Dissociation of Recruitment and Activation of the Small G-protein Rac during Fcγ Receptor-mediated Phagocytosis
2006; Elsevier BV; Volume: 281; Issue: 13 Linguagem: Inglês
10.1074/jbc.m513731200
ISSN1083-351X
AutoresCéline Cougoule, Saiko Hoshino, Anna Dart, Jenson Lim, Emmanuelle Caron,
Tópico(s)Cellular Mechanics and Interactions
ResumoRho-family proteins play a central role in most actin-dependent processes, including the control and maintenance of cell shape, adhesion, motility, and phagocytosis. Activation of these GTP-binding proteins is tightly regulated spatially and temporally; however, very little is known of the mechanisms involved in their recruitment and activation in vivo. Because of its inducible, restricted signaling, phagocytosis offers an ideal physiological system to delineate the pathways linking surface receptors to actin remodeling via Rho GTPases. In this study, we investigated the involvement of early regulators of Fcγ receptor signaling in Rac recruitment and activation. Using a combination of receptor mutagenesis, cellular, molecular, and pharmacological approaches, we show that Src family and Syk kinases control Rac and Vav function during phagocytosis. Importantly, both the immunoreceptor tyrosine-based activation motif within Fcγ receptor cytoplasmic domain and Src kinase control the recruitment of Vav and Rac. However, Syk activity is dispensable for Vav and Rac recruitment. Moreover, we show that Rac and Cdc42 activities coordinate F-actin accumulation at nascent phagosomes. Our results provide new insights in the understanding of the spatiotemporal regulation of Rho-family GTPase function, and of Rac in particular, during phagocytosis. We believe they will contribute to a better understanding of more complex cellular processes, such as cell adhesion and migration. Rho-family proteins play a central role in most actin-dependent processes, including the control and maintenance of cell shape, adhesion, motility, and phagocytosis. Activation of these GTP-binding proteins is tightly regulated spatially and temporally; however, very little is known of the mechanisms involved in their recruitment and activation in vivo. Because of its inducible, restricted signaling, phagocytosis offers an ideal physiological system to delineate the pathways linking surface receptors to actin remodeling via Rho GTPases. In this study, we investigated the involvement of early regulators of Fcγ receptor signaling in Rac recruitment and activation. Using a combination of receptor mutagenesis, cellular, molecular, and pharmacological approaches, we show that Src family and Syk kinases control Rac and Vav function during phagocytosis. Importantly, both the immunoreceptor tyrosine-based activation motif within Fcγ receptor cytoplasmic domain and Src kinase control the recruitment of Vav and Rac. However, Syk activity is dispensable for Vav and Rac recruitment. Moreover, we show that Rac and Cdc42 activities coordinate F-actin accumulation at nascent phagosomes. Our results provide new insights in the understanding of the spatiotemporal regulation of Rho-family GTPase function, and of Rac in particular, during phagocytosis. We believe they will contribute to a better understanding of more complex cellular processes, such as cell adhesion and migration. Reorganization of the actin cytoskeleton drives cell shape changes during processes such as cell adhesion, migration and chemotaxis, bacterial invasion, immune synapse formation, and phagocytosis (1Cougoule C. Wiedemann A. Lim J. Caron E. Semin. Cell Dev. Biol. 2004; 15: 679-689Crossref PubMed Scopus (46) Google Scholar, 2Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3796) Google Scholar). Generally induced locally, these morphological changes originate from the activation of a given set of cell surface receptors and the induction of signaling pathways that activate Rho family GTP-binding proteins, which control localized actin polymerization. Understanding how Rho-GTPase signaling is locally activated is therefore crucial to many essential aspects of molecular cell biology.Rho proteins cycle between inactive GDP-bound and active