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Previous Article | Next Article 
Molecular and Cellular Biology, January 2007, p. 312-323, Vol. 27, No. 1
0270-7306/07/$08.00+0 doi:10.1128/MCB.00985-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
,
Daniel D. Billadeau,2
Lawrence A. Quilliam,3 and
Mary C. Dinauer1,4*
Herman B. Wells Center for Pediatric Research, Department of Pediatrics, Riley Hospital for Children, Indiana University School of Medicine, 1044 West Walnut Street, R4 451A, Indianapolis, Indiana 46202,1 Division of Developmental Oncology Research, Department of Immunology, Mayo Clinic, 13-11C Guggenheim, 200 First Street Southwest, Rochester, Minnesota 55905,2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, and Walther Cancer Institute, 635 Barnhill Drive, MS-4053, Indianapolis, Indiana 46202,3 Departments of Microbiology and Immunology and Medical and Molecular Genetics, Indiana University School of Medicine, 1044 West Walnut Street, R4 402C, Indianapolis, Indiana 462024
Received 2 June 2006/ Returned for modification 10 July 2006/ Accepted 9 October 2006
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecular events associated with NADPH oxidase assembly and activation are partially defined. Phagocyte activation by soluble or particulate inflammatory mediators initiates signaling cascades that lead to p47phox phosphorylation and Rac activation to assemble the active oxidase complex. Phosphorylation of p47phox unmasks a pair of Src homology 3 (SH3) domains that mediate its translocation to the flavocytochrome via a proline-rich sequence on p22phox (18). The p47phox subunit functions as an adaptor protein to recruit p67phox via a high-affinity interaction between a proline-rich region and SH3 domain in the C termini of p47phox and p67phox, respectively (18). p67phox is a target of Rac-GTP and contains an "activation" domain that regulates flavocytochrome b (29). This domain is just distal to the N-terminal portion of p67phox that binds to the switch 1 effector domain of Rac-GTP (18, 24, 25). Current experimental evidence supports a model in which complex formation between p67phox and membrane-anchored Rac-GTP is required to optimally position p67phox for activation of electron transport through flavocytochrome b (12, 18, 36).
The Rac GTPase translocates to the plasma membrane independently of p67phox and p47phox (13, 15, 20) and is required for NADPH oxidase activity. In resting cells, Rac-GDP is present as a complex with Rho-GDP disassociation inhibitor (Rho-GDI), a negative regulator of Rho family GTPases, but this complex rapidly dissociates, and Rac-GTP forms in stimulated cells. This process is facilitated by activation of guanine nucleotide exchange factors (GEFs) and is accompanied by translocation of Rac to the plasma membrane with kinetics similar to p47phox and p67phox (13). The catalytic activity of the oxidase is dependent on interactions between Rac-GTP and p67phox (12, 24, 25), as mentioned above. Phagocytic leukocytes express Rac1 and Rac2 (13), two closely related isoforms that interact with the Rac binding domain of p67phox with similar affinity (25). The hematopoietic cell-restricted Rac2 is the preferred isoform to regulate neutrophil NADPH oxidase activity in response to most agonists (13, 22, 26, 34, 44), whereas Rac1 appears to play a more important role in macrophages and human monocytes (43, 46).
