Activation of Abl by Helicobacter pylori: A Novel Kinase for CagA and Crucial Mediator of Host Cell Scattering
2007; Elsevier BV; Volume: 132; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2007.01.050
ISSN1528-0012
AutoresIna Tammer, Sabine Brandt, Roland Hartig, Wolfgang König, Steffen Backert,
Tópico(s)Gastrointestinal disorders and treatments
ResumoBackground & Aims: The pathogenesis of Helicobacter pylori (Hp)-associated diseases depends on a specialized type IV secretion system. This type IV secretion system injects the cytotoxin-associated gene A (CagA) effector protein into target cells where CagA becomes phosphorylated on tyrosine residues by Src. Src then is inactivated rapidly, suggesting the presence of another host tyrosine kinase to ensure constant CagA phosphorylation in sustained Hp infections. We aimed to identify this kinase. Methods: By using the AGS gastric epithelial cell model, we performed a detailed functional characterization of Abl tyrosine kinase in signaling during Hp infections. Results: We showed that Abl kinase is activated and a novel crucial mediator of Hp infections. First, Abl-specific inhibitors SKI-DV2-43 or STI571 (Gleevec, Novartis) and knockdown of c-Abl/Abl-related gene Arg by small hairpin and interfering RNAs efficiently inhibit CagA phosphorylation and cell scattering. Second, during infection, Abl is activated rapidly by autophosphorylation at Y-412. Third, both Abl and Src phosphorylated Y-899, Y-918, and Y-972 of CagA. Fourth, we found that the Abl substrate CrkII is phosphorylated at Y-221 in vivo. Fifth, overexpression of kinase-dead Abl (K290M) blocked Hp-induced actin cytoskeletal rearrangements. We further showed that sustained activity of Abl is required to maintain CagA in a phosphorylated state. Moreover, phosphorylated CagA forms a physical complex with Abl and activated CrkII in vivo. Conclusions: We propose a model in which Hp has evolved a mechanism to use at least 2 tyrosine kinases, Abl and Src, for CagA phosphorylation and subsequent actin-cytoskeletal rearrangements leading to cell scattering and elongation. Background & Aims: The pathogenesis of Helicobacter pylori (Hp)-associated diseases depends on a specialized type IV secretion system. This type IV secretion system injects the cytotoxin-associated gene A (CagA) effector protein into target cells where CagA becomes phosphorylated on tyrosine residues by Src. Src then is inactivated rapidly, suggesting the presence of another host tyrosine kinase to ensure constant CagA phosphorylation in sustained Hp infections. We aimed to identify this kinase. Methods: By using the AGS gastric epithelial cell model, we performed a detailed functional characterization of Abl tyrosine kinase in signaling during Hp infections. Results: We showed that Abl kinase is activated and a novel crucial mediator of Hp infections. First, Abl-specific inhibitors SKI-DV2-43 or STI571 (Gleevec, Novartis) and knockdown of c-Abl/Abl-related gene Arg by small hairpin and interfering RNAs efficiently inhibit CagA phosphorylation and cell scattering. Second, during infection, Abl is activated rapidly by autophosphorylation at Y-412. Third, both Abl and Src phosphorylated Y-899, Y-918, and Y-972 of CagA. Fourth, we found that the Abl substrate CrkII is phosphorylated at Y-221 in vivo. Fifth, overexpression of kinase-dead Abl (K290M) blocked Hp-induced actin cytoskeletal rearrangements. We further showed that sustained activity of Abl is required to maintain CagA in a phosphorylated state. Moreover, phosphorylated CagA forms a physical complex with Abl and activated CrkII in vivo. Conclusions: We propose a model in which Hp has evolved a mechanism to use at least 2 tyrosine kinases, Abl and Src, for CagA phosphorylation and subsequent actin-cytoskeletal rearrangements leading to cell scattering and elongation. Helicobacter pylori (Hp) is the major causative agent in the development of chronic gastritis, duodenal ulcer, and gastric carcinoma in human beings.1Peek Jr, R.M. Blaser M.J. Helicobacter pylori and gastrointestinal tract adenocarcinomas.Nat Rev Cancer. 