Insulin signaling pathways regulating PKCBeta splicing

调节 PKCβ 剪接的胰岛素信号通路

• 批准号: 6914129
• 负责人: DENISE Ratzlaff COOPER
• 金额: $ 1.39万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-15 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6914129
• 关键词: RNA splicing; antisense nucleic acid; arginine; biological signal transduction; gene mutation; genetic regulation; genetic regulatory element; hormone regulation /control mechanism; human genetic material tag; insulin; phosphatidylinositol 3 kinase; protein kinase C; serine; tissue /cell culture; transcription factor

DESCRIPTION(provided by applicant): Insulin responsiveness in muscle, fat, and liver occurs via a complex network of signaling pathways that regulate metabolic processes including protein synthesis, glycogen synthesis, and glucose uptake. We reported that insulin rapidly regulated splicing of the pre-mRNA for PKCbetaII by enhanced exon inclusion. PKCBetaII and its alternatively spliced product, PKCBetaI, are involved in insulin signaling and have distinct functions in signaling pathways. Insulin regulation of alternative splicing occurs via activation of phosphatidylinositol 3-kinase (PI3-kinase) and possibly Akt and cPKC. Both kinases fulfill numerous roles in insulin signaling including nuclear actions. The splicing of PKCBetaII mRNA requires serine/arginine rich (SR) proteins that interact with the pre-mRNA to activate splice site selection for exon inclusion. We found that SRp40, one SR protein, is phosphorylated following insulin treatment in a P13-kinase dependent manner. Several kinases activated by P13-kinase and its products could phosphorylate SR proteins. The regulation of SR protein phosphorylation in relation to exon inclusion is a novel observation since phosphorylation of SR proteins by growth factor signaling pathways constitutes a new form of regulating splicing in addition to tissue specific- and developmental/cell cycle dependent- regulation. We hypothesize that Akt, PKC, and perhaps other kinases activated by PI3-kinase may phosphorylate SR proteins in response to insulin in skeletal muscle, fat and liver to regulate splice site selection. We will examine 1) the cis-elements identified by scanner linking mutagenesis of an insulin-responsive heterologous minigene we have developed to study exon inclusion (i.e., 216 bp exon encoding the C-terminal 52 amino acids of PKCBII and flanking intron sequences) in vivo in cell culture, 2) if SR proteins are substrates for Akt2 and cPKC isozymes, 3) the mechanism of splice site selection by developing in vitro splicing assays where HeLa cell nuclear extracts are supplemented with nuclear extracts from insulin-treated cells to track splicing intermediates, and 4) whether PKCBII alternative splicing can be redirected using antisense oliogonucleotides to probe functional elements in vivo. Elucidation of the signaling pathways that regulate splicing will become more important as an increasing number of proteins derived from alternative splicing are shown to have opposing effects on metabolic processes. The mechanism by which insulin regulates a process utilized to confer diversity in many biological systems represents a fundamental signaling event. The regulation of alternative splicing of PKCBetaII mRNA provides a molecular link between insulin activation of PI3-kinase and the post-transcriptional regulation of gene expression. The receptor signaling pathways involved in alternative pre-mRNA splicing may provide for a greater diversity in proteomic complexity than previously recognized.

描述（由申请人提供）：肌肉、脂肪和 肝脏通过复杂的信号通路网络发生， 代谢过程，包括蛋白质合成、糖原合成和 葡萄糖摄取我们报道了胰岛素可以快速调节 通过增强的外显子包含来表达PKC β II的前体mRNA。PKCBetaII及其 选择性剪接产物PKC β I参与胰岛素信号传导， 在信号通路中有不同的功能。胰岛素调节 选择性剪接通过激活磷脂酰肌醇3-激酶而发生 （PI 3-激酶）和可能的Akt和cPKC。这两种激酶都在 胰岛素信号传导包括核作用。PKC β II mRNA的剪接 需要富含丝氨酸/精氨酸（SR）的蛋白质与前体mRNA相互作用， 激活剪接位点选择以包含外显子。我们发现SRp 40，一个SR 蛋白质，在胰岛素处理后在P13-激酶中被磷酸化 依赖的方式。P13-激酶及其产物激活的几种激酶 可以磷酸化SR蛋白。SR蛋白磷酸化的调控 与外显子包含有关是一个新的观察，因为 SR蛋白通过生长因子信号通路构成一种新的 除了组织特异性和发育/细胞外， 周期依赖-调节。我们假设Akt、PKC和其他可能的 由PI 3-激酶激活的激酶可响应于 胰岛素在骨骼肌、脂肪和肝脏中调节剪接位点选择。我们 将检查1）通过扫描仪连接诱变鉴定的顺式元件， 我们已经开发了一个胰岛素应答异源小基因， 包含（即，216 bp外显子编码PKCBII的C-末端52个氨基酸 和侧翼内含子序列），2）如果SR蛋白是 Akt 2和cPKC同工酶的底物，3）剪接位点的机制 通过开发体外剪接测定进行选择，其中HeLa细胞核 提取物补充有来自胰岛素处理的细胞的核提取物， 轨道剪接中间体，以及4）PKCB II选择性剪接是否可以 使用反义寡核苷酸重定向以探测 vivo.阐明调节剪接的信号通路将成为 更重要的是，越来越多的蛋白质来源于替代品， 剪接显示对代谢过程具有相反的影响。的 胰岛素调节用于赋予生物多样性的过程的机制。 许多生物系统代表了基本的信号事件。的 PKC β II mRNA可变剪接的调节提供了一种分子联系， 胰岛素对PI 3-激酶的激活与转录后 基因表达的调控。受体信号通路参与 选择性前mRNA剪接可以提供蛋白质组学的更大多样性， 比以前认识到的复杂。