The specific GEFs that regulate the NADPH oxidase are not well defined. Recent studies indicate that P-Rex1, a GEF that is activated by phosphoinositol-3,4,5-phosphate and Gß
subunits of heterotrimeric G proteins, participates in chemoattractant-mediated activation of superoxide production (14, 40, 41). Additional studies suggest that Vav1, a hematopoietic cell-specific isoform of the Vav family of Rho/Rac GEFs, which are activated by tyrosine phosphorylation (37), also plays an important role in regulating chemoattractant-induced NADPH oxidase activity. Vav1 undergoes tyrosine phosphorylation in murine neutrophils stimulated with formyl-Met-Leu-Phe (fMLP), and superoxide production by fMLP-stimulated neutrophils from Vav–/– mice is attenuated by approximately threefold compared to stimulated neutrophils from wild-type mice (23). However, overall levels of activated Rac1 and Rac2 are similar in fMLP-stimulated Vav1–/– and wild-type neutrophils (16, 23). Additional studies in COS-7 cells stably expressing transgenic NADPH oxidase subunits found that a constitutively active form of Vav1 stimulates translocation of p67phox and p47phox to the plasma membrane and activates superoxide production (32). Moreover, constitutively active Vav1 stimulates NADPH oxidase activity more efficiently than Vav2 or Tiam1 (32). Taken together, these data suggest that Vav1 may regulate a specific pool of Rac in proximity to the NADPH oxidase and are consistent with the concept that GEFs are not functionally redundant but act in the context of specific downstream signaling pathways (35).
Three structurally related Vav isoforms have been identified in mammalian cells (37, 38). Vav1 expression is predominantly limited to the hematopoietic lineage, whereas Vav2 and Vav3 are widely expressed. Typical of other Rho family GEFs, all three Vav family members have a Dbl homology (DH) region that mediates guanine nucleotide exchange. This is flanked by domains that regulate GEF activity and others that mediate protein-protein interactions. These include a calponin homology domain and an acidic region N-terminal to the DH domain, and at the C-terminal side are a pleckstrin homology (PH) domain, a cysteine-rich region also referred to as the zinc finger (ZF) domain, and three juxtaposed Src homology domains (SH3-SH2-SH3) (37). Vav1 facilitates guanine nucleotide exchange for Rho family GTPases with some preference for Rac (10, 38). Phosphorylation of tyrosines within the acidic domain of Vav family GEFs relieves autoinhibitory intramolecular interactions involving the N-terminal portion of the protein that block access to the DH domain, thereby activating GEF activity. The PH domain also appears to regulate GEF activity via binding of phosphatidylinositols (19, 37). Vav family GEFs are activated by ligation of a variety of receptors, including the T-cell and B-cell antigen receptors, growth factor receptors, integrins, and chemokine receptors (37, 38). Genetic deletion of Vav1 family proteins leads to a variety of defects in T- and B-cell development and function, along with other phenotypic changes including leukocyte adhesion and chemokine signaling (37, 38).
This study further examines the role of Vav1 in regulation of the NADPH oxidase. The results show that p67phox, a target of Rac-GTP in the oxidase complex, interacts with Vav1, which activates nucleotide exchange on Rac and enhances superoxide production. The studies presented here suggest that Vav1 plays an important role as a NADPH oxidase-targeted GEF and provide new molecular insights into the function of p67phox in the oxidase complex and the regulation of Vav1 exchange activity, which act in a positive feedback loop for amplification of superoxide production in response to chemoattractants.
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MATERIALS AND METHODS |
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Cell lines and cDNA constructs. PLB-985 myelomonocytic cells, COS-7 cells, and derivative COSphox cells expressing NAPDH oxidase subunits gp91phox, p22phox, p67phox, and p47phox were maintained as previously described (32, 33, 47). Transient transfection of COS-7 and COSphox cells was also as previously described (32). A total of 1 to 2 µg of plasmid DNA was used for a 100-mm plate of cells, and 0.5 µg was used for a 60-mm plate. Transfected cells were generally harvested 24 h after transfection. For experiments involving stimulation with epidermal growth factor (EGF; final concentration, 50 ng/ml), 24 h after transfection, cells were cultured in medium with 0.1% serum for an additional 18 to 22 h prior to study.
