Direct Involvement of the Small GTPase Rac in Activation of the Superoxide-producing NADPH Oxidase Nox1
2006; Elsevier BV; Volume: 281; Issue: 31 Linguagem: Inglês
10.1074/jbc.m513665200
ISSN1083-351X
AutoresKei Miyano, Noriko Ueno, Ryu Takeya, Hideki Sumimoto,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoActivation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22phox at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22phox: the Noxa1-Noxo1 and Noxo1-p22phox interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1. Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22phox at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22phox: the Noxa1-Noxo1 and Noxo1-p22phox interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1. Although reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; Nox, NAD(P)H oxidase; PRR, proline-rich region; TPR, tetratricopeptide repeat; Noxo1, Nox organizer 1; Noxa1, Nox activator 1; Pak-PBD, the p21-binding domain of the protein kinase Pak; GTPγS, guanosine 5′-(γ-thio)triphosphate; GDPβS, guanosine 5′-(β-thio)diphosphate; siRNA, small interfering RNA; RNAi, RNA interference; HA, hemagglutinin; CHO, Chinese hamster ovary; SH3, Src homology domain 3; GST, glutathione S-transferase. were previously considered to be by-products in aerobic metabolism, it has recently been accepted that ROS are also produced as true products by specialized enzymes, thereby participating in a variety of biological processes including host defense, hormone biosynthesis, oxygen sensing, and signal transduction (1Bokoch G.M. Knaus U.G. Trends Biochem. Sci. 2003; 28: 502-508Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 2Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2492) Google Scholar, 3Geiszt M. Leto T.L. J. Biol. Chem. 2004; 279: 51715-51718Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 4Werner E. J. Cell Sci. 2004; 117: 143-153Crossref PubMed Scopus (76) Google Scholar, 5Sumimoto H. Miyano K. Takeya R. Biochem. Biophys. Res. Commun. 2005; 338: 677-686Crossref PubMed Scopus (255) Google Scholar). Enzymes dedicated to ROS production include members of the NAD(P)H oxidase (Nox) family, which are membrane-spanning flavocytochromes that reduce molecular oxygen to superoxide with electron derived from NAD(P)H (1Bokoch G.M. Knaus U.G. Trends Biochem. Sci. 2003; 28: 502-508Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 2Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2492) Google Scholar, 3Geiszt M. Leto T.L. J. Biol. Chem. 2004; 279: 51715-51718Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 4Werner E. J. Cell Sci. 2004; 117: 143-153Crossref PubMed Scopus (76) Google Scholar, 5Sumimoto H. Miyano K. Takeya R. Biochem. Biophys. Res. Commun. 2005; 338: 677-686Crossref PubMed Scopus (255) Google Scholar). The best characterized member of the family is gp91phox, also termed Nox2, that functions as the catalytic subunit of the phagocyte NADPH oxidase (phox) (6Quinn M.T. Gauss K.A. J. Leukocyte Biol. 2004; 76: 760-781Crossref PubMed Scopus (392) Google Scholar, 7Nauseef W.M. Histochem. Cell Biol. 2004; 122: 277-291Crossref PubMed Scopus (324) Google Scholar, 8Cross A.R. Segal A.W. Biochim. Biophys. Acta. 2004; 1657: 1-22Crossref PubMed Scopus (370) Google Scholar, 9Groemping Y. Rittinger K. Biochem. J. 2005; 386: 401-416Crossref PubMed Scopus (447) Google Scholar). This oxidase plays a crucial role in host defense, which is evident from recurrent and life-threatening infections that occur in patients with chronic granulomatous disease, whose phagocytes genetically lack the superoxide producing activity (6Quinn M.T. Gauss K.A. J. Leukocyte Biol. 2004; 76: 760-781Crossref PubMed Scopus (392) Google Scholar, 7Nauseef W.M. Histochem. Cell Biol. 2004; 122: 277-291Crossref PubMed Scopus (324) Google Scholar, 8Cross A.R. Segal A.W. Biochim. Biophys. Acta. 2004; 1657: 1-22Crossref PubMed Scopus (370) Google Scholar, 9Groemping Y. Rittinger K. Biochem. J. 2005; 386: 401-416Crossref PubMed