Activation of the Phagocyte NADPH Oxidase Protein p47

Activation of phagocyte NADPH oxidase requires interaction between p47 and p22. p47 in resting phagocytes does not bind p22. Phosphorylation of serines in the p47 C terminus enables binding to the p22 C terminus by inducing a conformational change in p47 that unmasks the SH3A domain. We report that an arginine/lysine-rich region in the p47 C terminus binds the p47 SH3 domains expressed in tandem (SH3AB) but does not bind the individual Nterminal SH3A and C-terminal SH3B domains. Peptides matching amino acids 301–320 and 314–335 of the p47 arginine/lysine-rich region block the p47 SH3AB/p22 phox C-terminal and p47 SH3AB/p47 phox Cterminal 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 with aspartic acid substitutions to mimic the effects of phosphorylations at serines 310 and 328 bind the p22 proline-rich region in contrast to wild-type p47. We conclude that the p47 SH3A domain-binding site is blocked by an interaction between the p47 SH3AB domains and the C-terminal arginine/lysine-rich region. Phosphorylation of serines in the p47 C terminus disrupts this interaction leading to exposure of the SH3A domain, binding to p22 , and activation of the NADPH oxidase.