Expression constructs included wild-type and mutant murine Vav1 cDNAs (Y203F/L213Q, K404A/R422G, and C529S) in pCDNA3 (4, 5) and Myc-tagged human Rac1, Rac1(G30S), and Rac2 in pRK5 (32). Other point mutants [murine Vav1(W495) and p67phox(R102E)] were generated by site-directed mutagenesis (QuikChange II; Stratagene) and truncated derivatives of p67phox by using internal restriction sites. cDNAs for expression of p67phox, an N-terminal fragment of p67phox encoding amino acids 1 to 299, and C-terminal Myc-tagged p67phox were gifts from D. Lambeth (Emory University Medical School, Atlanta, GA) and were subcloned into pcDNA3. p67phox was also subcloned into pGEX-2T-1 (Amersham Biosciences, NJ), and a fragment of Vav encompassing the DH, PH, and ZF domains (amino acids 186 to 545) was subcloned into pQE30 (QIAGEN, Valenica, CA). Plasmids for expression of murine Vav1 with an N-terminal enhanced green fluorescent protein (EFGP) tag and human p67phox with a C-terminal enhanced yellow fluorescent protein (EYFP) tag were constructed using pcDNA3-Vav1 and pEGFP-C3 (BD Biosciences Clontech, San Diego, CA) or pcDNA3-hp67phox (D. Lambeth, Emory University Medical School, Atlanta, GA) and pEYFP-N1 (BD Biosciences Clontech), respectively. Details of all plasmid constructs are available upon request. The expression vector for a 6.4-kDa fragment from p21-activated kinase 3 containing the p21-binding domain (PBD) for Cdc42/Rac and tagged with GST was from R. Cerione (Cornell University, Ithaca, NY).
Preparation of human neutrophils and PLB-985 granulocytes. Blood was collected from healthy donors by venipuncture, and neutrophils were isolated as described previously (20). To differentiate PLB-985 into granulocyte-like cells, 5 x 105 cells were taken from a log-phase culture and plated at 0.5 x 105 cells/ml in the presence of 0.65% N, N-dimethyl-formamide or 1.25% dimethyl sulfoxide for 6 days. Neutrophils and PLB-985 granulocytes were washed with cold endotoxin-free phosphate-buffered saline (PBS), and treated with the protease inhibitor diisofluorophosphate on ice for 10 min prior to use in the assays below.
NADPH oxidase activity. Superoxide production was measured in a quantitative kinetic assay based on the superoxide dismutase-inhibitable reduction of cytochrome c (32). In human neutrophils and PLB-985 granulocytes stimulated with 10 µM fMLP, superoxide release was measured by an isoluminol chemiluminescence assay in 96-well plates using an Lmax microplate luminometer (Molecular Devices, Sunnyvale, CA) (26).
Rac activation assays. Affinity precipitation and pull-down assays for Rac were performed essentially as described previously (3, 26, 45). Diisofluorophosphate-treated human neutrophils or differentiated PLB-985 granulocytes were suspended in 0.5 ml of PBS and stimulated at 37°C with fMLP (final concentration, 10 µM) or dimethyl sulfoxide as a vehicle control. Cell lysates were stored, if needed, at –80°C or analyzed immediately.
In vitro interaction of Vav1 with p67phox. In vitro binding assays were performed as described previously (11). A GST-tagged full-length p67phox and His-tagged Vav1 fragment encompassing the DH, PH, and ZF domains [His-Vav1 (DH-PH-ZF)] were each expressed in bacteria and purified by glutathioine-Sepharose-4B (Amersham Bioscience) or Ni-nitrilotriacetic acid (NTA) resin (QIAGEN), respectively, according to the manufacturer's protocol. For in vitro GST pull-down assays, 50 pmol of immobilized GST-p67phox was incubated in binding buffer [50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 0.5% NP-40, 0.5 mM dithiothreitol, and 0.1% bovine serum albumin (BSA), 20 µg/ml chymostatin, 2 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride] with different amounts of His-Vav1 (DH-PH-ZF) for 1 h at room temperature with shaking. After incubation, the glutathione (GSH)-Sepharose beads were extensively washed with binding buffer and then the bound His-Vav1 (DH-PH-ZF) protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by immunoblotting with anti-His antibody.
