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• W1985033865 abstract "Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91phox, is activated by Rac1. When Nox1 is co-expressed along with its regulatory subunits NOXO1 and NOXA1, significant ROS generation is seen. Herein, co-expression of constitutively active Rac1(G12V), but not wild-type Rac1, resulted in marked further stimulation of activity. Decreased Rac1 expression using small interfering RNA reduced Nox1-dependent ROS. CDC42(G12V) failed to increase activity, and small interfering RNA directed against CDC42 failed to decrease activity, pointing to specificity for Rac. TPR domain mutants of NOXA1 that interfere with Rac1 binding were ineffective in supporting Nox1-dependent ROS generation. Immunoprecipitation experiments demonstrated a complex containing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) could not substitute for Rac1(G12V) in such a complex. Nox1 formed a complex with Rac1(G12V) that was independent of NOXA1 and NOXO1, consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V) interaction with NOXA1 was enhanced by Nox1 and NOXO1, suggesting cooperative binding. A model is presented comparing activation by regulatory subunits of Nox1 versus gp91phox (Nox2) in which Rac1 activation provides a major trigger that acutely activates Nox1-dependent ROS generation. Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91phox, is activated by Rac1. When Nox1 is co-expressed along with its regulatory subunits NOXO1 and NOXA1, significant ROS generation is seen. Herein, co-expression of constitutively active Rac1(G12V), but not wild-type Rac1, resulted in marked further stimulation of activity. Decreased Rac1 expression using small interfering RNA reduced Nox1-dependent ROS. CDC42(G12V) failed to increase activity, and small interfering RNA directed against CDC42 failed to decrease activity, pointing to specificity for Rac. TPR domain mutants of NOXA1 that interfere with Rac1 binding were ineffective in supporting Nox1-dependent ROS generation. Immunoprecipitation experiments demonstrated a complex containing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) could not substitute for Rac1(G12V) in such a complex. Nox1 formed a complex with Rac1(G12V) that was independent of NOXA1 and NOXO1, consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V) interaction with NOXA1 was enhanced by Nox1 and NOXO1, suggesting cooperative binding. A model is presented comparing activation by regulatory subunits of Nox1 versus gp91phox (Nox2) in which Rac1 activation provides a major trigger that acutely activates Nox1-dependent ROS generation. Rho family GTPases are implicated in innate immunity, regulation of cell shape and migration, and mitogenic regulation (1Jaffe A.B. Hall A. Annu. Rev. Cell Dev. Biol. 2005; 21: 247-269Crossref PubMed Scopus (2377) Google Scholar, 2Niedergang F. Chavrier P. Curr. Top. Microbiol. Immunol. 2005; 291: 43-60PubMed Google Scholar, 3Zohn I.M. Campbell S.L. Khosravi-Far R. Rossman K.L. Der C.J. Oncogene. 1998; 17: 1415-1438Crossref PubMed Scopus (320) Google Scholar). Rac1 and Rac2 participate in the regulation of ROS generation in several cell types (4Archer H. Bar-Sagi D. Methods Mol. Biol. 2002; 189: 67-73PubMed Google Scholar, 5Joneson T. Bar-Sagi D. J. Biol. Chem. 1998; 273: 17991-17994Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), especially in the neutrophil, where Rac2 provides one of several “triggers” for activation of the phagocyte respiratory burst oxidase, a superoxide-generating NADPH-oxidase that participates in host defense against invading microbes. In addition to regulation of ROS 3The abbreviations used are: ROS, reactive oxygen species; JNK, c-Jun N-terminal kinase; HA, hemagglutinin; CAT, chloramphenicol acetyltransferase; RIPA, radioimmune precipitation; GTPγS, guanosine 5′-3-O-(thio)triphosphate; siRNA, small interfering RNA; PMA, phorbol 12-myristate 13-acetate; EGFP, enhanced green fluorescent protein; WT, wild type. production in phagocytes, there is growing evidence for Rac1 regulation of ROS generation in other cell types. For example, Ras-transformed fibroblasts overproduce superoxide, and ROS generation is inhibited by a dominant negative mutant form of Rac1 (6Irani K. Xia Y. Zweier J. Sollott S. Der C. Rearon E. Sundaresan M. Finkel T. Goldschmidt-Clermont P. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1441) Google Scholar); also, stimuli that increase Rac1-GTP in gastric epithelial cells increase ROS production (7Kawahara T. Kohjima M. Kuwano Y. Mino H. Teshima-Kondo S. Takeya R. Tsunawaki S. Wada A. Sumimoto H. Rokutan K. Am. J. Physiol. 2005; 288: C450-C457Crossref PubMed Scopus (120) Google Scholar). Mutationally activated Rac1 induces ROS formation (5Joneson T. Bar-Sagi D. J. Biol. Chem. 1998; 273: 17991-17994Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 7Kawahara T. Kohjima M. Kuwano Y. Mino H. Teshima-Kondo S. Takeya R. Tsunawaki S. Wada A. Sumimoto H. Rokutan K. Am. J. Physiol. 2005; 288: C450-C457Crossref PubMed Scopus (120) Google Scholar, 8Sundaresan 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 (441) Google Scholar), and second site mutations showed that Rac1 activation of ROS production correlates with mitogenic stimulation, but not with actin polymerization or JNK activation by Rac1 (5Joneson T. Bar-Sagi D. J. Biol. Chem. 1998; 273: 17991-17994Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Rac1-regulated ROS production is also linked to neuronal differentiation (9Suzukawa K. Miura K. Mitsushita J. Resau J. Hirose K. Crystal R. Kamata T. J. Biol. Chem. 2000; 275: 13175-13178Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), to growth and induction of cyclin D1 in airway smooth muscle (9Suzukawa K. Miura K. Mitsushita J. Resau J. Hirose K. Crystal R. Kamata T. J. Biol. Chem. 2000; 275: 13175-13178Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), to shear stress-induced protein phosphorylations in vascular endothelium (10Yeh L.H. Park Y.J. Hansalia R.J. Ahmed I.S. Deshpande S.S. Goldschmidt-Clermont P.J. Irani K. Alevriadou B.R. Am. J. Physiol. 1999; 276: C838-C847Crossref PubMed Google Scholar), and to platelet-derived growth factor-induced proliferation in vascular smooth muscle (11Kong G. Lee S. Kim K.S. J. Korean Med. Sci. 2001; 16: 712-718Crossref PubMed Scopus (23) Google Scholar). Despite a clear association between Rac1 and ROS production in a variety of cells, the ROS-generating target(s) of Rac1 have not been convincingly elucidated, and the source has been speculated to be either mitochondria or an unknown Nox enzyme. This study presents evidence in a model cell system that Nox1 can mediate Rac1-induced ROS generation in nonphagocytic cells. The phagocyte NADPH-oxidase provides a paradigm for regulation of ROS generation by Rac (12Dinauer M.C. Curr. Opin. Hematol. 2003; 10: 8-15Crossref PubMed Scopus (111) Google Scholar). The catalytic subunit gp91phox contains one FAD, two hemes, and an NADPH binding site (13Vignais P.V. Cell Mol. Life Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (634) Google Scholar) and is associated in the membrane with p22phox, which provides both stabilization and a docking site for regulatory subunits. Together, gp91phox and p22phox constitute flavocytochrome b558, which functions catalytically in neutrophils and monocytes in conjunction with the regulatory subunits p47phox, p67phox, p40phox and the small GTPase Rac2. In vitro, both Rac1 and Rac2 support NADPH-oxidase activity (14Abo A. Pick E. Hall A. Totty N. Teahan C.G. Segal A.W. Nature. 1991; 353: 668-670Crossref PubMed Scopus (767) Google Scholar, 15Knaus U.G. Heyworth P.G. Evans T. Curnutte J.T. Bokoch G.M. Science. 1991; 254: 1512-1515Crossref PubMed Scopus (545) Google Scholar), but in phagocytes from Rac1 or Rac2 knock-out animals, the system shows greater specificity for Rac2 (16Glogauer M. Marchal C.C. Zhu F. Worku A. Clausen B.E. Foerster I. Marks P. Downey G.P. Dinauer M. Kwiatkowski D.J. J. Immunol. 2003; 170: 5652-5657Crossref PubMed Scopus (255) Google Scholar, 17Kim C. Dinauer M.C. J. Immunol. 