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 肝脏通过调节的复杂信号通路网络发生 代谢过程，包括蛋白质合成，糖原合成和 葡萄糖吸收。我们报道胰岛素迅速调节了 通过增强的外显子包容性的PKCBETAII前MRNA。 PKCBETAII及其 或者，剪接的产品PKCBETAI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 替代剪接是通过激活磷脂酰肌醇3-激酶发生的 （PI3-激酶）以及AKT和CPKC。两种激酶在 胰岛素信号传导，包括核作用。 PKCBETAII mRNA的剪接 需要与前MRNA相互作用的丝氨酸/精氨酸蛋白（SR）蛋白 激活剪接位点选择外显子包含。我们发现SRP40，一个SR 蛋白质在p13-激酶中胰岛素处理后磷酸化。 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蛋白磷酸化的调节 关于外显子包容性是一种新的观察结果，因为 生长因子信号通路的SR蛋白构成了一种新形式 除了组织特异性和发育/细胞外，还调节剪接 循环依赖性调节。我们假设AKT，PKC，也许还有其他 由PI3-激酶激活的激酶可能会磷酸化SR蛋白响应于 骨骼肌，脂肪和肝脏中的胰岛素以调节剪接部位选择。我们 将检查1）通过连接诱变的扫描仪确定的顺式元素 我们已经开发出一种研究外显子的胰岛素反应性异源微基因 包含（即216 bp外显子编码PKCBII的C末端52氨基酸 在细胞培养中体内内含子序列），2）如果SR蛋白为 AKT2和CPKC同工酶的底物，3）剪接位点的机理 通过开发HeLa细胞核的体外剪接测定方法的选择 提取物补充了胰岛素处理的细胞中的核提取物 跟踪剪接中间体，4）PKCBII是否可以替代剪接 使用反义橄榄核苷酸重定向以探测功能元件 体内。阐明调节剪接的信号通路将变成 更重要的是，越来越多的蛋白质从替代品中得出 剪接显示对代谢过程有相反的影响。这 胰岛素调节用于赋予多样性的过程的机制 许多生物系统代表着一个基本信号事件。这 PKCBETAII mRNA的替代剪接的调节提供了分子链接 PI3-激酶的胰岛素激活与转录后的激活之间 基因表达的调节。涉及的受体信号通路 替代的前MRNA剪接可能可以提供更大的蛋白质组学多样性 比以前认识的复杂性。