For in vitro Ni-NTA pull-down assays, purified His-Vav (DH-PH-ZF) and GST-p67phox were incubated in binding buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 20 mM KCl, 1 mM dithiothreitol, 5 mM imidazole, and 500 µg/ml BSA) with nickel agarose beads for 1 h at room temperature with shaking. After incubation, the nickel agarose beads were extensively washed with buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, and 50 mM imidazole), and bound GST-p67phox was subjected to SDS-PAGE and detected by immunoblotting with anti-p67phox antibody.
Immunoprecipitation and immunoblotting. Immunoprecipitations were carried out as previously described (42). Briefly, cells were lysed in ice-cold immunoprecipitation lysis buffer [20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 1% Triton X-100, 0.1% BSA, 20 µg/ml chymostatin, 2 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 1 mM 4-(2-aminoethyl benzenesulfonyl fluoride] for 10 min with gentle mixing. Cleared cell lysates were incubated for 2 h at 4°C with the indicated antibodies and for an additional 2 h with protein A-Sepharose beads. Pellets were washed three times with ice-cold immunoprecipitation wash buffer and once with ice-cold PBS and then resuspended in 2x SDS sample buffer. Eluted immunoprecipitates and an aliquot of whole-cell lysate were separated by SDS-PAGE and analyzed by immunoblotting, with signals detected with enhanced chemiluminescence (ECL; Amersham Bioscience) (33).
Confocal microscopy. Twenty-four hours after transfection with indicated plasmids, COSphox cells were split and replated on glass slides and incubated overnight. Cells were then fixed with 4% paraformaldehyde (pH 7.6) and permeabilized with 0.1% Triton X-100 in PBS. After incubation for 30 min in 3% BSA, the cells were incubated for 1 h at room temperature with rabbit anti-Vav1 antibody and Myc MAb (for Rac1 or p67phox), followed by appropriate fluorescence-labeled secondary antibodies or rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR) for 30 min. Slides were washed three times with PBS and mounted with 50% glycerol and 1% DABCO in PBS or DAPI (4',6'-diamidino-2-phenylindole) II counterstain (32-804831; Vysis, Downers Grove, IL). Images were captured with a ZEISS LSM 510 fluorescence microscope at a magnification if x100. The extent of ruffling, as defined by the existence of irregular membrane folds around a part or all of the margin of the cells, was scored using the following scale: –, no ruffling present; +, ruffling confined to one or several noncontiguous areas of the cell surface; or ++, diffuse ruffling over at least 75% of the cell surface. These membrane folds were outlined by F-actin staining in the submembrane cytoskeleton, although ruffling was readily visible even in the absence of F-actin staining due to staining or fluorescence of Vav and/or p67phox. Ruffling was scored following transfection of cDNAs for EGFP-Vav1 alone or with those for Myc-tagged p67phox or Myc-tagged Rac1, and following transfection of EFYP-p67phox alone, with Myc-tagged Rac1, or with Myc-tagged Rac1 and Vav1. For each combination studied, experiments were performed three to five times, and at least 20 randomly chosen cells expressing the indicated protein(s) from transfected cDNAs, as visualized by fluorescent tag or indirect immunofluorescence staining, were scored in each experiment.
Guanine nucleotide exchange analysis.
Guanine nucleotide exchange assays were conducted as described previously (28). Briefly, fluorescence spectroscopic analysis of N-methylanthraniloyl (mant)-GTP incorporation into GST-Rac1 purified from bacteria was carried out at 25°C using a Perkin-Elmer Life Sciences LS 50B spectrometer. Flag-tagged Vav1 was immunoprecipitated from COS-7 cells 24 h following transfection with plasmids for expression of Flag-tagged Vav1 alone or in combination with p67phox and Rac. Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) and incubated with Anti-FLAG M2 Affinity Gel (Sigma, St. Louis, MO) for 2 h with gentle agitation. The resin-protein complexes were washed three times, and the proteins were eluted with FLAG peptide through gentle shaking for 30 min at 4°C. Exchange reaction assay mixtures contained 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 50 mg/ml BSA, 1% glycerol, 400 nM mant-GTP (Molecular Probes), and 2 µM Rac1 or Rac2. Assays were allowed to equilibrate with continuous stirring prior to addition of immunoprecipitation complexes as agonist. Relative fluorescence (excitation 
, 360 nm; emission , 440 nm) was monitored, and experiments were performed in duplicate.
