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• W2042341745 abstract "Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b558 and p67phox oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b558 that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b558in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation. Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b558 and p67phox oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b558 that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b558in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation. The process by which cells produce reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; cyt b558, flavocytochrome b558; WASP, Wiscott-Aldrich syndrome protein; PBD, p21 Rho GTPase-binding domain of p21-activated kinase; CRIB, Cdc42 and Rac-interactive binding domain; GST, glutathione S-transferase; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PIPES, 1,4-piperazinediethanesulfonic acid; fMLF, formylmethionylleucylphenylalanine. 1The abbreviations used are: ROS, reactive oxygen species; cyt b558, flavocytochrome b558; WASP, Wiscott-Aldrich syndrome protein; PBD, p21 Rho GTPase-binding domain of p21-activated kinase; CRIB, Cdc42 and Rac-interactive binding domain; GST, glutathione S-transferase; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PIPES, 1,4-piperazinediethanesulfonic acid; fMLF, formylmethionylleucylphenylalanine. has gained much interest because of the diverse functions attributed to this class of molecules. In nonphagocytic cells, oxidants affect a variety of cellular processes, including transcription factor activation, proliferation, transformation, and apoptosis. In neutrophils and other phagocytes, oxidants play an important role in cellular innate immune responses. A critical component of the bactericidal activity of phagocytes is the NADPH oxidase, also referred to as “phox” (phagocytic oxidase) (1Babior B.M. Lambeth J.D. Nauseef W. Arch. Biochem. Biophys. 2002; 15: 342-344Crossref Scopus (702) Google Scholar, 2Bokoch G.M. Knaus U.G. Trends Biochem. Sci. 2003; 28: 502-508Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 3Vignais P.V. Cell Mol. Life Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (620) Google Scholar), which generates superoxide anion and, subsequently, a number of other ROS. The phagocyte NADPH oxidase is a multiprotein system whose activity is regulated by the RhoGTPase Rac2 in human cells (4Knaus U.G. Heyworth P.G. Evans T. Curnutte J.T. Bokoch G.M. Science. 1991; 254: 1512-1515Crossref PubMed Scopus (540) Google Scholar, 5Bokoch G.M. Diebold B.A. Blood. 2001; 100: 2692-2696Crossref Scopus (279) Google Scholar, 6Kim C. Dinauer M.C. J. Immunol. 2001; 166: 1223-1232Crossref PubMed Scopus (170) Google Scholar). Electrons are transferred from NADPH to molecular oxygen through the action of an integral membrane flavocytochrome b558 (cyt b558), composed of subunits gp91phox and p22phox. In addition to Rac2, the activity of the NADPH oxidase is regulated by the cytosolic components p47phox, p67phox, and p40phox, which exist as a heterotrimeric complex in the cytosol of unstimulated neutrophils (7Lapouge K. Smith S.J. Groemping Y. Rittinger K. J. Biol. Chem. 2002; 277: 10121-10128Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In a separate cytosolic complex are Rac2 (or Rac1 in certain species) and GDP dissociation inhibitor (8Chuang T.H. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1993; 268: 26206-26211Abstract Full Text PDF PubMed Google Scholar). When neutrophils are activated, a series of interrelated regulatory events take place. p47phox becomes phosphorylated and mediates translocation of the p47phox/p67phox/p40phox complex to the plasma membrane (3Vignais P.V. Cell Mol. Life Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (620) Google Scholar). Rac2 and GDP dissociation inhibitor dissociate, followed by the guanine nucleotide exchange factor-mediated exchange of GTP for GDP and membrane localization of Rac2 (9Bokoch G.M. Bohl B.P. Chuang T.H. J. Biol. Chem. 1994; 269: 31674-31679Abstract Full Text PDF PubMed Google Scholar). At the membrane, p67phox and Rac2 interact with cyt b558 to form the functional NADPH oxidase complex. We have recently shown that Rac2 regulates NADPH oxidase activity via a two-step mechanism involving an initial functional interaction with cyt b558 to catalyze electron transfer to bound FAD, followed by a subsequent interaction with p67phox that results in electron transfer to the cyt b558-bound heme (10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar). The formation of ROS in nonphagocytic cells also involves Nox (NADPH oxidase) enzymes (11Lambeth J.D. Cheng G. Arnold R.S. Edens W.A. Trends Biochem. Sci. 2000; 25: 459-461Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 12Geiszt M. Witta J. Baffi J. Lekstrom K. Leto T.L. FASEB J. 2003; 17: 1502-1504Crossref PubMed Scopus (407) Google Scholar, 13Cheng G. Cao Z. Xu X. van Meir E.G. Lambeth J.D. Gene (Amst.). 