Activation of the Phagocyte NADPH Oxidase Protein p47 phox

Activation of phagocyte NADPH oxidase requires interaction between p47 phox and p22 phox . p47 phox in 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 SH3Adomain. We report that an arginine/lysine-rich region in the p47 phox C terminus binds the p47 phox SH3 domains expressed in tandem (SH3AB) but does not bind the individual N-terminal SH3A and C-terminal SH3Bdomains. Peptides matching amino acids 301–320 and 314–335 of the p47 phox arginine/lysine-rich region block the p47 phox SH3AB/p22 phox C-terminal and p47 phox SH3AB/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 SH3A domain-binding site is blocked by an interaction between the p47 phox SH3AB 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 SH3Adomain, binding to p22 phox , and activation of the NADPH oxidase.