Functional antagonism between Helicobacter pylori CagA and vacuolating toxin VacA in control of the NFAT signaling pathway in gastric epithelial cells.

Chronic infection with cagA-positive Helicobacter pylori is associated with the development of atrophic gastritis, peptic ulcers, and gastric adenocarcinoma. The cagA gene product CagA is injected into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Translocated CagA disturbs cellular functions by physically interacting with and deregulating intracellular signaling transducers through both tyrosine phosphorylation-dependent and -independent mechanisms. To gain further insights into the pathophysiological activities of CagA in gastric epithelial cells, we executed a genome-wide screening of CagA-responsive genes by using DNA microarray and identified nuclear factor of activated T cells (NFAT) transcription factors whose binding sites were overrepresented in the promoter regions of CagA-activated genes. Results of reporter assays confirmed that CagA was capable of activating NFAT in a manner independent of CagA phosphorylation. Expression of CagA in gastric epithelial cells provoked translocation of NFATc3, a member of the NFAT family, from the cytoplasm to the nucleus and activated an NFAT-regulated gene, p21WAF1/Cip1. CagA-mediated NFAT activation was abolished by inhibiting calcineurin or phospholipase Cgamma activity. Furthermore, treatment of cells with H. pylori VacA (vacuolating toxin), which inhibits NFAT activity in T lymphocytes, counteracted the ability of CagA to activate NFAT in gastric epithelial cells. These findings indicate that the two major H. pylori virulence factors inversely control NFAT activity. Considering the pleiotropic roles of NFAT in cell growth and differentiation, deregulation of NFAT, either positively or negatively, depending on the relative exposure of cells to CagA and VacA, may contribute to the various disease outcomes caused by H. pylori infection.

EPIYA motif is a membrane-targeting signal of Helicobacter pylori virulence factor CagA in mammalian cells.

Helicobacter pylori contributes to the development of peptic ulcers and atrophic gastritis. Furthermore, H. pylori strains carrying the cagA gene are more virulent than cagA-negative strains and are associated with the development of gastric adenocarcinoma. The cagA gene product, CagA, is translocated into gastric epithelial cells and localizes to the inner surface of the plasma membrane, in which it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif. Tyrosine-phosphorylated CagA specifically binds to and activates Src homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) at the membrane, thereby inducing an elongated cell shape termed the hummingbird phenotype. Accordingly, membrane tethering of CagA is an essential prerequisite for the pathogenic activity of CagA. We show here that membrane association of CagA requires the EPIYA-containing region but is independent of EPIYA tyrosine phosphorylation. We further show that specific deletion of the EPIYA motif abolishes the ability of CagA to associate with the membrane. Conversely, reintroduction of an EPIYA sequence into a CagA mutant that lacks the EPIYA-containing region restores membrane association of CagA. Thus, the presence of a single EPIYA motif is necessary for the membrane localization of CagA. Our results indicate that the EPIYA motif has a dual function in membrane association and tyrosine phosphorylation, both of which are critically involved in the activity of CagA to deregulate intracellular signaling, and suggest that the EPIYA motif is a crucial therapeutic target of cagA-positive H. pylori infection.

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.