A CARBOXY-TERMINAL DELETION MUTANT OF PROTEIN-KINASE C-BETA-II INHIBITS INSULIN-STIMULATED 2-DEOXYGLUCOSE UPTAKE IN L6 RAT SKELETAL-MUSCLE CELLS

Alternative splicing of pre-mRNA encoding the carboxy-terminal (C-term inal) exons of protein kinase C beta (PKC beta) leads to the expressio n of two protein isoforms, PKC beta I and PKC beta II, with the potent ial for different functions. PKC beta II expression is regulated by in sulin via alternative mRNA splicing. A physiological consequence of it s activation was investigated in L6 rat skeletal muscle cells expressi ng GLUT4 transporters, a cell line in which PKC is involved in glucose transport. We examined the contribution of PKC beta II for insulin-st imulated [H-3]2-deoxyglucose uptake by constructing three PKC beta II C-terminal deletion mutants designated M216, M217, and M218. When tran siently expressed in COS1 cells, M217, with nine amino acids deleted, demonstrated autophosphorylation activity 10-fold less than full-lengt h PKC beta II. The mutants M218, with 13 amino acids deleted, and M216 , with 52 amino acids deleted, demonstrated no autophosphorylation act ivity and are kinase negative. When transiently expressed in L6 myotub es, M217 inhibited insulin-stimulated 2-deoxyglucose uptake by 45% (wi th a 45% transfection efficiency) whereas M216 and M218, kinase-negati ve mutants, had no effect compared with cells transfected with control plasmid. Cotransfection of full-length PKC beta II with M217 was able to rescue the inhibition of insulin-stimulated 2-deoxyglucose uptake as compared with cotransfection of M217 with the control plasmid, sugg esting that M217 acts as a dominant-negative. In contrast, cotransfect ion of full-length PKC beta I, the other alternatively spliced form, d id not rescue inhibition of insulin-stimulated 2-deoxyglucose uptake b y M217. To further demonstrate the involvement of PKC, specifically PK C beta II, in insulin-stimulated 2-deoxyglucose uptake, we used two in hibitors, CG41251 (a specific PKC inhibitor) and CG53353 (a PKC beta I I-specific inhibitor at 1 mu M). Both inhibited insulin-stimulated 2-d eoxyglucose uptake 50-60% in L6 myotubes. We conclude that M217 may ac t as a specific PKC beta II dominant-negative and that PKC beta II is more specific for insulin-stimulated 2-deoxyglucose uptake in these ce lls than PKC beta I.