2002; 2: 28-37Crossref PubMed Scopus (1487) Google Scholar, 2Blaser M.J. Atherton J.C. Helicobacter pylori persistence: biology and disease 1.J Clin Invest. 2004; 113: 321-333Crossref PubMed Scopus (766) Google Scholar, 3Rieder G. Merchant J.L. Haas R. Helicobacter pylori cag-type IV secretion system facilitates corpus colonization to induce precancerous conditions in Mongolian gerbils.Gastroenterology. 2005; 128: 1229-1242Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Virulent Hp strains harbor a type IV secretion system (T4SS) encoded by the cag pathogenicity island.1Peek Jr, R.M. Blaser M.J. Helicobacter pylori and gastrointestinal tract adenocarcinomas.Nat Rev Cancer. 2002; 2: 28-37Crossref PubMed Scopus (1487) Google Scholar, 2Blaser M.J. Atherton J.C. Helicobacter pylori persistence: biology and disease 1.J Clin Invest. 2004; 113: 321-333Crossref PubMed Scopus (766) Google Scholar, 3Rieder G. Merchant J.L. Haas R. Helicobacter pylori cag-type IV secretion system facilitates corpus colonization to induce precancerous conditions in Mongolian gerbils.Gastroenterology. 2005; 128: 1229-1242Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 4Backert S. Meyer T.F. Type IV secretion systems and their effectors in bacterial pathogenesis.Curr Opin Microbiol. 2006; 9: 207-217Crossref PubMed Scopus (292) Google Scholar, 5Segal E.D. Cha J. Lo J. Falkow S. Tompkins L.S. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori.Proc Natl Acad Sci U S A. 1999; 96: 14559-14564Crossref PubMed Scopus (672) Google Scholar The cytotoxin-associated gene A (CagA) is the only described effector protein that interferes with global actin cytoskeletal rearrangements involved in host cell scattering and elongation.2Blaser M.J. Atherton J.C. Helicobacter pylori persistence: biology and disease 1.J Clin Invest. 2004; 113: 321-333Crossref PubMed Scopus (766) Google Scholar, 3Rieder G. Merchant J.L. Haas R. Helicobacter pylori cag-type IV secretion system facilitates corpus colonization to induce precancerous conditions in Mongolian gerbils.Gastroenterology. 2005; 128: 1229-1242Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 4Backert S. Meyer T.F. Type IV secretion systems and their effectors in bacterial pathogenesis.Curr Opin Microbiol. 2006; 9: 207-217Crossref PubMed Scopus (292) Google Scholar, 5Segal E.D. Cha J. Lo J. Falkow S. Tompkins L.S. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori.Proc Natl Acad Sci U S A. 1999; 96: 14559-14564Crossref PubMed Scopus (672) Google Scholar, 6Higashi H. Tsutsumi R. Muto S. Sugiyama T. Azuma T. Asaka M. Hatakeyama M. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein.Science. 2002; 295: 683-686Crossref PubMed Scopus (855) Google Scholar, 7Selbach M. Moese S. Hurwitz R. Hauck C.R. Meyer T.F. Backert S. The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation.EMBO J. 2003; 22: 515-528Crossref PubMed Scopus (188) Google Scholar, 8Amieva M.R. Vogelmann R. Covacci A. Tompkins L.S. Nelson W.J. Falkow S. Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA.Science. 2003; 300: 1430-1434Crossref PubMed Scopus (620) Google Scholar Recent data in the gerbil infection model indicated that CagA is a major disease-associated factor.3Rieder G. Merchant J.L. Haas R. Helicobacter pylori cag-type IV secretion system facilitates corpus colonization to induce precancerous conditions in Mongolian gerbils.Gastroenterology. 2005; 128: 1229-1242Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 9Franco A.T. Israel D.A. Washington M.K. Krishna U. Fox J.G. Rogers A.B. Neish A.S. Collier-Hyams L. Perez-Perez G.I. Hatakeyama M. Whitehead R. Gaus K. O’Brien D.P. Romero-Gallo J. Peek Jr, R.M. Activation of beta-catenin by carcinogenic Helicobacter pylori.Proc Natl Acad Sci U S A. 2005; 102: 10646-10651Crossref PubMed Scopus (405) Google Scholar After translocation into gastric epithelial cells, CagA is phosphorylated (CagAPY) at C-terminal EPIYA repeats by Src family kinases (SFKs).10Stein M. Bagnoli F. Halenbeck R. Rappuoli R. Fantl W.J. Covacci A. c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs.Mol Microbiol. 