Insulin and IGF-1 activate protein kinase B (PKBα) through phosphorylation at Thr308 and Ser473. This activation is prevented by the PI 3-kinase inhibitor wortmannin. Mutations at these sites significantly reduce PKBα activity, indicating their importance. PKBα becomes phosphorylated at both Thr308 and Ser473 in response to insulin or IGF-1, and both phosphorylations are necessary for high activity. Phosphorylation of Thr308 is not dependent on Ser473 and vice versa. MAPKAP kinase-2 phosphorylates Ser473, partially activating PKBα. The double mutant PKBα with both residues mutated to Asp shows synergistic activation. Phosphorylation of Thr308 and Ser473 is essential for high PKBα activity in vivo. The regulation of PKBα differs between isoforms, with PKBγ possibly having distinct regulatory mechanisms. The study highlights the role of PI 3-kinase in PKBα activation and the importance of phosphorylation at these sites for its function.Insulin and IGF-1 activate protein kinase B (PKBα) through phosphorylation at Thr308 and Ser473. This activation is prevented by the PI 3-kinase inhibitor wortmannin. Mutations at these sites significantly reduce PKBα activity, indicating their importance. PKBα becomes phosphorylated at both Thr308 and Ser473 in response to insulin or IGF-1, and both phosphorylations are necessary for high activity. Phosphorylation of Thr308 is not dependent on Ser473 and vice versa. MAPKAP kinase-2 phosphorylates Ser473, partially activating PKBα. The double mutant PKBα with both residues mutated to Asp shows synergistic activation. Phosphorylation of Thr308 and Ser473 is essential for high PKBα activity in vivo. The regulation of PKBα differs between isoforms, with PKBγ possibly having distinct regulatory mechanisms. The study highlights the role of PI 3-kinase in PKBα activation and the importance of phosphorylation at these sites for its function.