Molecular and genetic studies imply Akt-mediated signaling promotes protein kinase CβII alternative splicing via phosphorylation of serine/arginine-rich splicing factor SRp40

Insulin regulates alternative splicing of PKC beta II mRNA by phosphorylation of SRp40 via a phosphatidylinositol 3-kinase pathway (Patel, N. A., Chalfant, C. E., Watson, J. E., Wyatt, J. R., Dean, N. M., Eichler, D. C., and Cooper, D. C. ( 2001) J. Biol. Chem. 276, 22648-22654). Transient transfection of constitutively active Akt2 kinase promotes PKC beta II exon inclusion. Serine/arginine-rich (SR) RNA-binding proteins regulating the selection of alternatively spliced exons are potential substrates of Akt kinase because many of them contain RXRXX(S/T) motifs. Here we show that Akt2 kinase phosphorylated SRp40 in vivo and in vitro. Mutation of Ser(86) on SRp(40) blocked in vitro phosphorylation. In control Akt2(+/+) fibroblasts, insulin treatment increased the phosphorylation of endogenous SR proteins, but their phosphorylation state remained unaltered by insulin in fibroblasts from Akt2(-/-) mice. Levels of PKC beta II protein were up-regulated by insulin in Akt2(+/+) cells; however, only very low levels of PKC beta II were detected in Akt2(-/-) cells and did not change following insulin treatment. Endogenous PKC beta I and -beta II mRNA levels in Akt2(+/+) and Akt2(-/-) gastrocnemius muscle tissues were compared using quantitative real time PCR. The results indicated a 54% decrease in the expression of PKC beta II levels in Akt(-/-), whereas PKC beta I levels remained unchanged in both samples. Further, transfection of Akt2(-/-) cells with a PKC beta II splicing minigene revealed defective beta II exon inclusion. Co-transfection of the mutated SRp40 attenuated beta II exon inclusion. This study provides in vitro and in vivo evidence showing Akt2 kinase directly phosphorylated SRp40, thereby connecting the insulin, PI 3-kinase/Akt pathway with phosphorylation of a site on a nuclear splicing protein promoting exon inclusion. This model is upheld in Akt2-deficient mice with insulin resistance leading to diabetes mellitus.