Statistical analysis. Statistical analysis was performed with Instat Vision 2.0 and Excel (Microsoft, Redmond, WA) software. Data are expressed as mean ± standard deviation (SD), and a P value of <0.05 was considered significant. The two-tailed Student's t test or one-way analysis of variance (ANOVA) followed by a Tukey-Kramer multiple comparison test was used to determine the difference between groups.
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Recombinant purified p67phox and Vav1 interact directly in vitro. To examine whether p67phox and Vav1 interacted directly, we performed binding assays using purified recombinant proteins. Initial studies using cotransfected COS-7 cells in which portions of Vav1 were expressed in combination with full-length p67phox showed that a fragment of Vav1 comprised of the DH, PH, and the ZF domains was sufficient to coimmunoprecipitate p67phox (data not shown). We next examined interactions between a His-tagged DH-PH-ZF Vav1 fragment and GST-tagged full-length p67phox. His-Vav1 DH-PH-ZF was coincubated in vitro with GST-tagged p67phox, and the mixtures were precipitated with glutathione-Sepharose (Fig. 2A and B) or Ni-NTA agarose (Fig. 2C). The results show that the His-tagged Vav1 fragment and GST-p67phox coprecipitated regardless of whether the protein mixtures were affinity-purified using glutathione or Ni-NTA. Control experiments showed that the interaction was specific, because His-Vav1 was not precipitated if GST was used instead of GSTp67phox (Fig. 2B). These results indicate that Vav1 can bind directly to p67phox via the DH-PH-ZF domains of Vav1.
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To investigate whether p67phox could regulate an interaction between Vav1 and Rac, we coexpressed Myc-Rac1 and Flag-Vav1 with or without Myc-p67phox in COS-7 cells. The amount of exogenous (Myc-tagged) and endogenous (nontagged) Rac1 and Vav1 was examined in immunoprecipitates isolated with an anti-Vav1 antibody. The results show that approximately threefold more Rac1 coimmunoprecipitated when Vav1 and Rac1 were coexpressed with exogenous p67phox (Fig. 3A) and that Vav1 forms a complex with both exogenous and endogenous Rac under these conditions (Fig. 3D). Similar results in this assay were seen upon coexpression of Vav1, p67phox, and Rac2 (Fig. 3B). Vav1 and other Rho family GEFs bind the nucleotide-free transition state of their GTPase targets (35), whereas p67phox binds Rac-GTP (25). As an approach to determine the nucleotide status of Rac in the immunoprecipitation complex, we repeated the immunoprecipitations of Myc-Rac1 and Flag-Vav1, with or without Myc-p67phox, in the presence of Mg2+, which stabilizes guanine nucleotide binding to Rac. The results showed that in the presence of Mg2+, Rac1 was no longer recovered in the immunoprecipitation complex (Fig. 3C versus A), suggesting that it is nucleotide-free Rac that otherwise coimmunoprecipitates with Vav1 in the presence of p67phox. Given that the nucleotide-free transition state of Rac should exist only fleetingly in vivo, it is likely that binding of apo-Rac to Vav is an indication that the exchange factor is in an activated or open conformation when bound to p67phox, which enables it to complex more efficiently in vitro with the nucleotide-free Rac generated by Mg2+ chelation upon cell lysis. Since nucleotide-free Rac does not interact directly with p67phox (25), these results indicate that p67phox enhances interactions between Vav1 and the nucleotide-free form of Rac.
