Insulin Signaling Pathways Regulating PKCBeta Splicing

描述（由申请人提供）：前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，对剪接调控的机制了解甚少。本实验室发现胰岛素调节蛋白激酶C-(II) （PKC(II)）在其靶组织中的选择性剪接：肌肉、脂肪、肝脏和具有功能胰岛素受体的细胞，如主动脉平滑肌细胞、胚胎成纤维细胞和HeLa细胞。我们确定了富含丝氨酸/精氨酸（SR）家族剪接蛋白的成员，这些剪接蛋白与pre-mRNA中的剪接增强子结合，以调节外显子包含，作为响应胰岛素的磷酸化因子。我们首先研究了剪接增强子SRp40，并发现Akt是一种通过精氨酸/丝氨酸（RS）结构域残基磷酸化来调节其功能的激酶。我们假设Akt通过调节其他SR蛋白激酶（如Clk，一个由四个双功能LAMMER激酶组成的家族）在剪接调节的几个步骤中作为分子开关。Clk1（也称为Clk/Sty）和Clk2等激酶使SR蛋白磷酸化，并改变它们在剪接体中的相互作用。揭示Akt如何调节Clk将为胰岛素的作用增加另一个层面的调节。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的选择。目前的目标将研究(1)Clk1和Clk2磷酸化在PKC（选择性剪接）中的作用，(2)确定SRp55在PKC（剪接）中的功能，以及(3)鉴定参与胰岛素激活剪接体的剪接体复合物和使用体外剪接测定来确定参与胰岛素调节剪接的顺式元件的核提取物剪接因子的消耗。胰岛素调节PKC（II）的剪接，PKC（II）是一种在多个水平上参与胰岛素反应的激酶，这一发现表明该途径还有其他靶基因也必须以类似的方式剪接。这个独特的系统将揭示激酶调控的本质，重点关注PKC（II）在剪接和糖尿病中的作用。鉴于糖尿病及其并发症问题的严重性，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。需要确定导致糖尿病发病的因素和确定新的潜在治疗靶点是一个优先事项。在胰岛素刺激其靶组织后，pre-mRNA的加工是一个鲜为人知的区域，在糖尿病状态下会发生改变。了解胰岛素受体信号通路，以确定胰岛素作用如何在胰岛素反应组织的细胞核中反映，将使我们能够确定通过胰岛素受体调节代谢功能导致胰岛素作用抵抗的信号的特异性。本研究旨在研究胰岛素信号级联中的一种新激酶Clk/Sty及其核底物SR蛋白，该蛋白可以修饰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.