2001; 269: 131-140Crossref PubMed Scopus (691) Google Scholar). Nox enzymes are a group of homologues of gp91, the large subunit of the cyt b558 in the phagocyte NADPH oxidase (also referred to as Nox2). Recent studies on the Nox proteins indicate that the regulation of ROS production in nonphagocytic cells may parallel in many ways that of the phagocyte NADPH oxidase system, including regulation by p22phox and by p47phox and p67phox or their homologues (14Geiszt M. Lekstrom K. Witta J. Leto T.L. J. Biol. Chem. 2003; 30: 20006-20012Abstract Full Text Full Text PDF Scopus (245) Google Scholar, 15Banfi B. Clark R.A. Steger K. Krause K.H. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Another similarity of ROS production by phagocytic and nonphagocytic cells is regulation by the Rac GTPase. The involvement of Rac2 in the NADPH oxidase of phagocytic cells has been confirmed by the generation of Rac2 and Bcr (a Rac GTPase-activating protein) null-mice (6Kim C. Dinauer M.C. J. Immunol. 2001; 166: 1223-1232Crossref PubMed Scopus (170) Google Scholar, 16Roberts A.W. Kim C. Zhen L. Lowe J.B. Kapur R. Petryniak B. Spaetti A. Pollock J.D. Borneo J.B. Bradford G.B. Atkinson S.J. Dinauer M.C. Williams D.A. Immunity. 1999; 10: 183-196Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 17Voncken J.W. van Schaick H. Kaartinen V. Deemer K. Coates T.B. Pattengale P. Dorseuil O. Bokoch G.M. Groffen J. Heisterkamp N. Cell. 1995; 80: 719-728Abstract Full Text PDF PubMed Scopus (155) Google Scholar) and through the use of Rac antisense oligonucleotides (18Dorseuil O. Vazquez A. Lang P. Bertoglio J. Gacon G. Leca G. J. Biol. Chem. 1992; 267: 20540-20542Abstract Full Text PDF PubMed Google Scholar). There is also substantial evidence that Rac1 is involved in controlling ROS production in nonphagocytic cells, although a direct link to Nox has not been reported (2Bokoch G.M. Knaus U.G. Trends Biochem. Sci. 2003; 28: 502-508Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). For example, transient expression of constitutively active Rac1 in NIH3T3 fibroblasts increased (O2·¯) production in Ras-transformed cells (19Sundaresan M. Yu Z.X. Ferrans V.J. Sulciner D.J. Gutkind J.S. Irani K. Goldschmidt-Clermont P.J. Finkel T. Biochem. J. 1996; 318: 379-382Crossref PubMed Scopus (437) Google Scholar). Rac1 and, specifically, the insert domain of Rac1 (amino acids 124–135), was necessary for (O2·¯) production and mitogenesis in fibroblasts (20Joneson T. Bar-Sagi D. J. Biol. Chem. 1998; 273: 17991-17994Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Rac1 expression in NIH3T3 cells also led to increased (O2·¯) production in response to various growth factors and hormones (e.g. platelet-derived growth factor and angiotensin II) (21Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 649-652Crossref PubMed Scopus (1418) Google Scholar). A direct regulation of Nox function by Rac GTPase has been proposed (10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar). Cross-talk between members of the Rho GTPase family (Rho, Rac, and Cdc42) plays an important role in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. Many such regulatory interactions between Cdc42, Rac, and Rho have been described in the context of cytoskeletal remodeling during motility, presumably resulting in the coordinated functioning of the cellular cytoskeletal elements to promote smooth, continuous motion (22Matozaki T. Nakanishi H. Takai Y. Cell Signal. 2000; 12: 515-524Crossref PubMed Scopus (116) Google Scholar, 23Burridge K. Science. 