Influence of EPIYA-Repeat Polymorphism on the Phosphorylation-Dependent Biological Activity of Helicobacter pylori CagA
2006; Elsevier BV; Volume: 130; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2005.12.038
ISSN1528-0012
AutoresMasanori Naito, Takeshi Yamazaki, Ryouhei Tsutsumi, Hideaki Higashi, Kazunori Onoé, Shiho Yamazaki, Takeshi Azuma, Masanori Hatakeyama,
Tópico(s)RNA modifications and cancer
ResumoBackground & Aims: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3′-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. Methods: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. Results: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA–SHP-2 interaction. Conclusions: EPIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis. Background & Aims: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3′-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. Methods: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. Results: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA–SHP-2 interaction. Conclusions: EPIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis. 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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 (862) Google Scholar, 26Tsutsumi 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 (254) Google Scholar Through the complex formation, CagA activates Csk, which in turn inhibits SFK activity by phosphorylating the C-terminal inhibitory tyrosine residues. Because SFKs are responsible for CagA phosphorylation,16Stein 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 (372) Google Scholar, 17Selbach 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 (351) Google Scholar CagA–Csk interaction is considered to attenuate tyrosine phosphorylation-dependent pathophysiologic activity of CagA.26Tsutsumi 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 (254) Google Scholar Such a feedback regulation may contribute to the long-term equilibrium between cagA-positive H pylori and the host without causing excess CagA toxicity. CagA is tyrosine-phosphorylated at multiple Glu-Pro-Ile-Tyr-Ala (EPIYA) sites present in the C-terminal region.18Higashi 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 (862) Google Scholar, 19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar, 20Hatakeyama M. Oncogenic mechanisms of the Helicobacter pylori CagA protein.Nat Rev Cancer. 2004; 4: 688-694Crossref PubMed Scopus (610) Google Scholar Because of frequent homologous recombination within the 3′-region of the cagA gene, the EPIYA-repeat region of CagA is highly divergent among various CagA species and is composed of various combinations of four discrete segments termed EPIYA-A, -B, -C, and -D.19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar, 20Hatakeyama M. Oncogenic mechanisms of the Helicobacter pylori CagA protein.Nat Rev Cancer. 2004; 4: 688-694Crossref PubMed Scopus (610) Google Scholar, 27Higashi H. Yokoyama K. Fujii Y. Ren S. Yuasa H. Saadat I. Kamiya-Murata N. Azuma T. Hatakeyama M. EPIYA motif is a membrane-targeting signal of Helicobacter pylori virulence factor CagA in mammalian cells.J Biol Chem. 2005; 280: 23130-23137Crossref PubMed Scopus (126) Google Scholar Thus, the EPIYA-repeat regions of prevalent CagA proteins from H pylori isolated in Western countries consist of EPIYA-A, EPIYA-B, and variable numbers of EPIYA-C segments (ABC-, ABCC-, or ABCCC-type CagA). In contrast, the EPIYA-repeat regions of prevalent East Asian CagA proteins consist of EPIYA-A, EPIYA-B, and EPIYA-D segments (ABD-type CagA). Each of the EPIYA segments contains a single EPIYA site (EPIYA-A, -B, -C, or -D site), which undergoes tyrosine phosphorylation.27Higashi H. Yokoyama K. Fujii Y. Ren S. Yuasa H. Saadat I. Kamiya-Murata N. Azuma T. Hatakeyama M. EPIYA motif is a membrane-targeting signal of Helicobacter pylori virulence factor CagA in mammalian cells.J Biol Chem. 2005; 280: 23130-23137Crossref PubMed Scopus (126) Google Scholar SHP-2 binds to the EPIYA-C and EPIYA-D sites of Western and East Asian CagA species, respectively, in a phosphorylation-dependent manner. The