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Coexpression of p67phox with Vav1 leads to Vav-mediated Rac activation. To investigate whether interactions between p67phox and Vav1 can stimulate nucleotide exchange on Rac, we conducted GST-PBD pull-down assays to quantify Rac-GTP in COSphox cells transiently transfected with plasmids encoding Vav1, p67phox, Vav1 plus p67phox or Rac, or Vav1 plus Rac and p67phox. COSphox cells express gp91phox, p22phox, p67phox, and p47phox as stable transgenes (33) and were used in these experiments in order to examine NADPH oxidase activity in parallel (see below). Each set of transient expression combinations was tested using Rac1 or Rac2, with similar results (Fig. 5A and B). A small but detectable amount of endogenous Rac-GTP was recovered in cells transfected with empty vector or with a plasmid encoding p67phox, and the level of GTP-bound endogenous Rac increased slightly upon cotransfection of Vav1 without or with p67phox or Rac plasmid (Fig. 5A and B). Similarly, a small amount of exogenous Rac-GTP was recovered from cells transfected with a Rac1 or Rac2 expression plasmid, which increased modestly when either GTPase was cotransfected with a plasmid encoding Vav 1 (see Fig. S1 in the supplemental material). However, upon transient cotransfection with cDNAs encoding p67phox, Vav1, and Rac, a three- to fourfold increase in activation of both exogenous and endogenous Rac was observed (Fig. 5A and B). Similar results were seen using COS-7 cells instead of COSphox cells (Fig. 5C and 6A). In addition, the p67phox(R102E) mutant, which has impaired binding to Rac-GTP, did not enhance formation of activated Rac when coexpressed with Vav1 and Rac in COS-7 cells (Fig. 5C). Thus, interactions between p67phox, Vav1, and Rac appear to promote formation of Rac-GTP. In combination with the enhanced association of p67phox and Vav1 upon the interaction between Rac-GTP and p67phox (Fig. 4C to G), these interactions can create a positive feedback loop for upregulation of Vav1 activity.
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p67phox-enhanced Rac activation requires Vav1 GEF activity but is not associated with increased tyrosine phosphorylation of Vav1. The introduction of point mutations in Vav1 DH, PH, or ZF domains, previously shown to disrupt Vav1 GEF activity (30, 48), resulted in decreased formation of endogenous and exogenous Rac1-GTP in COS-7 cells in the presence of p67phox (Fig. 6A). Similar results were seen when Rac2 was coexpressed with p67phox and the Vav1 mutants (not shown). These results demonstrate that the GEF activity of Vav1 itself is required for p67phox-enhanced formation of Rac-GTP rather than being mediated by another GEF or by regulation of a GTPase activating protein.
We also examined whether p67phox-stimulated activation of Rac in the presence of Vav1 was associated with increased tyrosine phosphorylation of Vav1. Vav1 activation is regulated by tyrosine phosphorylation in its acidic domain, which relieves autoinhibition by the N terminus of Vav1 (38). We therefore evaluated tyrosine phosphorylation of Vav1 expressed in COSphox cells with or without coexpression of transfected p67phox and Rac. These levels of tyrosine phosphorylation of Vav1 were compared to the level elicited by EGF, previously established to activate Vav1 (8). Cotransfection of Vav with p67phox and Rac did not result in an increase in phosphotyrosine staining of immunoprecipitated Vav1 in cells not stimulated with EGF (Fig. 6B), although their cotransfection is associated with an increase in activated Rac (Fig. 5A and B and 6A). The addition of EGF stimulated tyrosine phosphorylation on Vav1 by approximately twofold, an increase that was not affected by the presence of transfected p67phox and Rac (Fig. 6B). These data indicate that the interactions between p67phox, Vav1, and Rac and concomitant Rac activation are not associated with increased tyrosine phosphorylation on Vav. However, we cannot rule out the possibility that the stimulatory effect of p67phox on Vav1 activity is totally independent of Vav1 acidic domain phosphorylation due to the inability to fully eliminate Vav1 phosphorylation in serum-starved cells (Fig. 6B).
