Constitutive activation of protein kinase B and phosphorylation of p47(phox) by membrane-targeted phosphoinositide 3-kinase
Constitutive activation of protein kinase B and phosphorylation of p47(phox) by membrane-targeted phosphoinositide 3-kinase
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DOI：
10.1016/s0960-9822(02)70713-6
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发表时间：
1996-10-01
期刊：
影响因子：
9.2
通讯作者：
Thelen, M
中科院分区：
文献类型：
--
作者：
Didichenko, SA;Tilton, B;Thelen, M
背景：磷脂酰肌醇3-激酶（PI 3-kinase）的活性是有丝分裂信号和分泌反应所必需的。推测细胞活化导致PI 3-激酶从胞质溶胶易位至质膜，在质膜中激酶与其底物磷脂酰肌醇（4，5）-二磷酸相互作用。因此，膜靶向的，因此组成型活性激酶可以帮助阐明PI 3-激酶在细胞内signaling.Results的作用：膜靶向序列的Ha-Ras，包含棕榈酰化和法尼基化的共识序列，融合到的羧基末端的PI 3-激酶的催化亚基的p110 α。脂质锚将PI 3-激酶导向膜，并导致转染细胞中组成性升高的磷脂酰肌醇（3，4，5）-三磷酸水平。膜靶向PI 3-激酶的表达导致下游效应物的连续激活，例如蛋白激酶B（PKB，也称为Akt/RAC），其最近被证明调节糖原合成酶激酶-3。组成性激活的PKB被废除的具体PI 3-激酶抑制剂渥曼青霉素，和PKB激活是边缘的转染表达非膜靶向PI 3-激酶。在G蛋白偶联受体激动剂刺激下，吞噬细胞NADPH氧化酶的快速组装需要胞质因子p47（phox）的多重磷酸化。我们在这里表明，膜靶向PI 3-激酶的表达在单核细胞系GM-1的结果在一个渥曼青霉素敏感的连续磷酸化p47（phox）。结论：PI 3-激酶的目标，其首选的底物的网站导致组成性刺激独立的增强催化，是足以调节不同的信号转导通路。(C)Current Biology Ltd ISSN 0960-9822
Background: Phosphoinositide 3-kinase (PI 3-kinase) activity is required for mitogenic signaling and for secretary responses. Cell activation is presumed to cause the translocation of PI 3-kinase from the cytosol to the plasma membrane where the kinase interacts with its substrate phosphatidylinositol (4,5)-bisphosphate. Thus, a membrane-targeted and therefore constitutively active kinase could help elucidate the role of PI 3-kinase in intracellular signaling.Results: The membrane-targeting sequence of Ha-Ras, containing the consensus sequence for palmitoylation and farnesylation, was fused to the carboxyl terminus of p110 alpha, the catalytic subunit of PI 3-kinase. The lipid anchor directed PI 3-kinase to the membrane and led to constitutively elevated phosphatidylinositol (3,4,5)-trisphosphate levels in transfected cells. Expression of membrane-targeted PI 3-kinase resulted in the continuous activation of downstream effecters, such as protein kinase B (PKB, also known as Akt/RAC), which was recently shown to regulate glycogen synthase kinase-3. The constitutive activation of PKB was abolished by the specific PI 3-kinase inhibitor wortmannin, and PKB activation was marginal in transfectants expressing nonmembrane-targeted PI 3-kinase. Multiple phosphorylation of the cytosolic factor p47(phox) is required for the rapid assembly of the phagocyte NADPH oxidase upon stimulation with agonists of G-protein-coupled receptors. We show here that the expression of membrane-targeted PI 3-kinase in the monoblastic cell line GM-1 results in a wortmannin-sensitive continuous phosphorylation of p47(phox).Conclusions: Targeting of PI 3-kinase to the site of its preferred substrate leads to constitutive stimulus-independent enhanced catalysis and is sufficient to regulate different signal transduction pathways. (C) Current Biology Ltd ISSN 0960-9822