Insulin signaling pathways regulating PKCBeta splicing

调节 PKCβ 剪接的胰岛素信号通路

基本信息

批准号：
6704151
负责人：
DENISE Ratzlaff COOPER
金额：
$ 0.74万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-15 至 2005-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6704151
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

项目摘要

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、脂肪和肌肉中的胰岛素反应性肝脏通过复杂的信号通路网络发生调节代谢过程包括蛋白质合成、糖原合成等葡萄糖摄取。我们报道胰岛素快速调节剪接通过增强外显子包含来形成 PKCbetaII 的前 mRNA。 PKCβII 及其选择性剪接产物 PKCBeI 参与胰岛素信号传导和在信号通路中具有不同的功能。胰岛素调节通过激活磷脂酰肌醇 3-激酶发生选择性剪接（PI3-激酶）以及可能的 Akt 和 cPKC。两种激酶在以下方面发挥多种作用：胰岛素信号传导包括核作用。 PKCβII mRNA的剪接需要富含丝氨酸/精氨酸 (SR) 的蛋白质与前 mRNA 相互作用激活外显子包含的剪接位点选择。我们发现 SRp40，一种 SR 蛋白质，在胰岛素处理后在 P13 激酶中被磷酸化依赖方式。 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相关的是一个新的观察结果，因为 SR蛋白通过生长因子信号通路构成了一种新形式除了组织特异性和发育/细胞外还调节剪接周期依赖性调节。我们假设 Akt、PKC 以及其他可能 PI3 激酶激活的激酶可能会磷酸化 SR 蛋白以响应骨骼肌、脂肪和肝脏中的胰岛素调节剪接位点选择。我们将检查 1) 通过扫描仪连接突变识别的顺式元件我们开发用于研究外显子的胰岛素反应性异源小基因包含（即编码 PKCBII C 端 52 个氨基酸的 216 bp 外显子和侧翼内含子序列）体内细胞培养物中，2）如果SR蛋白是 Akt2 和 cPKC 同工酶的底物，3) 剪接位点机制通过开发体外剪接试验进行选择，其中 HeLa 细胞核提取物补充有胰岛素处理细胞的核提取物跟踪剪接中间体，以及 4) PKCBII 替代剪接是否可以使用反义寡核苷酸重定向以探测中的功能元件体内。阐明调节剪接的信号通路将成为更重要的是，越来越多的蛋白质源自替代品研究表明剪接对代谢过程具有相反的影响。这胰岛素调节用于赋予多样性的过程的机制许多生物系统代表基本的信号事件。这 PKCβII mRNA 选择性剪接的调节提供了分子联系 PI3激酶的胰岛素激活与转录后调节之间的关系基因表达的调控。受体信号通路参与选择性的前 mRNA 剪接可能会提供更大的蛋白质组多样性比以前认识到的复杂性。