Mechanism of activation of protein kinase B by insulin and IGF-1

Insulin activated endogenous protein kinase Ba (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBa was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBa was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBa molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBa was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBa by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBa activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBa becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).