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

Hideaki Higashi,Akihiro Nakaya,Ryouhei Tsutsumi,Kazuyuki Yokoyama,Yumiko Fujii,Susumu Ishikawa,Megumi Higuchi,Atsushi Takahashi,Yo Kurashima,Yasuhiro Teishikata,Shinya Tanaka,Takeshi Azuma,Masanori Hatakeyama

doi:10.1074/jbc.m309964200

Hideaki Higashi, Akihiro Nakaya + Show 11 more

Open Access

https://doi.org/10.1074/jbc.m309964200

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Abstract

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.

Highlights

Helicobacter pylori, a micro-aerophilic spiral-shaped bacterium that colonizes the human stomach, is thought to infect about half of the world’s population [1]
To elucidate the mechanism by which cytotoxin-associated gene A antigen (CagA) induces morphological change, we generated a series of AGSderived, stable transfectant clones that express HA-tagged, wild-type CagA, derived from H. pylori strain NCTC11637, or HA-tagged, phosphorylation-resistant CagA (PR CagA), in which the tyrosine residues constituting the five EPIYA motifs were substituted with alanines, under the control of a tetracycline-inducible gene expression system (Fig. 1A)
We identified an important role of SHP-2 in the morphogenetic activity of H. pylori CagA in gastric epithelial cells

Summary

The abbreviations used are

CagA, cytotoxin-associated gene A antigen; cagA, cytotoxin-associated gene A; SH2, Src homology 2; SHP-2, SH2 domain-containing protein-tyrosine phosphatase-2; siRNA, small interference RNA; Grb, growth factor receptor bound protein 2; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/Erk kinase; cag PAI, cytotoxin-associated gene pathogenicity island; Dox, doxycycline; PMA, phorbol 12-myristate 13-acetate; HA, hemagglutinin; Csk, C-terminal Src kinase; Gab, Grb2associated binder; WT, wild-type; PR, phosphorylation-resistant; rtTA, reverse tetracycline-dependent transactivator; PI3K, phosphatidylinositol 3-kinase; tet-on, tetracycline-inducible; HGF, hepatocyte growth factor; JNK, c-Jun N-terminal kinase; FCS, fetal calf serum. That the number of EPIYA motifs in CagA varies among different Western H. pylori isolates [8, 25, 26]. Western CagA having a greater number of repeatable EPIYA motifs exhibits stronger activity to bind SHP-2 and to induce the hummingbird phenotype [24]. Grb and c-Met have been reported to form physical complexes with CagA, their interactions are independent of CagA phosphorylation [37, 38]. These observations collectively suggest that the bacterial protein functions as a docking/scaffolding protein that recruits and deregulates multiple signaling molecules in the injected host cells [39]. We obtained evidence that SHP-2 is involved in the regulation of Erk MAPK activity by CagA

EXPERIMENTAL PROCEDURES

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DISCUSSION