2002; 43: 971-980Crossref PubMed Scopus (370) Google Scholar, 11Selbach M. Moese S. Hauck C.R. Meyer T.F. Backert S. Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.J Biol Chem. 2002; 277: 6775-6778Crossref PubMed Scopus (350) Google Scholar, 12Backert S. Moese S. Selbach M. Brinkmann V. Meyer T.F. Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells.Mol Microbiol. 2001; 42: 631-644Crossref PubMed Scopus (206) Google Scholar Phosphorylation of CagA is critical for signaling to the actin cytoskeleton and a large number of CagAPY binding partners have been described including the SH2 domain–containing signaling proteins Shp-2, Csk, and Crk.6Higashi H. Tsutsumi R. Muto S. Sugiyama T. Azuma T. Asaka M. Hatakeyama M. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein.Science. 2002; 295: 683-686Crossref PubMed Scopus (855) Google Scholar, 13Tsutsumi R. Higashi H. Higuchi M. Okada M. Hatakeyama M. Attenuation of Helicobacter pylori CagA × SHP-2 signaling by interaction between CagA and C-terminal Src kinase.J Biol Chem. 2003; 278: 3664-3670Crossref PubMed Scopus (251) Google Scholar, 14Suzuki M. Mimuro H. Suzuki T. Park M. Yamamoto T. Sasakawa C. Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion.J Exp Med. 2005; 202: 1235-1247Crossref PubMed Scopus (177) Google Scholar AGS gastric epithelial cells serve as a model system to study CagAPY-induced rearrangement of the actin cytoskeleton. Infected AGS cells elongate, a morphology that originally was referred to as the hummingbird phenotype.5Segal E.D. Cha J. Lo J. Falkow S. Tompkins L.S. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori.Proc Natl Acad Sci U S A. 1999; 96: 14559-14564Crossref PubMed Scopus (672) Google Scholar Later it was shown that the latter phenotype combines 2 successive events: (1) the induction of motility leading to cell scattering, and (2) host cell elongation.4Backert S. Meyer T.F. Type IV secretion systems and their effectors in bacterial pathogenesis.Curr Opin Microbiol. 2006; 9: 207-217Crossref PubMed Scopus (292) Google Scholar Intriguingly, after 3–4 hours of infection, CagAPY induces the inactivation of Src by interaction with Src itself and Csk, a tyrosine kinase that negatively regulates SFKs.7Selbach M. Moese S. Hurwitz R. Hauck C.R. Meyer T.F. Backert S. The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation.EMBO J. 2003; 22: 515-528Crossref PubMed Scopus (188) Google Scholar, 13Tsutsumi R. Higashi H. Higuchi M. Okada M. Hatakeyama M. Attenuation of Helicobacter pylori CagA × SHP-2 signaling by interaction between CagA and C-terminal Src kinase.J Biol Chem. 2003; 278: 3664-3670Crossref PubMed Scopus (251) Google Scholar Inactivation of Src coincides with tyrosine dephosphorylation of the actin binding proteins cortactin, ezrin, vinculin, and focal adhesion kinase, which contribute to host cell scattering and elongation.2Blaser M.J. Atherton J.C. Helicobacter pylori persistence: biology and disease 1.J Clin Invest. 2004; 113: 321-333Crossref PubMed Scopus (766) Google Scholar, 4Backert S. Meyer T.F. Type IV secretion systems and their effectors in bacterial pathogenesis.Curr Opin Microbiol. 2006; 9: 207-217Crossref PubMed Scopus (292) Google Scholar, 7Selbach M. Moese S. Hurwitz R. Hauck C.R. Meyer T.F. Backert S. The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation.EMBO J. 2003; 22: 515-528Crossref PubMed Scopus (188) Google Scholar, 15Selbach M. Moese S. Backert S. Jungblut P.R. Meyer T.F. The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin.Proteomics. 2004; 4: 2961-2968Crossref PubMed Scopus (72) Google Scholar, 16Tsutsumi R. Takahashi A. Azuma T. Higashi H. Hatakeyama M. Focal adhesion kinase is a substrate and downstream effector of SHP-2 complexed with Helicobacter pylori CagA.Mol Cell Biol. 2006; 26: 261-276Crossref PubMed Scopus (163) Google Scholar The finding that SFK members are able to phosphorylate CagA in vivo and in vitro highlights the importance of SFKs in Hp infections.10Stein M. Bagnoli F. Halenbeck R. Rappuoli R. Fantl W.J. Covacci A. c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs.Mol Microbiol. 