GTP-bound conformations. Only the latter can bind downstream effectors. Conversion to the GTP-bound form is catalyzed by guanine nucleotide exchange factors (GEFs), 3The abbreviations used are: GEF, guanine nucleotide exchange factor; FcγR, Fcγ receptor; ITAM, immunoreceptor tyrosine-based activation motif; SRBC, sheep red blood cells; PBS, phosphate-buffered saline; GFP, green fluorescent protein; WT, wild-type. 3The abbreviations used are: GEF, guanine nucleotide exchange factor; FcγR, Fcγ receptor; ITAM, immunoreceptor tyrosine-based activation motif; SRBC, sheep red blood cells; PBS, phosphate-buffered saline; GFP, green fluorescent protein; WT, wild-type. whereas inactivation back to the GDP-bound form is mediated by GTPase-activating proteins. Inducible activation of Rho GTPases requires their targeting to membranes, where they will interact with both GEFs and downstream targets (3Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2038) Google Scholar). Although Rho proteins can bind to membranes directly, by virtue of their posttranslationally added prenylation, their subcellular localization is also regulated, as Rho GTP-binding proteins are held soluble and inactive in the cytosol through an interaction between their prenyl moiety and the effector region of GDP dissociation inhibitor proteins (4Dransart E. Morin A. Cherfils J. Olofsson B. J. Biol. Chem. 2005; 280: 4674-4683Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The mechanisms by which Rho proteins are recruited and activated in particular regions of the cells to control actin polymerization are still poorly understood. Furthermore, the deciphering of these mechanisms is complicated by several factors, for example by the fact that different Rho-family members can be involved in a given cellular process, leading to synergistic or antagonistic cross-talk, as shown during cell migration or adherens junction formation (5Noren N.K. Niessen C.M. Gumbiner B.M. Burridge K. J. Biol. Chem. 2001; 276: 33305-33308Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 6Sander E.E. ten Klooster J.P. van Delft S. van der Kammen R.A. Collard J.G. J. Cell Biol. 1999; 147: 1009-1022Crossref PubMed Scopus (732) Google Scholar). Because of its spatiotemporally restricted and inducible signaling, the phagocytic uptake of model particles offers an ideal biological system to delineate the pathways linking the localized ligation of surface receptors to actin remodeling via Rho proteins (1Cougoule C. Wiedemann A. Lim J. Caron E. Semin. Cell Dev. Biol. 2004; 15: 679-689Crossref PubMed Scopus (46) Google Scholar).Phagocytosis is the mechanism by which cells take up large particles (>0.5 μm) into an intracellular compartment, the phagosome (7Aderem A. Underhill D.M. Annu. Rev. Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2050) Google Scholar). Phagocytic uptake is initiated by the direct or opsonin-mediated recognition of ligands exposed on the particle surface by specific receptors present at the surface of phagocytic cells (e.g. macrophages, neutrophils, and receptor-transfected cells). Receptor clustering triggers intracellular signaling pathways that lead to the reorganization of the actin cytoskeleton, which is essential for particle uptake. The receptor for the Fc portion of immunoglobulins (FcγR) is the best studied phagocytic receptor (8Castellano F. Chavrier P. Caron E. Semin. Immunol. 2001; 13: 347-355Crossref PubMed Scopus (129) Google Scholar, 9Greenberg S. Grinstein S. Curr. Opin. Immunol. 2002; 14: 136-145Crossref PubMed Scopus (434) Google Scholar, 10Wiedemann A. Lim J. Caron E. Rosales C. Molecular Mechanisms of Phagocytosis. Landes Bioscience, Georgetown, TX2004: 72-84Google Scholar). Binding of IgG-opsonized particles to FcγR induces the activation of Src kinases, which phosphorylate two tyrosines in the receptor immunoreceptor tyrosine-based activation motif (ITAM) domain (11Ghazizadeh S. Bolen J.B. Fleit H.B. J. Biol. Chem. 1994; 269: 8878-8884Abstract Full Text PDF PubMed Google Scholar). ITAM phosphorylation is crucial for the formation of actin-rich cups and thus for particle uptake, as shown in experiments using