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Insulin Regulates Protein Kinase CßII Alternative Splicing in Multiple Target Tissues: Development of a Hormonally Responsive Heterologous Minigene

Department of Biochemistry and Molecular Biology (N.A.P., H.S.A., K.M., D.R.C.), College of Medicine, University of South Florida, and The Research Service (J.E.W., D.R.C.), James A. Haley Veterans Hospital, Tampa, Florida 33612; Department of Biochemistry (C.E.C.), Virginia Commonwealth University and Hunter Holmes McGuire Veterans Medical Center, Richmond, Virginia 23298-0614; Institute of Cell Signaling (T.S.P.), University of Nottingham Medical School, Queen’s Medical Center, Nottingham NG7 2UH, United Kingdom; and University of Rochester (J.S.), School of Medicine and Dentistry, Rochester, New York 14642

Address all correspondence and requests for reprints to: Denise R. Cooper, Ph.D. J. A. Haley Veterans Hospital (VAR 151), 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail: dcooper{at}hsc.usf.edu.

Cells respond to external signals like insulin to alter metabolic pathways in response to varying physiological environments. Insulin stimulates the protein kinase C ß (PKCß) isozymes and preferentially switches the expression to PKCßII isozyme, which is shown to have a crucial role in glucose uptake, cellular proliferation, and differentiation. We have developed an insulin-responsive PKCßII heterologous minigene to identify cis-elements in vivo in eukaryotes by cloning the PKCßII exon and its flanking intronic sequences into the splicing vector pSPL3. The transfected minigene mimicked the endogenous insulin response of PKCßII 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 PKCßII 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.

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