Scopus (447) Google Scholar). gp91phox, stably complexed with the membrane-integrated protein p22phox, is dormant in resting cells, but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal ROS. Activation of gp91phox/Nox2 requires the small GTPase Rac and the two specific adaptor proteins p47phox and p67phox, each containing two SH3 domains. These proteins, albeit existing in the cytoplasm of resting phagocytes, are targeted upon cell stimulation to the membrane to interact with the gp91phox-p22phox complex, which allows gp91phox to transport electrons for superoxide production (10Koshkin V. Lotan O. Pick E. Biochim. Biophys. Acta. 1997; 1319: 139-146Crossref PubMed Scopus (51) Google Scholar, 11Cross A.W. Erickson R.W. Curnutte J.T. J. Biol. Chem. 1999; 274: 15519-15525Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 12Diebold B.A. Bokoch G.M. Nat. Immunol. 2001; 2: 211-215Crossref PubMed Scopus (239) Google Scholar, 13Nisimoto 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, 14Sarfstein R. Gorzalczany Y. Mizrahi A. Berdichevsky Y. Molshanski-Mor S. Weinbaum C. Hirshberg M. Dagher M.-C. Pick E. J. Biol. Chem. 2004; 279: 16007-16016Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 15Hashida S. Yuzawa S. Suzuki N.N. Fujioka Y. Takikawa T. Sumimoto H. Inagaki F. Fujii H. J. Biol. Chem. 2004; 279: 26378-2638610Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 16Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar). The stimulus-induced membrane targeting of p47phox and subsequent activation of gp91phox require the interaction between p47phox and p22phox, which is mediated via binding of the p47phox SH3 domains to the p22phox C-terminal proline-rich region (PRR) (16Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar, 17Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar, 18Groemping Y. Lapouge K. Smerdon S.J. Rittinger K. Cell. 2003; 113: 345-355Abstract Full Text Full Text PDF Scopus (318) Google Scholar, 19Nobuhisa I. Takeya R. Ogura K. Ueno N. Kohda D. Inagaki F. Sumimoto H. Biochem. J. 2006; 396: 183-192Crossref PubMed Scopus (32) Google Scholar): the substitution of Gln for Pro-156 in the PRR of p22phox (P156Q), a mutation found in a patient with chronic granulomatous disease (20Dinauer M.C. Pierce E.A. Erickson R.W. Muhlebach T.J. Messner H. Orkin S.H. Seger R.A. Curnutte J.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11231-11235Crossref PubMed Scopus (83) Google Scholar), leads to a defective binding to p47phox (16Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar, 17Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar). Upon cell stimulation, p67phox translocates to the membrane via its association with p47phox (21Leusen J.H. Bolscher B.G. Hilarius P.M. Weening R.S. Kaulfersch W. Seger R.A. Roos D. Verhoeven A.J. J. Exp. Med. 1994; 180: 2329-2334Crossref PubMed Scopus (90) Google Scholar, 22Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar, 23Leusen J.H. Fluiter K. Hilarius P.M. Roos D. Verhoeven A.J. Bolscher B.G. J. Biol. Chem. 1995; 270: 11216-11221Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24Morozov I. Lotan O. Joseph G. Gorzalczany Y. Pick E. J. Biol. Chem. 1998; 273: 15435-15444Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 25Kami K. Takeya R. Sumimoto H. Kohda D. EMBO J. 2002; 21: 4268-4276Crossref PubMed Scopus (149) Google Scholar), which is crucial for oxidase activation (26Mizuki K. Takeya R. Kuribayashi F. Nobuhisa I. Kohda D. Nunoi H. Takeshige K. Sumimoto H. Arch. Biochem. Biophys. 2005; 444: 185-194Crossref PubMed Scopus (31) Google Scholar). At the membrane, p67phox directly interacts with GTP-bound Rac. This small GTPase is recruited to the membrane independently of p47phox or p67phox, and binds to the p67phox N-terminal region of about 200 amino acid residues, containing four tetratricopeptide repeat (TPR) motifs (27Koga H. Terasawa H. Nunoi H. Takeshige K. Inagaki F. Sumimoto H. J. Biol. Chem. 1999; 274: 25051-25060Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 28Lapouge K. Smith S.J.M. Walker P.A. Gamblin S.J. Smerdon S.J. Rittinger K. Mol. Cell. 2000; 6: 899-907Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). A mutant p67phox carrying the substitution of Glu for Arg-102 in the third TPR neither interacts with Rac nor supports superoxide production by gp91phox (27Koga H. Terasawa H. Nunoi H. Takeshige K. Inagaki F. Sumimoto H. J. Biol. Chem. 1999; 274: 25051-25060Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Thus the Rac-p67phox interaction plays an essential role in activation of gp91phox (27Koga H. Terasawa H. Nunoi H. Takeshige K. Inagaki F. Sumimoto H. J. Biol. Chem. 1999; 274: 25051-25060Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 28Lapouge K. Smith S.J.M. Walker P.A. Gamblin S.J. Smerdon S.J. Rittinger K. Mol. Cell. 2000; 6: 899-907Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 29Price 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, 30Biberstine-Kinkade K.J. Yu L. Stull N. LeRoy B. Bennett S. Cross A. Dinauer M.C. J. Biol. Chem. 2002; 277: 30368-30374Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 31Miyano K. Ogasawara S. Han C.H. Fukuda H. Tamura M. Biochemistry. 2001; 40: 14089-14097Crossref PubMed Scopus (38) Google Scholar). In addition, deletion of a region C-terminal to the Rac-binding domain in p67phox (amino acid residues 200-212), so-called an activation domain, or the V204A substitution results in an impaired activation of gp91phox (32Hata K. Takeshige K. Sumimoto H. Biochem. Biophys. Res. Commun. 1997; 241: 226-231Crossref PubMed Scopus (36) Google Scholar, 33Han 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). Taken together, Rac binding to the p67phox TPR domain seems to induce a conformational change of the activation domain in p67phox, thereby activating gp91phox (14Sarfstein R. Gorzalczany Y. Mizrahi A. Berdichevsky Y. Molshanski-Mor S. Weinbaum C. Hirshberg M. Dagher M.-C. Pick E. J. Biol. Chem. 2004; 279: 16007-16016Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 33Han 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, 34Alloul N. Gorzalczany Y. Itan M. Sigal N. Pick E. Biochemistry. 2001; 40: 14557-14566Crossref PubMed Scopus (58) Google Scholar, 35Gorzalczany Y. Alloul N. Sigal N. Weinbaum C.K. Pick E. J. Biol. Chem. 2002; 277: 18605-18610Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Nox1, the first identified gp91phox homologue in mammals, is abundantly expressed in colon epithelial cells and vascular smooth muscle cells (36Suh 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, 37Bánfi B. Maturana A. Jaconi S. Arnaudeau S. Laforge T. Sinha B. Ligeti E. Demaurex N. Krause K.-H. Science. 2000; 287: 138-142Crossref PubMed Scopus (251) Google Scholar). Recent studies have shown that Nox1 participates in host defense at the colon (38Geiszt M. Lekstrom K. Brenner S. Hewitt S.M. Dana R. Malech H.L. Leto T.L. J. Immunol. 2003; 171: 299-306Crossref PubMed Scopus (167) Google Scholar, 39Kawahara T. Kuwano Y. Teshima-Kondo S. Takeya R. Sumimoto H. Kishi K. Tsunawaki S. Hirayama T. Rokutan K. J. Immunol. 2004; 172: 3051-3058Crossref PubMed Scopus (123) Google Scholar) and angiotensin II-mediated hypertension (40Matsuno K. Yamada H. Iwata K. Jin D. Katsuyama M. Matsuki M. Takai S. Yamanishi K. Miyazaki M. Matsubara H. Yabe-Nishimura C. Circulation. 2005; 112: 2677-2685Crossref PubMed Scopus (405) Google Scholar, 41Dikalova A. Clempus R. Lassegue B. Cheng G. McCoy J. Dikalov S. San Martin A. Lyle A. Weber D.S. Weiss D. Taylor W.R. Schmidt H.H. Owens G.K. Lambeth J.D. Griendling K.K. Circulation. 2005; 112: 2668-2676Crossref PubMed Scopus (360) Google Scholar). This non-phagocytic oxidase forms a heterodimer with p22phox (42Takeya 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, 43Ambasta R.K. Kumar P. Griendling K.K. Schmidt H.H. Busse R. Brandes R.P. J. Biol. Chem. 2004; 279: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 44Hanna I.R. Hilenski L.L. Dikalova A. Taniyama Y. Dikalov S. Lyle A. Quinn M.T. Lassegue B. Griendling K.K. Free Radic. Biol. Med. 2004; 37: 1542-1549Crossref PubMed Scopus (72) Google Scholar, 45Kawahara T. Ritsick D. Cheng G. Lambeth J.D. J. Biol. Chem. 2005; 280: 31859-31869Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), and can be activated by p47phox and p67phox but to a much lesser extent than gp91phox/Nox2 (42Takeya 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, 46Bánfi B. Clark R.A. Steger K. Krause K.H.J. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 47Geiszt 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). Activation of Nox1 requires the regulatory proteins Noxo1 (Nox organizer 1) and Noxa1 (Nox activator 1), novel respective homologues of p47phox and p67phox, but not cell stimulants (42Takeya 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, 46Bánfi B. Clark R.A. Steger K. Krause K.H.J. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 47Geiszt 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, 48Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Noxo1 is known to bind to p22phox via its tandem SH3 domains, whereas Noxa1 interacts with Noxo1 by binding of the Noxa1 SH3 domain to the Noxo1 C terminus (Refs. 40Matsuno K. Yamada H. Iwata K. Jin D. Katsuyama M. Matsuki M. Takai S. Yamanishi K. Miyazaki M. Matsubara H. Yabe-Nishimura C. Circulation. 2005; 112: 2677-2685Crossref PubMed Scopus (405) Google Scholar; see Fig. 1). The roles of these interactions in Nox1 activation, however, have remained to be elucidated. Rac is considered to be involved in ROS production also in non-phagocytic cells (1Bokoch G.M. Knaus U.G. Trends Biochem. Sci. 2003; 28: 502-508Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 4Werner E. J. Cell Sci. 2004; 117: 143-153Crossref PubMed Scopus (76) Google Scholar, 49Gregg D. Rauscher F.M. Goldschmidt-Clermont P.J. Am. J. Physiol. 2003; 285: C723-C734Crossref PubMed Scopus (110) Google Scholar). This small GTPase, however, does not seem to be directly involved in activation of non-phagocytic oxidases such as Nox3 (50Ueno N. Takeya R. Miyano K. Kikuchi H. Sumimoto H. J. Biol. Chem. 2005; 280: 23328-23339Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), Nox4 (51Martyn K.D. Frederick L.M. von Loehneysen K. Dinauer M.C. Knaus U.G. Cell. Signal. 2006; 18: 69-82Crossref PubMed Scopus (625) Google Scholar), and Duox, a distantly related member of the Nox family (52Fortemaison N. Miot F. Dumont J.E. Dremier S. Eur. J. Endocrinol. 2005; 152: 127-133Crossref PubMed Scopus (28) Google Scholar). On the other hand, it has been reported that blockade of molecules acting upstream of Rac activation decreases superoxide production in Nox1-expressing cells, suggesting the involvement of Rac (53Park H.S. Lee S.H. Park D. Lee J.S. Ryu S.H. Lee W.J. Rhee S.G. Bae Y.S. Mol. Cell. Biol. 2004; 24: 4384-4394Crossref PubMed Scopus (201) Google Scholar, 54Kawahara 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). However, it has remained unknown how Rac functions in Nox1 activation. We have previously demonstrated that GTP-loaded Rac directly interacts with the N-terminal TPR domain of Noxa1 as well as that of p67phox; the interaction is abolished by substitution of Glu for Arg-103 (Ref. 42Takeya 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; see Fig. 1). In the present study, we show that electropermeabilized HeLa cells, which ectopically express Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner. The production is decreased when a mutant Noxa1(R103E) is expressed instead of the wild-type one, indicative of the role for Rac. Consistent with this, Nox1-dependent superoxide production by intact HeLa cells is decreased by depletion or sequestration of Rac, but enhanced by expression of the constitutively active Rac1(Q61L). We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22phox: the Noxa1-Noxo1 and Noxo1-p22phox interactions are both required for the superoxide producing activity of Nox1. Rac functions