Insulin Signaling Pathways Regulating PKCBeta Splicing

前信使核糖核酸转录本的选择性剪接是产生多肽多样性的一种广泛的方法 来自单一基因。然而，超过60%的人类基因是通过选择性剪接表达的， 剪接调控的机制还知之甚少。这个实验室发现，胰岛素调节 蛋白激酶C-II(PKC？II)在其靶组织：肌肉、脂肪、肝脏和在具有 功能性胰岛素受体，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)的剪接蛋白家族的成员，它们与剪接结合 前信使核糖核酸中的增强子在因子被磷酸化时调节外显子包涵体 胰岛素。我们首先研究了SRp40，一种剪接增强剂，并确定Akt是一种调节其 通过精氨酸/丝氨酸(RS)结构域残基的磷酸化发挥作用。我们假设Akt会采取行动 作为一个分子开关，通过同时调节其他SR蛋白激酶，在几个步骤中调节剪接 例如CLK，一个由四个双功能Lammer激酶组成的家族。诸如CLK1(也称为CLK/Sty)之类的激酶 和Clk2使SR蛋白磷酸化并改变它们在剪接体中的相互作用。揭开Akt是如何 调节CLK将为胰岛素的作用增加另一个水平的调节。这项研究的长期目标是 为了确定胰岛素是如何通过激活不同的激酶和 拼接因子。目前的目标将研究(1)CLK1和ClK2在蛋白激酶C中的磷酸化作用 选择性剪接，(2)确定SRp55在PKC剪接中的功能，以及(3)识别剪接体 参与胰岛素激活剪接体的复合体和核剪接因子的耗竭 提取物使用体外剪接试验来确定参与胰岛素调节剪接的顺式元件。这个 发现胰岛素调节PKC？II的剪接，PKC？II是参与胰岛素反应的一种激酶 水平，这表明该途径的其他靶基因也必须拼接在 类似的方式。这个独特的系统将揭示激酶调节的本质，重点是在 剪接和糖尿病。项目叙事 考虑到糖尿病及其并发症所遇到的问题的严重性，理解 胰岛素的作用对医疗保健有巨大的影响，因为它是与疾病相关的第六大原因 美国的死亡事件。需要确定导致糖尿病发病的因素并确定新的潜在因素 治疗目标是当务之急。胰岛素刺激靶基因后前信使核糖核酸的加工 组织是一个知之甚少的区域，在糖尿病状态下会发生变化。对胰岛素的理解 受体信号通路，目的是确定胰岛素的作用如何反映在核内 胰岛素反应组织将使我们能够确定通过胰岛素传递信号的特异性 调节代谢功能的受体，导致胰岛素抵抗的作用。这份提案是专门设计的 研究胰岛素信号转导通路中的新激酶CLK/Sty及其核底物SR 蛋白质，它可以改变RNA的加工过程来改变基因的表达。胰岛素中的SR蛋白会发生变化 抵抗。

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. Project Narrative 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.