ACTIVATION OF PI 3-KINASE IS REQUIRED FOR PDGF-INDUCED H2O2 PRODUCTION

自磷酸化血小板衍生生长因子受体通过募集含SH 2结构域的酶（包括磷脂酰肌醇3-激酶（PI 3 K）、Ras的GTP酶激活蛋白（GAP）、蛋白酪氨酸磷酸酶（SHP- 2）和磷脂酶C-γ 1（PLC γ 1））触发细胞内信号级联。在表达各种PDGF受体突变体的HepG 2细胞中测量PDGF依赖性H2 O2产生，其中激酶活性所必需的Lys 635被改变为Arg，或用于结合PI 3 K的Tyr残基（Tyr 740和Tyr 751）、GAP（Tyr 771）、SHP-2（Tyr 1009）和PLC γ 1（Tyr 1021）以各种组合被突变为Phe。激酶缺陷型受体不能产生H2 O2。PI 3 K结合位点的消除也废除了H2 O2的生产，而H2 O2的生产略有增强时，GAP，SHP-2，PLC-γ 1的结合位点被删除。在四种效应酶的结合位点中，单独存在PI 3 K结合位点足以产生H2 O2。在表达野生型受体或仅含有PI 3 K结合位点的受体的细胞中，PDGF诱导的H2 O2产生被PI 3 K抑制剂LY 294002或Rac 1的显性负性形式N17 Rac 1的过表达完全阻断。这些结果表明，一个产品的PI 3激酶是必要的激活假定的NADPH氧化酶在非吞噬细胞与PDGF刺激和Rac 1提供PI 3 K产品和NADPH氧化酶之间的联系。- H2 O2、PDGF、血小板衍生生长因子;磷酸肌醇3-激酶、Rac、NADPH氧化酶

Autophosphorylated platelet-derived growth factor receptor triggers intracellular signaling cascades by recruiting SH2 domain-containing enzymes that include phosphatidylinositol 3-kinase (PI3K), the GTPase- activating protein of Ras (GAP), the protein tyrosine phosphatase (SHP- 2), and phospholipase C-gamma1 (PLCgamma1). PDGF-dependent H2O2 production was measured in HepG2 cells expressing various PDGF receptor mutants in which the Lys635 essential for kinase activity is changed to Arg or the Tyr residues for the binding of PI3K (Tyr740 and Tyr751), GAP (Tyr 771), SHP-2 (Tyr 1009), and PLCgamma1 (Tyr 1021) were mutated to Phe in various combinations. The kinase deficient receptor failed to produce H2O2. Elimination of PI3K binding site also abrogated H2O2 production, whereas H2O2 production was slightly enhanced when the binding sites for GAP, SHP-2, and PLC-gamma1 were removed. Among the binding sites for the four effector enzymes, the presence of PI3K binding site alone was sufficient for H2O2 production. The PDGF-induced H2O2 production in cells expressing wild-type receptor or the receptor containing only the PI3K binding site was completely blocked by a PI3K inhibitor, LY294002 or by overexpression of a dominant negative form of Rac1, N17Rac1. These results suggest that a product of PI3-kinase is necessary for the activation of putative NADPH oxidase in nonphagocytice cells stimulated with PDGF and that Rac1 provides the linkage between the PI3K product and NADPH oxidase. - H2O2, PDGF, platelet-derived growth factor; phosphoinositide 3-kinase, Rac, NADPH oxidase