Activation of the phagocyte NADPH oxidase protein p47phox -: Phosphorylation controls SH3 domain-dependent binding to p22phox

Activation of phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox), p47(phox);, resting phagocytes does not bind p22(phox). Phosphorylation of serines in the p47(phox) C terminus enables binding to the p22(phox) C terminus by inducing a conformational change in p47(phox) that unmasks the SH3(A) domain. We report that an arginine/lysine-rich region in the p47phox C terminus binds the p47(phox) SH3 domains expressed in tandem (SH3(AB)) but does not bind the individual N-terminal SH3(A) and C-terminal SH3(B) domains. Peptides matching amino acids 301-320 and 314-335 of the p47(phox) arginine/lysine-rich region block the p47(phox) SH3(AB)/p22(phox) C-terminal and p47(phox) SH3(AB)/p47(phox) C-terminal binding and inhibit NADPH oxidase activity in vitro, Peptides with phosphoserines substituted for serines 310 and 328 do not block binding and are poor inhibitors of oxidase activity. Mutated full-length p47(phox) with aspartic acid substitutions to mimic the effects of phosphorylations at serines 310 and 328 bind the p22(phox) proline-rich region in contrast to wild-type p47(phox), We conclude that the p47(phox) SH3(A) domain-binding site is blocked by an interaction between the p47(phox) SH3(AB) domains and the C-terminal arginine/lysine-rich region. Phosphorylation of serines in the p47(phox) C terminus disrupts this interaction leading to exposure of the SH3(A) domain, binding to p22(phox) and activation of the NADPH oxidase.