Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent.
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.
Price
Marianne O
MO
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.
Atkinson
Simon J
SJ
Knaus
Ulla G
UG
Dinauer
Mary C
MC
D000818:Animals; D030801:Animals, Genetically Modified; D019556:COS Cells; D004790:Enzyme Induction; D005455:Fluorescent Antibody Technique; D006801:Humans; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000235:genetics; D010586:Phagocytes / Q000201:enzymology; D021381:Protein Transport; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D013481:Superoxides; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein