Artigo Acesso aberto Revisado por pares

Phosphoinositide 3-Kinase-dependent and -independent Activation of the Small GTPase Rac2 in Human Neutrophils

1999; Elsevier BV; Volume: 274; Issue: 25 Linguagem: Inglês

10.1074/jbc.274.25.18055

ISSN

1083-351X

Autores

Takashi Akasaki, Hirofumi Koga, Hideki Sumimoto,

Tópico(s)

Neutrophil, Myeloperoxidase and Oxidative Mechanisms

Resumo

The small GTPase Rac participates in various cellular events such as cytoskeletal reorganization. It has remained, however, largely unknown about intracellular signaling pathways for Rac activation because of the lack of a simple and reliable assay to estimate the activation. Here we describe a novel method to detect the GTP-bound, active Rac in cells by pulling it down with the Rac-binding domain of the protein kinase PAK. Experiments using this method reveal that stimulation of human neutrophils with the Gi-coupled receptor agonistsN-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4) leads to a rapid and transient increase in the GTP-bound state of Rac2, whereas phorbol myristate acetate (PMA) causes a slow but more sustained activation of Rac2. Pretreatment of cells with pertussis toxin results in defective activation of Rac2 in response to fMLP and LTB4, indicating that coupling of the receptors to Gi plays a crucial role in the activation. Furthermore, the phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 block Rac2 activation elicited by the receptor agonists, but not that by PMA. Thus the Gi-coupled receptors likely mediate Rac2 activation via PI3K, whereas PMA activates Rac2 in a PI3K-independent manner. The small GTPase Rac participates in various cellular events such as cytoskeletal reorganization. It has remained, however, largely unknown about intracellular signaling pathways for Rac activation because of the lack of a simple and reliable assay to estimate the activation. Here we describe a novel method to detect the GTP-bound, active Rac in cells by pulling it down with the Rac-binding domain of the protein kinase PAK. Experiments using this method reveal that stimulation of human neutrophils with the Gi-coupled receptor agonistsN-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4) leads to a rapid and transient increase in the GTP-bound state of Rac2, whereas phorbol myristate acetate (PMA) causes a slow but more sustained activation of Rac2. Pretreatment of cells with pertussis toxin results in defective activation of Rac2 in response to fMLP and LTB4, indicating that coupling of the receptors to Gi plays a crucial role in the activation. Furthermore, the phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 block Rac2 activation elicited by the receptor agonists, but not that by PMA. Thus the Gi-coupled receptors likely mediate Rac2 activation via PI3K, whereas PMA activates Rac2 in a PI3K-independent manner. The small GTPases Rac1 and Rac2 are members of the Rho subfamily of the Ras-related GTP-binding proteins and serve as a molecular switch cycling between an active GTP-bound and an inactive GDP-bound state (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5165) Google Scholar, 2Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2079) Google Scholar, 3Narumiya S. J. Biochem. 1996; 120: 215-228Crossref PubMed Scopus (359) Google Scholar). In the active state, Rac interacts with a variety of effector proteins to elicit cellular responses, including cytoskeletal reorganization and gene activation (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5165) Google Scholar, 2Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2079) Google Scholar, 3Narumiya S. J. Biochem. 1996; 120: 215-228Crossref PubMed Scopus (359) Google Scholar). Growth factor or integrin-induced Rac activation in such responses is thought to require PtdIns(3,4,5)P3, 1The abbreviations used are: PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphoinositide 3-kinase; fMLP, N-formyl-methionyl-leucyl-phenylalanine; LTB4, leukotriene B4; Gi, the Gi class of heterotrimeric GTP-binding protein; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; GST, glutathioneS-transferase; RBD, Rac-binding domain; PTX, pertussis toxin; GTPγS, guanosine 5′-3-O-(thio)-triphosphate; PAGE, polyacrylamide gel electrophoresis; Giα, the a subunit of Gi; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology. 1The abbreviations used are: PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphoinositide 3-kinase; fMLP, N-formyl-methionyl-leucyl-phenylalanine; LTB4, leukotriene B4; Gi, the Gi class of heterotrimeric GTP-binding protein; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; GST, glutathioneS-transferase; RBD, Rac-binding domain; PTX, pertussis toxin; GTPγS, guanosine 5′-3-O-(thio)-triphosphate; PAGE, polyacrylamide gel electrophoresis; Giα, the a subunit of Gi; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology.that is generated from PtdIns(4,5)P2 by PI3K (4Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1216) Google Scholar), based on experiments using cells overexpressing constitutively active and dominant negative forms of Rac, and on those of microinjection of the mutant Rac proteins to cells (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5165) Google Scholar, 2Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2079) Google Scholar, 3Narumiya S. J. Biochem. 1996; 120: 215-228Crossref PubMed Scopus (359) Google Scholar, 4Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1216) Google Scholar). A major problem in this field has been the lack of the assay to evaluate activation of endogenous Rac. In human neutrophils, Rac is considered to be involved in activation of the phagocyte NADPH oxidase. The oxidase, dormant in resting cells, is activated during phagocytosis of invading microorganisms to produce superoxide, a precursor of microbicidal oxidants, thereby playing an important role in host defense (5Chanock S.J. el Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar, 6DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (455) Google Scholar, 7Sumimoto H. Ito T. Hata K. Mizuki K. Nakamura R. Kage Y. Sakaki Y. Nakamura M. Takeshige K. Hamasaki N. Mihara K. Membrane Proteins: Structure, Function & Expression Control. Kyushu University Press, Fukuoka/S. Karger AG, Basel1997: 235-245Google Scholar). The activation, at a cell level, can be mimicked by soluble stimuli (5Chanock S.J. el Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar, 6DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (455) Google Scholar, 7Sumimoto H. Ito T. Hata K. Mizuki K. Nakamura R. Kage Y. Sakaki Y. Nakamura M. Takeshige K. Hamasaki N. Mihara K. Membrane Proteins: Structure, Function & Expression Control. Kyushu University Press, Fukuoka/S. Karger AG, Basel1997: 235-245Google Scholar, 8Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar) including the chemotactic peptide fMLP and LTB4, which bind to their own Gi-coupled receptors on the plasma membrane (8Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar, 9Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (845) Google Scholar, 10Sumimoto H. Takeshige K. Minakami S. Biochim. Biophys. Acta. 1984; 803: 271-277Crossref PubMed Scopus (78) Google Scholar), and PMA, an activator of PKC (11Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2346) Google Scholar). The NADPH oxidase is also activated with anionic amphiphiles such as arachidonic acid in a cell-free system reconstituted with five polypeptides; the membrane-bound catalytic core cytochrome b 558 comprising the two subunits gp91 phox and p22 phox, and the three cytosolic signaling proteins p47 phox, p67 phox, and Rac (5Chanock S.J. el Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar, 6DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (455) Google Scholar, 7Sumimoto H. Ito T. Hata K. Mizuki K. Nakamura R. Kage Y. Sakaki Y. Nakamura M. Takeshige K. Hamasaki N. Mihara K. Membrane Proteins: Structure, Function & Expression Control. Kyushu University Press, Fukuoka/S. Karger AG, Basel1997: 235-245Google Scholar). In the system, solely the GTP-bound Rac, but not the GDP-bound protein, is able to induce superoxide production (12Abo A. Pick E. Hall A. Totty N. Teahan C.G. Segal A.W. Nature. 