Andjelković M, Alessi D R, Meier R, Fernandez A, Lamb N J, Frech M, Cron P, Cohen P, Lucocq J M, Hemmings B A
Friedrich Miescher-Institut, Maulbeerstrasse 66, CH-4056 Basel, Switzerland.
J Biol Chem. 1997 Dec 12;272(50):31515-24. doi: 10.1074/jbc.272.50.31515.
We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBalpha was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBalpha was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis demonstrated that m/p-PKBalpha activity was due to phosphorylation on Thr308 and Ser473, that are normally induced on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBalpha activity. The pleckstrin homology (PH) domain of m/p-PKBalpha 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, PKBalpha was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. Our 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.
我们研究了亚细胞定位在蛋白激酶B（PKB）激活调节中的作用。将来自Lck酪氨酸激酶的肉豆蔻酰化/棕榈酰化基序连接到蛋白激酶B的N端，以改变其亚细胞定位。肉豆蔻酰化/棕榈酰化（m/p）-PKBα与转染细胞的质膜相关，而野生型激酶主要位于胞质溶胶中。与未刺激的野生型酶相比，m/p-PKBα的活性高60倍，并且不能被生长因子或磷酸酶抑制剂进一步刺激。体内32P标记和诱变表明，m/p-PKBα的活性归因于Thr308和Ser473的磷酸化，这两个位点通常在细胞用胰岛素或胰岛素样生长因子-1（IGF-1）刺激后在PKB上被诱导。磷酸肌醇3激酶（PI3-K）的显性负性形式不影响m/p-PKBα的活性。m/p-PKBα的普列克底物蛋白同源（PH）结构域对于其激活或Thr308和Ser473的磷酸化不是必需的，这表明该结构域可能作为膜靶向模块。与此观点一致，IGF-1刺激后几分钟内PKBα易位到质膜。这种易位需要PH结构域并且对渥曼青霉素敏感。我们的结果表明，PI3-K活性是PKB易位到质膜所必需的，在质膜上其通过与胰岛素或IGF-1诱导相同位点的磷酸化而激活。激活后，激酶从膜上脱离并易位到细胞核。