hck/lyn/fgr-deficient mice, FcγR mutants, or Src kinase inhibitors (12Fitzer-Attas C.J. Lowry M. Crowley M.T. Finn A.J. Meng F. DeFranco A.L. Lowell C.A. J. Exp. Med. 2000; 191: 669-682Crossref PubMed Scopus (195) Google Scholar, 13Greenberg S. Chang P. Silverstein S.C. J. Exp. Med. 1993; 177: 529-534Crossref PubMed Scopus (168) Google Scholar, 14Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1781) Google Scholar, 15Majeed M. Caveggion E. Lowell C.A. Berton G. J. Leukocyte Biol. 2001; 70: 801-811PubMed Google Scholar). Phosphorylated ITAM motifs act as docking sites for a non-receptor tyrosine kinase, Syk, which undergoes autophosphorylation and whose activation is necessary for phagocytosis (16Kiefer F. Brumell J. Al-Alawi N. Latour S. Cheng A. Veillette A. Grinstein S. Pawson T. Mol. Cell Biol. 1998; 18: 4209-4220Crossref PubMed Google Scholar, 17Crowley M.T. Costello P.S. Fitzer-Attas C.J. Turner M. Meng F. Lowell C. Tybulewicz V.L. DeFranco A.L. J. Exp. Med. 1997; 186: 1027-1039Crossref PubMed Scopus (404) Google Scholar). Moreover, surface expression of a FcγR/Syk chimera is sufficient to stimulate particle uptake (18Greenberg S. Chang P. Wang D.C. Xavier R. Seed B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1103-1107Crossref PubMed Scopus (112) Google Scholar). If Syk activation clearly lies downstream of Src kinase activity as demonstrated in hck/lyn/fgr-deficient macrophages (12Fitzer-Attas C.J. Lowry M. Crowley M.T. Finn A.J. Meng F. DeFranco A.L. Lowell C.A. J. Exp. Med. 2000; 191: 669-682Crossref PubMed Scopus (195) Google Scholar, 17Crowley M.T. Costello P.S. Fitzer-Attas C.J. Turner M. Meng F. Lowell C. Tybulewicz V.L. DeFranco A.L. J. Exp. Med. 1997; 186: 1027-1039Crossref PubMed Scopus (404) Google Scholar), its role in actin polymerization downstream of FcγR is still controversial. On one hand, actin polymerized normally at nascent phagosomes in macrophages derived from Syk-deficient mice (16Kiefer F. Brumell J. Al-Alawi N. Latour S. Cheng A. Veillette A. Grinstein S. Pawson T. Mol. Cell Biol. 1998; 18: 4209-4220Crossref PubMed Google Scholar). On the other hand, actin polymerization was impaired in FcγR-transfected DT40 lymphocytes engineered to lack Syk (19Cox D. Chang P. Kurosaki T. Greenberg S. J. Biol. Chem. 1996; 271: 16597-16602Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), whereas piceatannol, a specific inhibitor of Syk (20Oliver J.M. Burg D.L. Wilson B.S. McLaughlin J.L. Geahlen R.L. J. Biol. Chem. 1994; 269: 29697-29703Abstract Full Text PDF PubMed Google Scholar), blocked actin accumulation at nascent phagosomes in murine macrophages (15Majeed M. Caveggion E. Lowell C.A. Berton G. J. Leukocyte Biol. 2001; 70: 801-811PubMed Google Scholar). Nevertheless, taken together, these data show that Src kinase activity and ITAM-dependent signaling are essential for actin polymerization and particle uptake during FcγR-mediated phagocytosis.Rho GTP-binding proteins play a critical role during FcγR-mediated uptake, as inhibition of either Rac or Cdc42 blocks phagocytosis (19Cox D. Chang P. Kurosaki T. Greenberg S. J. Biol. Chem. 1996; 271: 16597-16602Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (799) Google Scholar, 22Massol P. Montcourrier P. Guillemot J.C. Chavrier P. EMBO J. 1998; 17: 6219-6229Crossref PubMed Scopus (198) Google Scholar). Rac and Cdc42 are activated and recruited to forming phagosomes in response to IgG-particle challenge (21Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (799) Google Scholar, 23Patel J.C. Hall A. Caron E. Mol. Biol. Cell. 2002; 13: 1215-1226Crossref PubMed Scopus (103) Google Scholar, 24Niedergang F. Colucci-Guyon E. Dubois T. Raposo G. Chavrier P. J. Cell Biol. 2003; 161: 1143-1150Crossref PubMed Scopus (149) Google Scholar). Elegant fluorescence resonance energy transfer studies have recently confirmed that active forms of Rac and Cdc42 accumulate rapidly, coincidentally with F-actin, underneath bound IgG-opsonized particles (25Hoppe A.D. Swanson J.A. Mol. Biol. Cell. 2004; 15: 3509-3519Crossref PubMed Scopus (274) Google Scholar). It is thought that each of these two Rho proteins controls the local recruitment