1991; 353: 668-670Crossref PubMed Scopus (756) Google Scholar, 13Knaus U.G. Heyworth P.G. Evans T. Curnutte J.T. Bokoch G.M. Science. 1991; 254: 1512-1515Crossref PubMed Scopus (540) Google Scholar, 14Mizuno T. Kaibuchi K. Ando S. Musha T. Hiraoka K. Takaishi K. Asada M. Nunoi H. Matsuda I. Takai Y. J. Biol. Chem. 1992; 267: 10215-10218Abstract Full Text PDF PubMed Google Scholar), probably via binding to p67 phox (15Diekmann D. Abo A. Johnston C. Segal A.W. Hall A. Science. 1994; 265: 531-533Crossref PubMed Scopus (344) Google Scholar, 16Nisimoto Y. Freeman J.L.R. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 17Mizuki K. Kadomatsu K. Hata K. Ito T. Fan Q.-W. Kage Y. Fukumaki Y. Sakaki Y. Takeshige K. Sumimoto H. Eur. J. Biochem. 1998; 251: 573-582Crossref PubMed Scopus (67) Google Scholar). This implicates that Rac functions as a switch for the oxidase activation, although the oxidase activation also requires Rac-independent events such as stimulus-induced conformational change of p47 phox that leads to its interaction with p22 phox (18Sumimoto 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 (254) Google Scholar, 19Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (240) Google Scholar, 20Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 21Hata K. Ito T. Takeshige K. Sumimoto H. J. Biol. Chem. 1998; 273: 4232-4236Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In Epstein-Barr virus-transformed B lymphocytes or HL60 leukemic cells, introduction of Rac antisense oligonucleotides or expression of a dominant negative form of Rac2 is shown to partially inhibit superoxide production (22Dorseuil O. Vazquez A. Lang P. Bertoglio J. Gacon G. Leca G. J. Biol. Chem. 1992; 267: 20540-20542Abstract Full Text PDF PubMed Google Scholar,23Gabig T.G. Crean C.D. Mantel P.L. Rosli R. Blood. 1995; 85: 804-811Crossref PubMed Google Scholar), suggesting a role of Rac on the oxidase activation at a cell level. However, stimulus-dependent activation of Rac has not been demonstrated. Activation-specific probes for small GTPases have recently been constructed, which allows determination of the activity of endogenous Ras and Rap1 (24Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 25de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar, 26Franke B. Akkerman J.-W.N. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (362) Google Scholar) and hemagglutinin-tagged Rac1 and Cdc42 (27Bagrodia S. Taylor S.J. Jordon K.A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) without radioactive in vivo labeling. Using a similar procedure, we have developed here a novel assay to estimate activation of endogenous Rac in cells, by pulling down the GTP-bound, active Rac with the Rac-binding domain (RBD) of the protein kinase PAK2 expressed as a GST fusion. This assay is based on the finding that PAK2-RBD binds to the GTP-bound Rac with a high affinity, whereas the affinity for the GDP-bound protein is undetectably low (28Zhang B. Chernoff J. Zheng Y. J. Biol. Chem. 1998; 273: 8776-8782Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Using this method, we show here that Rac2, the predominant isoform in human neutrophils (29Heyworth P.G. Bohl B.P. Bokoch G.M. Curnutte J.T. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar), is rapidly and transiently converted to the GTP-bound, active state, in response to the Gi-coupled receptor agonists fMLP and LTB4. The activation appears to require PI3K that is known to be stimulated by the Gi signaling in neutrophils (8Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar,30Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar, 31Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar, 32Ding J. Vlahos C.J. Liu R. Brown R.F. Badwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 33Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar). On the other hand, PMA induces a slow but more sustained activation of Rac2, which is dependent on PKC but independent of PI3K. fMLP and wortmannin were purchased from Wako Chemical (Osaka, Japan), LTB4 from Cayman Chemical (Ann Arbor, MI), PMA and pertussis toxin (PTX) from Research Biochemicals International (Natick, MA), and LY294002 and GF109203X from Biomol Research Laboratories (Plymouth Meeting, PA). Other chemicals used were of the highest purity commercially available. We isolated a DNA fragment encoding the Rac-binding domain of human PAK2 (PAK2-RBD; amino acids 66–147) from total RNA of the neuroblastoma cell line SH-SY5Y by reverse transcriptase-polymerase chain reaction, according to the protocol of the manufacturer (Perkin Elmer). The polymerase chain reaction product was subcloned into pGEX-4T (Amersham Pharmacia Biotech), and subjected to DNA sequencing for the confirmation of precise construction. The GST fusion protein was expressed in E. coli BL21 cells and purified by glutathione-Sepharose-4B (18Sumimoto 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 (254) Google Scholar, 20Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 21Hata K. Ito T. Takeshige K. Sumimoto H. J. Biol. Chem. 1998; 273: 4232-4236Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The DNA fragments encoding human Cdc42 and RhoA were obtained by reverse transcriptase-polymerase chain reaction and cloned into pGEX-2T (Amersham Pharmacia Biotech). The cDNAs for human Rac1 and Rac2 subcloned into pGEX-2T were prepared as described previously (17Mizuki K. Kadomatsu K. Hata K. Ito T. Fan Q.-W. Kage Y. Fukumaki Y. Sakaki Y. Takeshige K. Sumimoto H. Eur. J. Biochem. 1998; 251: 573-582Crossref PubMed Scopus (67) Google Scholar). The identities of all the constructs were verified by DNA sequencing. The GST fusion proteins were prepared as described above, followed by cleavage with thrombin, according to the protocol of the manufacturer (Amersham Pharmacia Biotech). Recombinant Rac1 or Rac2 was incubated for 30 min at 30 °C in 90 μl of a nucleotide-exchange buffer (137 mmNaCl, 2.7 mm KCl, 2 mm EDTA, 4.3 mmNa2HPO4, and 1.4 mmKH2PO4, pH 7.0) in the presence of 11 mm GTPγS, GTP, GDP, ATP, CTP, or UTP. The exchange reaction was terminated by the addition of 10 μl of 100 mm MgCl2. The nucleotide-loaded proteins were incubated at 4 °C with GST-PAK2-RBD and glutathione-Sepharose-4B beads in buffer A (20 mm Hepes, pH 7.4, 142.5 mm NaCl, 1% Nonidet P-40, 10% glycerol, 4 mmEGTA, 4 mm EDTA). After washing the beads three times with buffer A, the samples were subjected to 12% SDS-PAGE, and stained with Coomassie Brilliant Blue. Human neutrophils were isolated from fresh venous blood of healthy volunteers by dextran sedimentation, hypotonic lysis, and the Conray/Ficoll method (10Sumimoto H. Takeshige K. Minakami S. Biochim. Biophys. Acta. 1984; 803: 271-277Crossref PubMed Scopus (78) Google Scholar, 18Sumimoto 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 (254) Google Scholar). More than 98% of the cells were neutrophils in the preparation. The neutrophils (2.8 × 107 cells) in 700 μl of Hepes-buffered saline (HBS; 20 mm Hepes, pH 7.4, 135 mm NaCl, 5 mm KCl, 2 mm glucose, 1 mm MgSO4, and 0.6 mmCaCl2) were preincubated in the presence (for stimulation with fMLP or LTB4) or absence (for stimulation with PMA) of cytochalasin B (5 μg/ml) at 37 °C for 5 min and subsequently stimulated with indicated concentrations of fMLP, LTB4, or PMA. Where indicated, human neutrophils were preincubated for 2 h at 37 °C in the presence or absence of PTX (8 μg/ml), followed by stimulation with fMLP, LTB4, or PMA. The reaction was stopped by the addition of the same volume of the lysis buffer (20 mm Hepes, pH 7.4, 150 mm NaCl, 2% Nonidet P-40, 20% glycerol, 8 mm EGTA, 8 mm EDTA, 80 μm p-amidinophenylmethanesulfonyl fluoride (hydrochloride), 100 μg/ml of aprotinin, and 200 μg/ml each of leupeptin, chymostatin, and pepstatin A). The lysate was centrifuged for 20 s at 12,000 ×g, and the supernatant was incubated on ice for 3 min with GST-PAK2-RBD, which had been freshly coupled with glutathione-Sepharose-4B beads. Proteins complexed with the beads were recovered by centrifugation, washed two