The study investigates the role of subcellular localization in the regulation of protein kinase B (PKB) activation. By attaching a myristoylation/palmitoylation motif from the Lck tyrosine kinase to the N terminus of PKBα, the researchers found that this modified form of PKBα was associated with the plasma membrane, whereas wild-type PKBα was mostly cytosolic. The activity of the myristoylated/palmitoylated (m/p)-PKBα was 60-fold higher than that of the unstimulated wild-type enzyme and could not be further increased by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis studies showed that the activity of m/p-PKBα was due to phosphorylation on Thr308 and Ser473, which are normally induced by insulin or insulin-like growth factor-1 (IGF-1) stimulation. A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBα activity, suggesting that PI3-K activity is not required for its activation. The pleckstrin homology (PH) domain of m/p-PKBα was not essential for its activation or phosphorylation on Thr308 and Ser473, indicating that this domain may serve as a membrane-targeting module. Consistent with this, PKBα translocated to the plasma membrane within minutes after stimulation with IGF-1, and this translocation required the PH domain and was sensitive to wortmannin. These results indicate that PI3-K activity is required for the translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of Thr308 and Ser473. Following activation, the kinase detached from the membrane and translocated to the nucleus. The study also found that the membrane-targeted form of PKBα was activated in unstimulated cells to a level greater than that attained by wild-type PKBα after stimulation with insulin, IGF-1, or vanadate. The activation of m/p-PKBα resulted from its phosphorylation on Thr308 and Ser473, the same residues that are normally induced by insulin and IGF-1. The findings suggest that forced membrane localization is sufficient for full activation of PKBα through stoichiometric phosphorylation of these residues by upstream kinases. Transient membrane association is crucial for the physiological activation of PKBα, and correct subcellular localization is essential for its activation and appropriate inactivation by phosphatases.The study investigates the role of subcellular localization in the regulation of protein kinase B (PKB) activation. By attaching a myristoylation/palmitoylation motif from the Lck tyrosine kinase to the N terminus of PKBα, the researchers found that this modified form of PKBα was associated with the plasma membrane, whereas wild-type PKBα was mostly cytosolic. The activity of the myristoylated/palmitoylated (m/p)-PKBα was 60-fold higher than that of the unstimulated wild-type enzyme and could not be further increased by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis studies showed that the activity of m/p-PKBα was due to phosphorylation on Thr308 and Ser473, which are normally induced by insulin or insulin-like growth factor-1 (IGF-1) stimulation. A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBα activity, suggesting that PI3-K activity is not required for its activation. The pleckstrin homology (PH) domain of m/p-PKBα was not essential for its activation or phosphorylation on Thr308 and Ser473, indicating that this domain may serve as a membrane-targeting module. Consistent with this, PKBα translocated to the plasma membrane within minutes after stimulation with IGF-1, and this translocation required the PH domain and was sensitive to wortmannin. These results indicate that PI3-K activity is required for the translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of Thr308 and Ser473. Following activation, the kinase detached from the membrane and translocated to the nucleus. The study also found that the membrane-targeted form of PKBα was activated in unstimulated cells to a level greater than that attained by wild-type PKBα after stimulation with insulin, IGF-1, or vanadate. The activation of m/p-PKBα resulted from its phosphorylation on Thr308 and Ser473, the same residues that are normally induced by insulin and IGF-1. The findings suggest that forced membrane localization is sufficient for full activation of PKBα through stoichiometric phosphorylation of these residues by upstream kinases. Transient membrane association is crucial for the physiological activation of PKBα, and correct subcellular localization is essential for its activation and appropriate inactivation by phosphatases.