EPIYA-D site exhibits greater SHP-2–binding activity and therefore stronger ability to induce cell elongation known as the hummingbird phenotype than does the EPIYA-C site.19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar Consequently, H pylori strains carrying East Asian CagA elicit stronger mucosal inflammation and are more closely associated with gastric carcinoma than are those carrying Western CagA.28Azuma T. Yamazaki S. Yamakawa A. Ohtani M. Muramatsu A. Suto H. Ito Y. Dojo M. Yamazaki Y. Kuriyama M. Keida Y. Higashi H. Hatakeyama M. Association between diversity in the Src homology 2 domain-containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer.J Infect Dis. 2004; 189: 820-827Crossref PubMed Scopus (186) Google Scholar Furthermore, Western H pylori strains carrying CagA with multiple EPIYA-C sites, which bind SHP-2 more effectively than those having a single EPIYA-C site, are more frequently isolated from patients with gastric adenocarcinoma.29Argent R.H. Kidd M. Owen R.J. Thomas R.J. Limb M.C. Atherton J.C. Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori.Gastroenterology. 2004; 127: 514-523Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar Hence, the degree of CagA to deregulate SHP-2 appears to play an important role in determining oncogenic potential of individual H pylori cagA-positive strain. In this work, we investigated the influence of EPIYA-repeat polymorphism on CagA activities to bind SHP-2 and Csk as well as to induce the hummingbird phenotype and found that the degree of phosphorylation-dependent CagA activities is variably altered by the diversity of EPIYA repeats. Our results provide a functional link between the EPIYA-repeat polymorphism of CagA and the virulence of individual cagA-positive H pylori. A gene encoding the ABDD-type CagA of F75 East Asian strain19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar was synthesized and was cloned into pSP65SRα. EPIYA-repeat variants of East Asian CagA were generated from the ABDD-type CagA by using a Chameleon site-directed mutagenesis kit (Stratagene, La Jolla, CA). Similarly, EPIYA-repeat variants of Western CagA were generated from the gene encoding ABCCC-type CagA of NCTC11637 origin.18Higashi 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 (862) Google Scholar A membrane-targeting mutant of Csk was made by adding the myristoylation signal sequence from avian c-Src to the N-terminus and was cloned into pSP65SRα.30Chow L.M. Fournel M. Davidson D. Veillette A. Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk.Nature. 1993; 365: 156-160Crossref PubMed Scopus (237) Google Scholar AGS human gastric epithelial cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and at 37° C in 5% CO2. Cells (2.0 × 106 cells/10-cm2 dish) were transfected with 30 μg of plasmids by using LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Antihemagglutinin (anti-HA) monoclonal antibody 3F10 (Roche Applied Science, Indianapolis, IN) was used as primary antibody for immunoprecipitation. Anti-HA polyclonal antibody Y-11 (Santa Cruz Biotechnology, Santa Cruz, CA), anti–SHP-2 polyclonal antibody C-18 (Santa Cruz Biotechnology), anti-Csk polyclonal antibody C-20 (Santa Cruz Biotechnology), antiphosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Charlottesville, VA), anti–phospho-Src (Tyr-416) polyclonal antibody (Cell Signaling, Danvers, MA), anti–c-Src polyclonal antibody N-16 (Santa Cruz Biotechnology) and anti–Myc-epitope monoclonal antibody 9E10 were used as primary antibodies for immunoblotting. For immunoprecipitation, cells were harvested 36 hours after transfection and total cell lysates were prepared as described previously.18Higashi 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 (862) Google Scholar Total cell lysates and immunoprecipitates were subjected to SDS-PAGE. Protein transferred to polyvinylidene difluoride membrane filters (Millipore, Billerica, MA) were incubated in primary antibodies and then visualized by using Western blot chemiluminescence reagent (PerkinElmer Life Sciences, Wellesley, MA). Intensities of chemiluminescence on the