The roles of Akt (protein kinase B) and the atypical lambda isoform of protein kinase C (PKClambda), both of which act downstream of phosphoinositide 3-kinase, in the activation of glycogen synthase and phosphorylation of 4E-BP1 (PHAS-1) in response to insulin were investigated. A mutant Akt (Akt-AA) in which the phosphorylation sites targeted by growth factors are replaced by alanine was shown to inhibit insulin-induced activation of both Akt and glycogen synthase in L6 myotubes. Expression of a mutant Akt in which Lys179 in the kinase domain was replaced by aspartate also inhibited insulin-induced activation of glycogen synthase but had no effect on insulin activation of endogenous Akt. A kinase-defective mutant of PKClambda (lambdaDeltaNKD), which prevents insulin-induced activation of PKClambda, did not affect the activation of glycogen synthase by insulin. Insulin-induced phosphorylation of 4E-BP1 was inhibited by Akt-AA in Chinese hamster ovary cells. However, lambdaDeltaNKD had no effect on 4E-BP1 phosphorylation induced by insulin. These data suggest that Akt, but not PKClambda, is required for insulin activation of glycogen synthase and for insulin-induced phosphorylation of 4E-BP1.