PSF Regulates the Porcine P450scc IFGRE

描述(申请人提供)：我们的工作重点是IGF-I对P-450胆固醇侧链裂解(P450scc)基因表达的调节，P450scc是类固醇合成途径中的限速酶。我们鉴定了猪P450scc基因的IGF-I反应元件(IGFRE)，它与Sp1(刺激物)和多嘧啶结合蛋白(PTB)相关的剪接因子(PSF，阻遏物)结合。 在这个提案中，我们将研究PSF对IGFRE活性的调节机制。研究将在稳定的猪颗粒细胞系JC-410细胞中进行。我们假设PSF对IGFRE的抑制涉及一种阻止IGF-I刺激的Sp1磷酸化的机制。在特定的目标1中，我们将确定IGF-I介导的Sp1磷酸化位点，已经证明IGF-I刺激Sp1上苏氨酸739的磷酸化，这是一个已知的ERK1/2磷酸化位点。在特定的目标2中，我们将研究ERK1/2信号通路，作为至少一个通过Sp1磷酸化来调节IGF-I刺激IGFRE的部分机制。最后，我们将研究蛋白激酶C(PKC)IOTA作为PSF-Sp1相互作用的调节器来调节IGFRE(特定目标3)。 我们将确定IGF-I通过Sp1、PSF和PKC IOTA相互作用来调控P450scc基因表达的基本机制。这些知识将增加我们的理解，并为激素控制卵巢类固醇激素生成的新概念提供基本背景。

DESCRIPTION (provided by applicant): Our work focuses on IGF-I regulation of gene expression for P-450 cholesterol side-chain cleavage (P450scc), the rate-limiting enzyme in the steroidogenic pathway. We identified an IGF-I response element (IGFRE) of the porcine P450scc gene that binds Sp1(stimulator) and polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF, repressor). In this proposal we will investigate mechanisms of PSF regulation of IGFRE activity. Studies will be conducted in a stable porcine granulosa cell line, the JC-410 cells. We hypothesize that PSF repression of the IGFRE involves a mechanism that prevents IGF-I-stimulated phosphorylation of Sp1. In specific aim 1 we will determine IGF-I-mediated Sp1 phosphorylation sites having already demonstrated that IGF-I stimulates phosphorylation of threonine 739 on Sp1, a known erk 1/2 phosphorylation site. In specific aim 2 we will investigate the erk 1/2 signaling pathway as at least a partial mechanism for regulating IGF-I stimulation of the IGFRE through Sp1 phosphorylation. Finally, we will investigate protein kinase C (PKC) iota as a modulator of PSF-Sp1 interactions regulating the IGFRE (specific aim 3). We will determine the fundamental mechanisms whereby IGF-I controls P450scc gene expression through interactions of Sp1, PSF, and PKC iota. This knowledge will increase our understanding and provide the basic background for novel concepts on hormonal control of ovarian steroidogenesis.