2002; 43: 971-980Crossref PubMed Scopus (370) Google Scholar, 11Selbach M. Moese S. Hauck C.R. Meyer T.F. Backert S. Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.J Biol Chem. 2002; 277: 6775-6778Crossref PubMed Scopus (350) Google Scholar However, CagA phosphorylation is not fully abrogated in Src/Yes/Fyn (SYF) knockout fibroblasts,11Selbach M. Moese S. Hauck C.R. Meyer T.F. Backert S. Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.J Biol Chem. 2002; 277: 6775-6778Crossref PubMed Scopus (350) Google Scholar suggesting that CagA also may be phosphorylated by other tyrosine kinases. In particular, because redundancy exists among the host tyrosine kinases, it often is not clear which kinase(s) is/are involved in phosphorylation of CagA in vivo. In this study, we show that Hp infection profoundly activates Abl, another nonreceptor tyrosine kinase that is known to regulate cell morphogenesis and motility.17Feller S.M. Knudsen B. Hanafusa H. c-Abl kinase regulates the protein binding activity of c-Crk.EMBO J. 1994; 13: 2341-2351Crossref PubMed Scopus (326) Google Scholar, 18Hernandez S.E. Krishnaswami M. Miller A.L. Koleske A.J. How do Abl family kinases regulate cell shape and movement?.Trends Cell Biol. 2004; 14: 36-44Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 19Sionov R.V. Coen S. Goldberg Z. Berger M. Bercovich B. Ben Neriah Y. Ciechanover A. Haupt Y. c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination.Mol Cell Biol. 2001; 21: 5869-5878Crossref PubMed Scopus (87) Google Scholar, 20Burton E.A. Oliver T.N. Pendergast A.M. Abl kinases regulate actin comet tail elongation via an N-WASP-dependent pathway.Mol Cell Biol. 2005; 25: 8834-8843Crossref PubMed Scopus (53) Google Scholar, 21Abassi Y.A. Vuori K. Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling.EMBO J. 2002; 21: 4571-4582Crossref PubMed Scopus (66) Google Scholar Hp strains P1, P12, 26695, G27, and the production of isogenic cagA, cagE, cagL, and virB11 knockout mutants were described.7Selbach M. Moese S. Hurwitz R. Hauck C.R. Meyer T.F. Backert S. The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation.EMBO J. 2003; 22: 515-528Crossref PubMed Scopus (188) Google Scholar, 11Selbach M. Moese S. Hauck C.R. Meyer T.F. Backert S. Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.J Biol Chem. 2002; 277: 6775-6778Crossref PubMed Scopus (350) Google Scholar, 12Backert S. Moese S. Selbach M. Brinkmann V. Meyer T.F. Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells.Mol Microbiol. 2001; 42: 631-644Crossref PubMed Scopus (206) Google Scholar AGS and MKN-28 gastric epithelial cells and MCF-7 breast cancer epithelial cells were cultivated using RPMI-1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Karlsruhe, Germany).5Segal E.D. Cha J. Lo J. Falkow S. Tompkins L.S. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori.Proc Natl Acad Sci U S A. 1999; 96: 14559-14564Crossref PubMed Scopus (672) Google Scholar, 6Higashi H. Tsutsumi R. Muto S. Sugiyama T. Azuma T. Asaka M. Hatakeyama M. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein.Science. 2002; 295: 683-686Crossref PubMed Scopus (855) Google Scholar, 7Selbach M. Moese S. Hurwitz R. Hauck C.R. Meyer T.F. Backert S. The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation.EMBO J. 2003; 22: 515-528Crossref PubMed Scopus (188) Google Scholar, 8Amieva M.R. Vogelmann R. Covacci A. Tompkins L.S. Nelson W.J. Falkow S. Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA.Science. 2003; 300: 1430-1434Crossref PubMed Scopus (620) Google Scholar, 9Franco A.T. Israel D.A. Washington M.K. Krishna U. Fox J.G. Rogers A.B. Neish A.S. Collier-Hyams L. Perez-Perez G.I. Hatakeyama M. Whitehead R. Gaus K. O’Brien D.P. Romero-Gallo J. Peek Jr, R.M. Activation of beta-catenin by carcinogenic Helicobacter pylori.Proc Natl Acad Sci U S A. 2005; 102: 10646-10651Crossref PubMed Scopus (405) Google Scholar, 10Stein M. Bagnoli F. Halenbeck R. Rappuoli R. Fantl W.J. Covacci A. c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs.Mol Microbiol. 