2001; 166: 1223-1232Crossref PubMed Scopus (173) Google Scholar), which is selectively expressed in phagocytic cells. In naive neutrophils not exposed to bacteria or inflammatory mediators, flavocytochrome b558 is catalytically inactive, and its regulatory subunits p47phox and p67phox are located in the cytosol. Similarly, Rac in resting cells is complexed to GDP and is associated in the cytosol with the inhibitor protein RhoGDI (18DerMardirossian C. Bokoch G.M. Trends Cell Biol. 2005; 15: 356-363Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Upon exposure of cells to chemical activators or bacteria, p47phox (and possibly other subunits) becomes phosphorylated, triggering conformational changes that result in its translocation to the membrane and assembly with the flavocytochrome. Similarly, cell activation results in activation of one or more guanine nucleotide exchange factors, causing exchange of GDP for GTP on Rac, dissociation from RhoGDI, and translocation to the membrane where Rac binds to the flavocytochrome. Both Rac and p47phox contain binding sites for different regions of p67phox, which also translocates to the membrane, where it is oriented and held in place with the assistance of Rac and p47phox. The “activation domain” of p67phox (19Han C.-H. Freeman J.L.R. Lee T. Motalebi S.A. Lambeth J.D. J. Biol. Chem. 1998; 273: 16663-16668Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 20Nisimoto Y. Motalebi S. Han C.-H. Lambeth J.D. J. Biol. Chem. 1999; 274: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) is essential for activating electron flow within the flavocytochrome, thereby turning on NADPH-oxidase activity. The net result of cell activation, then, is to induce subunit assembly and activation of the enzyme. Nox1 is the first member to be described of a family of homologs of gp91phox (21Suh Y.-A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1284) Google Scholar) that now numbers seven members in humans (22Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2492) Google Scholar). Nox1-dependent ROS generation can be reconstituted in cells by co-transfection with the regulatory subunits NOXO1 and NOXA1 (23Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 24Banfi B. Clark R.A. Steger K. Krause K.H. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 25Geiszt M. Lekstrom K. Witta J. Leto T.L. J. Biol. Chem. 2003; 278: 20006-20012Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 26Takeya R. Ueno N. Kami K. Taura M. Kohjima M. Izaki T. Nunoi H. Sumimoto H. J. Biol. Chem. 2003; 278: 25234-25246Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). NOXO1 is a homolog of p47phox, and NOXA1 is a homolog of p67phox. Unlike p47phox and p67phox, NOXO1 and NOXA1 are co-localized with Nox1 in membranes in unstimulated cells (23Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) and therefore do not require cell activation for assembly at the membrane. This is consistent with the structure of NOXO1, which lacks an autoinhibitory region (AIR) that is present in p47phox and is the target of regulatory phosphorylations. In naive phagocytes, the AIR binds internally to the tandem Src homology 3 region (bis-Src homology 3) of p47phox, blocking its interaction with p22phox. Phosphorylation of AIR upon phagocyte activation relieves this inhibition, permitting association and assembly to occur. Therefore, the absence of the AIR in NOXO1 probably accounts in part for the ability of NOXO1 to assemble with Nox1 in resting cells (23Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In addition, the PX domain of NOXO1 binds to phospholipids that are present in naive cells, allowing localization of NOXO1 to the membrane (23Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Whereas less is known about NOXA1, this protein has an activation domain that is highly homologous to that present in p67phox. NOXA1 also contains a TPR domain that associates with Rac1 in yeast two-hybrid and pull-down assays (26Takeya R. Ueno N. Kami K. Taura M. Kohjima M. Izaki T. Nunoi H. Sumimoto H. J. Biol. Chem. 2003; 278: 25234-25246Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). However, whether the NOXA1-Rac1 complex is functional in Nox1-dependent ROS production has not been demonstrated. Interestingly, in reconstitution studies using transfected cells (23Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 24Banfi B. Clark R.A. Steger K. Krause K.H. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 26Takeya R. Ueno N. Kami K. Taura M. Kohjima M. Izaki T. Nunoi H. Sumimoto H. J. Biol. Chem. 2003; 278: 25234-25246Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar), co-expression of Nox1, NOXO1, and NOXA1 results in the production of relatively high levels of reactive oxygen in several cell types without the need to co-express a small GTPase such as Rac1. In contrast, when gp91phox is expressed in HEK293 cells along with its regulatory subunits, there is an absolute requirement for co-expression of activated Rac in order to observe reactive oxygen generation (27Cheng G. Ritsick D.R. Lambeth J.D. J. Biol. Chem. 2004; 279: 34250-34255Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The above results call into question whether a small GTPase is indeed required for Nox1-dependent activity. Whereas it is possible that Nox1 can function in the absence of a small GTPase, another hypothesis is that very low levels of endogenous activated small GTPases are sufficient for Nox1 activity, making it unnecessary to co-transfect an activated GTPase. The present studies were therefore designed to investigate whether Nox1 activity requires the small GTPase Rac1 for optimal ROS generation. Cells—HEK293H cells (Invitrogen) were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Vectors Encoding Nox Enzymes and Regulatory Proteins—Cloning and subcloning of Nox1, Nox2 (gp91phox), NOXO1, NOXA1, p47phox, and p67phox were previously described (23Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 27Cheng G. Ritsick D.R. Lambeth J.D. J. Biol. Chem. 2004; 279: 34250-34255Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). (Myc)2-Rac1(G12V), Myc-Rac1 wild-type, and (HA)3-CDC42(G12V), in pcDNA3.1 were from the University of Missouri-Rolla cDNA Resource Center (Rolla, MO). (HA)3-Rac1(G12V) was constructed by amplifying HA and Rac1(G12V) separately and inserting these PCR products into pcDNA3.1. (HA)3-NOXA1 was constructed by inserting the NOXA1 PCR product into pCMV5-(HA)3. Myc-NOXO1 was constructed by inserting the PCR product of NOXO1 into the vector, pRK5(Myc). pRK5-Myc-PAK1 WT was kindly provided by Dr. Gary Bokoch (Scripps Research Institute). NOXA1(D68A) in pCMV-Sport6 was constructed by amplifying NOXA1 in pCMV-Sport6 using an SP6 primer and primer 1 (5′-AAG TTG GCC ACT CCT CGC TGG AAG AAG CCA ACC GCC ATG CAG GTGGCC TTG GTC ACG GCT TG-3′). Primer 1 introduces an MscI site (underlined) and changes (italics) codon 68 in NOXA1 from aspartic acid (D) to alanine (A). The PCR product was digested with XhoI and MscI. The digested fragment was inserted into XhoI and MscI sites of pCMV-Sport6/NOXA1 to replace the wild-type region. NOXA1(R103E) and NOXA1(D109A) in pCMV-Sport6 were made using an analogous strategy. NOXA1(D109A) also has a E100G mutation accidentally introduced by PCR. Transient Transfections—HEK293H cells were plated at 4 × 105 cells/well in 6-well plates and grown overnight in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin to reach to 40–50% confluence. Cells were transfected with pcDNA3.1 or vectors harboring the insert gene using Fugene 6 according to the manufacturer's instructions. After 48 h, cells were removed from the well and washed twice with cold Hanks' balanced salt solution containing calcium and magnesium. The cells were then pelleted at 1000 × g for 5 min and resuspended in Hanks' balanced salt solution. Measurement of Reactive Oxygen Species—Reactive oxygen was measured using luminol chemiluminescence as previously described (27Cheng G. Ritsick D.R. Lambeth J.D. J. Biol. Chem. 2004; 279: 