in Nox1 activation via its interaction with Noxa1: this GTPase likely acts by inducing a conformational change of the activation domain of Noxa1; it also facilitates membrane localization of Noxa1 in the absence of sufficient interaction between Noxa1 and Noxo1. Plasmid Construction—The human cDNAs encoding Nox1, gp91phox/Nox2, p22phox, Noxo1, Noxa1, p47phox, p67phox, Rac1, and the p21-binding domain of the protein kinase Pak (Pak-PBD; amino acids 66-147) were prepared as previously described (27Koga H. Terasawa H. Nunoi H. Takeshige K. Inagaki F. Sumimoto H. J. Biol. Chem. 1999; 274: 25051-25060Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 40Matsuno K. Yamada H. Iwata K. Jin D. Katsuyama M. Matsuki M. Takai S. Yamanishi K. Miyazaki M. Matsubara H. Yabe-Nishimura C. Circulation. 2005; 112: 2677-2685Crossref PubMed Scopus (405) Google Scholar, 48Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 55Akasaki T. Koga H. Sumimoto H. J. Biol. Chem. 1999; 274: 18055-18059Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The cDNA for human RhoGDI (56Leffers H. Nielsen M.S. Andersen A.H. Honore B. Madsen P. Vandekerckhove J. Celis J.E. Exp. Cell Res. 1993; 209: 165-174Crossref PubMed Scopus (81) Google Scholar) were prepared by PCR using a mixture of human brain cDNAs from Human Multiple Tissue cDNA Panel I (BD Biosciences) as a template. Mutations leading to the indicated amino acid substitutions were introduced by PCR-mediated site-directed mutagenesis. The DNA fragments were ligated to the indicated expression vectors. All the constructs were sequenced for confirmation of their identities. Transfection of cDNAs Encoding Nox1, gp91phox, p22phox, and Cytosolic Regulatory Proteins in HeLa, Caco-2, and CHO Cells—The cDNAs for Nox1 and gp91phox were ligated to the mammalian expression vector pcDNA3.0 (Invitrogen). The cDNA encoding Rac1, p22phox, Noxo1, p47phox, Noxa1, and p67phox ligated to the mammalian expression vector pEF-BOS (57Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar): Rac1 was constructed for expression as a myc-tagged protein; p22phox as a protein without a tag; Noxo1 and p47phox as an HA-tagged protein; and Noxa1 and p67phox as a myc-tagged protein. Transfection of HeLa cells with the cDNAs was performed using Lipofectamine (Invitrogen), whereas FuGENE 6 Transfection Reagent (Roche Diagnostics) was used for transfection of CHO cells (58Takeya R. Ueno N. Sumimoto H. Methods Enzymol. 2005; 406: 456-468Crossref Scopus (27) Google Scholar). When indicated, Nox1 was expressed as a FLAG-tagged protein using the vector pEF-BOS. Transfection of the human colon cancer Caco-2 cells with cDNAs for Noxo1 and Noxa1 was performed with Cell Line Nucleofector® Kit T (Amaxa) using the Nucleofector® apparatus (Amaxa), according to the manufacturer's instruction. Estimation of Oxidase Proteins Expressed in HeLa, Caco-2, and CHO Cells—Total cell lysates of HeLa, Caco-2, and CHO cells were used for estimation of expression of Noxo1, Noxa1, p47phox, and p67phox. The lysates were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore), and probed with an anti-HA monoclonal antibody (Covance Research Products), an anti-myc monoclonal antibody (Roche Diagnostics), an anti-FLAG monoclonal antibody (Sigma), anti-p22phox polyclonal antibodies (Santa Cruz Biotechnology), an anti-Rac monoclonal antibody (BD Biosciences), or an anti-Cdc42 monoclonal antibody (BD Transduction Laboratories). The blots were developed using ECL Plus (Amersham Biosciences) for visualization of the antibodies, as previously described (42Takeya 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, 50Ueno N. Takeya R. Miyano K. Kikuchi H. Sumimoto H. J. Biol. Chem. 2005; 280: 23328-23339Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Superoxide Production by Electropermeabilized HeLa Cells Expressing Nox1, Noxo1, and Noxa1—The transfected HeLa cells were cultured for 30 h, and harvested by incubation with trypsin/EDTA for 1 min at 37 °C. Cell permeabilization