Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells

We have investigated glycogen synthase (GS) activation in L6hIR cells expre ssing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and in sulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3<alpha> (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein pho sphatase I activity. PDK1 (a phosphatidylinositol. trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no diff erence in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta acti vity by either a PKC zeta antisense or a dominant negative mutant also redu ced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and ins ulin stimulation of GS (p < 0.002), similar to A-kt/PKB inhibition. In L6hI R, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with G SK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alon e, these events did not significantly affect GSK3 alpha and -beta activitie s. Inhibition of PKC activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and preven ted full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insu lin activation of GS in the L6hIR skeletal muscle cells. In these cells, in sulin inhibition of GSK3<alpha> and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.