34250-34255Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Mammalian Two-hybrid Assay—The pM cloning vector (Clontech) contains a GAL4 DNA binding domain, and the vector of pVP16 (Clontech) encodes an activation domain from Herpes simplex virus V16 protein. Both pM and pVP16 were modified by adding KpnI sites in the multiple cloning sites upstream of their BamHI site. pG5CAT expresses a fusion protein consisting of five consensus GAL4 binding sites fused to chloramphenicol acetyltransferase (CAT). pM3-VP16 is a positive control vector that expresses a fusion of the GAL4-BD and the VP16-AD. Full-length NOXA1 and its mutants, including NOXA1(D68A), NOXA1(R103E), and NOXA1(E100G/D109A), were subcloned into KpnI and BamHI sites of pM, whereas Rac1(G12V) and CDC42(G12V) were subcloned into KpnI and BamHI sites of pVP16. For detecting protein-protein interactions, HEK293H cells were co-transfected with 0.2 μg of pG5CAT along with 0.5 μg of pM (or its derivatives) plus 0.5 μg of pVP16 (or its derivatives) as indicated. Cells were harvested after 48 h and stored at –80 °C. CAT activity was assayed using the Fast CAT (deoxy)chloramphenicol acetyltransferase assay kit (Molecular Probes, Inc.). Briefly, frozen cells were resuspended in 100 μl of 0.25 m Tris-HCl, pH 7.4, and lysed with three freeze-thaw cycles and centrifuged at 12,000 rpm for 5 min at 4 °C. 60 μl of supernatant was mixed with 10 μl of FAST CAT substrate solution and preincubated at 37 °C for 5 min. Then 10 μl of freshly prepared 9 mm acetyl-CoA was added and incubated at 37 °C for 2–3 h. The reaction was stopped by adding 1 ml of ice-cold ethyl acetate. The liquid phases were separated by centrifugation at 12,000 rpm for 3 min. 900 μl of ethyl acetate containing the reaction product was dried under vacuum, and the residue was redissolved in 20μl of ethyl acetate. A 5-μl aliquot was applied to a silica gel-60 thin layer chromatography plate (Merck), and separated using 85:15 (v/v) chloroform/methanol. The separated substrate and product were visualized under UV light and photographed using an Alphaimager™ (Alpha Innotech Corp.). Immunoprecipitation—HEK293H cells grown to ∼50% confluence on 10-cm plates were transfected with 5 μg of each plasmid indicated (Fig. 5), using FuGene 6. After 48 h, the cells were harvested by washing twice with Hanks' balanced salt solution. The cells were lysed in 600 μl of RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm EDTA) with protease inhibitor mixture (Sigma). MgCl2 (60 mm) was included in the RIPA buffer for those experiments where Rac1 WT or endogenous Rac1 binding was examined. The lysate was centrifuged at 13,000 rpm for 15 min at 4 °C, and 1.5 mg protein of cleared lysate was used for each immunoprecipitation. The lysates were incubated overnight at 4 °C with primary antibodies with end-over-end rotation. The next morning, either 15 μl of protein G-Sepharose beads (1:1 slurry; Sigma) or 20 μl of streptavidinagarose beads (Molecular Probes) was added to the mixtures and rotated for an additional 2.5 h at 4 °C. Beads were pelleted by centrifuging at 1,500 × g for 2 min and washed three times with cold RIPA buffer. The pellets were resuspended in 25 μl of RIPA buffer or RIPA buffer with 20 mm biotin. The immunocomplexes were analyzed by Western blotting and visualized by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Pierce). Antibodies—3 mg each of chicken anti-NOXO1 and anti-NOXA1 antibodies (23Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 27Cheng G. Ritsick D.R. Lambeth J.D. J. Biol. Chem. 2004; 279: 34250-34255Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) were separately biotinylated using the DSB-X Biotin Protein Labeling Kit (Molecular Probes) according to the manufacturer's instructions and were used to immunoprecipitate untagged NOXO1 and NOXA1, respectively. Anti-Nox1 E39.1 monoclonal antibody was previously described (23Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) and was the kind gift of Jackie Papkoff at diaDexus (S. San Francisco, CA). Anti-green fluorescent