Vilhardt Frederik, Plastre Olivier, Sawada Makoto, Suzuki Kazuo, Wiznerowicz Maciej, Kiyokawa Etsuko, Trono Didier, Krause Karl-Heinz
Biology of Ageing Laboratory, Department of Geriatrics, Geneva University Hospitals, Switzerland.
J Biol Chem. 2002 Nov 1;277(44):42136-43. doi: 10.1074/jbc.M200862200. Epub 2002 Aug 30.
Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia (cell line and primary culture) were transduced with lentiviral expression vectors. Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin). These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits. Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway. The following results suggest a key role for Rac in the priming effect of Nef. (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes. Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli.
Nef是一种多功能的HIV蛋白，可激活Vav/Rac/p21激活激酶（PAK）信号通路。鉴于该通路在吞噬细胞NADPH氧化酶激活中的潜在作用，我们研究了HIV-1 Nef蛋白对吞噬细胞呼吸爆发的影响。用慢病毒表达载体转导小胶质细胞（细胞系和原代培养物）。Nef的表达本身不会激活NADPH氧化酶，但会导致对多种刺激（钙离子载体、甲酰肽、内毒素）的反应大幅增强。这些效应不是由吞噬细胞NADPH氧化酶亚基的上调引起的。缺乏与Vav和PAK相互作用相关基序的Nef突变体无法重现野生型Nef的效应，表明Vav/Rac/PAK信号通路发挥了作用。以下结果表明Rac在Nef的启动效应中起关键作用。（i）艰难梭菌毒素B使Rac失活消除了Nef的效应。（ii）在转导了Nef的细胞中，活化的Rac1比例增加，并且（iii）显性阳性Rac1（V12）突变体模拟了Nef的效应。据我们所知，这些结果是对完整吞噬细胞中Rac激活对NADPH氧化酶影响的首次分析。Rac激活不足以刺激吞噬细胞NADPH氧化酶；然而，它显著增强了NADPH氧化酶对其他刺激的反应。
