Helicobacter pylori CagA Induces Ras-independent Morphogenetic Response through SHP-2 Recruitment and Activation*

The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wild-type or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the “hummingbird” phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA (siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.

[1]  M. Borodovsky,et al.  cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Sheila M. Thomas,et al.  Regulation of Early Events in Integrin Signaling by Protein Tyrosine Phosphatase SHP-2 , 1999, Molecular and Cellular Biology.

[3]  C. Maroun,et al.  The Tyrosine Phosphatase SHP-2 Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase and Epithelial Morphogenesis Downstream from the Met Receptor Tyrosine Kinase , 2000, Molecular and Cellular Biology.

[4]  N. Sasaki,et al.  Helicobacter pylori infection and the development of gastric cancer. , 2001, The New England journal of medicine.

[5]  T. Azuma,et al.  The CagA protein of Helicobacter pylori is translocated into epithelial cells and binds to SHP-2 in human gastric mucosa. , 2003, The Journal of infectious diseases.

[6]  H. Schaeffer,et al.  Mitogen-Activated Protein Kinases: Specific Messages from Ubiquitous Messengers , 1999, Molecular and Cellular Biology.

[7]  R. Rappuoli,et al.  c‐Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs , 2002, Molecular microbiology.

[8]  S. Falkow,et al.  Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Selbach,et al.  Src Is the Kinase of the Helicobacter pylori CagA Protein in Vitro and in Vivo * , 2002, The Journal of Biological Chemistry.

[10]  R. Hunt,et al.  Meta-analysis of the relationship between Helicobacter pylori seropositivity and gastric cancer. , 1998, Gastroenterology.

[11]  C. Marshall,et al.  Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation , 1995, Cell.

[12]  T. Fujioka,et al.  Development of Helicobacter pylori-induced gastric carcinoma in Mongolian gerbils. , 1998, Cancer research.

[13]  Helicobacter pylori Caga Protein Can Be Tyrosine Phosphorylated in Gastric Epithelial Cells , 2000 .

[14]  N. Ahn,et al.  Determination of v-Mos-catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase by ESI/MS. , 1995, Biochemistry.

[15]  B. Mayer,et al.  Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins , 1995, Molecular and cellular biology.

[16]  T. Schöneberg,et al.  B‐Raf/Rap1 signaling, but not c‐Raf‐1/Ras, induces the histidine decarboxylase promoter in Helicobacter pylori infection , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  M. Selbach,et al.  The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c‐Src inactivation , 2003, The EMBO journal.

[18]  M. Naumann,et al.  Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus , 2000, Cellular microbiology.

[19]  R. Rappuoli,et al.  Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[20]  N. Perrimon,et al.  Opposing actions of CSW and RasGAP modulate the strength of Torso RTK signaling in the Drosophila terminal pathway. , 1998, Molecular cell.

[21]  A. Saltiel,et al.  Expression of catalytically inactive Syp phosphatase in 3T3 cells blocks stimulation of mitogen-activated protein kinase by insulin. , 1994, The Journal of biological chemistry.

[22]  C. Sasakawa,et al.  Grb2 is a key mediator of helicobacter pylori CagA protein activities. , 2002, Molecular cell.

[23]  D. Chopin,et al.  Epithelial Cell Plasticity in Development and Tumor Progression , 2004, Cancer and Metastasis Reviews.

[24]  Danesh.  Helicobacter pylori infection and gastric cancer: systematic review of the epidemiological studies , 1999, Alimentary pharmacology & therapeutics.

[25]  B. Neel,et al.  Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[26]  R. Rappuoli,et al.  Helicobacter pylori virulence and genetic geography. , 1999, Science.

[27]  W. Birchmeier,et al.  Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response , 2003, The Journal of cell biology.

[28]  M. Hengartner,et al.  The Caenorhabditis elegans SH2 domain-containing protein tyrosine phosphatase PTP-2 participates in signal transduction during oogenesis and vulval development. , 1998, Genes & development.

[29]  Z. Zhao,et al.  Concerted Activity of Tyrosine Phosphatase SHP-2 and Focal Adhesion Kinase in Regulation of Cell Motility , 1999, Molecular and Cellular Biology.

[30]  T. Hunter,et al.  Oncogenic kinase signalling , 2001, Nature.

[31]  D. Graham,et al.  Variants of the 3′ Region of the cagA Gene inHelicobacter pylori Isolates from Patients with DifferentH. pylori-Associated Diseases , 1998, Journal of Clinical Microbiology.