protein polyclonal antibody was purchased from ANASPEC, Inc. (San Jose, CA). Anti-Myc and anti-HA monoclonal antibodies were from Cell Signaling Technologies, Inc. (Beverly, MA). Anti-Rac1 and anti-CDC42 antibodies were from Upstate Biotechnology, Inc. (Charlottesville, VA). Rac Activation Assay—The endogenous GTP-associated form of Rac1 was detected using Rac/CDC42 Assay kit (Upstate Biotechnology), following the manufacturer's protocol. Similar to the above immunoprecipitation, HEK293H cells grown to confluence on 10-cm plates were harvested by washing twice with Hanks' balanced salt solution. The cells were lysed in 1× Mg2+ lysis buffer supplied by the manufacturer and supplemented with protease inhibitor mixture. The lysate was centrifuged at 13,000 rpm for 15 min at 4 °C. A total of four samples were prepared for each condition described in the legend to Fig. 1A. Two equal aliquots of the lysate were preloaded with either GTPγSor GDP as controls. A third equal aliquot was not preloaded. All three aliquots were then shaken with glutathione S-transferase-PAK1-(67–150) bound to glutathione-agarose beads at 4 °C for 1 h. The fourth sample was composed of one-twenty-fifth of the volume of lysate that was used in the other three samples for the pull-down and was used directly for Western blotting. Beads were pelleted by centrifuging at 1,500 × g for 2 min and washed three times with cold Mg2+ lysis buffer provided by the kit. The pellets were resuspended in 40 μl of SDS-PAGE loading buffer and subjected to Western blotting. Rac1-GTP was visualized using an anti-Rac1 monoclonal antibody supplied with the kit (Upstate Biotechnology). Western Blot Analysis—Cells were lysed in RIPA buffer with protease inhibitor mixture (Sigma). Lysate (60 μg of protein) was resolved by 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane using a semidry electrophoretic transfer cell (Bio-Rad) at 15 V for 1 h. In some experiments, 40 μg of cell lysate was resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using the Tank Transfer System (Bio-Rad). The proteins were detected using standard Western blotting and visualized by chemiluminescence as described above. Blots were stripped and reprobed as necessary. RNA Interference of Rac1 and CDC42—Nonspecific randomized control siRNA and Rac1 siRNA were purchased from Upstate USA, Inc. (catalog no. M-003560), whereas siRNA of CDC42 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (catalog no. sc-29256). 200 pmol of control siRNA, Rac1 siRNA, and/or CDC42 siRNA were co-transfected into HEK293H cells in a 6-well plate with 0.5 μg each of Nox1, NOXO1, and NOXA1 using X-tremeGENE siRNA transfection reagent (Roche Applied Science) according to the manufacturer's instruction. After 72 h, the cells were harvested as described above. An aliquot of the cells was subject to luminol assay, and the remainder was used for Western blotting. Stimulation of Nox1-dependent ROS Production by Rac1—We previously reported that co-transfection of Nox1, NOXO1, and NOXA1 resulted in significant generation of ROS (23Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In these earlier experiments, we failed to observe stimulation of Nox1-dependent ROS production by Rac1(G12V). Because these experiments used high concentrations of plasmids encoding Nox1, NOXO1, and NOXA1, this may have artifactually saturated Nox1 with its regulatory subunits, preventing further activation by Rac1. We therefore used relatively low concentrations of each plasmid in the present study. As shown in Fig. 1A, whereas significant ROS generation was still seen without co-expressed Rac1 and without phorbol 12-myristate, 13-acetate (PMA) stimulation (Fig. 1A, lane 2), activity was increased slightly by expression of wild-type Rac1 (lane 3) and was further increased by expression of Rac1(G12V) (lane 4). To test whether there may have been endogenous Rac1-GTP present in these cells that might account for partial activation of Nox1 in the absence of Rac1(G12V), the content of endogenous Rac1GTP was evaluated