title: Molecular and genetic studies imply Akt-mediated signaling promotes protein kinase CbetaII alternative splicing via phosphorylation of serine/arginine-rich splicing factor SRp40.
abstract: Insulin regulates alternative splicing of PKCbetaII 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 PKCbetaII 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 Ser86 on SRp40 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 PKCbetaII protein were up-regulated by insulin in Akt2(+/+) cells; however, only very low levels of PKCbetaII were detected in Akt2(-/-) cells and did not change following insulin treatment. Endogenous PKCbetaI and -betaII 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 PKCbetaII levels in Akt(-/-), whereas PKCbetaI levels remained unchanged in both samples. Further, transfection of Akt2(-/-) cells with a PKCbetaII splicing minigene revealed defective betaII exon inclusion. Co-transfection of the mutated SRp40 attenuated betaII 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.
authors.affiliation: Department of Biochemistry, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
authors.forename: Niketa A
authors.initials: NA
authors.lastname: Patel
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kaneko
authors.forename: Hercules S
authors.initials: HS
authors.lastname: Apostolatos
authors.forename: Sun Sik
authors.initials: SS
authors.lastname: Bae
authors.forename: James E
authors.initials: JE
authors.lastname: Watson
authors.forename: Karen
authors.initials: K
authors.lastname: Davidowitz
authors.forename: David S
authors.initials: DS
authors.lastname: Chappell
authors.forename: Morris J
authors.initials: MJ
authors.lastname: Birnbaum
authors.forename: Jin Q
authors.initials: JQ
authors.lastname: Cheng
authors.forename: Denise R
authors.initials: DR
authors.lastname: Cooper
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D064546:Protein Kinase C beta; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D016601:RNA-Binding Proteins; D051381:Rats; D012694:Serine / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D007328:Insulin; D007527:Isoenzymes; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D012694:Serine; D001120:Arginine; C494918:AKT1 protein, human; C494921:AKT2 protein, human; C494920:Akt1 protein, rat; C494922:Akt2 protein, mouse; C494923:Akt2 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D064546:Protein Kinase C beta