The role of translocation in the activation and function of protein kinase B (PKB) was investigated. By attaching the myristoylation/palmitylation motif from the Lck tyrosine kinase to the N terminus of PKB, the subcellular localization of PKB was altered. Myristoylated/palmitylated (m/p)-PKBα was localized to the plasma membrane, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBα was 60-fold higher than that of the unstimulated wild-type enzyme and could not be further stimulated by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis showed that m/p-PKBα activity was due to phosphorylation on Thr308 and Ser473, which are normally induced by insulin or IGF-1. A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBα activity. The pleckstrin homology (PH) domain of m/p-PKBα was not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBα was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. The results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation, the kinase detached from the membrane and translocated to the nucleus. Growth factors elicit cellular responses by activating phosphoinositide 3-kinase (PI3-K). Protein kinase B (PKB), also known as RAC protein kinase or c-Akt, was recognized as a downstream target of PI3-K. Three mammalian isoforms of PKB have been identified, termed PKBα, -β, and -γ. All three isoforms contain a pleckstrin homology (PH) domain at the N terminus, followed by a catalytic domain related to protein kinases A and C, and a C-terminal regulatory region. PKBα was found to mediate insulin- and IGF-1-induced cellular responses. PKBα is the cellular homologue of the oncogene product v-Akt. Cloning of v-akt revealed that it was created by fusion of viral Gag sequences to the N terminus of mouse PKBα. Overexpression of PKBα or -β is associated with some human ovarian, pancreatic, and breast carcinomas. PKBα is activated by a variety of growth factors and phosphatase inhibitors through a phosphorylation mechanism. The activation of PKBThe role of translocation in the activation and function of protein kinase B (PKB) was investigated. By attaching the myristoylation/palmitylation motif from the Lck tyrosine kinase to the N terminus of PKB, the subcellular localization of PKB was altered. Myristoylated/palmitylated (m/p)-PKBα was localized to the plasma membrane, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBα was 60-fold higher than that of the unstimulated wild-type enzyme and could not be further stimulated by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis showed that m/p-PKBα activity was due to phosphorylation on Thr308 and Ser473, which are normally induced by insulin or IGF-1. A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBα activity. The pleckstrin homology (PH) domain of m/p-PKBα was not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBα was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. The results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation, the kinase detached from the membrane and translocated to the nucleus. Growth factors elicit cellular responses by activating phosphoinositide 3-kinase (PI3-K). Protein kinase B (PKB), also known as RAC protein kinase or c-Akt, was recognized as a downstream target of PI3-K. Three mammalian isoforms of PKB have been identified, termed PKBα, -β, and -γ. All three isoforms contain a pleckstrin homology (PH) domain at the N terminus, followed by a catalytic domain related to protein kinases A and C, and a C-terminal regulatory region. PKBα was found to mediate insulin- and IGF-1-induced cellular responses. PKBα is the cellular homologue of the oncogene product v-Akt. Cloning of v-akt revealed that it was created by fusion of viral Gag sequences to the N terminus of mouse PKBα. Overexpression of PKBα or -β is associated with some human ovarian, pancreatic, and breast carcinomas. PKBα is activated by a variety of growth factors and phosphatase inhibitors through a phosphorylation mechanism. The activation of PKB