of the Arp2/3 complex, thereby actin polymerization at phagosomes (26May R.C. Caron E. Hall A. Machesky L.M. Nat. Cell Biol. 2000; 2: 246-248Crossref PubMed Scopus (265) Google Scholar). Nevertheless, the role of Rac and Cdc42 in actin polymerization at forming phagosomes is still controversial, as two independent studies have reported that expression of dominant negative Rac or Cdc42 did not completely block F-actin accumulation at nascent phagosomes, although each individually inhibited FcγR-mediated phagocytosis (19Cox D. Chang P. Kurosaki T. Greenberg S. J. Biol. Chem. 1996; 271: 16597-16602Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 22Massol P. Montcourrier P. Guillemot J.C. Chavrier P. EMBO J. 1998; 17: 6219-6229Crossref PubMed Scopus (198) Google Scholar). Another unresolved issue is whether Rac and Cdc42 are recruited in their active form or locally activated. Our published data support the latter, as the inactive (N17) form of Rac is recruited to nascent phagosomes (21Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (799) Google Scholar). Furthermore, the recruitment of Rac (either N17 or wild-type) to nascent phagosomes occurs in the absence of Vav-exchange activity, despite the fact that Vav activity is necessary for Rac activation and phagocytosis (23Patel J.C. Hall A. Caron E. Mol. Biol. Cell. 2002; 13: 1215-1226Crossref PubMed Scopus (103) Google Scholar).Intriguingly, whether and how the tyrosine kinase and Rho signaling pathways are connected to control actin polymerization and phagocytosis downstream of FcγR has not yet been elucidated. Because activation of Src and Syk kinases corresponds to the first signaling events detected after binding of IgG-opsonized particles, they are good candidates to control small GTPase function during phagocytosis. Src and Syk kinases have been described to phosphorylate Vav and enhance Vav exchange factor activity in vitro as well as during T- and B-cell receptor signaling (27Han 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 (710) Google Scholar, 28Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (244) Google Scholar). In line with this, we have shown that Syk is still recruited to nascent phagosomes in macrophages expressing dominant negative Vav, suggesting that Vav acts downstream of initial FcγR signaling (23Patel J.C. Hall A. Caron E. Mol. Biol. Cell. 2002; 13: 1215-1226Crossref PubMed Scopus (103) Google Scholar).Herein, we show that early FcγR signaling controls recruitment and Vav-dependent activation of Rac and actin polymerization. Remarkably, the kinase activity of Src, but not Syk, controls Rac and Vav recruitment to nascent phagosomes, whereas Syk activity is essential for Rac activation, and therefore, for particle uptake. We also show that Rac activation is dispensable for actin polymerization at nascent phagosomes although necessary to coordinate with Cdc42-actin polymerization and engulfment during FcγR-mediated phagocytosis.EXPERIMENTAL PROCEDURESDNA Constructs—Eucaryotic pRK5 expression vectors encoding human FcγRIIA (FcγR), myc-tagged N17Rac, N17Cdc42, Wasp (amino acids 201–310), Vav wild-type (Vav-WT), VavC (comprising just the carboxyl-terminal SH3-SH2-SH3 domains), VavΔ342-348 (containing a 6-amino-acid deletion in the catalytic DH domain, VavΔDH), pEGFP-p59HckWT and pEGFP-RacWT have been previously described (21Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (799) Google Scholar, 23Patel J.C. Hall A. Caron E. Mol. Biol. Cell. 2002; 13: 1215-1226Crossref PubMed Scopus (103) Google Scholar, 29Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Hemagglutinin-tagged pCMV-VavΔ342-348 was a kind gift from Charles Abrams (University of Pennsylvania, Philadelphia, PA).Plasmids expressing truncated versions of the FcγR were generated from the pRK5-FcγRIIA template by inverse PCR using the Expand Long Template PCR System (Roche Applied Science) using the following primers: 5′-tcagatcttcatttcctgcagtagatcaa-3′ and 5′-gaagatcttctagagtcgacctgcag-3′ for FcγRΔ240; 5′-tcagatcttcattggtttcttcaagttgt-3′ and 5′-gaagatcttctagagtcgacctgcag-3′ for FcγRΔ275. The resulting PCR products, which contained terminal