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 du ring phagocytosis to produce superoxide, a precursor of microbicidal o xidants. The activated oxidase is a complex of membrane-integrated cyt ochrome b(558), composed of 91-kDa (gp91(phox)) and 22-kDa (p22(phox)) subunits, and two cytosolic factors (p47(phox) and p67(phox)), each c ontaining two Src homology 3 (SH3) domains. Here we show that the regi on of the tandem SH3 domains of p47(phox) (p47-SH3) expressed as a glu tathione S-transferase fusion protein inhibits the superoxide producti on in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dod ecyl sulfate, activators of the oxidase in vitro, cause exposure of p4 7-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 granuloma tous disease. Based on the observations, we propose a signal-transduci ng mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.