway. Approach: The purpose of this application is to utilize a phospho-proteomic approach to identify PKA substrates in GCs. We will obain a purified preparation of rat GCs, place them in culture, pretreat cells without and with a selective PKA inhibitor peptide, myristoylated-PKI, then treat GCs without and with FSH for 15 min. Expected Results: Results should elucidate proteins whose phosphorylation is stimulated by FSH and inhibited by PKI. We will confirm results by standard cell biological and biochemical approaches. Significance: As PKA is fundamental to the function of most cells, results from these studies should provide a better understanding of how PKA integrates cellular signaling pathways.