Price Marianne O, Atkinson Simon J, Knaus Ulla G, Dinauer Mary C
Herman B Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology), James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, Indiana 46202, USA.
J Biol Chem. 2002 May 24;277(21):19220-8. doi: 10.1074/jbc.M200061200. Epub 2002 Mar 14.
Transient expression of constitutively active Rac1 derivatives, (G12V) or (Q61L), was sufficient to induce phagocyte NADPH oxidase activity in a COS-7 cell model in which human cDNAs for essential oxidase components, gp91(phox), p22(phox), p47(phox), and p67(phox), were expressed as stable transgenes. Expression of constitutively active Rac1 in "COS(phox)" cells induced translocation of p47(phox) and p67(phox) to the membrane. Furthermore, translocation of p47(phox) was induced in the absence of p67(phox) expression, even though Rac does not directly bind p47(phox). Rac effector domain point substitutions (A27K, G30S, D38A, Y40C), which can selectively eliminate interaction with different effector proteins, impaired Rac1V12-induced superoxide production. Activation of endogenous Rac1 by expression of constitutively active Rac-guanine nucleotide exchange factor (GEF) derivatives was sufficient to induce high level NADPH oxidase activity in COS(phox) cells. The constitutively active form of the hematopoietic-specific GEF, Vav1, was the most effective at activating superoxide production, despite detection of higher levels of Rac1-GTP upon expression of constitutively active Vav2 or Tiam1 derivatives. These data suggest that Rac can play a dual role in NADPH oxidase activation, both by directly participating in the oxidase complex and by activating signaling events leading to oxidase assembly, and that Vav1 may be the physiologically relevant GEF responsible for activating this Rac-regulated complex.
组成型活性Rac1衍生物(G12V)或(Q61L)的瞬时表达足以在COS-7细胞模型中诱导吞噬细胞NADPH氧化酶活性,在该模型中,必需氧化酶成分的人类cDNA,即gp91(phox)、p22(phox)、p47(phox)和p67(phox),被表达为稳定转基因。在“COS(phox)”细胞中组成型活性Rac1的表达诱导了p47(phox)和p67(phox)向膜的转位。此外,即使Rac不直接结合p47(phox),在没有p67(phox)表达的情况下也诱导了p47(phox)的转位。Rac效应器结构域点突变(A27K、G30S、D38A、Y40C)可选择性消除与不同效应蛋白的相互作用,损害Rac1V12诱导的超氧化物产生。通过组成型活性Rac-鸟嘌呤核苷酸交换因子(GEF)衍生物的表达激活内源性Rac1足以在COS(phox)细胞中诱导高水平的NADPH氧化酶活性。造血特异性GEF的组成型活性形式Vav1在激活超氧化物产生方面最有效,尽管在组成型活性Vav2或Tiam1衍生物表达后检测到更高水平的Rac1-GTP。这些数据表明,Rac在NADPH氧化酶激活中可发挥双重作用,既通过直接参与氧化酶复合物,也通过激活导致氧化酶组装的信号事件,并且Vav1可能是负责激活这种Rac调节复合物的生理相关GEF。