Ras effector-homolog region on Rac regulates protein associations in the neutrophil respiratory burst oxidase complex

Abstract

Rac, a small molecular weight GTPase in the Ras superfamily, participates in the activation of the multicomponent superoxide-generating NADPH oxidase of human neutrophils. Rac is 30% identical to Ras overall, but is 75% identical within the sequence corresponding to the effector region of Ras, which regulates mitogenesis through interactions with the protein kinase Raf1. We investigated the role of this region in Rac1 using site-directed mutagenesis. In a cell-free semirecombinant NADPH oxidase system, mutants in the 26, 33, 38, and 45 amino acids showed 20-110-fold reduced binding to the oxidase complex as judged by EC50 values and reduced (44-80%) maximal activities in superoxide generation. Only the GTP gamma S-bound form associated, since the GDP-bound form of Rac neither activated alone nor competed with GTP gamma S-Rac. EC50 values for neither p47-phox nor p67-phox were affected when mutant Racs were used in place of Rac. Data indicate direct binding of the Rac effector region to one or more components of the respiratory burst oxidase. Results indicate a general role for conserved effector-equivalent regions in small GTPases in the regulation of protein-protein interactions.

Rac “Insert Region” Is a Novel Effector Region That Is Implicated in the Activation of NADPH Oxidase, but Not PAK65

“…Titrations with cytosolic components p47 phox and p67 phox in the semirecombinant cell-free system demonstrate that the Rac insert region mutations have a modest but reproducible effect to increase the EC 50 (26). Thus, the kinetic consequences of mutations in these two effector regions are significantly different.…”

“…The amino acid sequence of Rac in this region is highly homologous to that in Ras, suggesting conservation of the effector binding function of this region. Site-specific mutations (25)(26)(27) and chimeric proteins of Rac/Cdc42 (28) assayed for the ability to support superoxide generation demonstrate that this region is important for the interactions within the NADPH oxidase complex. Rac can bind directly to p67 phox , and mutations in amino acids within this region eliminate this interaction (27).…”

“…Mutagenesis-Site-directed mutations were made in Rac using a modification of the Kunkel single-strand mutagenesis method as described by Freeman et al (26). All Rac and Rac mutant proteins contain a Cys 189 to Ser mutation to increase the stability of the protein during storage as described by Kreck (11).…”

Regulation of the Neutrophil Respiratory Burst Oxidase

“…Rac contains an additional region, the insert region, that is important for assembly (54) and that was proposed to interact directly with the cytochrome (39). Thus, both Rac and p47 phox are the targets of "assembly signals," including guanine nucleotide exchange and phosphorylation, and both provide binding sites for p67…”

Bioorganic Studies in AIDS: Synthetic Antifungals Against Pneumocystis carinii Based on the Multivalency Concept

“…Activated human neutrophils exert microbicidal activity through two types of mechanisms: one in which the intact neutrophils' H 2 O 2 -myeloperoxidase-Cl -system mediates the production of HOCl, which in turn chlorinates endogenous amines to yield a new group of powerful oxidizing agents, chloramines, and the other in which granule-poor cytoplasts have a • NO-dependent cytotoxic mechanism [24]. Of further interest is how Rac1 and Rac2, the small GTP-dependent factors, activated upon exposure to microorganisms, regulate O 2 -• generation in both macrophages and neutrophils [25]. According to the oxidoredox hypothesis [16], multivalent drugs containing oxidoredox pharmacophores could mimic the action of macrophage and neutrophil oxidants or could interact with macrophage/neutrophilderived oxidants in specific ways to modulate the physiological concentrations of the latter.…”