immunoblotted membrane were quantitated by using a luminescence image analyzer (LAS1000, FUJIFILM, Tokyo, Japan). AGS cells were seeded into 35-mm2 dishes (1.2 × 105 cells/dish) and 8 μg of plasmids was transfected into cells. Cell morphology was examined by light microscopy. Based on the structure of the EPIYA-repeat region, most of the Western CagA proteins are categorized as the ABCn-type (where n indicates the number of EPIYA-C sites),27Higashi H. Yokoyama K. Fujii Y. Ren S. Yuasa H. Saadat I. Kamiya-Murata N. Azuma T. Hatakeyama M. EPIYA motif is a membrane-targeting signal of Helicobacter pylori virulence factor CagA in mammalian cells.J Biol Chem. 2005; 280: 23130-23137Crossref PubMed Scopus (126) Google Scholar, 28Azuma T. Yamazaki S. Yamakawa A. Ohtani M. Muramatsu A. Suto H. Ito Y. Dojo M. Yamazaki Y. Kuriyama M. Keida Y. Higashi H. Hatakeyama M. Association between diversity in the Src homology 2 domain-containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer.J Infect Dis. 2004; 189: 820-827Crossref PubMed Scopus (186) Google Scholar, 29Argent R.H. Kidd M. Owen R.J. Thomas R.J. Limb M.C. Atherton J.C. Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori.Gastroenterology. 2004; 127: 514-523Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar whereas prevalent East Asian CagA species belong to the ABD-type (Figure 1). Notably, however, a small number of East Asian CagA proteins show complicated variations in the EPIYA-repeat region. The presence of duplicated EPIYA-A and/or EPIYA-B segments in some of these CagA variants suggested that the EPIYA-repeat polymorphism of CagA not only affects CagA–SHP-2 interaction but also CagA–Csk interaction. To address the effect of EPIYA diversity on CagA function, we generated a series of EPIYA-repeat variants of CagA that represent reported variations in the order and number of EPIYA segments (Figure 1). All of the CagA constructs generated were C-terminal HA-tagged. To investigate the effect of EPIYA-repeat polymorphism on the level of CagA tyrosine phosphorylation, AGS human gastric epithelial cells were transfected with each of the CagA variants and cell lysates prepared were immunoblotted with an anti-HA antibody or an anti-phosphotyrosine antibody. The protein bands detected by chemiluminescence were quantitated with the luminescence image analyzer. The intensity of the image obtained by the luminescence image analyzer is directly proportional to the light intensity, indicating that it is broader in dynamic range and has better linearity and is therefore more accurate than densitometric analysis for quantitation. In Western CagA proteins, the level of CagA tyrosine phosphorylation was proportional to the number of EPIYA-C sites as reported previously (Figure 2A).19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar In East Asian CagA species, CagA tyrosine phosphorylation was correlated to the number of EPIYA-D sites (Figure 2B). Because EPIYA-A and EPIYA-B sites were also tyrosine-phosphorylated, although less effectively, CagA with more EPIYA-A and/or EPIYA-B sites underwent higher levels of tyrosine phosphorylation than did CagA with less EPIYA-A and/or EPIYA-B sites (Figure 2B). Next, to investigate the effect of EPIYA-repeat polymorphism on the complex formation between CagA and SHP-2, lysates of cells expressing each of the EPIYA-repeat variants of CagA were immunoprecipitated with an anti-HA antibody. The anti-HA immunoprecipitates were then immunoblotted with an anti–SHP-2 antibody. The amount of SHP-2 coprecipitated with CagA was proportional to the number of EPIYA-C sites in Western CagA as previously reported (Figure 3A, left).19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar Quantitation analysis revealed that ABCC-type CagA and ABCCC-type CagA, respectively, coprecipitated 4-fold and 8-fold greater amounts of SHP-2 than the ABC-type CagA did (Figure 3A, right). In East Asian CagA species, those having 2 EPIYA-D sites coprecipitated 8-fold greater amounts of SHP-2 than did CagA with a single EPIYA-D site (Figure 3B). These results indicate that the presence of multiple EPIYA-C or -D sites synergistically potentiates the ability of CagA to bind SHP-2. The effect of EPIYA-repeat polymorphism on the complex formation between CagA and Csk was next investigated. Consistent with the observation that Csk binds to the EPIYA-A or EPIYA-B site of CagA in gastric epithelial cells,21Tsutsumi R. Takahashi A. Azuma A. 