Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase.

Abstract

Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.

Rac2 Is an Essential Regulator of Neutrophil Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activation in Response to Specific Signaling Pathways

“…For example, Rac1 has been shown to bind to and stimulate p21-activated kinase 1 activity more efficiently that Rac2 (53). In addition, studies using the yeast two-hybrid system have suggested that the affinity of Rac2 for p67 phox is higher than that for Rac1 (19), and Rac2 is more active than Rac1 in stimulating NADPH oxidase activity in the presence of neutrophil cytosol (28). Another potential mechanism for functional defects in rac2 Ϫ/Ϫ cells is that the overall level of Rac-GTP might be a rate-limiting step in the activation of downstream functions, and signaling cascades triggered by different neutrophil agonists may vary in the amount of activated Rac generated.…”

“…In the yeast two-hybrid system, p67 phox has higher affinity for Rac2 than for Rac1 (19). Recombinant isoprenylated Rac1 and Rac2 were equipotent in a cell-free NADPH oxidase assay using purified flavocytochrome b and recombinant cytosolic phox proteins, but Rac2 was more active than Rac1 when neutrophil cytosol was added (28).…”

Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils

“…To mimic the potential effect of cytosolic RacGAPs, we first added these proteins to the semirecombinant cell-free O2 •--generation system. In previous studies it has been shown that addition of Bcr, ARHGAP1 or ARHGAP35 decreased O2 •--production in crude cell-free systems [39,40]. However, these studies were carried out under different experimental conditions so that a direct comparison of individual RacGAP effects was not possible.…”

“…Due to their complex domain structure [32,34] and extensive regulation [30,35], RacGAPs could fine tune Nox activity. In fact, a role for RacGAPs in the regulation of assembly and function of the Nox2 complex has been indicated in previous in vitro studies [36][37][38][39][40] and in genetically modified animals [41][42][43]. These were all isolated studies and comparative aspects have not been raised.…”

“…In human neutrophils the RacGAPs Bcr, ARHGAP35 and ARHGAP1 have been identified by immunoblotting [40] and all three proteins decreased O2 •--production when they were added to a cell-free system [39,40]. However, the contribution of endogenous, membrane-bound RacGAPs has not been investigated.…”