Insulin regulates splicing of pre-mRNA for protein kinase C␤II (PKC␤II) 1 by enhanced exon inclusion in rat skeletal muscle myotubes (L6) (1, 2). Alternative splicing of nuclear pre-mRNA allows for the formation of multiple RNA isoforms from a single primary RNA transcript. It is a widespread mechanism controlling gene expression in higher eukaryotes (3), and an estimated 30,000 human genes generate over 100,000 protein isoforms with distinct functions by alternative splicing (4, 5). Alternative splicing can be controlled developmentally or by tissue specificity (6 -9) and is a well studied mechanism. Our laboratory has demonstrated hormonal regulation of PKC␤II alternative splicing, and mechanisms for exon recognition involve the regulated phosphorylation of the serine/arginine-rich (SR) protein SRp40 (10, 11).A family of splicing factors, SR proteins, are highly phosphorylated RNA-binding proteins involved in the alternative splicing process (12-14). Ten highly conserved SR proteins consist of one or two N-terminal ribonucleoprotein-type RNA-binding domains and a C-terminal SR domain that promotes proteinprotein interactions within the splicing complex. SR proteins are required for constitutive splicing for the formation of the early prespliceosomal complex to stabilize U1 small nuclear ribonucleoprotein at the 5Ј splice site and in spliceosome formation to mediate interactions between U1 small nuclear ribonucleoprotein and U2AF, bound to the 5Ј and 3Ј splice sites (15)(16)(17). SR proteins also function in alternative splicing in vivo in a concentration-dependent manner by modulating 5Ј splice site choice (18,19). The SR domains direct SR proteins to localize to speckles within the nucleus (20), and some SR proteins can "shuttle" between the nucleus and cytoplasm depending on their phosphorylation state (21). The function of SR proteins in hormone-regulated alternative splicing has not been elucidated.Our studies focused on SRp40 because its expression was known to be regulated by insulin (22). Predictions by SELEX and reports by other groups identified a binding site for SRp40 in the downstream intron of the rat PKC␤ gene (23). We found that modified antisense oligonucleotides (RNase H-resistant) blocked the interaction of SRp40 with the PKC␤II intron and

Serine/arginine-rich (SR) proteins play essential roles in the constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich (RS) domain by SR protein kinases such as Cdc2-like kinases (Clk/Sty) modulates their subcellular localization and activation. However, it remains unclear how these kinases and their target SR proteins are regulated by extracellular signals. Regulation of protein kinase C betaII (PKCbetaII) pre-mRNA alternative splicing via exon inclusion by Akt2, a central kinase in insulin action, involves phosphorylation of SR proteins. Here we showed that Akt2, in response to insulin, resulted in phosphorylation of Clk/Sty, which then altered SR protein phosphorylation in concert with Akt2. Insulin-stimulated PKCbetaII pre-mRNA splicing was blocked by Clk/Sty and phosphatidylinositol-3-kinase inhibitors, and diabetic Akt2-null mouse tissues had impaired phospho-Clk/Sty, SR protein phosphorylation, and PKCbetaII expression. Furthermore, we observed that Akt2 phosphorylated several SR proteins distinct from Clk/Sty in response to insulin. Akt2-catalyzed phosphorylation of Clk/Sty and SR proteins revealed a role for both kinases in splicing regulation indicating dual functions for Akt2 in response to insulin in this pathway.