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 (164) Google Scholar the amount of Csk coprecipitated with CagA was proportional to the number of EPIYA-A and EPIYA-B sites in both Western CagA (Figure 4A) and East Asian CagA (Figure 4B), except for the cases with ABDABD-type CagA and ABDBD-type CagA. These 2 East Asian CagA variants coprecipitated significantly larger amounts of Csk compared with other CagA species (Figure 4B). Because they exhibited the highest level of tyrosine phosphorylation among the CagA variants (Figure 2B), we examined the possibility that the increased level of CagA tyrosine phosphorylation affected the Csk-binding activity. To this end, a tyrosine residue within the first EPIYA-D site of ABDABD-type CagA was replaced by phenylalanine residue. The resulting ABdABD-type CagA showed levels of tyrosine phosphorylation and SHP-2 binding similar to those observed with ABABD-type CagA in AGS cells (Figure 5). However, the CagA mutant still retained the ability to coprecipitate Csk to a level comparable to that of Csk coprecipitated by ABDABD-type CagA. These results excluded the role of tyrosine-phosphorylated EPIYA-D in the CagA-Csk interaction and suggested that the presence of 2 EPIYA-B segments, both of which are directly followed by EPIYA-D segments, confers strong Csk-binding activity to the tyrosine-phosphorylated EPIYA-B sites.Figure 5Effect of EPIYA-D phosphorylation on CagA–Csk interaction. Lysates prepared from AGS cells transfected with the indicated CagA variant or empty vector were immunoprecipitated with anti-HA antibody. ABdABD-type CagA was made from ABDABD-type CagA by replacing the tyrosine residue in the first EPIYA-D site with phenylalanine. The immunoprecipitates (IP) and total cell lysates (TCL) were immunoblotted with the indicated antibodies (upper). Relative amounts of tyrosine-phosphorylated (pY) CagA (lower left), bound SHP-2 (lower middle), and bound Csk (lower right) were calculated from the immunoblotting data (IB), defining the values in ABD-type CagA as 1. Experiments were performed in triplicate, and error bars indicate 2× SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Comparison of the SHP-2–binding activity confirmed that ABD-type East Asian CagA interacts with SHP-2 more strongly than does ABC-type Western CagA (Figure 6A).19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar On the other hand, ABC-type CagA was found to bind Csk more efficiently than ABD-type CagA (Figure 6A). Given that CagA binds to SHP-2 and Csk in a mutually exclusive manner,26Tsutsumi 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 (254) Google Scholar the observation indicated that CagA–SHP-2 interaction affects CagA–Csk complex formation. To address this possibility, we replaced the tyrosine residue in the EPIYA-C site of ABC-type CagA or in the EPIYA-D site of ABD-type CagA with alanine residue. The resulting ABc-type and ABd-type CagA mutants bound Csk at comparable levels (Figure 6B). Thus, degree of CagA-Csk interaction is affected by the degree of strength of the CagA–SHP-2 interaction. To determine whether elevated Csk activity is capable of inhibiting tyrosine phosphorylation of CagA through SFK inhibition, we generated a Csk mutant that possesses a membrane-targeting signal derived from avian c-Src at the N-terminus (Myr-Csk). The Csk mutant associates with the plasma membrane and acts as a constitutively active form of Csk.30Chow L.M. Fournel M. Davidson D. Veillette A. Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk.Nature. 