BglII restriction sites were digested and religated overnight at 16 °C. To generate the FcγR(Y/F)2 mutant, tyrosines 282 and 298 were sequentially substituted with phenylalanines on pRK5-FcγRIIA by reverse PCR, using the QuikChange site-directed mutagenesis kit (Stratagene). We used the following combinations of primers (mutation underlined): 5′-gctgacggcggcttcatgactctgaacccC-3′ and 5′-ggggttcagagtcatgaagccgccgtcagc-3′ to introduce the Y282F substitution; 5′-cgatgataaaaacatcttcctgactcttcc-3′ and 5′-ggaagagtcaggaagatgtttttatcatcg-3′ for the Y298F mutation. Mutagenesis products were transformed into One Shot TOP10 chemically competent Escherichia coli (Invitrogen), according to the manufacturer's instructions. All constructions were checked by DNA sequencing (MWG), amplified, and prepared for transfection using the Qiagen maxi-prep kit.Antibodies and Drugs—Mouse monoclonal anti-Rac (clone23A8) and anti-phosphotyrosine (clone 4G10) antibodies were purchased from Upstate Biotechnology, the anti-myc antibody (clone 9E10) was from Santa Cruz Biotechnology, the rabbit anti-Vav polyclonal antibody was from Transduction Laboratories, and the rat monoclonal anti-hemagglutinin antibody (clone 3F10) was from Roche Applied Science. All conjugated secondary antibodies (donkey) were purchased from Jackson ImmunoResearch Laboratories. PP2 and piceatannol were purchased from Calbiochem-Biosciences and dissolved in dimethyl sulfoxide (Me2SO). The maximum final concentration of Me2SO never exceeded 0.1% (v/v) in vehicle- or drug-treated cells.Cell Culture and Transfection—Cells from the murine macrophage J774.A1 and simian kidney fibroblast COS-7 cell lines were maintained in complete medium, Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (PAA laboratories), and penicillin/streptomycin (100 units/ml and 100 μg/ml, respectively, Invitrogen). COS-7 cells were seeded on coverslips in 6-cm dishes (105cells/dish) and transfected with the calcium/phosphate protocol as described previously (29Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, DNA/calcium phosphate precipitates (10 μg of DNA/400 μl of calcium phosphate/6-cm dishes containing 3.6 ml of fresh complete medium) were added onto the cells for 16–18 h, washed, and incubated in fresh complete medium for an additional 6 h.Drug Treatments and Phagocytosis Assay—Transfected COS-7 were transferred to 10 mm Hepes-buffered serum-free Dulbecco's modified Eagle's medium (SFM) for a period of 18 h and then incubated with drugs for 30 min. Drugs were maintained in the medium during a challenge with IgG-opsonized sheep red blood cells (SRBC, Cappel) prepared as previously described (21Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (799) Google Scholar, 23Patel J.C. Hall A. Caron E. Mol. Biol. Cell. 2002; 13: 1215-1226Crossref PubMed Scopus (103) Google Scholar). Briefly, 0.4 μl of SRBC were opsonized with rabbit anti-SRBC IgG during 30 min in 1 ml of gelatin veronal buffer (Sigma) and washed once with gelatin veronal buffer. IgG-SRBC were allowed to adhere for 15 min at 4 °C and synchronized phagocytosis was induced for 15 min at 37 °C.Immunofluorescence—Cells were washed once with phosphate-buffered saline (PBS) and fixed in cold 4% (w/v) paraformaldehyde for 15 min at 4 °C. Free aldehyde groups were neutralized in 13.3 mg/ml of NH4Cl/PBS for 10 min. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min and blocked with 1% bovine serum albumin/PBS for 15 min. For immunostaining, cells were incubated for 30 min with antibodies diluted in 1% bovine serum albumin/PBS supplemented with excess human IgG (Sigma) to prevent nonspecific binding of the antibodies to Fcγ receptors. FITC-, rhodamine- and Cy5-conjugated donkey anti-rabbit IgG were used to detect opsonized SRBC. Myc-tagged constructs were visualized using mouse monoclonal anti-myc followed by fluorescein isothiocyanate-conjugated anti-mouse IgG. F-actin was stained using Alexa546-conjugated phalloidin (Molecular Probes). Coverslips were mounted in Mowiol mountant (Calbiochem), and images were captured using a Zeiss LSM 510 confocal microscope.Determination of Rac Activity Levels—The Cdc42/Rac