NADPH oxidase is activated in COSphox cells upon cotransfection of p67phox, Vav1, and Rac. Since previous studies showed that constitutively activated Rac1-V12 is sufficient to activate NADPH oxidase in COSphox cells (32), we examined whether cotransfection of plasmids for expression of p67phox, Vav1, and Rac-WT (where WT is wild type) also led to activation of superoxide production in COSphox cells. Compared to COSphox cells transfected with either empty vector or vector containing the p67phox cDNA, expression of Vav1 or Vav1 plus additional p67phox or Rac1 consistently resulted in small but detectable increases in superoxide production by COSphox cells (Fig. 7A and B). Although not statistically significant, this stimulation of NADPH oxidase activity is consistent with the small increases in exogenous Rac1-GTP detected using pull-down assays under these conditions (Fig. 5A). In the absence of overexpressed Rac, the synergistic effect seen on superoxide production by COSphox cells following coexpression of Vav1 plus additional p67phox compared to Vav1 alone (Fig. 7B) is more evident compared to the effect on Rac-GTP levels (Fig. 5A and B) or ruffling (Fig. 7D) (see below). It may be that the effect of the feed-forward loop utilizing endogenous Rac and transfected p67phox and Vav is more readily detectable for superoxide production, since the Rac/p67phox/Vav complex is linked to the NADPH oxidase via p67phox. Cotransfection of COSphox cells with plasmids for increased expression of p67phox and Rac1, along with Vav1, produced a two- to threefold increase in NADPH oxidase activity in the absence of any additional agonist (Fig. 7A and B). Similar results were obtained using Rac2 instead of Rac1 (not shown). Thus, Rac activation upon cotransfection of p67phox, Vav1, and Rac stimulates NADPH oxidase activity, which correlates with the level of Rac-GTP (Fig. 5A).
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DISCUSSION |
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In human neutrophils and PLB-985 granulocytes, complex formation between p67phox, Vav1, and Rac2, but not Rac1, is stimulated by fMLP (Fig. 1B and C). These data establish that Vav1 is an oxidase target GEF and is linked to p67phox in neutrophils and provide further evidence that fMLP-initiated NADPH oxidase activation in neutrophils is mediated by a process that preferentially utilizes Rac2. The presence of Rac2 but not Rac1 in this complex is consistent with the marked defects in superoxide production in Rac2-deficient murine neutrophils, despite increased activation of Rac1 (22, 26, 34, 44), whereas NADPH oxidase activity is normal in the genetic absence of Rac1 (17). In transfection assays in COS-7 cells, the interaction of p67phox with Vav1 promotes nucleotide exchange on both Rac1 and Rac2. However, it is likely that in fMLP-stimulated neutrophils, preference for the Rac2 isoform is conferred by constraints in the activation or localization of Rac2 that promote efficient interaction between p67phox, Rac2, and Vav1.
As an essential regulator of the NADPH oxidase, p67phox has a well-characterized role in activating electron transport through flavocytochrome b, an activity that is dependent upon Rac-GTP (12, 18, 36). This study identifies two unexpected additional functions for p67phox, showing that it both interacts with a Rac GEF and enhances its nucleotide exchange activity. It also maps the Vav-interacting region of p67phox to the C-terminal region of p67phox, a site distinct from the Rac-GTP binding domain in the N-terminal portion of p67phox. However, the binding of Rac-GTP potentiates the interaction between p67phox and Vav1, as this interaction is inhibited by overexpression of Rho-GDI or by mutations of Rac and p67phox that specifically disrupt their association (Fig. 4C to E and 5B). Conversely, wild-type p67phox enhances the coimmunoprecipitation of Rac and Vav1 (Fig. 3A and B) and stimulates Vav-mediated activation of Rac (Fig. 5A and B). Thus, we propose that in fMLP-stimulated neutrophils, Rac-GTP binds to p67phox, which in turn promotes the interaction between p67phox and Vav1, followed by further activation of Rac and NADPH oxidase activity. In this model (Fig. 8), p67phox acts as a crucial link in a positive feedback loop for activation of Rac.