1993; 365: 156-160Crossref PubMed Scopus (237) Google Scholar Coexpression studies of abD-type CagA, which has tyrosine-to-phenylalanine substitutions in both EPIYA-A and EPIYA-B sites and thus does not bind Csk, together with Myr-Csk revealed that the level of tyrosine phosphorylation at the EPIYA-D site as well as the amount of SHP-2 associated with CagA were significantly reduced in AGS cells expressing Myr-Csk (Figure 7). The result indicated that elevated Csk activity inhibits SFK-mediated CagA phosphorylation and subsequent CagA–SHP-2 complex formation. CagA-activated SHP-2 induces an elongated cell shape, termed the hummingbird phenotype.18Higashi 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 (862) Google Scholar, 22Higashi H. Nakaya A. Tsutsumi R. Yokoyama K. Fujii Y. Ishikawa S. Higuchi M. Takahashi A. Kurashima Y. Teishikata Y. Tanaka S. Azuma T. Hatakeyama M. Helicobacter pylori CagA induces Ras-independent morphogenetic response through SHP-2 recruitment and activation.J Biol Chem. 2004; 279: 17205-17216Crossref PubMed Scopus (231) Google Scholar Thus, CagA with greater ability to bind SHP-2 exhibits stronger activity to induce the hummingbird phenotype than does CagA with less SHP-2–binding activity.19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar On the other hand, elevated Csk activity inhibits SFK and subsequent CagA–SHP-2 complex formation (Figure 7). These results collectively indicate that stimulation of Csk by CagA down-regulates CagA-SHP-2 signaling. To investigate whether such a feedback regulation of the CagA activity indeed operates in cells expressing CagA, we directly compared the ability of ABD-type CagA, ABDD-type CagA, and ABDABD-type CagA to induce the hummingbird phenotype. When expressed in AGS cells, ABDD-type CagA and ABDABD-type CagA bound more SHP-2 than did ABD-type CagA (Figure 3B). As shown in Figure 8A, kinetic studies of the hummingbird phenotype induction by these CagA species revealed that, at 15 hours after transfection, 15% of AGS cells transfected with ABD-type CagA exhibited the hummingbird phenotype, whereas 20% of cells transfected with ABDD-type CagA or ABDABD-type CagA developed the hummingbird phenotype. At 17 hours after transfection, 20% of the transfected cells showed the hummingbird phenotype in all CagA cases. At this time point, the level of ABD-type CagA expression was greater than that of ABDD- or ABDABD-type CagA (Figure 8B). Thus, CagA with greater SHP-2–binding activity induced the hummingbird phenotype significantly earlier than did CagA with less SHP-2–binding activity. Next duration of the hummingbird phenotype with these CagA species was examined. At 48 hours after transfection, the number of cells with the hummingbird phenotype induced by ABDABD-type CagA, which bound more Csk than did ABD-type CagA or ABDD-type CagA (Figure 4B), was significantly less than that induced by ABD- or ABDD-type CagA (Figure 8A). When the ratio of tyrosine-phosphorylated CagA to total CagA was compared at 15 and 48 hours after transfection, CagA tyrosine phosphorylation was found to decrease more rapidly in cells expressing ABDABD-type CagA than in cells expressing ABD-type CagA or ABDD-type (Figure 8B). These results indicated that CagA that binds more Csk down-regulates CagA tyrosine phosphorylation and thereby attenuates CagA–SHP-2 interaction more strongly than does CagA that binds less Csk. To determine whether stronger attenuation of CagA-SHP-2 signaling by ABDABD-type CagA was due to greater inhibition of SFK activity, we examined the level of active SFKs in cells expressing each of these CagA species with the use of anti–p-Src (Tyr-416) antibody, which specifically recognizes active forms of SFKs. As shown in Figure 8C, ABDABD-type CagA inhibited SFK kinase activity more strongly than did ABD- or ABDD-type CagA. From these observations, we concluded that CagA–Csk interaction causes inhibition of SFKs, which in turn results in reduced levels of CagA phosphorylation and subsequent downregulation of CagA–SHP-2 interaction. In the present work, we demonstrated that the level of tyrosine phosphorylation, the degree of SHP-2 binding activity, and the magnitude of the hummingbird phenotype–inducing activity are proportional to the number of EPIYA-C and EPIYA-D sites in Western CagA and East Asian CagA, respectively. Thus, the EPIYA-D site of East Asian CagA is the functionally equivalent of the EPIYA-C site of Western CagA in terms of CagA–SHP-2 interaction.19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar, 20Hatakeyama M. Oncogenic mechanisms of the Helicobacter pylori CagA protein.Nat Rev Cancer. 