Vav Proteins in Neutrophils Are Required for FcγR-Mediated Signaling to Rac GTPases and Nicotinamide Adenine Dinucleotide Phosphate Oxidase Component p40(phox)

Utomo, Ahmad; Culleré, Xavier; Glogauer, Michael; Swat, Wojciech; Mayadas, Tanya N.

doi:10.4049/jimmunol.177.9.6388

Cited by 76 publications

(68 citation statements)

References 58 publications

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“…6), suggesting that there is selective pressure to maintain this residue. Functional activation of neutrophils and macrophages via integrins or Fcγ receptors (FcγR) is markedly impaired in mice lacking both Vav1 and Vav3 (23)(24)(25), consistent with the finding that these are the main Vav isoforms expressed by these cells (23,24). Vav1 residues E509, N510, E556, and G559 in the ZF domain, predicted to interact with NCF2 His-389 (Fig.…”

Section: Resultssupporting

confidence: 63%

“…7, IgG bead-elicited ROS responses for either the Q389 or A389 mutation were reduced by approximately twofold compared with WT NCF2, whereas there was no effect on PMAelicited ROS release. The differential effect of NCF2 H389 substitutions on PMA-vs. IgG bead-elicited ROS release is especially relevant, because it is consistent with the lack of participation of any Vav isoform in PMA-elicited NADPH oxidase activity, whereas Vav1 and Vav3 are essential for FcγR-elicited NADPH oxidase activity (25). Furthermore, these results are consistent with previous results in which an R395W mutation reduced the PMA-induced (Vav-independent) NADPH oxidase activity and caused CGD (16), supporting the notion that amino acid 395 is involved in the binding to NCF4 rather than to Vav1.…”

Section: Resultsmentioning

confidence: 95%

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Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase

Jacob

Eisenstein

Dinauer

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

153

119

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Systemic lupus erythematosus (SLE), the prototypic systemic autoimmune disease, is a debilitating multisystem autoimmune disorder characterized by chronic inflammation and extensive immune dysregulation in multiple organ systems, resulting in significant morbidity and mortality. Here, we present a multidisciplinary approach resulting in the identification of neutrophil cytosolic factor 2 (NCF2) as an important risk factor for SLE and the detailed characterization of its causal variant. We show that NCF2 is strongly associated with increased SLE risk in two independent populations: childhood-onset SLE and adult-onset SLE. The association between NCF2 and SLE can be attributed to a single nonsynonymous coding mutation in exon 12, the effect of which is the substitution of histidine-389 with glutamine (H389Q) in the PB1 domain of the NCF2 protein, with glutamine being the risk allele. Computational modeling suggests that the NCF2 H389Q mutation reduces the binding efficiency of NCF2 with the guanine nucleotide exchange factor Vav1. The model predicts that NCF2/H389 residue interacts with Vav1 residues E509, N510, E556, and G559 in the ZF domain of Vav1. Furthermore, replacing H389 with Q results in 1.5 kcal/ mol weaker binding. To examine the effect of the NCF2 H389Q mutation on NADPH oxidase function, site-specific mutations at the 389 position in NCF2 were tested. Results show that an H389Q mutation causes a twofold decrease in reactive oxygen species production induced by the activation of the Vav-dependent Fcγ receptor-elicited NADPH oxidase activity. Our study completes the chain of evidence from genetic association to specific molecular function.F ine localization of the polymorphisms responsible for genotype-phenotype correlations is emerging as a difficult hurdle in the implementation and interpretation of genetic association studies. Candidate gene studies and, more recently, genome-wide association studies (GWAS), have begun to elucidate the complex genetic profile of systemic lupus erythematosus (SLE) with identification of ∼30 risk loci (1-3). However, for almost all these identified loci, the causal polymorphism that leads to lupus susceptibility has not been discovered. GWAS have been praised for representing an "agnostic" approach that is unbiased by prior assumptions regarding genetic association with the disease. However, such an approach typically ignores all valuable prior information collected over decades about the pathogenesis and genetic basis of diseases that have been previously studied. This inevitably leads to the inclusion of regions (and additional SNPs) that have little to no possibility of being associated with a disease, increasing the number of tests. More tests mean a more stringent multiple testing correction and a reduction of power or a greater number of subjects to overcome the reduction of power. To avoid this reduction of power, we have developed a two-step bioinformatics-driven design that increases the power of gene association studies using a partial Bayesian approach. The...

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Section: Resultssupporting

confidence: 63%

Section: Resultsmentioning

confidence: 95%

Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase

Jacob

Eisenstein

Dinauer

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

153

119

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“…As with zymosan stimulation, V123 neutrophils produced very little ROS (90% less than WT) when stimulated with either serum-opsonized S. aureus or IgG- opsonized SRBCs (Fig. 2), supporting previous findings that the Vav family mediates integrin-and FcR-dependent ROS formation (10,29). To conclude, in contrast to GPCR-dependent ROS formation, there is no cooperation between the P-Rex and Vav families in the control of particle-induced ROS formation.…”