2002; 43: 971-980Crossref PubMed Scopus (370) Google Scholar, 11Selbach M. Moese S. Hauck C.R. Meyer T.F. Backert S. Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.J Biol Chem. 2002; 277: 6775-6778Crossref PubMed Scopus (350) Google Scholar, 12Backert S. Moese S. Selbach M. Brinkmann V. Meyer T.F. Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells.Mol Microbiol. 2001; 42: 631-644Crossref PubMed Scopus (206) Google Scholar, 13Tsutsumi R. Higashi H. Higuchi M. Okada M. Hatakeyama M. Attenuation of Helicobacter pylori CagA × SHP-2 signaling by interaction between CagA and C-terminal Src kinase.J Biol Chem. 2003; 278: 3664-3670Crossref PubMed Scopus (251) Google Scholar Infections were performed routinely with serum-starved (12 h) cells using a multiplicity of infection of 100. The Abl tyrosine kinase inhibitors imatinib mesylate (STI571; Novartis, Nuernberg, Germany) and SKI-DV-43 (kindly provided by Nikolas von Bubnoff), as well as AG1478, AG1295, and PP2 (Calbiochem, Schwalbach, Germany) were dissolved in Me2SO and added to the cells 30 minutes before infection. After infection the cells were harvested in ice-cold phosphate-buffered saline (PBS) containing 1 mmol/L Na3VO4 (Sigma, Taufkirchen, Germany). The pSilencer2.1-U6-Hygro vector system (Ambion, Austin, TX) was used to clone the c-Abl small hairpin RNA (shRNA) and a scrambled shRNA sequence as negative control. Transfection of the plasmids was performed using Effectene (Qiagen, Hilden, Germany). Stable cell lines were selected in 200 μg/mL hygromycin (Invitrogen). The Abl-related gene (Arg) small interfering RNA (siRNA) oligonucleotide was transfected for 48 hours according to the manufacturer’s instructions (Santa Cruz, CA). A total of 1010 wild-type (wt) Hp cells were lysed in 200 μL ice-cold kinase buffer (25 mmol/L HEPES pH 7.0, 150 mmol/L NaCl, 10 mmol/L MgCl2, 1% Nonidet-P-40, 5 mmol/L dithiothreitol, 1 mmol/L Na3VO4, COMPLETE protease inhibitors [Roche, Basel, Switzerland]). A total of 1 × 107 SYF or SYF + c-Src cells22Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. Src family kinases are required for integrin but not PDGFR signal transduction.EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (647) Google Scholar were stimulated with 50 μmol/L Na3VO4/H2O2 for 1 hour and harvested in 1 mL ice-cold kinase buffer. A total of 25 μL of cell lysate was incubated with 25 μL of Hp lysate and 1 μmol/L of adenosine triphosphate for 30 minutes at 30°C.11Selbach M. Moese S. Hauck C.R. Meyer T.F. Backert S. Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.J Biol Chem. 2002; 277: 6775-6778Crossref PubMed Scopus (350) Google Scholar A total of 1010Hp cells expressing either wt CagA or phosphorylation-deficient mutant CagAY899/912/972F were harvested in 1 mL of kinase buffer as described previously.15Selbach M. Moese S. Backert S. Jungblut P.R. Meyer T.F. The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin.Proteomics. 2004; 4: 2961-2968Crossref PubMed Scopus (72) Google Scholar A total of 5 U of recombinant human c-Src (Upstate, Charlottesville, VA) or c-Abl (NEB, Frankfurt/M., Germany) and 1 μmol/L of adenosine triphosphate were mixed with 30 μL of the Hp lysate and incubated for 30 minutes at 30°C as described earlier. Plasmids expressing wt CagA or CagAY899/912/972F were described.12 Mouse wt c-Abl (pCMV-c-Abl-IV), kinase-defective c-Abl (pCMV-c-Abl-K290H),19 and constitutive-active c-Abl-P242/249E (Abl-PP)20 were kindly provided by Ygal Haupt and Anne-Marie Pendergast. Wt CrkII, CrkII-R38V, CrkII-W169L, and CrkII-Y221F mutants in pCAGGS vector were a generous gift from Kristiina Vuori.21 All constructs were transfected into AGS using LipofectAMINE 2000 (Invitrogen). After 36 hours the cells were either subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis or fluorescence microscopy. Transfected CrkII was visualized by a rabbit α-CrkII antibody (Santa