Insulin regulates protein kinase CbetaII alternative splicing in multiple target tissues: development of a hormonally responsive heterologous minigene.
Cells respond to external signals like insulin to alter metabolic pathways in response to varying physiological environments. Insulin stimulates the protein kinase C beta (PKCbeta) isozymes and preferentially switches the expression to PKCbetaII isozyme, which is shown to have a crucial role in glucose uptake, cellular proliferation, and differentiation. We have developed an insulin-responsive PKCbetaII heterologous minigene to identify cis-elements in vivo in eukaryotes by cloning the PKCbetaII exon and its flanking intronic sequences into the splicing vector pSPL3. The transfected minigene mimicked the endogenous insulin response of PKCbetaII alternative splicing in five distinct cell types, i.e. L6 skeletal muscle, 3T3-L1 pre-adipocytes, HepG2 human hepatoma cells, A10 vascular smooth muscle cells, and murine embryonic fibroblasts within 30 min of insulin stimulation. Sequential deletions of the flanking introns in the minigene demonstrated that insulin regulated elements within the 5'-intron flanking the PKCbetaII exon. Mutational studies indicated the SRp40 binding site promotes splice site selection. In these cases, splicing appears to be regulated by a phosphatidylinositol 3-kinase signaling pathway because LY294002 and wortmannin, its specific inhibitors, blocked exon inclusion. Cotransfection with constitutively active Akt2 kinase mimicked insulin action. Signal-dependent regulation of splicing by insulin is unique from tissue-specific and developmentally regulated mechanisms previously reported and serves as a prototype for studies of alternative splicing involving protein phosphorylation.
Patel
Niketa A
NA
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, USA.
Apostolatos
Hercules S
HS
Mebert
Konrad
K
Chalfant
Charles E
CE
Watson
James E
JE
Pillay
T S
TS
Sparks
Janet
J
Cooper
Denise R
DR
D017667:Adipocytes / Q000378:metabolism; D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism; D011493:Protein Kinase C / Q000096:biosynthesis / Q000235:genetics*; D064546:Protein Kinase C beta; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D022821:RNA Splice Sites; D051381:Rats
D007328:Insulin; D011518:Proto-Oncogene Proteins; D022821:RNA Splice Sites; C494921:AKT2 protein, human; C494923:Akt2 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D064546:Protein Kinase C beta