RAS EFFECTOR-HOMOLOG REGION ON RAC REGULATES PROTEIN ASSOCIATIONS IN THE NEUTROPHIL RESPIRATORY BURST OXIDASE COMPLEX
RAS EFFECTOR-HOMOLOG REGION ON RAC REGULATES PROTEIN ASSOCIATIONS IN THE NEUTROPHIL RESPIRATORY BURST OXIDASE COMPLEX
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DOI：
10.1021/bi00249a031
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发表时间：
1994-11-15
期刊：
影响因子：
2.9
通讯作者：
LAMBETH, JD
中科院分区：
文献类型：
--
作者：
FREEMAN, JLR;KRECK, ML;LAMBETH, JD
Rac是Ras超家族中的一种小分子量GT3，参与人中性粒细胞多组分超氧化物生成NADPH氧化酶的激活。Rac与Ras总体上有30%相同，但在对应于Ras的效应子区域的序列内有75%相同，Ras的效应子区域通过与蛋白激酶Raf 1的相互作用来调节有丝分裂。我们研究了该地区的作用，在Rac 1使用定点突变。在无细胞半重组NADPH氧化酶系统中，26、33、38和45个氨基酸中的突变体显示出与氧化酶复合物的结合减少20-110倍，如通过EC（50）值判断的，并且减少（44-80%）超氧化物生成的最大活性。只有GTP γ S结合形式相关，因为GDP结合形式的Rac既不单独激活也不与GTP γ S-Rac竞争。当使用突变体Rac代替Rac时，p47-phox和p67-phox的EC（50）值均不受影响。数据表明Rac效应区与呼吸爆发氧化酶的一种或多种组分直接结合。结果表明小GTP酶中保守的效应等效区在调节蛋白质-蛋白质相互作用中的一般作用。
Rac, a small molecular weight GTPase in the Ras superfamily, participates in the activation of the multicomponent superoxide-generating NADPH oxidase of human neutrophils. Rac is 30% identical to Ras overall, but is 75% identical within the sequence corresponding to the effector region of Ras, which regulates mitogenesis through interactions with the protein kinase Raf1. We investigated the role of this region in Rac1 using site-directed mutagenesis. In a cell-free semirecombinant NADPH oxidase system, mutants in the 26, 33, 38, and 45 amino acids showed 20-110-fold reduced binding to the oxidase complex as judged by EC(50) values and reduced (44-80%) maximal activities in superoxide generation. Only the GTP gamma S-bound form associated, since the GDP-bound form of Rac neither activated alone nor competed with GTP gamma S-Rac. EC(50) values for neither p47-phox nor p67-phox were affected when mutant Racs were used in place of Rac. Data indicate direct binding of the Rac effector region to one or more components of the respiratory burst oxidase, Results indicate a general role for conserved effector-equivalent regions in small GTPases in the regulation of protein-protein interactions.