TI - Mechanism of activation of protein kinase B by insulin and IGF-1 . AB - Insulin activated endogenous protein kinase B alpha ( also known as RAC/Akt kinase ) activity 12-fold in L6 myotubes , while after transfection into 293 cells PKBalpha was activated 20 - and 50-fold in response to insulin and IGF-1 respectively . In both cells , the activation of PKBalpha 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 PKBalpha molecules were analysed after transfection into 293 cells . The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2 . These experiments demonstrated that activation of PKBalpha 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 PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa . We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1 -stimulated cells by an upstream kinases .