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.
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