THE RAC TARGET NADPH OXIDASE P67(PHOX) INTERACTS PREFERENTIALLY WITH RAC2 RATHER THAN RAC1

NADPH oxidase is a plasma membrane enzyme of phagocytes generating sup eroxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme minimally requires assembly at the membrane with flavocytochrome b(558) of cytosolic components p47(phox), p67(phox) a nd Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small GTPa se proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidas e activation in vitro; however, Rac2 is largely predominant in human p hagocytes. Here, using the yeast two hybrid system, we provide data de monstrating in vivo interactions between human p47(phox), p67(phox), a nd Rac proteins. Rac proteins interact with p67(phox) in a GTP-depende nt manner, but do not interact with p47(phox). Moreover, Rac effector site mutants, which are known to be inactive in NADPH oxidase, lose th eir interaction with p67(phox); Rac2L61 mutant, which has an increased NADPH oxidase affinity, shows an increased affinity for p67(phox). Fi nally, we observe that p67(phox) interacts 6-fold better with Rac2 tha n with Rac1. We also show a strong intracellular interaction between p 47(phox) and p67(phox). These results indicate that activated Rac can regulate NADPH oxidase by interacting with p67(phox) and that Rac2 is the main p67(phox)-interacting GTPase in human cells.