Cruz) and with TRITC-conjugated goat α-rabbit antibody (Sigma). Cells were analyzed using the Leica (Solms, Germany) DMRE7 fluorescence microscope. Western blots were probed with α-phosphotyrosine PY-99 monoclonal antibody, a goat α-Arg polyclonal antibody (pAB), an α-c-CrkII monoclonal antibody (Santa Cruz), a goat α-GST pAB (Amersham Biosciences, Buckinghamshire, UK), a rabbit α-CagA pAB (Austral Biologicals, San Ramon, CA), an α-c-Abl monoclonal antibody or an α-c-Abl-PY-412 pAB (Sigma). Rabbit α-c-Src-PY-416, α-c-Src-PY-527, and α-Crk-II-PY-221 pABs were purchased from NEB. An α-c-CagA-PY pAB was produced as described14Suzuki M. Mimuro H. Suzuki T. Park M. Yamamoto T. Sasakawa C. Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion.J Exp Med. 2005; 202: 1235-1247Crossref PubMed Scopus (177) Google Scholar and a goat α-glyceraldehyde-3-phosphate dehydrogenase pAB (Santa Cruz) served as loading control. Western blots and band intensities were measured and quantified with the Lumi-Imager F1 (Roche). A total of 1 × 107 AGS cells were washed with cold PBS and lysed for 30 minutes at 4°C in lysis buffer (20 mmol/L Tris pH 72, 150 mmol/L NaCl, 5 mmol/L ethylenediaminetetraacetic acid, 1% Triton-X-100, 10% glycerol, 1 mmol/L Na3VO4, COMPLETE). Lysates were precleared with protein G–Sepharose (Amersham Biosciences) for 2 hours at 4°C. Polyclonal α-CrkII or α-c-Abl AB (2 μg each) was added to the supernatants and incubated overnight at 4°C. Immune complexes were precipitated by the addition of protein G–Sepharose for 2 hours and washed once with lysis buffer and 3 times with 0.5 × PBS. All data were evaluated using the Student t test with SigmaStat statistical software (version 2.0; Statcon, Witzenhausen, Germany), with significance set at a P value of .05 or less (*) and a P value of .005 (**) or less. The availability of potent and relatively specific Abl kinase inhibitors SKI-DV2-4323 or STI57124 has allowed us to test the hypothesis that Abl participates in Hp-induced actin cytoskeletal rearrangements. In an initial experiment, AGS gastric epithelial cells were treated with different tyrosine kinase inhibitors before infection with Hp. Compared with noninfected AGS cells, noninhibited cells infected with Hp for 4 hours displayed the typical scattering phenotype characterized by the loss of cell-to-cell contacts and drastic cellular elongation (Figure 1A). Incubation of AGS cells with SKI-DV2-43 or STI571 before infection significantly reduced cell scattering and elongation (Figure 1B). Similarly, Hp-induced cell scattering of other epithelial cells such as MKN-28 and MCF-7 also was blocked by SKI-DV2-43 and STI571, whereas numerous controls including Me2SO (mock), AG1295 (inhibitor of the platelet-derived growth factor receptor), and AG1478 (inhibitor of the epidermal growth factor receptor) did not affect the cellular phenotype (Figure 1C and data not shown). These data indicate that Abl kinases might play a role in Hp-induced cell scattering of epithelial cells. To test whether the presence of SKI-DV2-43 or STI571 also influenced the phosphorylation of CagA, protein samples were subjected to immunoblotting with a phospho-specific antibody and an antibody detecting total CagA protein on the blot. As shown in Figure 1D, both inhibitors significantly reduced the CagAPY signal at 4 hours after infection, however, phosphorylation was not abrogated fully. Similar effects have been reported with the Src-specific inhibitor PP2 in our earlier study.11 The strong reduction in the amount of CagAPY was not attributed to a bactericidal effect of the inhibitors because no effect on the viability of Hp was apparent (not shown). These observations suggest that, besides SFKs, Abl also may play a role in the phosphorylation of CagA. To determine by a more direct approach whether Abl is important for Hp infection, we generated stable c-Abl–deficient AGS cells using a specific shRNA expression construct. Knockdown of c-Abl was very efficient and was reduced significantly, but did not eliminate CagA phosphorylation