Role of translocation in the activation and function of protein kinase B
Role of translocation in the activation and function of protein kinase B
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DOI：
10.1074/jbc.272.50.31515
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发表时间：
1997-12-12
影响因子：
4.8
通讯作者：
Hemmings, BA
中科院分区：
文献类型：
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作者：
Andjelkovic, M;Alessi, DR;Hemmings, BA
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。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末端以改变其亚细胞位置。豆蔻酰化/棕榈酰化（m/p）-PKB α与转染细胞的质膜相关，而野生型激酶主要是胞浆。与未受刺激的野生型酶相比，m/p-PKB α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P-32标记和诱变证明m/p-PKB α活性是由于Thr（308）和Ser（473）上的磷酸化，这通常是在用胰岛素或胰岛素样生长因子-1（IGF-1）刺激细胞后在PKB上诱导的。磷酸肌醇3-激酶（PI 3-K）的显性负性形式不影响m/p-PKB α活性。m/p-PKB α的普列克底物蛋白同源（PH）结构域对于其在Thr（308）和Ser（473）上的活化或磷酸化是不需要的，这表明该结构域可以作为膜靶向模块。与此观点一致，PKB α在IGF-1刺激后几分钟内易位到质膜。这种易位需要PH结构域，并且对渥曼青霉素敏感。我们的研究结果表明，PI 3-K活性是所需的PKB易位到质膜，在那里它的激活发生通过磷酸化的相同的网站，诱导胰岛素或IGF-1。激活后，激酶从膜上脱落并易位到细胞核。
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)-PKB alpha was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKB alpha 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 P-32 labeling and mutagenesis demonstrated that m/p-PKB alpha activity was due to phosphorylation on Thr(308) and Ser(473), 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-PKB alpha activity. The pleckstrin homology (PH) domain of m/p-PKB alpha was not required for its activation or phosphorylation on Thr(308) and Ser(473), suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKB alpha 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.