1999; 283: 2028-2029Crossref PubMed Scopus (52) Google Scholar, 24Ridley A. J. Cell Sci. 2001; 114: 2713-2722Crossref PubMed Google Scholar). Cross-talk in Rho GTPase signaling occurs through a number of mechanisms. Individual Rho GTPase family members can modulate the activity of guanine nucleotide exchange factors and/or GTPase-activating proteins that control the activity of other Rho GTPases. In addition, the ability of multiple RhoGTPase family members to interact with common effectors also allows for cross-talk. In motile fibroblasts, Rac1 prevents the phosphorylation of myosin light chain through its effector p21-activated kinase, which phosphorylates and decreases the activity of myosin light chain kinase, thus decreasing the contractile force exerted by Rho action (25Sanders L.C. Matsumura F. Bokoch G.M. de Lanerolle P. Science. 1999; 283: 2083-2085Crossref PubMed Scopus (496) Google Scholar). This process serves to balance the protrusive forces generated by Rac and Cdc42, and the contractile forces initiated by Rho, a critical requirement for directional cell movement. To date, such complex interplay between Rho GTPases has not been described for Rac-mediated formation of ROS. In this paper, we identify cross-talk between Rac2 (and Rac1) and Cdc42 in regulation of ROS production by the phagocyte NADPH oxidase. We show that this inhibitory interaction results from a competition between the active Rac2 (or Rac1) oxidase-regulatory component and the oxidase-inactive Cdc42 for binding to flavocytochrome b558. This antagonistic cross-talk between these Rho GTPase family members provides a novel mechanism by which oxidant production may be regulated in neutrophils and in other nonphagocytic cells. Proteins—Recombinant p47phox and nonprenylated Rho GTPases were expressed and purified as a glutathione S-transferase (GST) fusion protein in Escherichia coli (10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar). p67phox and prenylated Rho GTPases were expressed and purified as GST fusion proteins in baculovirus-infected Sf9 cells as previously described (10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar, 26Uhlinger D.J. Inge K.L. Kreck M.L. Tyagi S.R. Neckelmann N. Lambeth J.D. Biochem. Biophys. Res. Commun. 1992; 186: 509-516Crossref PubMed Scopus (33) Google Scholar). GST fusion proteins were cleaved with thrombin for use in enzyme assays. Rho GTPases were quantified and preloaded with guanine nucleotides as reported (27Heyworth P.G. Knaus U.G. Xu X. Uhlinger D.J. Conroy Bokoch G.M. Curnutte J.T. Mol. Biol. Cell. 1993; 4: 261-269Crossref PubMed Scopus (111) Google Scholar). Cytochrome b558 was purified from human neutrophil membranes, relipidated, and reflavinated as reported to generate flavocytochrome b558, abbreviated as cyt b558 throughout (10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar). Mutagenesis—The deletion mutant, Cdc42 Δ124–135 P136A, was prepared using standard overlapping PCR and site-directed mutagenesis. Proline 136 was mutated to Ala, as was previously done for Rac2 Δ124–135 (10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar, 28Freeman J.L. Abo A. Lambeth J.D. J. Biol. Chem. 1996; 271: 19794-19801Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) to avoid possible structural perturbations. Cdc42 K27A,G30S and Rac2 A27K,G30S were prepared using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA). In Vitro Binding Assay Using Purified Proteins—30–50 pmol of GST-Rac1, GST-Rac2, GST-Cdc42, or GST-RhoA (preloaded with either GTPγS or GDP) or an equivalent amount of GST were incubated with 5 pmol of cyt b558 and 30 μl of glutathione-Sepharose beads in 0.5 ml of Relax buffer (10 mm PIPES, pH 7.3, 100 mm KCl, 3 mm NaCl, 3.5 mm MgCl2) for 18 h with constant inversion at 4 °C. The beads were collected by brief centrifugation and washed with Relax buffer before the addition of SDS-PAGE sample buffer. The precipitated proteins were subjected to SDS-PAGE and Western blot analysis using a monoclonal anti gp91phox antibody that was kindly supplied by Dr. Mark Quinn (Montana State University). Cell-free Assay for Superoxide Production—Superoxide production was determined as the rate of superoxide dismutase inhibitable cytochrome c reduction (29Bromberg Y. Pick E. J. Biol. Chem. 1985; 260: 13539-13545Abstract Full Text PDF PubMed Google Scholar), as in Ref. 10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar. Cell-free assays typically contained cyt b558 (2 nm), p47phox (140 nm), p67phox (50 nm), prenylated Rac1 or Rac2-GTPγS (15 nm), FAD (200 nm), cytochrome c (100 μm), and SDS (60 μm). Nonprenylated Rac1-GTPγS was used at a concentration of 130 nm when substituted for prenylated Rac1-GTPγS. The mixture (145 μl) was incubated for 5 min at 25 °C before the addition of NADPH (0.2 mm). An extinction coefficient at 550 nm of 21 mm–1 cm–1 was used to calculate the rate of cytochrome c reduction. Whole Cell Assay for Superoxide Production—Neutrophils were isolated from human venous blood donated by healthy donors as previously described (27Heyworth P.G. Knaus U.G. Xu X. Uhlinger D.J. Conroy Bokoch G.M. Curnutte J.T. Mol. Biol. Cell. 1993; 4: 261-269Crossref PubMed Scopus (111) Google Scholar). 