interactive-binding domain of Pak1 (PAK-CRIB) fused to glutathione S-transferase (GST) was prepared for GTPase pull-down assays as previously described (23Patel J.C. Hall A. Caron E. Mol. Biol. Cell. 2002; 13: 1215-1226Crossref PubMed Scopus (103) Google Scholar). J774.A1 macrophages seeded at 107 cells/14-cm dish were starved in 12 ml of 10 mm Hepes-buffered SFM for 2 h and challenged with 50 μl of IgG-SRBC/plate for 20 min at 37 °C. Cells were then scraped on ice in 800 μl of pull-down buffer (PD buffer, 50 mm Tris-HCl, pH7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 10 mm MgCl2, 1 μg/ml protease inhibitor mixture (Roche Applied Science), 1 mm phenylmethylsulfonyl fluoride) per plate. Lysates were cleared by centrifugation at 10,000 rpm for 1 min at 4 °C. 700 μl of supernatant were used to assess the levels of active Rac using the Pak-CRIB pull-down method (30Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). Briefly, lysates were incubated for 45 min at 4 °C with 10 mg of a 50% slurry of PAK-CRIB-GST coupled to glutathione-agarose beads to precipitate GTP-loaded GTPases. Beads were subsequently washed three times in cold, modified PD buffer (without deoxycholate or SDS) and resuspended in 20 μl of 2× sample buffer. Equal amounts of beads and total cell lysates were analyzed by SDS-PAGE and immunoblotting, using a monoclonal anti-Rac antibody. Fold activation of Rac was assessed, relative to the level of Rac in the total cell lysate, by quantification of autoradiographic exposures using ImageJ software.Analysis of Vav Phosphorylation Level—J774.A1 macrophages seeded at 2 × 106 cells/10-cm dish were starved in 10 mm Hepes-containing SFM during 1.5 h, treated with the drugs for an additional 30 min, then challenged with 15 μl of IgG-SRBC in 3 ml of cold Hepes-buffered SFM for 15 min at 4 °C. Cells were washed once to remove unbound particles, reincubated at 37 °C for 10 min, washed again on ice with 10 ml of ice-cold PBS, and scraped in 200 μl of lysis buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, protease inhibitor mixture 1 μg/ml, 1 mm Na3VO4-, 1 mm NaF). Whole cell lysates were analyzed by SDS-PAGE and immunoblotting, using a monoclonal anti-phosphotyrosine antibody. Total Vav levels were assessed after membrane stripping and immunoblotting. Fold phosphorylation of Vav was related to the level of Vav in the total cell lysate, by quantification of autoradiographic exposures using the ImageJ software.Scoring—Binding and phagocytosis indices were determined by counting the number of particles respectively associated to and internalized by 100 cells, whether J774.A1 or FcγR-expressing COS-7 cells. Protein recruitment to nascent phagosomes was scored blindly by confocal microscopy and is expressed as the percent positive phagosomes out of a population of >100 phagosomes, corresponding to an average of 20 cells/coverslip.Statistics—A paired t test was used to determine the statistical significance of the results. Data sets were considered different for p values < 0.05.RESULTSSrc and Syk kinase activities are necessary for FcγR-mediated phagocytosis (12Fitzer-Attas C.J. Lowry M. Crowley M.T. Finn A.J. Meng F. DeFranco A.L. Lowell C.A. J. Exp. Med. 2000; 191: 669-682Crossref PubMed Scopus (195) Google Scholar, 15Majeed M. Caveggion E. Lowell C.A. Berton G. J. Leukocyte Biol. 2001; 70: 801-811PubMed Google Scholar, 16Kiefer F. Brumell J. Al-Alawi N. Latour S. Cheng A. Veillette A. Grinstein S. Pawson T. Mol. Cell Biol. 1998; 18: 4209-4220Crossref PubMed Google Scholar). Moreover, the first detectable signaling events after particle binding are Src-mediated tyrosine phosphorylation of the FcγR ITAM domain and the accumulation to nascent phagosomes of several phosphorylated proteins, including Src kinases themselves and the Src substrate Syk (31Cox D. Greenberg S. Semin. Immunol. 