2004; 4: 688-694Crossref PubMed Scopus (610) Google Scholar We also showed that the CagA–SHP-2 interaction is synergistic rather than additive with regard to the number of EPIYA-C or EPIYA-D sites. The observation indicates that simultaneous interaction between the 2 Src homology 2 domains of a single SHP-2 protein and the 2 tyrosine-phosphorylated EPIYA-C or EPIYA-D sites in a CagA molecule dramatically stabilizes CagA–SHP-2 complex formation. We also found that the ability of CagA to interact with Csk is proportional to the number of EPIYA-A and EPIYA-B sites. Notably, certain East Asian CagA proteins such as ABDABD-type CagA and ABDBD-type CagA exhibit extremely strong activities to bind Csk compared to other CagA species. These CagA variants are characterized by the presence of 2 EPIYA-B segments, each of which is directly connected by the EPIYA-D segment. The structure made by the 2 EPIYA-B/EPIYA-D stretches may confer strong Csk-binding affinity to the EPIYA-B sites of CagA. If Csk can form a homodimer, as are the cases with other protein kinases, such a Csk homodimer might form a stable complex with CagA carrying the 2 EPIYA-B/EPIYA-D stretches. Whereas the EPIYA-A or EPIYA-B site of Western CagA shows the ability to bind Csk comparable to the ability of that of East Asian CagA, ABC-type Western CagA binds Csk more efficiently than does ABD-type East Asian CagA. This is because stronger SHP-2 binding of ABD-type CagA to ABC-type CagA more competitively inhibits CagA–Csk interaction. CagA that binds more SHP-2 exhibits stronger activity to induce the hummingbird phenotype.19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar At the same time, CagA that binds more Csk attenuates the hummingbird phenotype more efficiently. These observations suggest functional interplay between the CagA–SHP-2 complex and the CagA–Csk complex. Indeed, our result showing that stronger CagA–Csk interaction shortens the duration of the hummingbird phenotype provides evidence for the presence of a feedback regulatory mechanism of CagA–SHP-2 signaling by the CagA–Csk interaction. Because CagA–Csk interaction also depends on CagA tyrosine phosphorylation,21Tsutsumi R. Takahashi A. Azuma A. 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 (164) Google Scholar, 26Tsutsumi 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 (254) Google Scholar such a feedback regulatory loop may generate oscillation of deregulated SHP-2 activity in cells expressing CagA. Given that ABD-type East Asian CagA binds SHP-2 more strongly but Csk less effectively than does ABC-type Western CagA, East Asian CagA may cause a greater magnitude of oscillation of SHP-2 activity, which may contribute to its more virulent nature. H pylori strains carrying East Asian CagA are more closely associated with gastric carcinoma than those carrying Western CagA.28Azuma T. Yamazaki S. Yamakawa A. Ohtani M. Muramatsu A. Suto H. Ito Y. Dojo M. Yamazaki Y. Kuriyama M. Keida Y. Higashi H. Hatakeyama M. Association between diversity in the Src homology 2 domain-containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer.J Infect Dis. 2004; 189: 820-827Crossref PubMed Scopus (186) Google Scholar Among Western cagA-positive strains, those with CagA having multiple EPIYA-C sites are more frequently isolated from patients with gastric carcinoma.29Argent R.H. Kidd M. Owen R.J. Thomas R.J. Limb M.C. Atherton J.C. Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori.Gastroenterology. 