4 × 105 neutrophils were incubated with 100 μm cytochrome c in 200 μl of KRGH buffer (25 mm Hepes, pH 7.4, 1.2 mm KH2PO4, 118 mm NaCl, 4.7 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 5.5 mm dextrose) for 5 min at 37 °C. Superoxide production was induced with fMetLeuPhe (10–7m). For Cos-phox cells (a Cos-7 cell line that stably expresses the phagocytic NADPH oxidase components), 2.5 × 105 cells were incubated with 100 μm cytochrome c in 250 μl of PBSG buffer (phosphate-buffered saline supplemented with 0.9 mm CaCl2, 0.5 mm MgCl2, and 7.5 mm glucose). Cos-phox Cell Culture and Transfection—Cos-7 cells stably transfected with cDNA for p47phox, p67phox, gp91phox, and p22phox were maintained as previously described (30Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar). LipofectAMINE Plus (Invitrogen) was used to transfect 6 μg of total cDNA (pRK5m-RhoGTPases)/10-cm plate. Transfection efficiency (50%) was determined by transfecting cDNA containing green fluorescent protein and fluorescence-activated cell sorter analysis. The cells were harvested 21–24 h after transfection, and Western blot analysis was used to verify protein expression as described previously (30Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar) using Myc monoclonal antibody 9E10, Rac1 monoclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY), and a Cdc42 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Delivery of Proteins into Neutrophils Using Bioporter—pET23-wild type WASP-CRIB (amino acids 201–321) and non-Cdc42-binding mutant-WASP CRIB (F271C,H246D,H249D) were expressed as C-terminal His6-tagged fusion proteins. These constructs were kindly supplied by Dr. Klaus Hahn (The Scripps Research Institute). The proteins were expressed and purified from E. coli and quantified by BCA protein assay (Pierce). The proteins were delivered into neutrophils as previously described (31Gardiner E.M. Pestonjamasp K.N. Bohl B.P. Chamberlain C. Hahn K.M. Bokoch G.M. Curr. Biol. 2002; 12: 2029-2034Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Briefly, Bioporter reagent (Gene Therapy Systems, La Jolla, CA) was reconstituted with chloroform according to the manufacturer's instructions, and 2–3 μl was aliquoted into Eppendorf tubes and evaporated overnight. 100 μl of phosphate-buffered saline containing 30 μg of WASP-CBIB protein was used to rehydrate the Bioporter reagent. Neutrophils (3.0 × 106) in 900 μl of KRGH buffer were added, and the mixture was inverted for 1 h at 25 °C. Triplicate tubes were prepared for each protein, and bovine serum albumin was used in place of WASP CRIB as a reference for 100% activity. An aliquot (200 μl) of cells was removed after the incubation period, and cytochrome c (100 μm) and fMLF (10–7m) were added to measure the rate of superoxide production as described above. Transduction of Tat Fusion Proteins into Neutrophils—The pHis-Tat-HA-WASP-CRIB and pHis-Tat-HA-mutant WASP-CRIB (H246A, H249A,V250A,G251A,D253A) vectors were a kind gift from Dr. Jacques Bertoglio (INSERM, France). The proteins were expressed in E. coli and purified using Ni2+-agarose chromatography as described (32Haddad E. Zugaza J.L. Louache F. Debili N. Crouin C. Schwarz K. Fischer A. Vainchenker W. Bertoglio J. Blood. 