using the Rac binding domain of PAK (PAK-(67–150)). This binding domain interacts exclusively with the GTP form of Rac1 and CDC42. As shown in the Western blot in Fig. 1B (top), Rac1 was seen in both the total cell lysate (lane 1) and in the PAK1 pull-down complex from lysates of untreated cells (lane 3). Because the pull-down complex was concentrated 25-fold compared with lysate and gave about 5% of the staining on the Western blot, compared with the band seen in the lysate, one can estimate that ∼0.2% of endogenous Rac1 is in the GTP-bound form. This was not affected by co-expression of Nox1, NOXA1, and NOXO1 (Fig. 1B, second panel). Co-transfection of Myc-tagged wild-type Rac1 WT along with Nox1, NOXA1, and NOXO1 did not result in an increase in the level of activated endogenous Rac1, but some activated Myc-tagged Rac1 WT was seen (Fig. 1B, third panel from the top). Finally, when Myc-Rac1(G12V) was co-expressed along with Nox1, NOXA1, and NOXO1, the PAK1 pull-down revealed both Rac1(G12V) and an increased level of endogenous activated Rac1 (Fig. 1B, bottom). For unknown reasons, Rac1(T17N) failed to inhibit ROS generation when co-expressed with Nox1, NOXO1, and NOXA1, perhaps because this cell type may utilize a guanine nucleotide exchange factor that is insensitive to inhibition by Rac1(T17N). The concentration dependence for Rac1(G12V) activation of Nox1 with and without PMA stimulation is shown in Fig. 1C. Without PMA, Nox1-dependent ROS generation was stimulated by Rac1(G12V) by ∼3-fold at the highest concentrations tested (1,000 ng). Rac2, which is expressed exclusively in phagocytic cells (28Knaus U.G. Heyworth P.G. Kinsella B.T. Curnutte J.T. Bokoch G.M. J. Biol. Chem. 1992; 267: 23575-23582Abstract Full Text PDF PubMed Google Scholar), produced a similar -fold activation and dose dependence (data not shown). PMA caused up to 2-fold increase in basal activity in the absence of Rac1(G12V), and activity increased by a roughly constant amount throughout the Rac1(G12V) concentration range. We previously showed that Nox2, when co-expressed with p47phox, p67phox, and Rac1(G12V), can be activated by PMA (27Cheng G. Ritsick D.R. Lambeth J.D. J. Biol. Chem. 2004; 279: 34250-34255Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). As a control, we tested the dose dependence for activation of Nox2 by Rac1(G12V), as is shown in Fig. 1D. Nox2 activation absolutely required stimulation by PMA, and no basal activity was seen in the absence of Rac1(G12V). Thus, the quantity of endogenous Rac1-GTP in unstimulated cells is not sufficient to activate Nox2. The absolute requirement of PMA for Nox2, but not Nox1, activation is not surprising, since PMA-dependent phosphorylation of the AIR of p47phox and perhaps other components is well known (29Yuzawa S. Suzukik N.N. Fujioka Y. Ogura K. Sumimoto H. Inagaki F Genes Cells. 2004; 9: 443-456Crossref PubMed Scopus (55) Google Scholar, 30Yuzawa S. Ogura K. Horiuchi M. Suzuki N.N. Fujiolka Y. Kataoka M. Sumimoto H. Inagaki F. J. Biol. Chem. 2004; 279: 29752-29760Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), whereas the AIR is absent in NOXO1. Interestingly, in a transgenic COS7 cell line stably expressing gp91phox, p22phox, p47phox, and p67phox, stimulus-independent ROS production was induced by transfection of Rac1(G12V) (31Price M. Atkinson S.J. Knaus U.G. Dinauer M.C. J. Biol. Chem. 2002; 277: 19220-19228Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In these COSphox cells, transfection of Rac1(GV12) drove translocation of p47phox to the membrane, suggesting that Rac1 acts not only as a participant in the NADPH oxidase complex but also as a regulator of oxidase assembly. We were not able to compare the protein expression levels of Nox1 versus Nox2 in Fig. 1D due to the lack of an antibody that recognizes the same epit" @default.
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• W1985033865 title "Nox1-dependent Reactive Oxygen Generation Is Regulated by Rac1" @default.
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