[32]  R. Haas,et al.  Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. , 2000, Science.

[33]  D. Graham,et al.  Molecular epidemiology of Helicobacter pylori: separation of H. pylori from East Asian and non-Asian countries , 2000, Epidemiology and Infection.

[34]  R. Rappuoli,et al.  Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[35]  M. Selbach,et al.  Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells , 2001, Molecular microbiology.

[36]  Megumi Higuchi,et al.  Attenuation of Helicobacter pylori CagA·SHP-2 Signaling by Interaction between CagA and C-terminal Src Kinase* , 2003, The Journal of Biological Chemistry.

[37]  E. Mccleskey,et al.  Rap1 mediates sustained MAP kinase activation induced by nerve growth factor , 1998, Nature.

[38]  M. Hatakeyama.  Helicobacter pylori CagA--a potential bacterial oncoprotein that functionally mimics the mammalian Gab family of adaptor proteins. , 2003, Microbes and infection.

[39]  B. Neel,et al.  The "Gab" in signal transduction. , 2003, Trends in cell biology.

[40]  Shigeo Ohno,et al.  Protein Kinase C δ Activates the MEK-ERK Pathway in a Manner Independent of Ras and Dependent on Raf* , 1996, The Journal of Biological Chemistry.

[41]  Takeshi Azuma,et al.  Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[42]  C. Walsh,et al.  Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptor beta to Ras. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[43]  T. Pawson,et al.  SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. , 1993, Science.

[44]  G. Friedman,et al.  Risk for gastric cancer in people with CagA positive or CagA negative Helicobacter pylori infection. , 1997, Gut.

[45]  B. Roe,et al.  Analyses of the cag pathogenicity island of Helicobacter pylori , 1998, Molecular microbiology.

[46]  W. Birchmeier,et al.  Coupling of Gab1 to C-Met, Grb2, and Shp2 Mediates Biological Responses , 2000, The Journal of cell biology.

[47]  Jonathan A. Cooper,et al.  A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase , 1994, Molecular and cellular biology.

[48]  H. Nagai,et al.  Helicobacter pylori infection induces gastric cancer in mongolian gerbils. , 1998, Gastroenterology.

[49]  M. Kasuga,et al.  Role of SH-PTP2, a protein-tyrosine phosphatase with Src homology 2 domains, in insulin-stimulated Ras activation. , 1994, Molecular and cellular biology.

[50]  W. Birchmeier,et al.  How to make tubes: signaling by the Met receptor tyrosine kinase. , 2003, Trends in cell biology.

[51]  Takeshi Azuma,et al.  SHP-2 Tyrosine Phosphatase as an Intracellular Target of Helicobacter pylori CagA Protein , 2001, Science.

[52]  T. Pawson,et al.  Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp‐2 , 1997, The EMBO journal.

[53]  G. Feng,et al.  Protein-tyrosine Phosphatase Shp-2 Regulates Cell Spreading, Migration, and Focal Adhesion* , 1998, The Journal of Biological Chemistry.

[54]  S. Falkow,et al.  Disruption of the Epithelial Apical-Junctional Complex by Helicobacter pylori CagA , 2003, Science.

[55]  D. Hanahan,et al.  The Hallmarks of Cancer , 2000, Cell.

[56]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[57]  E. Kuipers,et al.  Helicobacter pylori and atrophic gastritis: importance of the cagA status. , 1996, Journal of the National Cancer Institute.

[58]  C. Qu.  Role of the SHP-2 tyrosine phosphatase in cytokine-induced signaling and cellular response. , 2002, Biochimica et biophysica acta.

[59]  M. Rugge,et al.  Patients younger than 40 years with gastric carcinoma , 1999, Cancer.

[60]  I. Hariharan,et al.  The Rap1 GTPase functions as a regulator of morphogenesis in vivo , 1999, The EMBO journal.

[61]  M. Blaser,et al.  Heightened inflammatory response and cytokine expression in vivo to cagA+ Helicobacter pylori strains. , 1995, Laboratory investigation; a journal of technical methods and pathology.

[62]  A. Ostman,et al.  Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases. , 2001, Trends in cell biology.

[63]  D. Graham,et al.  Relationship between the cagA 3' repeat region of Helicobacter pylori, gastric histology, and susceptibility to low pH. , 1999, Gastroenterology.

[64]  M. Blaser,et al.  Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. , 1995, Cancer research.