Molecular and Genetic Studies Imply Akt-mediated Signaling Promotes Protein Kinase CβII Alternative Splicing via Phosphorylation of Serine/Arginine-rich Splicing Factor SRp40

Niketa A. Patel, Satoshi Kaneko, Hercules S. Apostolatos, Sun Sik Bae, James E. Watson, Karen Davidowitz, David S. Chappell, Morris J. Birnbaum, Jin Q. Cheng, Denise R. Cooper
2005 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
more » ... 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.
doi:10.1074/jbc.m411485200 pmid:15684423 fatcat:djv35hfdxrhvxdcmuzhcgvny2y