Rac ''insert region'' is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65
Rac ''insert region'' is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65
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DOI：
10.1074/jbc.271.33.19794
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发表时间：
1996-08-16
影响因子：
4.8
通讯作者：
Lambeth, JD
中科院分区：
文献类型：
--
作者：
Freeman, JL;Abo, A;Lambeth, JD
小的GTdR Rac与胞质p47（phox）和p67（phox）以及膜相关黄细胞色素B（558）组装形成多组分呼吸爆发氧化酶。Rac区域（残基26-45）中的氨基酸突变，与Pas中的效应子区域同源，先前显示干扰Rac与氧化酶的结合。本文阐明了Rac中参与调节氧化酶活性的另外一个区域。Rho家族小GTP酶含有Pas中不存在的12个氨基酸的“插入"区域（残基124-135）。构建了该区域的点突变和缺失，并用于体外研究PAK 65和NADPH氧化酶的活化。表观结合常数突变体Rac蛋白对氧化酶的活性（基于EC（50）值）比野生型Rac高至少13-25倍。插入区相对于26-45效应区的突变导致不同的动力学结果，表明这两个蛋白区的不同作用：插入区而不是26-45效应区的突变导致p67（phox）的EC（50）增加。虽然26-45个氨基酸的效应区的突变显示PAK和NADPH氧化酶的激活显着减少，插入区突变不影响PAK的激活。我们提出，26-45效应区和插入区的组合使用提供了Rho家族GTP酶的多功能性，在其特异性的几个下游目标。
The small GTPase Rac assembles with the cytosolic p47(phox) and p67(phox) and the membrane-associated flavocytochrome b(558) to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Pas, was previously shown to interfere with Rac binding to the oxidase. Herein wc have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12-amino acid ''insert'' region (residues 124-135) that is not present in Pas, Point mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. Apparent binding constants (based on EC(50) values) of the mutant Rac proteins for the oxidase are at least 13-25-fold higher than for wild-type Rac, Mutations in the insert region versus the 26-45 effector region resulted in distinct kinetic consequences, pointing to different roles for these two protein regions: mutations in the insert region but not the 26-45 effector region resulted in an increase in the EC(50) for p67(phox). Although mutations in the 26-45 amino acid effector region showed markedly diminished activation of both PAK and the NADPH oxidase, insert region mutations did not affect activation of PAK. We propose that the combinatorial use of the 26-45 effector region and the insert region provides the Rho family GTPases with versatility in their specificity for several downstream targets.