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 (gp91(phox)) and 22-kDa (p22(phox)) subunits, and two cytosolic factors (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47(phox) (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 p22(phox) and p67(phox) but fails to interact with a mutant p22(phox) 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.
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Publication status||Published - Jun 7 1994|
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