and AGS cell elongation (Figure 2A–C). However, the Abl kinase family consists of 2 highly related proteins: c-Abl and Arg.17Feller S.M. Knudsen B. Hanafusa H. c-Abl kinase regulates the protein binding activity of c-Crk.EMBO J. 1994; 13: 2341-2351Crossref PubMed Scopus (326) Google Scholar, 18Hernandez S.E. Krishnaswami M. Miller A.L. Koleske A.J. How do Abl family kinases regulate cell shape and movement?.Trends Cell Biol. 2004; 14: 36-44Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 19Sionov R.V. Coen S. Goldberg Z. Berger M. Bercovich B. Ben Neriah Y. Ciechanover A. Haupt Y. c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination.Mol Cell Biol. 2001; 21: 5869-5878Crossref PubMed Scopus (87) Google Scholar, 20Burton E.A. Oliver T.N. Pendergast A.M. Abl kinases regulate actin comet tail elongation via an N-WASP-dependent pathway.Mol Cell Biol. 2005; 25: 8834-8843Crossref PubMed Scopus (53) Google Scholar, 21Abassi Y.A. Vuori K. Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling.EMBO J. 2002; 21: 4571-4582Crossref PubMed Scopus (66) Google Scholar Interestingly, silencing of Arg had a more pronounced effect on the CagAPY signal but not AGS cell elongation as compared with the c-Abl knockout (Figure 2A–C). However, knockout of both c-Abl and Arg lead to an almost complete blockade of host cell elongation (Figure 2A–C), whereas expression of a control shRNA oligonucleotide had no effect (Figure 2C, bottom). These data confirmed that c-Abl and Arg are involved in Hp-induced AGS cell elongation and CagA phosphorylation in vivo. To prove whether CagA can function as a substrate for Abl kinases in the absence of SFKs we used lysates of fibroblasts derived from c-src-/-, c-yes-/-, and c-fyn-/- (SYF) triple-knockout mice cells.21Abassi Y.A. Vuori K. Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling.EMBO J. 2002; 21: 4571-4582Crossref PubMed Scopus (66) Google Scholar As a control, SYF cells stably re-expressing c-Src were used (SYF + c-src) (Figure 3A). Because Hp was unable to translocate CagA into mouse fibroblasts,11Selbach M. Moese S. Hauck C.R. Meyer T.F. Backert S. Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.J Biol Chem. 2002; 277: 6775-6778Crossref PubMed Scopus (350) Google Scholar we first stimulated the cells with Na3VO4/H2O2 to induce Abl activity, and prepared cell lysates to perform in vitro CagA phosphorylation assays. As expected, SYF + c-src cells strongly induced the CagA phosphorylation (Figure 3A, left panels). Inhibition of Src by PP2 lead to an approximately 25% reduction of the CagAPY signal whereas inhibition of Abl by SKI-DV2-43 reduced the signal by approximately 70%. In contrast, SYF cell lysates also supported CagA phosphorylation but to a minor degree, and the CagAPY signal was abrogated completely by the presence of SKI-DV2-43 but not PP2 (Figure 3A, right panels). This indicated that both c-Src and Abl can phosphorylate CagA in cell lysates. To investigate the role of Abl further, we performed in vitro kinase assays using purified Abl (or Src as control) incubated with either wt CagA or a CagAY899/918/972F mutant in which the tyrosine residues in the known phosphorylation sites Y-899, Y-918, and Y-972 were replaced by phenylalanines.12 We detected very strong and similar levels of CagA phosphorylation with both recombinant Abl or Src when co-incubated with wt CagA (Figure 3B). As control, reactions without recombinant kinase were unable to phosphorylate CagA. Interestingly, incubation of either Abl or Src with the CagAY899/918/972F mutant revealed almost no detectable signal for CagAPY. These results surprisingly show that CagA and its EPIYA repeats serve as a substrate for both Abl and Src kinases. Next, we determined the activation status of both Abl and Src in a time course of AGS cell infection up to 8 hours. The results are summarized in Figure 4A. First, we tested an antibody that recognizes the phosphorylated tyrosine residue 412 (α-Abl-PY-412) in the Abl activation-loo
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