Insulin regulates alternative splicing of protein kinase C beta II through a phosphatidylinositol 3-kinase-dependent pathway involving the nuclear serine/arginine-rich splicing factor, SRp40, in skeletal muscle cells.
Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) betaII mRNA. In this report, we show that insulin regulates this exon inclusion (alternative splicing) via the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway through the phosphorylation state of SRp40, a factor required for insulin-regulated splice site selection for PKCbetaII mRNA. By taking advantage of a well known inhibitor of PI 3-kinase, LY294002, we demonstrated that pretreatment of L6 myotubes with LY294002 blocked insulin-induced PKCbetaII exon inclusion as well as phosphorylation of SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells mimicked insulin-induced exon inclusion. When antisense oligonucleotides targeted to a putative SRp40-binding sequence in the betaII-betaI intron were transfected into L6 cells, insulin effects on splicing and glucose uptake were blocked. Taken together, these results demonstrate a role for SRp40 in insulin-mediated alternative splicing independent of changes in SRp40 concentration but dependent on serine phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This switch in PKC isozyme expression is important for increases in the glucose transport effect of insulin. Significantly, insulin regulation of PKCbetaII exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCbetaII mRNA in L6 cells occurs in response to a metabolic change.
Patel
N A
NA
Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida 33612, USA.
Chalfant
C E
CE
Watson
J E
JE
Wyatt
J R
JR
Dean
N M
NM
Eichler
D C
DC
Cooper
D R
DR
D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers; D004789:Enzyme Activation; D005091:Exons; D007328:Insulin / Q000502:physiology*; D007527:Isoenzymes / Q000235:genetics*; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics*; D064546:Protein Kinase C beta; D051381:Rats
D017931:DNA Primers; D007328:Insulin; D007527:Isoenzymes; D003513:Cycloheximide; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D064546:Protein Kinase C beta