Insulin Receptor Substrate-2 Phosphorylation is Necessary for Protein Kinase Cζ Activation by Insulin in L6hIR Cells

Abstract

We have investigated glycogen synthase (GS) activation in L6hIR cells expressing 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 insulin 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 3α (GSK3α) and GSK3β inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKCζ in B2 and F4 cells. In L6hIR, inhibition of PKCζ activity by either a PKCζ antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3α and -β (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase Cζ (PKCζ) co-precipitation with GSK3α and β. PKCζ also phosphorylated GSK3α and -β. Alone, these events did not significantly affect GSK3α and -β activities. Inhibition of PKCζ activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3α and -β by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3α and -β requires dual phosphorylation by both Akt/PKB and PKCζ.