2001; 13: 339-345Crossref PubMed Scopus (75) Google Scholar). We used a pharmacological approach to investigate the role of Src and Syk kinases in the activation of Rac signaling. PP2, a selective inhibitor of Src-family kinases (14Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1781) Google Scholar), and piceatannol, a selective Syk inhibitor (20Oliver J.M. Burg D.L. Wilson B.S. McLaughlin J.L. Geahlen R.L. J. Biol. Chem. 1994; 269: 29697-29703Abstract Full Text PDF PubMed Google Scholar), were titrated for inhibition of FcγR-mediated phagocytosis in J774.A1 macrophages (data not shown). Compared with control, Me2SO-treated cells, macrophages treated with PP2 (10 μm), and piceatannol (75 μm) exhibited a marked reduction in their FcγR-dependent ability to phagocytose but were as competent in binding particles, confirming that the drugs specifically act on phagocytic signaling (Fig. 1A). Me2SO treatment did not affect the binding and phagocytic properties of any of the cell types we used (data not shown).We next examined the effect of these drugs on FcγR-induced Rac activation. Endogenous GTP-loaded Rac was precipitated from lysates of control, drug-treated and SRBC-challenged J774.A1 using the Rac/Cdc42 binding fragment of p21-activated kinases (PAK) fused to GST (GST-PAK-CRIB). Drug treatments per se had no reproducible effect on RacGTP levels in unchallenged cells (mean ± S.D. of 1.7 ± 0.8 for PP2 and 1.2 ± 1 for piceatannol). As already described for FcγR-transfected COS-7 cells (23Patel J.C. Hall A. Caron E. Mol. Biol. Cell. 2002; 13: 1215-1226Crossref PubMed Scopus (103) Google Scholar) and RAW 264.7 macrophages (24Niedergang F. Colucci-Guyon E. Dubois T. Raposo G. Chavrier P. J. Cell Biol. 2003; 161: 1143-1150Crossref PubMed Scopus (149) Google Scholar), challenge of J774.A1 macrophages with IgG-SRBC triggered a transient increase in the amount of GTP-loaded Rac in control Me2SO-treated cells after 10 min at 37 °C (Fig. 1B and data not shown). The SRBC-dependent, FcγR-mediated increase in RacGTP levels was blocked in PP2- and piceatannol-treated cells (Fig. 1B), indicating that Src and Syk kinase activities are necessary to trigger Rac activation during FcγR-mediated phagocytosis.Rac has been described to control actin polymerization at nascent phagosomes (26May R.C. Caron E. Hall A. Machesky L.M. Nat. Cell Biol. 2000; 2: 246-248Crossref PubMed Scopus (265) Google Scholar). We thus examined the effect of each Rac-inhibiting treatment on the accumulation of F-actin underneath bound SRBC. In J774.A1 macrophages, PP2, but not piceatannol, treatment blocked actin polymerization at nascent phagosomes (Fig. 2). These results were confirmed in FcγR-transfected COS-7 cells, a phagocytic model that recapitulates all steps and signaling pathways of phagocytosis analyzed so far (see above (21Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (799) Google Scholar, 23Patel J.C. Hall A. Caron E. Mol. Biol. Cell. 2002; 13: 1215-1226Crossref PubMed Scopus (103) Google Scholar, 32Downey G.P. Botelho R.J. Butler J.R. Moltyaner Y. Chien P. Schreiber A.D. Grinstein S. J. Biol. Chem. 1999; 274: 28436-28444Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 33Indik Z.K. Park J.G. Hunter S. Schreiber A.D. Blood. 1995; 86: 4389-4399Crossref PubMed Google Scholar, 34Lowry M.B. Duchemin A.M. Robinson J.M. Anderson C.L. J. Exp. Med. 1998; 187: 161-176Crossref PubMed Scopus (75) Google Scholar)). In FcγR-expressing COS-7 cells, both drugs prevented phagocytosis, although had neither had any significant effect on SRBC binding (Fig. 3A). Importantly, PP2, but not piceatannol, abrogated F-actin accumulation at nascent phagosomes, indicating that Src-family, but not Syk kinases, control F-actin accumulation at nascent phagosomes (Fig. 3B). It is well documented that expression of Src-like kinases and Syk kinases is not restricted to hematopoietic cells (35Yanagi S. Inatome R. Takano T. Yamamura H. Biochem. Biophys. Res. Commun. 2001; 288: 495-498Crossref PubMed Scopus (147) Google Scholar, 36Huang Z.Y. Hunter S. Kim M.K. Chien P. Worth R.G. Indik Z.K. Schreiber A.D. J. Leukocyte Biol. 2004; 76: 491-499Crossref PubMed Scopus (20) Google Scholar) and both kinase activities appear to be expressed in COS cells (33Indik Z.K. Park J.G. Hunter S. Schreiber A.D. Blood. 1995; 86: 4389-4399Crossref PubMed Google Scholar). The differential effect of piceatannol on
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