PHAGOCYTE NADPH OXIDASE P67-PHOX POSSESSES A NOVEL CARBOXYLTERMINAL BINDING-SITE FOR THE GTPASES RAC2 AND CDC42

Rac GTPases regulate activation of the phagocyte NADPH oxidase, a mult i-component enzyme complex that produces superoxide in response to hos t infection. GTP-bound Rac binds to the cytosol protein p67-phox enabl ing it to participate in oxidase assembly. Details of this interaction are poorly understood. Previous studies showed that Rac/p67-phox bind ing is GTP-dependent and that several Rad mutants lost the ability to activate the oxidase even though they still bound p67-phox. Using two hybrid and blot overlay binding methods, we identified a novel binding site in the p67-phox C-terminus that binds Rad, Rac2, and Cdc42, a re lated GTPase which does not activate the oxidase. Binding was independ ent of the GDP/GTP state. We also showed that GTP-Cdc42 binds p67-phox N-terminus similar to GTP-Rac. Therefore, Rac binding to p67-phox is not synonymous with NADPH oxidase activation, and Rac probably partici pates in other steps of oxidase activation in addition to binding p67- phox. (C) 1998 Academic Press.