CHARACTERIZATION OF THE EFFECTOR-SPECIFYING DOMAIN OF RAC INVOLVED INNADPH OXIDASE ACTIVATION

Production of microbicidal oxidants by phagocytic leukocytes requires activation of a latent NADPH oxidase by the coordinated assembly of a membrane-associated flavocytochrome b(558), with three cytosolic compo nents, p47(phox), p67(phox), and the low molecular weight GTP-binding protein Rac. Rac1 and Rac2 have 92% sequence identity and are both act ive in supporting the oxidase, while CDC42Hs, the closest relative to Pac with 70% sequence identity, only weakly supports oxidase activatio n in vitro, We have used CDC42Hs as a foil to identify residues in Pac that are critical for oxidase activation. Most of the divergent seque nces of CDC42Hs could be incorporated into Rac-CDC42Hs chimeric protei ns without affecting cell-free NADPH oxidase activity, However, incorp oration of the amino-terminal segment of CDC42Hs (residues 1-40), whic h differs from Rad by only four residues (positions 3, 27, 30, and 33) , resulted in a marked loss of oxidase activation capacity. Point muta genesis studies showed that this was due to changes at residues 27 and 30, but nob residues 3 and 33. Conversely, incorporation of the amino terminus of Rac1 (residues 1-40) into CDC42Hs increased its activity to that of Rac1, indicating that this terminus contains the effector-s pecifying domain of Rac. Taken together, these studies show that the d ifference in the activity between CDC42Hs and Rac1 is due entirely to differences in amino acids at position 27 and 30.