Sigal Natalia, Gorzalczany Yara, Sarfstein Rive, Weinbaum Carolyn, Zheng Yi, Pick Edgar
Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
J Biol Chem. 2003 Feb 14;278(7):4854-61. doi: 10.1074/jbc.M211011200. Epub 2002 Dec 9.
The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b(559) and four cytosolic components as follows: p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b(559) and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67(phox) but in the absence of amphiphile and p47(phox). We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67(phox)-Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg(2+) from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function.
吞噬细胞中产生超氧化物的NADPH氧化酶复合物由一种膜相关黄素细胞色素b(559)和四个胞质组分组成，如下：p47(phox)、p67(phox)、p40(phox)以及小GTP酶Rac(1或2)。氧化酶的激活是胞质组分与细胞色素b(559)组装的结果，并且在体外可通过暴露于作为激活剂的阴离子两亲物的膜和胞质组分混合物来模拟。我们报道Rac1的异戊二烯化使其具有在不存在两亲物和p47(phox)的情况下与p67(phox)协同支持氧化酶激活的能力。我们现在展示以下6点。1) Rac鸟嘌呤核苷酸交换因子Trio显著增强异戊二烯化的Rac1-GDP对氧化酶的激活作用。2) 这在不存在外源性GTP或任何其他GTP生成来源的情况下发生，表明Trio的作用不涉及Rac1上的GDP向GTP的交换。3) Trio不会增强异戊二烯化的Rac1-GTP、存在或不存在两亲物时未异戊二烯化的Rac1-GDP以及GDP结合形式的异戊二烯化[p67(phox)-Rac1]嵌合体对氧化酶的激活作用。4) 在结合Trio或通过核苷酸交换对Trio作出反应的能力方面存在缺陷的Rac1突变体，无法通过增强的氧化酶激活对Trio作出反应。5) 具有保守的Rac1结合能力但缺乏核苷酸交换活性的Trio突变体无法增强氧化酶激活。6) 从Rac1-GDP中置换Mg(2+)可模拟Trio的作用。这些结果揭示了一种由鸟嘌呤核苷酸交换因子激活Rac的新机制，并表明在不存在核苷酸交换的情况下，Trio诱导Rac1构象变化足以增强其效应子功能。
