CRYPTIC RAC-BINDING AND P21(CDC42HS RAC)-ACTIVATED KINASE PHOSPHORYLATION SITES OF NADPH OXIDASE COMPONENT P67(PHOX)/

Rad is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as w ell as cell growth and differentiation. Rac1 and Rac2 are specifically required for superoxide formation by components of the NADPH oxidase, In binding assays, Rad interacts directly with p67(phox), but not wit h the other oxidase components: cytochrome b, p40(phox), or p47(phox) (Prigmore, E., Ahmed, S., Best, A., Kozma, R., Manser, E., Segal, A. W ., and Lim, L. (1995) J. Biol. Chem. 270, 10717-10722), Here, the Rac1 /2 interaction with p67(phox) has been characterized further. Rac1 and Rac2 can bind to p67(phox) amino acid residues 170-199, and the N ter minus (amino acids 1-192) of p67(phox) can be used as a specific inhib itor of Rac signaling. Deletion of p67(phox) C-terminal sequences (ami no acids 193-526), the C-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif (amino acids 226-236) stimulates Rad bindi ng by similar to 8-fold. p21(Cdc42Hs/Rac)-activated kinase (PAK) phosp horylates p67(phox) amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-ter minal SH3 domain or the polyproline-rich motif, These data suggest a r ole for cryptic Rac-binding and PAK phosphorylation sites of p67(phox) in control of the NADPH oxidase.