times with buffer A, and resuspended in Laemmli sample buffer. The proteins were resolved by 12% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was probed with anti-Rac1 antibody (C-11) or anti-Rac2 antibody (C-11) (Santa Cruz Biotechnology), and detection was performed using horseradish peroxidase-conjugated donkey anti-rabbit antibody and the ECL plus detection kit (both from Amersham Pharmacia Biotech). Human neutrophils suspended in 1 ml of HBS were preincubated for 5 min at 37 °C and subsequently stimulated with PMA or fMLP. The superoxide-producing activity was determined, as described previously (10Sumimoto H. Takeshige K. Minakami S. Biochim. Biophys. Acta. 1984; 803: 271-277Crossref PubMed Scopus (78) Google Scholar, 18Sumimoto 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 (254) Google Scholar). To test the validity of the RBD of human PAK2 (amino acids 66 to 147) as a tool to identify the active GTP-bound state of Rac, we expressed and purified the domain as a GST fusion protein (GST-PAK2-RBD). Recombinant Rac1 and Rac2 preloaded with GTPγS were precipitated using glutathione-Sepharose-4B beads coupled to the fusion protein, washed three times, and analyzed by SDS-PAGE (Fig. 1). Under the conditions, the binding of the active Rac1/2 to GST-PAK2-RBD appeared stable because further washing did not affect the recovery of the GTPases (data not shown). The same results were obtained when GTP was loaded instead of GTPγS (data not shown). On the other hand, the beads coupled to GST-PAK2-RBD did not retain the wild-type Rac1 and Rac2 in GDP-bound states (Fig. 1). When Rac2 was loaded with ATP, CTP, or UTP, no interaction with the GST fusion protein was observed (data not shown). In addition, neither GTP- nor GTPγS-bound Rac interacted with the beads coupled to GST alone (data not shown). Thus PAK2-RBD specifically recognizes the GTP-bound, active state of Rac to form a stable complex and thereby can be used as an activation-specific probe for Rac. After human neutrophils were stimulated with the Gi-coupled receptor agonist fMLP and then lysed, Rac was precipitated with GST-PAK2-RBD bound to glutathione-Sepharose beads and identified by Western blot with a polyclonal antibody against Rac2. This antibody is specific to Rac2 and does not recognize other members of the Rho family GTPases such as Rac1, Cdc42, or RhoA (data not shown). As shown in Fig.2 A, stimulation with fMLP led to a rapid and transient increase in the amount of Rac2 that bound to PAK2-RBD: the increase occurred within 30 s and reached its maximum level at 1 min. The amounts of Rac2 detected were dependent on the concentration of fMLP (Fig. 2 B). Increases do not seem to be because of changes in Rac protein levels because the same amount of Rac2 was detected during the stimulation in the whole cell lysates by Western blot analysis (data not shown). At the maximal level, about 10–12% of total Rac2 could be precipitated with GST-PAK2-RBD, when estimated by Western blot using various amounts of the whole cell lysates (Fig. 2 C). In contrast with the GST fusion protein, GST alone bound to the beads failed to precipitate Rac2 in the fMLP-stimulated cells (data not shown). When an antibody specific to Rac1 was used instead of the anti-Rac2 antibody, no specific bands on Western blot were observed (data not shown). This is consistent with Rac2 being largely predominant (>96%) in human neutrophils (29Heyworth P.G. Bohl B.P. Bokoch G.M. Curnutte J.T. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar). Since PAK2-RBD associates exclusively with the GTP-bound state of Rac2, with no detectable affinity for the GDP-bound protein (Fig. 1), we conclude that fMLP induces a rapid and transient conversion of Rac2 to the GTP-bound active state. LTB4, another Gi-coupled receptor agonist, also caused a rapid and transient activation of Rac2 in a dose-dependent manner (Fig. 2, A and B). The maximal amount of the GTP-bound Rac2 formed by LTB4 (about 10% of total Rac2) was slightly less than that in response to fMLP (Fig.2 C). Human neutrophils, in response to fMLP, produce superoxide, the production which is catalyzed by the phagocyte NADPH oxidase that is activated upon cell stimulation (31Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar, 32Ding J. Vlahos C.J. Liu R. Brown R.F. Badwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 33Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar, 34Watson F. Robinson J. Edwards S.W. J. Biol. Chem. 1991; 266: 7432-7439Abstract Full Text PDF PubMed Google Scholar). LTB4 also triggers superoxide production but to a lesser extent (10Sumimoto H. Takeshige K. Minakami S. Biochim. Biophys. Acta. 1984; 803: 271-277Crossref PubMed Scopus (78) Google Scholar). The kinetics of the production by these agonists, i.e. rapid onset and short duration of 1–2 min (10Sumimoto H. Takeshige K. Minakami S. Biochim. Biophys. Acta. 1984; 803: 271-277Crossref PubMed Scopus (78) Google Scholar, 32Ding J. Vlahos C.J. Liu R. Brown R.F. Badwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 34Watson F. Robinson J. Edwards S.W. J. Biol. Chem. 1991; 266: 7432-7439Abstract Full Text PDF PubMed Google Scholar), bear a resemblance to those of Rac2 activation (Fig. 2 A), which suggests that the agonist-induced conversion of Rac2 to the active state is linked to the NADPH oxidase activation. We next tested whether PMA, a potent inducer of superoxide production (34Watson F. Robinson J. Edwards S.W. J. Biol. Chem. 1991; 266: 7432-7439Abstract Full Text PDF PubMed Google Scholar), affects states of Rac2 in neutrophils or not. As shown in Fig. 2 B, PMA induced Rac2 activation in a dose-dependent manner. The activation occurred slowly but in a more sustained manner: it was observed as early as 1–2 min and reached a maximum after 5–10 min, followed by a decrease at 20 min (Fig. 2 A). The time course also resembles that of PMA-triggered activation of the NADPH oxidase (Ref. 34Watson F. Robinson J. Edwards S.W. J. Biol. Chem. 1991; 266: 7432-7439Abstract Full Text PDF PubMed Google Scholar; and data not shown). Approximately 7–8% of total Rac2 was converted to the GTP-bound state by PMA at the maximum (Fig. 2 C). It is well documented that both fMLP and LTB4 receptors on human neutrophils are coupled to the Gi class of heterotrimeric G-proteins (8Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar, 9Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (845) Google Scholar). The finding that both agonists cause activation of Rac2 in neutrophils (Fig. 2) suggests a role for Gi. To clarify the involvement of Gi in Rac2 activation, we tested the effect of PTX, which catalyzes ADP-ribosylation of Giα to uncouple Gi from the receptors (8Bokoch G.M. Blood. 1995; 86: 1649-1660Crossref PubMed Google Scholar). The toxin treatment of human neutrophils resulted in a complete loss of the fMLP-induced superoxide production, while it did not affect the PMA-induced one (data not shown). In the PTX-treated cells, neither fMLP nor LTB4 was capable of activating Rac2 (Fig. 3), indicating that coupling of the receptors to Gi is required for the Rac2 activation. On the other hand, the treatment did not affect PMA-triggered activation of Rac2 (Fig. 3). In human neutrophils, fMLP activates PI3K to generate PtdIns(3,4,5)P3 (30Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar, 31Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar, 32Ding J. Vlahos C.J. Liu R. Brown R.F. Badwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 33Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar). PI3K inhibitors block not only the generation of PtdIns(3,4,5)P3, but also superoxide production induced by fMLP (31Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar, 32Ding J. Vlahos C.J. Liu R. Brown R.F. Badwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 33Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar). To investigate the role of PI3K in Rac2 activation, we used wortmannin and LY294002, that specifically inhibit PI3K by distinct mechanisms (35Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar, 36Wymann M.P. Bulgarelli-Leva G. Zvelebil M.J. Pirola