Section: The Vav Family Controls the Ros Response To Opsonized Particlessupporting

confidence: 67%

“…Loss of Rac2 activity in humans (D57N mutation) results in severe recurrent bacterial infections, and Rac2 D57N neutrophils are largely unable to chemotax to sites of infection, produce ROS, or secrete granule proteins (4,5). Rac2 2/2 mice cannot clear fungal infections (6), and, in Rac2 2/2 neutrophils, adhesion, chemotaxis (polarization and motility), ROS formation, phagocytosis, and degranulation are impaired (6)(7)(8)(9)(10). In contrast, conditional Rac1 2/2 neutrophils produce ROS normally and can move, but with poor directionality (11,12).…”

mentioning

confidence: 99%

P-Rex1 and Vav1 Cooperate in the Regulation of Formyl-Methionyl-Leucyl-Phenylalanine–Dependent Neutrophil Responses

Lawson

Donald

Anderson

et al. 2011

The Journal of Immunology

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G protein-coupled receptor (GPCR) activation elicits neutrophil responses such as chemotaxis and reactive oxygen species (ROS) formation, which depend on the small G protein Rac and are essential for host defense. P-Rex and Vav are two families of guanine-nucleotide exchange factors (GEFs) for Rac, which are activated through distinct mechanisms but can both control GPCR-dependent neutrophil responses. It is currently unknown whether they play specific roles or whether they can compensate for each other in controlling these responses. In this study, we have assessed the function of neutrophils from mice deficient in P-Rex and/or Vav family GEFs. We found that both the P-Rex and the Vav family are important for LPS priming of ROS formation, whereas particle-induced ROS responses and cell spreading are controlled by the Vav family alone. Surprisingly, fMLF-stimulated ROS formation, adhesion, and chemotaxis were synergistically controlled by P-Rex1 and Vav1. These responses were more severely impaired in neutrophils lacking both P-Rex1 and Vav1 than those lacking the entire P-Rex family, the entire Vav family, or both P-Rex1 and Vav3. P-Rex1/Vav1 (P1V1) double-deficient cells also showed the strongest reduction in fMLF-stimulated activation of Rac1 and Rac2. This reduction in Rac activity may be sufficient to cause the defects observed in fMLF-stimulated P1V1 neutrophil responses. Additionally, Mac-1 surface expression was reduced in P1V1 cells, which might contribute further to defects in responses involving integrins, such as GPCR-stimulated adhesion and chemotaxis. We conclude that P-Rex1 and Vav1 together are the major fMLFR -dependent Dbl family Rac-GEFs in neutrophils and cooperate in the control of fMLF-stimulated neutrophil responses.

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“…is regulated by phosphatidylinositol 3-kinase (18) and protein kinase C (18,19), and inhibition of protein kinase C attenuates tumor necrosis factor-␣-mediated serine phosphorylation of p47 phox and its interaction with gp91 phox at the cell periphery (19). In neutrophils and macrophages, Fc␥R-mediated activation of NADPH oxidase and ROS production is regulated by the Vav guanine exchange factor and the phosphatidylinositol 3-kinase-dependent phosphorylation of p40 phox (20). Hyperoxia-dependent activation of lung endothelial cell (EC) NADPH oxidase is also partly regulated by extracellular signal-regulated kinase (ERK) and p38 MAPK, suggesting the involvement of serine/threonine phosphorylation of the subcomponents of the enzyme in regulating ROS production (10).…”

mentioning

confidence: 99%

Regulation of Hyperoxia-induced NADPH Oxidase Activation in Human Lung Endothelial Cells by the Actin Cytoskeleton and Cortactin

Usatyuk

Romer

et al. 2007

Journal of Biological Chemistry

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Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxiainduced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47 phox , a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47 phox to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47phox . In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxiainduced activation of NADPH oxidase and ROS generation in human lung endothelial cells. production that is dependent on NADPH oxidase activation and independent of the mitochondrial electron transport or xanthine/xanthine oxidase systems (10). The mechanisms of NADPH oxidase activation are complex. In phagocytes, activation of NADPH oxidase requires serine phosphorylation of the cytosolic p47 phox , p67 phox , and p40 phox components, assembly of the phosphorylated subunits with Rac2, and translocation to the phagosomes for association with cytochrome b 558. Here, one-electron reduction of molecular O 2 to O 2 . occurs with NADPH as the electron donor (7). In leukocytes, formyl-Met-Leu-Phe-OH or phorbol ester stimulates phosphorylation of p47 phox at multiple serine residues through reactions involving several protein kinases such as protein kinase C, protein kinase A, and mitogen-activated protein kinases (14 -17). In HPAECs, tumor necrosis factor-␣-medi-*This work was supported by National Institutes of Health Grants RO1 HL 69909 (to V. N.), PO1 HL 58064 (to V. N. and J. G. N. G.) and DE13079-01 and AI061042 (to L. H. R.). The costs of publication...

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Vav Proteins in Neutrophils Are Required for FcγR-Mediated Signaling to Rac GTPases and Nicotinamide Adenine Dinucleotide Phosphate Oxidase Component p40(phox)

Cited by 76 publications

References 58 publications

Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase

Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase

P-Rex1 and Vav1 Cooperate in the Regulation of Formyl-Methionyl-Leucyl-Phenylalanine–Dependent Neutrophil Responses

Regulation of Hyperoxia-induced NADPH Oxidase Activation in Human Lung Endothelial Cells by the Actin Cytoskeleton and Cortactin

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