The study investigates the mechanism by which insulin and IGF-1 activate protein kinase B (PKBα). Insulin and IGF-1 stimulate PKBα activation by 12- to 50-fold in L6 myotubes and 293 cells, respectively. This activation is accompanied by phosphorylation of Thr308 and Ser473, which are critical for high-level PKBα activity. The phosphorylation of these residues is prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Mutations at these sites (ala or asp) reduce or eliminate PKBα activation. In vitro experiments using MAPKAP kinase-2 show that phosphorylation of Ser473 alone partially activates PKBα, while phosphorylation of Thr308 and Ser473 synergistically activates the enzyme. In 293 cells, mutation of either Thr308 or Ser473 to Ala significantly reduces PKBα activation by insulin or IGF-1, and the double mutant (Thr308/Asp473) cannot be activated further. These findings suggest that phosphorylation of both Thr308 and Ser473 is required for high-level PKBα activation. Additionally, the phosphorylation of Thr308 is not dependent on Ser473, and vice versa. The study also examines the role of other kinases in PKBα activation, concluding that the mechanism likely involves one or more protein kinases that phosphorylate Thr308 and Ser473, or the recruitment of PKBα to the plasma membrane by PIP3.The study investigates the mechanism by which insulin and IGF-1 activate protein kinase B (PKBα). Insulin and IGF-1 stimulate PKBα activation by 12- to 50-fold in L6 myotubes and 293 cells, respectively. This activation is accompanied by phosphorylation of Thr308 and Ser473, which are critical for high-level PKBα activity. The phosphorylation of these residues is prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Mutations at these sites (ala or asp) reduce or eliminate PKBα activation. In vitro experiments using MAPKAP kinase-2 show that phosphorylation of Ser473 alone partially activates PKBα, while phosphorylation of Thr308 and Ser473 synergistically activates the enzyme. In 293 cells, mutation of either Thr308 or Ser473 to Ala significantly reduces PKBα activation by insulin or IGF-1, and the double mutant (Thr308/Asp473) cannot be activated further. These findings suggest that phosphorylation of both Thr308 and Ser473 is required for high-level PKBα activation. Additionally, the phosphorylation of Thr308 is not dependent on Ser473, and vice versa. The study also examines the role of other kinases in PKBα activation, concluding that the mechanism likely involves one or more protein kinases that phosphorylate Thr308 and Ser473, or the recruitment of PKBα to the plasma membrane by PIP3.