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The modulation of Rac exchange by p67phox, an effector of Rac-GTP, adds to the emerging evidence that Rho family GTPases can form signaling complexes with their upstream GEFs and immediate downstream effectors through direct or indirect scaffolding interactions (35). These complexes can promote efficient effector activation and contribute to signaling specificity. In most cases, the GEF acts as a scaffold to localize Rac effectors (6, 7, 9, 31). However, amplification of GEF activity by assembly of a GEF-GTPase-effector complex, such as described here, has been shown in only a few reports; WASP, an effector of Cdc42, recruits intersectin, a Cdc42-specific GEF, to a signaling complex and activates Cdc42 in vitro (21). Similarly, Gß binds to PAK1 via PIX
, a Cdc42 GEF, and activates Cdc42, which in turn activates PAK1 to regulate neutrophil polarization (27).
In summary, we report here that p67phox, the NADPH oxidase subunit that is a target of activated Rac, both associates with and enhances the activity of a guanine nucleotide exchange factor, Vav1, thus creating a positive feedback loop for local amplification of Rac activation and subsequently NADPH oxidase activity. The results provide new mechanistic insights into the regulation of superoxide production by neutrophils. Additional studies are required to elucidate the molecular details by which the binding of p67phox to Vav1 activates Vav1 exchange activity and to identify other GEFs that activate Rac in context of the oxidase and to examine their association with p67phox or other NADPH oxidase subunits.
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ACKNOWLEDGMENTS |
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We thank Xiaohong Li and Shari Upchurch for assistance with manuscript preparation.
Wenyu Ming performed the majority of experiments, wrote the first draft of the manuscript, prepared all figures, and participated in all additional aspects of manuscript preparation. Shijun Li performed experiments and reviewed the manuscript. Daniel D. Billadeau provided key reagents along with reviewing and editing the manuscript. Lawrence A. Quilliam provided input into experimental design, manuscript preparation, and editing of the manuscript. Mary C. Dinauer oversaw all aspects of this project and manuscript preparation.
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FOOTNOTES |
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Published ahead of print on 23 October 2006.
Supplemental material for this article is available at http://mcb.asm.org/.
Present address: Department of Urology, Stanford University Medical Center, 300 Pasteur Dr., Stanford, CA 94305.
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REFERENCES |
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4). (B) Kinetics of NADPH oxidase activity in COSphox cells transiently transfected for expression of indicated proteins or empty vector, as measured by reduction of cytochrome c. (C and D) Confocal microscopy analysis of COSphox cells transiently cotransfected for expression of p67phox-EYFP or Vav1-EGFP, along with additional cDNAs, as indicated. Cells were fixed and stained with rhodamine-labeled phalloidin or indicated antibodies. DAPI staining used to visualize the nucleus. Scale bar, 10 µm. (C) Vav1-EGFP was coexpressed with or without Myc-Rac1. Cells were stained with Myc MAb (for exogenous Rac1), followed by Alexa Fluor-633 goat anti-mouse antibody. p67phox-EYFP was coexpressed with Vav1 and/or Myc-tagged Rac1; cells were stained with rabbit anti-Vav1 antibody and Alexa Fluor-555 goat anti-rabbit antibody and with Myc MAb and Alexa Fluor-633 goat anti-mouse antibody. Merged images were generated from the red and green signals, and regions where these overlap appear orange or yellow. Images shown are representative fields from three to five independent experiments. Scale bar, 10 µm. (D) The bar graph shows the relative frequency of COSphox cells with various ruffling scores (–, +, and ++; see Materials and Methods) for cells expressing the indicated proteins following transient transfection. For each ruffling score, representative images of rhodamine-labeled phalloidin-stained cells are shown in the panel above the bar graph. Data are the means ± SD of three to five separate experiments; to determine the difference between groups for the frequency of cells with ++ ruffling, data were analyzed using ANOVA followed by a Tukey-Kramer multiple comparison test. **, P < 0.001 versus all other groups.