Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1
Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1
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DOI：
10.1074/jbc.m513665200
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发表时间：
2006-08-04
影响因子：
4.8
通讯作者：
Sumimoto, Hideki
中科院分区：
文献类型：
--
作者：
Miyano, Kei;Ueno, Noriko;Sumimoto, Hideki
非吞噬细胞产生超氧化物的NADPH氧化酶Nox 1与p22（phox）在膜上复合，需要其调节可溶性蛋白Noxo 1和Noxa 1的激活。然而，小型GTQRac的作用仍有待澄清。在这里，我们表明，Rac直接参与Nox 1激活通过与Noxa 1相互作用。电透化HeLa细胞，异位表达Nox 1，Noxo 1，和Noxa 1，产生超氧化物在GTP依赖的方式，这是废除的突变体Noxa 1（R103 E），缺陷的Rac结合的表达。在表达Nox 1的HeLa和Caco-2细胞中的超氧化物产生通过Rac的耗尽或隔离而减少;另一方面，它通过组成型活性Rac 1（Q61 L）的表达而增强，但不通过具有A27 K取代的突变Rac 1的表达而增强，所述突变Rac 1缺乏与Noxa 1的结合。我们还表明，Nox 1激活需要Noxa 1，这通常是通过Noxa 1结合到Noxo 1介导的膜招聘，Noxo 1是一种与Nox 1伴侣p22 phox相连的蛋白质：Noxa 1-Noxo 1和Noxo 1-p22 phox相互作用都是Nox 1活性所必需的。Rac可能促进Noxa 1的膜定位：尽管Noxa 1（W 436 R），在Noxo 1结合中有缺陷，既不与膜结合也不激活Nox 1，但W 436 R取代的效果通过Rac 1（Q61 L）的表达而恢复。Rac-Noxa 1相互作用也在不同于Noxa 1定位的步骤中起作用，因为结合缺陷型Noxa 1（R103 E）尽管靶向膜，但不支持Nox 1产生超氧化物。此外，在活化结构域中携带Ala取代瓦尔-205的突变体Noxa 1（预期其在Rac结合后经历构象变化）完全定位于膜，但不能活化Nox 1。
Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22(phox) at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22phox: the Noxa1-Noxo1 and Noxo1-p22phox interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1( Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1( R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.