Freeman J L, Kreck M L, Uhlinger D J, Lambeth J D
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
Biochemistry. 1994 Nov 15;33(45):13431-5. doi: 10.1021/bi00249a031.
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 EC50 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. EC50 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.
Rac是Ras超家族中的一种小分子量GTP酶，参与人类中性粒细胞多组分超氧化物生成NADPH氧化酶的激活。Rac与Ras的整体序列一致性为30%，但在与Ras效应区相对应的序列中一致性为75%，Ras通过与蛋白激酶Raf1相互作用调节有丝分裂。我们使用定点诱变研究了该区域在Rac1中的作用。在无细胞半重组NADPH氧化酶系统中，26、33、38和45位氨基酸的突变体与氧化酶复合物的结合能力降低了20 - 110倍（根据EC50值判断），超氧化物生成的最大活性降低了44% - 80%。只有GTPγS结合形式能结合，因为GDP结合形式的Rac既不能单独激活，也不能与GTPγS - Rac竞争。当使用突变体Rac替代Rac时，p47 - phox和p67 - phox的EC50值均未受影响。数据表明Rac效应区与呼吸爆发氧化酶的一个或多个组分直接结合。结果表明小GTP酶中保守的效应器等效区域在调节蛋白质 - 蛋白质相互作用中具有普遍作用。
