Insulin Signaling Pathways Regulating PKCBeta Splicing

描述（由申请人提供）：前体mRNA转录物的选择性剪接是从单个基因产生多肽多样性的广泛手段。超过60%的人类基因通过选择性剪接表达，然而，剪接调控机制知之甚少。该实验室发现，胰岛素在其靶组织中调节蛋白激酶C-（II）（PKC（II））的选择性剪接：肌肉、脂肪、肝脏和具有功能性胰岛素受体的细胞，如主动脉平滑肌细胞、胚胎成纤维细胞和HeLa细胞。我们鉴定了富含丝氨酸/精氨酸（SR）家族剪接蛋白的成员，其结合前体mRNA中的剪接增强子以调节外显子包含，作为响应于胰岛素而磷酸化的因子。我们首先研究了剪接增强子SRp 40，并将Akt鉴定为通过精氨酸/丝氨酸（RS）结构域中残基的磷酸化来调节其功能的激酶。我们假设Akt作为一个分子开关，在剪接调控的几个步骤，也调节其他SR蛋白激酶，如Clk，一个家庭的四个双功能LAMMER激酶。激酶如Clk 1（也称为Clk/Sty）和Clk 2磷酸化SR蛋白，并改变它们在剪接体中的相互作用。解开Akt如何调节Clk将为胰岛素作用增加另一个调节水平。该研究的长期目标是确定胰岛素如何通过激活各种激酶和剪接因子来调节核剪接位点选择。目前的目标是研究（1）Clk 1和Clk 2磷酸化在PKC（选择性剪接）中的作用，（2）确定SRp 55在PKC（剪接）中的功能，（3）鉴定参与胰岛素激活剪接体的剪接体复合物，并使用体外剪接测定法从核提取物中耗尽剪接因子，以确定参与胰岛素调节剪接的顺式元件。胰岛素调节PKC（II）（一种在多个水平参与胰岛素应答的激酶）剪接的发现表明，该途径的其他靶基因也必须以类似的方式剪接。独特的系统将揭示激酶调节的本质，重点是剪接和糖尿病中的PKC（II）。鉴于糖尿病及其并发症所遇到的问题的严重性，了解胰岛素的作用对医疗保健有着巨大的影响，因为它是美国疾病相关死亡的第六大原因。确定糖尿病发病的因素和确定新的潜在治疗靶点是当务之急。胰岛素刺激其靶组织后前mRNA的加工是一个知之甚少的领域，在糖尿病状态下会发生改变。了解胰岛素受体信号通路的目的是确定胰岛素作用如何反映在胰岛素反应组织的细胞核中，这将使我们能够确定通过胰岛素受体调节代谢功能的信号传导的特异性，从而导致胰岛素作用抵抗。该提案旨在研究胰岛素信号级联中的新激酶Clk/Sty及其核底物SR蛋白，其修饰RNA加工以改变基因表达。SR蛋白在胰岛素抵抗中发生改变。

DESCRIPTION (provided by applicant): Alternative splicing of pre-mRNA transcripts is a widespread means for producing polypeptide diversity from a single gene. Over 60% of human genes are expressed through alternative splicing, however, mechanisms of splicing regulation are poorly understood. This lab discovered that insulin regulates the alternative splicing of protein kinase C-(II (PKC(II) in its target tissues: muscle, fat, liver and in cells with functional insulin receptor such as aortic smooth muscle cells, embryonic fibroblasts, and HeLa cells. We identified members of the Serine/Arginine-rich (SR) family splicing proteins that bind to splicing enhancers in the pre-mRNA to regulate exon inclusion as the factors phosphorylated in response to insulin. We first studied SRp40, a splicing enhancer and identified Akt as a kinase that regulated its function via phosphorylation of residues in the arginine/serine (RS) domain. We hypothesize that Akt acts as a molecular switch in splicing regulation at several steps by also regulating other SR protein kinases such as Clk, a family of four dual function LAMMER kinases. Kinases such as Clk1 (also called Clk/Sty) and Clk2 phosphorylate SR proteins and alter their interactions in the spliceosome. Unraveling how Akt regulates Clk will add another level of regulation to insulin action. The long-term goal of the research is to determine how insulin regulates nuclear splice site selection via the activation of various kinases and splicing factors. The current aims will investigate (1) the roles of Clk1 and Clk2 phosphorylation in PKC( alternative splicing, (2) determine how SRp55 functions in PKC( splicing, and (3) identify spliceosome complexes involved in the insulin activated spliceosome and depletion of splicing factors from nuclear extracts using in vitro splicing assays to define cis-elements involved in insulin regulated splicing. The discovery that insulin regulates splicing of PKC(II, a kinase involved in insulin responses at multiple levels, indicates that there are also other target genes of this pathway that must also be spliced in a similar manner. The unique system will reveal the nature of kinase regulation, focusing on PKC(II in splicing and diabetes. PUBLIC HEALTH RELEVANCE Given the magnitude of the problems encountered with diabetes and its complications, understanding insulin action has an immense impact on healthcare since it is the sixth leading cause of disease-related death in the US. The need to define the factors contributing to diabetes onset and identify new potential therapeutic targets is a priority. The processing of pre-mRNA following insulin stimulation of its target tissues is a poorly understood area that is altered in the diabetic state. Understanding the insulin receptor signaling pathways with the goal of defining how insulin action is reflected in the nucleus of insulin responsive tissues will allow us to determine the specificity of signaling through the insulin receptor to regulate metabolic functions causing resistance to insulin action. This proposal is designed to investigate a new kinase in the insulin signaling cascade, Clk/Sty, and its nuclear substrates, SR proteins, which modify RNA processing to alter gene expression. SR proteins are altered in insulin resistance.