Participation of the small molecular weight GTP-binding protein Rac1 in cell-free activation and assembly of the respiratory burst oxidase. Inhibition by a carboxyl-terminal Rac peptide.
NADPH-dependent superoxide generation was activated by anionic amphiphiles plus GTP gamma S in a cell-free system consisting of plasma membranes plus recombinant p47-phox, p67-phox, and the small GTP-binding protein Rac1. Rac1 was expressed in Escherichia coli both as the native form and as a mutant form (Rac1(C189S)) lacking the prenylation site. When preloaded with GTP gamma S, both Rac proteins supported activity to a level comparable to that seen using cytosol. A peptide corresponding to the carboxyl-terminal region of Rac1 was used to investigate oxidase assembly and activation. Rac1(178-188), but not several control peptides, inhibited activity. The peptide inhibited competitively (Ki = 15 microM) with respect to Rac1(C189S), while inhibition was noncompetitive or mixed with respect to p47-phox and p67-phox. This indicated specific inhibition of the interaction of the Rac protein with its target, possibly cytochrome b558. The peptide was effective only when added prior to activation with arachidonic acid, suggesting that it affects assembly rather than activity. Consistent with this possibility, the peptide prevented translocation of p47-phox and p67-phox to the plasma membrane. Thus, Rac plays a central role in the assembly of the neutrophil NADPH oxidase.
Kreck
M L
ML
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
Uhlinger
D J
DJ
Tyagi
S R
SR
Inge
K L
KL
Lambeth
J D
JD
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000502:physiology*; D009252:NADPH Dehydrogenase / Q000502:physiology*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000502:physiology*; D013329:Structure-Activity Relationship; D013481:Superoxides / Q000378:metabolism; D020744:rac GTP-Binding Proteins
D003573:Cytochrome b Group; D046911:Macromolecular Substances; D010455:Peptides; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C022709:superoxide-forming enzyme; D009252:NADPH Dehydrogenase; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins