Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase.
The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.
Sumimoto
H
H
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Kage
Y
Y
Nunoi
H
H
Sasaki
H
H
Nose
T
T
Fukumaki
Y
Y
Ohno
M
M
Minakami
S
S
Takeshige
K
K
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D016718:Arachidonic Acid / Q000494:pharmacology; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D019255:NADPH Oxidases; D010586:Phagocytes / Q000201:enzymology*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D016897:Respiratory Burst; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D016718:Arachidonic Acid; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase