ASSEMBLY AND ACTIVATION OF THE PHAGOCYTE NADPH OXIDASE - SPECIFIC INTERACTION OF THE N-TERMINAL SRC HOMOLOGY-3 DOMAIN OF P47(PHOX) WITH P22(PHOX) IS REQUIRED FOR ACTIVATION OF THE NADPH OXIDASE

The phagocyte NADPH oxidase is activated during phagocytosis to produc e superoxide, a precursor of microbicidal oxidants. The activation inv olves assembly of membrane-integrated cytochrome b(558) comprising gp9 1(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) a nd p67(phox)), each containing two Src homology 3 (SH3) domains, and t he small G protein Rac. In the present study, we show that the N-termi nal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail o f p22(phox) with high affinity (K-D = 0.34 mu M). The binding is speci fic to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro(156)-co ntaining proline-rich sequence but not other putative SH3 domain-bindi ng sites of p22(phox). Replacement of Trp(193) by Arg in the N-termina l SH3 domain completely abrogates the association with p22(phox). A mu tant p47(phox) with this substitution is incapable of supporting super oxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal S H3 domain of p47(phox) and the proline-rich region of p22(phox) is ess ential for activation of the NADPH oxidase.