2004; 127: 514-523Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar Although a possibility remains that such Western H pylori strains are positively selected under conditions that predispose gastric carcinoma, the selected variants are biologically more active and thus more potent in inducing gastric mucosal damage. These clinical observations indicate that H pylori strains possessing CagA with stronger SHP-2 activity are more critically involved in the development of gastric carcinoma. However, such H pylori strains can also be isolated from patients with atrophic gastritis or peptic ulcers (Figure 1).19Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (486) Google Scholar, 27Higashi H. Yokoyama K. Fujii Y. Ren S. Yuasa H. Saadat I. Kamiya-Murata N. Azuma T. Hatakeyama M. EPIYA motif is a membrane-targeting signal of Helicobacter pylori virulence factor CagA in mammalian cells.J Biol Chem. 2005; 280: 23130-23137Crossref PubMed Scopus (126) Google Scholar, 28Azuma T. Yamazaki S. Yamakawa A. Ohtani M. Muramatsu A. Suto H. Ito Y. Dojo M. Yamazaki Y. Kuriyama M. Keida Y. Higashi H. Hatakeyama M. Association between diversity in the Src homology 2 domain-containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer.J Infect Dis. 2004; 189: 820-827Crossref PubMed Scopus (186) Google Scholar, 29Argent R.H. Kidd M. Owen R.J. Thomas R.J. Limb M.C. Atherton J.C. Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori.Gastroenterology. 2004; 127: 514-523Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar Accordingly, we consider that most if not all cagA-positive H pylori strains are capable of inducing atrophic gastritis and peptic ulcers, depending on host genetic factors such as cytokine gene polymorphisms,3El-Omar E.M. Carrington M. Chow W.H. McColl K.E. Bream J.H. Young H.A. Herrera J. Lissowska J. Yuan C.C. Rothman N. Lanyon G. Martin M. Fraumeni Jr, J.F. Rabkin C.S. Interleukin-1 polymorphisms associated with increased risk of gastric cancer.Nature. 2000; 404: 398-402Crossref PubMed Scopus (2073) Google Scholar, 4Machado J.C. Figueiredo C. Canedo P. Pharoah P. Carvalho R. Nabais S. Castro-Alves C. Campos M.L. Van-Doorn L.J. Caldas C. Seruca R. Carneiro F. Sobrinho-Simoes M. A proinflammatory genetic profile increases the risk for chronic atrophic gastritis and gastric carcinoma.Gastroenterology. 2003; 125: 364-371Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar and only a portion of H pylori strains that possess CagA proteins with stronger SHP-2–binding activities can direct progression of multistep gastric carcinogenesis. Extending this idea, host genetic polymorphisms that potentiate the interaction between CagA and SHP-2 or Csk may also affect the oncogenic role of cagA-positive H pylori. Indeed, it has recently been reported that the G/A single nucleotide polymorphism in the PTPN11 gene that encodes SHP-2 is a risk factor for gastric atrophy, a precancerous mucosal change, in individuals infected with H pylori cagA-positive strains.31Goto Y. Ando T. Yamamoto K. Tamakoshi A. El-Omar E. Goto H. Hamajima N. Association between serum pepsinogens and polymorphism of PTPN11 encoding SHP-2 among Helicobacter pylori seropositive Japanese.Int J Cancer. 2006; 118: 203-208Crossref PubMed Scopus (43) Google Scholar From the bacterial point of view, a critical role of CagA may be to provoke gastric mucosal damages that result in increase in gastric pH, providing better environmental conditions for H pylori settlement. The observed CagA EPIYA-repeat polymorphism might have been developed to fine tune the individual CagA activity within certain ranges in both magnitude and duration so as to ensure long-term colonization of H pylori, without causing acute and fatal damages to the host. SHP-2 and Csk bind tyrosine-phosphorylated CagA via distinct EPIYA sites. Because Csk acts as an inhibitor of CagA–SHP-2 signaling, the degree of individual CagA to deregulate SHP-2 cannot be simply determined by the total number of EPIYA sites or by the net level of CagA tyrosine phosphorylation. Obviously, CagA with more EPIYA-C and EPIYA-D sites and, at the same time, with fewer EPIYA-A and EPIYA-B sites is biologically more active in deregulating SHP-2 in both magnitude and duration. Accordingly, EPIYA-repeat polymorphism of CagA substantially influences the degree of virulence as well as the oncogenic potential of individual cagA-positive H pylori strain.
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