AN OMICS APPROACH TO INDENTIFY PKA TARGETS IN GRANULOSA CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 女性的生育需要卵母细胞、支持颗粒细胞(GC)和组成卵泡的囊膜细胞的受控成熟。卵泡的生长是一个动态的过程，需要精细的调控。卵泡在腔前阶段被抑制，直到它们被垂体激素卵泡刺激素(FSH)刺激。作为对FSH的反应，GCs产生类固醇、蛋白质激素和生长因子，调节下丘脑/脑垂体轴和子宫容受性，促进卵母细胞成熟和卵泡发育到排卵前表型。所有文献记载的对FSH的反应都是通过cAMP及其主要的细胞内靶标cAMP依赖的蛋白激酶(PKA)介导的。GCS提供了最好的细胞模型之一，其响应是由PKA协调的。PKA通过磷酸化直接调节转录的底物或通过调节其靶标调节转录的途径来完成这种整合功能。在PKA激活的众多途径中，磷脂酰肌醇-3激酶(PI-3K)通路被认为是GC生存、增殖和分化的基础。我们已经证明，PKA磷酸化一种未知的底物，促进胰岛素受体底物-1的酪氨酸磷酸化，从而指导PI-3K途径的激活。方法：本应用的目的是利用磷酸蛋白质组学方法来鉴定GC中的PKA底物。我们将制备一种纯化的大鼠GCs，将它们放入培养中，在没有和有选择性PKA抑制肽的情况下对细胞进行预处理，然后在没有FSH和有FSH的情况下处理GCs 15min。预期结果：结果应阐明其磷酸化被FSH刺激而被PKI抑制的蛋白质。我们将通过标准的细胞生物学和生化方法来确认结果。意义：由于PKA是大多数细胞功能的基础，这些研究的结果应该能更好地理解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.