L. Vanhaesebroeck B. Waterfield M.D. Panayotou G. Mol. Cell. Biol. 1996; 16: 1722-1733Crossref PubMed Scopus (628) Google Scholar). As shown in Fig.4, A and B, fMLP-induced Rac2 activation was dose-dependently inhibited by wortmannin and by LY294002, respectively. Both inhibitors blocked fMLP-triggered superoxide production (Refs. 31Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar, 32Ding J. Vlahos C.J. Liu R. Brown R.F. Badwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 33Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar; and data not shown) and PtdIns(3,4,5)P3 generation (31Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar, 32Ding J. Vlahos C.J. Liu R. Brown R.F. Badwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 33Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar, 35Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar) in the same dose-dependent manner. LTB4-elicited activation of Rac2 was also sensitive to wortmannin or LY294002 (Fig.4 C). Thus the PI3K activity appears to be essential for the Gi-mediated activation of Rac2. PMA is incapable of activating PI3K in human neutrophils (30Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar). Consistent with this, the PI3K inhibitors failed to affect PMA-induced Rac2 activation (Fig. 4 D) as well as superoxide production (data not shown). We therefore conclude that PMA activates Rac2 independently of PI3K. To study the role of PKC in PMA-induced Rac 2 activation, we tested the effect of the bisindolylmaleimide derivative GF109203X, a potent and selective inhibitor of PKC (37Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar). In the presence of 0.1 μm GF109203X, the PMA-elicited superoxide production by neutrophils was reduced by about 50%, whereas fMLP was capable of fully triggering the production (Ref. 38Wenzel-Seifert K. Schächtele C. Seifert R. Biochem. Biophys. Res. Commun. 1994; 200: 1536-1543Crossref PubMed Scopus (49) Google Scholar; and data not shown). At a higher concentration (1.0 μm), where the PMA-elicited superoxide production was completely prevented, the fMLP-induced one was only partially (by about 30%) blocked (Ref. 38Wenzel-Seifert K. Schächtele C. Seifert R. Biochem. Biophys. Res. Commun. 1994; 200: 1536-1543Crossref PubMed Scopus (49) Google Scholar; and data not shown). As shown in Fig. 5, GF109203X effectively inhibited the increase in the amount of GTP-bound Rac2 in neutrophils stimulated by PMA but not by fMLP. Thus PMA appears to form the GTP-bound Rac2 by activating PKC, which is not likely involved in fMLP-triggered activation of the GTPase. In the present study, we have developed a novel method to directly detect the GTP-bound active Rac that is converted from the GDP-bound state in intact cells. Using this method, we show that the Gi-coupled receptor agonists fMLP and LTB4elicit a rapid and transient activation of Rac2 in human neutrophils and that the activation is mediated via not only Gi but also PI3K. On the other hand, PMA causes a slow but more sustained activation of Rac2 in a PI3K-independent manner. Recent studies have suggested that PI3K is located upstream of Rac in growth factor or integrin-induced cytoskeletal reorganization (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5165) Google Scholar, 2Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2079) Google Scholar, 3Narumiya S. J. Biochem. 1996; 120: 215-228Crossref PubMed Scopus (359) Google Scholar, 4Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1216) Google Scholar). This suggestion is, however, largely based on experiments using a constitutively active or a dominant negative form of Rac, but not on those evaluating extent of activation of Rac. The only exception has been a study by Hawkins et al. (39Hawkins P.T. Eguinoa A. Qiu R.