Kohn A D, Takeuchi F, Roth R A
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305, USA.
J Biol Chem. 1996 Sep 6;271(36):21920-6. doi: 10.1074/jbc.271.36.21920.
Akt is a serine/threonine kinase that is stimulated by receptor tyrosine kinases and contains a pleckstrin homology domain. One model proposed to explain this activation suggests that receptor tyrosine kinases stimulate a phosphatidylinositol 3-kinase whose lipid products directly activate Akt kinase by interacting with its pleckstrin homology domain. In the present study, we show, in three cell types, that Akt does not require its pleckstrin homology domain to respond to either insulin or platelet-derived growth factor. Moreover, attachment of the src myristoylation signal to target Akt, without its pleckstrin homology domain, to the membrane constitutively activates Akt by causing an increase in its basal level of phosphorylation. This constitutively active form of Akt can also activate p70(S6K), indicating that the pleckstrin homology domain is not necessary for downstream interactions. Fusion of the inter src homology 2 domain from the p85 regulatory subunit of the phosphatidylinositol 3-kinase to Akt also constitutively activated Akt and induced an association with the lipid kinase. Phosphorylation of this fusion protein still critically contributes toward its increased activity. The sum of these results indicates that the primary mechanism of Akt activation is via protein phosphorylation.
Akt是一种丝氨酸/苏氨酸激酶，受受体酪氨酸激酶刺激，含有一个普列克底物蛋白同源结构域。提出的一种解释这种激活的模型表明，受体酪氨酸激酶刺激一种磷脂酰肌醇3激酶，其脂质产物通过与其普列克底物蛋白同源结构域相互作用直接激活Akt激酶。在本研究中，我们在三种细胞类型中表明，Akt对胰岛素或血小板衍生生长因子的反应不需要其普列克底物蛋白同源结构域。此外，将src肉豆蔻酰化信号连接到没有其普列克底物蛋白同源结构域的靶向Akt上，使其定位于膜上，通过增加其基础磷酸化水平来组成性激活Akt。这种组成性激活形式的Akt也能激活p70(S6K)，表明普列克底物蛋白同源结构域对于下游相互作用不是必需的。将磷脂酰肌醇3激酶p85调节亚基的src同源结构域2与Akt融合也能组成性激活Akt，并诱导其与脂质激酶结合。这种融合蛋白的磷酸化对其活性增加仍起关键作用。这些结果的总和表明，Akt激活的主要机制是通过蛋白质磷酸化。