was performed by the method of Grinstein and Furuya (59Grinstein S. Furuya W. J. Biol. Chem. 1988; 263: 1779-1783Abstract Full Text PDF PubMed Google Scholar) with minor modifications. Briefly, the cells were suspended in an electroporation buffer (140 mm KCl, 1.0 mm MgCl2, 0.2 mm CaCl2, 1.0 mm EGTA, 1.0 mm NADPH, and 10 mm Hepes, pH 7.4) containing 0.5 mm GTPγS or 2.5 mm GDPβS, and transferred to a Bio-Rad Gene Pulser and permeabilized with a discharge of 5 kV/cm from a 25-μF capacitor. After being washed with the electroporation buffer, the cells were tested for estimation of the superoxide producing activity. The activity was determined by superoxide dismutase-inhibitable chemiluminescence with an enhancer-containing luminol-based detection system (DIOGENES; National Diagnostics), as previously described (42Takeya 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, 50Ueno N. Takeya R. Miyano K. Kikuchi H. Sumimoto H. J. Biol. Chem. 2005; 280: 23328-23339Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 58Takeya R. Ueno N. Sumimoto H. Methods Enzymol. 2005; 406: 456-468Crossref Scopus (27) Google Scholar). The chemiluminescence was assayed at 37 °C using a luminometer (Auto Lumat LB953; EG&G Berthold). Superoxide Production by Cells Expressing Nox1 or gp91phox— The transfected cells were cultured for 30 h, and harvested by incubation with trypsin/EDTA for 1 min at 37 °C. After being washed with Hepes-buffered saline (120 mm NaCl, 5 mm KCl, 5 mm glucose, 1 mm MgCl2, 0.5 mm CaCl2, and 17 mm Hepes, pH 7.4), the cells were suspended at the concentration of 8 × 105 cells/ml (HeLa and CHO cells) or 2 × 105 cells/ml (Caco-2 cells), and preincubated for 5 min at 37 °C. The superoxide producing activity was determined by superoxide dismutase-inhibitable chemiluminescence with DIOGENES. The chemiluminescence was measured for 10 min at 37 °C using the luminometer. For estimation of superoxide production by gp91phox/Nox2, the transfected cells were stimulated with phorbol 12-myristate 13-acetate (Research Biochemicals International) at 200 ng/ml (50Ueno N. Takeya R. Miyano K. Kikuchi H. Sumimoto H. J. Biol. Chem. 2005; 280: 23328-23339Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 58Takeya R. Ueno N. Sumimoto H. Methods Enzymol. 2005; 406: 456-468Crossref Scopus (27) Google Scholar). RNA Interference (RNAi) for Knockdown of Rac1 and Cdc42— As double strand small interfering RNA (siRNA) targeting Rac1, the 25-nucleotide modified synthetic RNA (RAC1 Validated Stealth® RNAi) was purchased from Invitrogen and tentatively named Rac1 siRNA number 3. The sequences were as follows: 5′-AGGGUCUAGCCAUGGCUAAGGAGAU-3′ (sense) and 5′-AUCUCCUUAGCCAUGGCUAGACCCU-3′ (antisense). The following RNA, designated as Rac1 siRNA number 1, was designed for a coding region of Rac1: 5′-UCCGUGCAAAGUGGUAUCCUGAGGU-3′ (sense) and 5′-ACCUCAGGAUACCACUUUGCACGGA-3′ (antisense). The sequence of Rac1 siRNA number 2 corresponded to that of a 3′-untranslated region: 5′-CUUGGAACCUUUGUACGCUUUGCUC-3′ (sense) and 5′-GAGCAAAGCGUACAAAGGUUCCAAG-3′ (antisense). As a negative control for Rac1 siRNAs numbers 1 and 2 (cont 1), Medium GC Duplex of Stealth® RNAi Negative Control Duplexes (Invitrogen) was used. As a negative control for Rac1 siRNA number 3 (cont 2), the following RNA was used: 5′-AGGUGCUACGCAUGCGUAAGAGGAU (sense) and 5′-AUCCUCUUACGCAUGCGUAGCACCU-3′ (antisense). As siRNA targeting Cdc42, two different RNA duplexes (CDC42 Validated Stealth® RNAi) were purchased from Invitrogen: 5′-CCUCUACUAUUGAGAAACUUGCCAA-3′ (sense) and 5′-UUGGCAAGUUUCUCAAUAGUAGAGG-3′ (antisense) as Cdc42 siRNA number 1; and 5′-UCCUUUCUUGCUUGUUGGGACUCAA-3′ (sense) and 5′-UUGAGUCCCAACAAGCAAGAAAGGA-3′ (antisense) as Cdc42 siRNA number 2. As a negative control for Cdc42 siRNAs, Low GC Duplex of Stealth® RNAi Negative Control Duplexes (Invitrogen) was used. The sequence of Rac3 siRNA corresponded to that of a coding region: 5′-CCUCCUUCGAGAAUGUUCGUGCCAA-3′ (sense) and 5′-UUGGCACGAACAUUCU
Referência(s)