-G. Stokoe D. Cooke F.T. Walters R. Wennström S. Claesson-Welsh L. Evans T. Symons M. Stephens L. Curr. Biol. 1995; 5: 393-403Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar), showing that, in [32P]Pi-preloaded endothelial cells that stably overexpress an epitope-tagged Rac1, platelet-derived growth factor stimulates an increase in the [32P]GTP content of this Rac protein to a significant but small extent (about 1.7-fold), which is inhibited by wortmannin. The present study, using the novel method, demonstrates that fMLP and LTB4 cause a drastic increase in the GTP-bound, active state of "endogenous" Rac2, and that this increase is effectively blocked by PI3K inhibitors. The blockade does not seem to be due to nonspecific effects, because the inhibitors do not affect PMA-induced activation of Rac2. Thus PI3K likely functions upstream of Rac2 in the Gi-coupled receptor signaling pathway in human neutrophils. The mechanism by which PI3K activates Rac2 in neutrophils is presently unknown. The Rac GTPases are directly activated by a family of GEFs related to the oncogene product Dbl that enhance the exchange of bound GDP for GTP on Rac (40Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar). All known Dbl-related molecules have a PH domain (40Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar), and some PH domains directly interact with PtdIns(4,5)P2 and PtdIns(3,4,5)P3, a substrate and a product of PI3K, respectively (41Hirata M. Kanematsu T. Takeuchi H. Yagisawa H. Jpn. J. Pharmacol. 1998; 76: 255-263Crossref PubMed Scopus (31) Google Scholar). Recent studies have shown that Rac activation by GEFs, such as Vav and Sos, appears to be regulated by binding of the lipids to the PH domain (42Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (708) Google Scholar, 43Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (387) Google Scholar). A similar regulation may occur in the PI3K-mediated activation of Rac2 in human neutrophils. In addition to the PI3K-dependent mechanism, GEFs for Rac can be regulated by posttranslational modifications such as phosphorylation by a protein kinase. It has been reported that tyrosine-phosphorylated Vav, but not the unphosphorylated protein, enhances GDP/GTP exchange on Rac (44Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (676) Google Scholar). In this context, it should be noted that PMA, a direct activator of PKC, increases the GTP-bound, active state of Rac2, independently of PI3K. Phosphorylation by PKC might regulate GEF(s) to activate Rac2 in neutrophils. Activation of Rac in stimulated neutrophils has been postulated to occur because the GTP-bound Rac, but not the GDP-bound protein, can activate the phagocyte NADPH oxidase in vitro (12Abo A. Pick E. Hall A. Totty N. Teahan C.G. Segal A.W. Nature. 1991; 353: 668-670Crossref PubMed Scopus (756) Google Scholar, 13Knaus U.G. Heyworth P.G. Evans T. Curnutte J.T. Bokoch G.M. Science. 1991; 254: 1512-1515Crossref PubMed Scopus (540) Google Scholar, 14Mizuno T. Kaibuchi K. Ando S. Musha T. Hiraoka K. Takaishi K. Asada M. Nunoi H. Matsuda I. Takai Y. J. Biol. Chem. 1992; 267: 10215-10218Abstract Full Text PDF PubMed Google Scholar). However, stimulus-dependent activation of Rac has not been demonstrated. This study, using the novel method, shows that three stimulants for the NADPH oxidase activation in vivo, fMLP, LTB4, and PMA, are all capable of activating Rac2 in human neutrophils. The kinetics of Rac2 activation by the stimulants correspond well with those of superoxide production. Although it is well documented that PI3K inhibitors block superoxide production triggered by fMLP but not that by PMA (31Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar, 32Ding J. Vlahos C.J. Liu R. Brown R.F. Badwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 33Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar), it has remained elusive how PI3K functions in the oxidase activation. As described above, PI3K is required for activation of Rac2 in the fMLP signaling, whereas PMA activates Rac2 in a PI3K-independent manner. Thus the present findings support the idea that Rac2 serves as a switch for the NADPH oxidase activation. We are grateful to Drs. K. Takeshige and D. Kang (Kyushu University) for encouragement and discussion.

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