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.
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