2001; 97: 33-37Crossref PubMed Scopus (164) Google Scholar, 33Han H. Fuortes M. Nathan C. J. Exp. Med. 2003; 197: 63-75Crossref PubMed Scopus (48) Google Scholar) under nondenaturing conditions. The proteins were used immediately after removing imidazole by dialysis. Neutrophils (2 × 106) were incubated with 100 μg of Tat-WASP-CRIB or Tat-mutWASP-CRIB protein in 1 ml of KRGH buffer at 37 °C for 30 min. After 30 min, 0.2 ml of the cells were assayed for superoxide production. Cdc42 and Rac2 Interact with Cyt b558 from Human Neutrophils—We previously reported that recombinant Rac2 interacted with cyt b558in vitro as determined using changes in the fluorescence of Rac-bound mant-GppNHp (10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar). In support of this result, we observed that cyt b558 binds to prenylated GST-Rac2 in pull-down assays (Fig. 1A). The interaction of cyt b558 with Rac2 was only slightly enhanced when Rac2 was loaded with GTP versus GDP. Binding did not occur to GST alone or to GST-RhoA. Prenylated GST-Rac1 behaved similarly when substituted for Rac2 (data not shown). Interestingly, however, we observed that cyt b558 bound to prenylated GST-Cdc42 in a manner that was also insensitive to the nucleotide state of Cdc42. This was unexpected, because Cdc42 has been shown to be unable to activate the NADPH oxidase (27Heyworth P.G. Knaus U.G. Xu X. Uhlinger D.J. Conroy Bokoch G.M. Curnutte J.T. Mol. Biol. Cell. 1993; 4: 261-269Crossref PubMed Scopus (111) Google Scholar, 34Kwong C.H. Malech H.L. Rotrosen D. Leto T.L. Biochemistry. 1993; 32: 5711-5717Crossref PubMed Scopus (93) Google Scholar, 35Kwong C.H. Adams A.G. Leto T.L. J. Biol. Chem. 1995; 270: 19868-19872Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Both GST-Rac2 and GST-Cdc42 pulled down the intact cyt b558 heterodimer (Fig. 1B). Cyt b558 could be detected in these assays using GST-Rac2:cyt b558 ratios in the range of 2–10. In the cell-free assays a Rac:cyt b558 ratio of 5 was required for maximal activity using prenylated Rac2 or Rac1 (Fig. 1C). Mutation of the Switch 1 Region of Cdc42 Enables Activation of the NADPH Oxidase—Kwong et al. (35Kwong C.H. Adams A.G. Leto T.L. J. Biol. Chem. 1995; 270: 19868-19872Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) reported that by mutating Cdc42 at two residues (K27A and S30G) in the Switch I domain, Cdc42 could now mimic Rac1 in activating the NADPH oxidase in a cell-free system. The amino acids at positions 27 and 30 have been shown to be critical for the interaction between Rac and p67phox in the derived crystal structure (36Lapouge K. Smith S.J. Walker P.A. Gamblin S.J. Smerdon S.J. Rittinger K. Mol. Cell. 2000; 6: 899-907Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), and these residues are conserved in Rac1 and Rac2 but not Cdc42 or RhoA. We verified that the K27A,S30G Cdc42 mutant was as effective as Rac2 in supporting NADPH oxidase activity in our system (Fig. 2). This result implies that if any other domain(s) of Rac are important for NADPH oxidase function, then those domains must be functionally conserved in Cdc42, because mutating only the two Switch I residues enables Cdc42 to be fully active in the oxidase system. One such oxidase regulatory region is the insert domain of Rac. As noted previously, this region is involved in regulation of NADPH oxidase activity (10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar, 28Freeman J.L. Abo A. Lambeth J.D. J. Biol. Chem. 1996; 271: 19794-19801Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 37Nisimoto Y. Freeman J.L. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and we have shown that it mediates binding to cyt b558 (10Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (235) Google Scholar). Fig. 3 demonstrates that the insert region (amino acids 124–135) of prenylated Rac2 and Cdc42 was necessary for their interaction with cyt b558. We therefore hypothesized that Cdc42 might compete with Rac for binding to cyt b558 through this region and thus inhibit superoxide production through the formation of a nonfunctional NADPH oxidase complex.Fig. 3The insert domain of Rac2 and Cdc42 is required for their interaction with cytochrome b558. Prenylated GST-Rho GTPases (50 pmol) were preloaded with either GTPγS or GDP and incubated with cyt b558 (5 pmol) purified from human neutrophil membranes. The GST fusion proteins were pulled down with glutathione-Sepharose, and the precipitated proteins were subjected to SDS-PAGE and Western blot analysis using an anti-gp91phox antibody. Column a, GST-Rac2 WT-GTPγS; column b, GST-Rac2 WT-GDP; column c, GST-Rac2 Δins-GTPγS; column d, GST-Rac2 Δins-GDP; column e, GST-Cdc42 WT-GTPγS; column f, GST-Cdc42 WT-GDP; column g, GST-Cdc42 Δins-GTPγS; column h, GST-Cdc42 Δins-GDP.View Large Image Figure ViewerDownload (PPT) Cdc42 Inhibits Activation