SIGNAL-TRANSDUCTION PATHWAYS INVOLVED IN THE REGULATION OF PROTEIN-SYNTHESIS BY INSULIN IN L6 MYOBLASTS

The phosphorylation states of three proteins implicated in the action of insulin on translation were investigated, i.e., 70-kDa ribosomal pr otein S6 kinase (p70(S6k)), eukaryotic initiation factor (eIF) 4E, and the eIF-4E binding protein 4E-BP1. Addition of insulin caused a stimu lation of protein synthesis in L6 myoblasts in culture, an effect that was blocked by inhibitors of phosphatidylinositide-3-OH kinase (wortm annin), p70(S6k) (rapamycin), and mitogen-activated protein kinase (MA P kinase) kinase (PD-98059). The stimulation of protein synthesis was accompanied by increased phosphorylation of p70(S6k), an effect that w as blocked by rapamycin and wortmannin but not PD-98059. Insulin cause d dephosphorylation of eIF-4E, an effect that appeared to be mediated by the p70(S6k) pathway. Insulin also stimulated phosphorylation of 4E -BP1 as well as dissociation of the 4E-BP1 . eIF-4E complex. Both rapa mycin and wortmannin completely blocked the insulin-induced changes in 4E-BP1 phosphorylation and association of 4E-BP1 and eIF-4E; PD-98059 had no effect on either parameter. Finally, insulin stimulated format ion of the active eIF-4G . eIF-4E complex, an effect that was not prev ented by any of the inhibitors. Overall, the results suggest that insu lin stimulates protein synthesis in L6 myoblasts in part through utili zation of both the p70(S6k) and MAP kinase signal transduction pathway s.