Molecular and Genetic Studies Imply Akt-mediated Signaling Promotes Protein Kinase CβII Alternative Splicing via Phosphorylation of Serine/Arginine-rich Splicing Factor SRp40
Journal of Biological Chemistry
2001) J. Biol. Chem. 276, 22648 -22654). Transient transfection of constitutively active Akt2 kinase promotes PKC␤II exon inclusion. Serine/arginine-rich (SR) RNAbinding 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 SRp40 blocked in vitro phosphorylation. In control Akt2(؉/؉) fibroblasts, insulin
... broblasts, 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␤II protein were up-regulated by insulin in Akt2(؉/؉) cells; however, only very low levels of PKC␤II were detected in Akt2(؊/؊) cells and did not change following insulin treatment. Endogenous PKC␤I and -␤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␤II levels in Akt(؊/؊), whereas PKC␤I levels remained unchanged in both samples. Further, transfection of Akt2(؊/؊) cells with a PKC␤II splicing minigene revealed defective ␤II exon inclusion. Co-transfection of the mutated SRp40 attenuated ␤II exon inclusion. This study provides in vitro and in vivo evidence showing Akt2 kinase directly phosphorylated SRp40, thereby connecting the insulin, PI 3kinase/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.