Insulin signaling pathways regulating PKCBeta splicing

描述(申请人提供)：肌肉、脂肪和脂肪的胰岛素反应性 肝脏通过一个复杂的信号网络来调节 代谢过程包括蛋白质合成、糖原合成和 葡萄糖摄取。我们报告说，胰岛素快速调节剪接 通过增强外显子包涵体表达PKCbetaII的前mRNA。PKCBetaII及其ITS 另一种剪接产物PKCBetaI参与胰岛素信号转导和 在信号通路中有不同的功能。胰岛素对心脏功能的调节作用 选择性剪接通过激活磷脂酰肌醇3-激酶而发生 (PI3-激酶)，可能还有Akt和cPKC。这两种蛋白在蛋白中扮演多种角色 胰岛素信号包括核反应。PKCBetaII基因的剪接 需要与前mRNA相互作用的富含丝氨酸/精氨酸(SR)的蛋白质来 激活外显子包含的剪接位点选择。我们发现SRp40，一个SR 在胰岛素治疗后，蛋白质在P13-激酶中被磷酸化 依赖的态度。P13-激酶及其产物激活的几种激酶 可以使SR蛋白磷酸化。肌浆网蛋白磷酸化的调控 与外显子包涵体的关系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一种新的形式 除了组织特异性和发育/细胞外还调节剪接 依赖周期的调节。我们假设Akt、PKC，或许还有其他 由PI3-激酶激活的激酶可能使SR蛋白磷酸化 骨骼肌、脂肪和肝脏中的胰岛素调节剪接位点的选择。我们 将检查1)通过扫描仪鉴定的顺式元件，这些顺式元件与 我们开发的研究外显子的胰岛素反应异源微型基因 包涵体(即编码PKCBII C端52个氨基酸的216个碱基的外显子 和侧翼内含子序列)，2)如果SR蛋白 Akt2和cPKC同工酶的底物，3)剪接位点的作用机制 HeLa细胞核区体外剪接试验的筛选 提取液补充胰岛素处理细胞的核提取液以 跟踪剪接中间体，以及4)PKCBII替代剪接是否可以 重定向使用反义寡核苷酸来探测功能元件 活着。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自于替代 剪接被证明对新陈代谢过程有相反的影响。这个 胰岛素调节过程中的一种机制，该过程用于在 许多生物系统代表着一个基本的信号事件。这个 调节PKCBetaII mRNA的选择性剪接提供了一种分子联系 胰岛素激活PI3-激酶与转录后的关系 基因表达的调控。受体信号转导途径参与 替代的前信使核糖核酸剪接可能在蛋白质组中提供更大的多样性 比之前认识到的更复杂。

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.