of the NADPH Oxidase in a Cell-free System—We tested our hypothesis initially using the well established, semi-recombinant NADPH oxidase cell-free system. Purified cyt b558 from neutrophil membranes, reconstituted with FAD, was incubated with recombinant p47phox, p67phox, Rac2-GTP, and varying concentrations of Cdc42 in the presence of SDS as activator. As shown in Fig. 4A (curve a), using prenylated Rho GTPases, inclusion of Cdc42-GTP in this system inhibited the rate of superoxide production. Cdc42 in the GDP-bound form was also inhibitory but to a slightly lesser degree (curve b), consistent with somewhat decreased binding to cyt b558. When a 5-fold higher concentration of Rac was used in the assay, the degree of inhibition by Cdc42-GTP was decreased (curve c). In contrast to Cdc42, RhoA-GTP over the same concentration range was not inhibitory (curve d). To test the role of the Cdc42 insert domain in this inhibitory effect, we made a deletion mutant of prenylated Cdc42 (Cdc42 Δins) that lacks the insert domain (amino acids 124–135) and used this in place of wild type Cdc42 in our assays. Cdc42 Δins in the GTP bound form was not inhibitory at any of the concentrations tested (Fig. 4B), indicating that the cyt b558-binding insert domain of Cdc42 is required for competition with Rac2 for cyt b558 binding. Prenylated Cdc42WT was also inhibitory when either prenylated Rac1-GTPγS (Fig. 4C) or nonprenylated Rac1-GTPγS (Fig. 4D) was substituted for prenylated Rac2, indicating that Cdc42 also competes with Rac1. The insert domain of prenylated Cdc42 was also required for its ability to inhibit Rac1-dependent superoxide production by the cell-free system (Fig. 4, C and D) Nonprenylated Cdc42 was not inhibitory in any of the cell-free assays. This may reflect the need for the strong association of Cdc42 with the plasma membrane afforded by the additional prenyl group to effectively compete with the higher affinity Rac binding to cyt b558. Rac and Cdc42 Antagonize Each Other at the Level of Cyt b558 Binding—To determine whether the competition between Rac and Cdc42 in the cell-free system occurs because of competition for cyt b558 binding, we included increasing amounts of untagged, prenylated Cdc42 in a GST-Rac2 pull-down of cyt b558. As shown in Fig. 5, the amount of cyt b558 pulled down by GST-Rac2 decreased as the amount of Cdc42 in the assay increased. A similar result was observed when untagged, prenylated Rac2 was included in the pull-down of cyt b558 by GST-Cdc42 and when Rac1 (prenylated or nonprenylated) was substituted for Rac2. Cdc42 Inhibits Rac-induced Oxidant Production in Cos7-phox Cells—To assess whether Cdc42 was capable of inhibiting NADPH oxidase activity in vivo, we used a genetically engineered Cos7 cell line (Cos-phox cells) (30Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar) that stably express the required components of the NADPH oxidase (gp91phox, p22phox, p47phox, and p67phox). This cell line expresses endogenous Rac1 and generates little or no superoxide under nonstimulated conditions (Fig. 6, bar A). However, as reported (30Price M.O. McPhail L.C. Lambeth J.D. Han C.H. Knaus U.G. Dinauer M.C. Blood. 2002; 99: 2653-2661Crossref PubMed Scopus (101) Google Scholar), transfection of constitutively active Rac1Q61L into these cells resulted in superoxide production without any additional stimulation (bar B). Co-transfection of Rac1Q61L plus Cdc42Q61L into these cells resulted in ∼50% inhibition of the superoxide formation observed with Rac1Q61L alone (bar C). This level of inhibition is consistent with the efficiency of transfection of both of these Rho GTPases in the Cos-phox cells, which we determined to be ∼50%. The hypothesis that the insert domain of Cdc42 is involved in the inhibition of oxidase activity was further supported by the observation that transfection of Cdc42Q61LΔins along with Rac1Q61 L resulted in only ∼10% inhib" @default.
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• W2042341745 date "2004-07-01" @default.
• W2042341745 modified "2023-10-01" @default.
• W2042341745 title "Antagonistic Cross-talk between Rac and Cdc42 GTPases Regulates Generation of Reactive Oxygen Species" @default.
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• W2042341745 doi "https://doi.org/10.1074/jbc.m313891200" @default.
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