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Summary Helicobacter pylori East Asian CagA is more closely associated with gastric cancer than Western CagA. Here we show that, upon tyrosine phosphorylation, the East Asian CagA-specific EPIYA-D segment binds to the N-SH2 domain of pro-oncogenic SHP2 phosphatase two orders of magnitude greater than Western CagA-specific EPIYA-C. This high-affinity binding is achieved via cryptic interaction between Phe at the +5 position from phosphotyrosine in EPIYA-D and a hollow on the N-SH2 phosphopeptide-binding floor. Also, duplication of EPIYA-C in Western CagA, which increases gastric cancer risk, enables divalent high-affinity binding with SHP2 via N-SH2 and C-SH2. These strong CagA bindings enforce enzymatic activation of SHP2, which endows cells with neoplastic traits. Mechanistically, N-SH2 in SHP2 is in an equilibrium between stimulatory "relaxed" and inhibitory "squeezed" states, which is fixed upon high-affinity CagA binding to the "relaxed" state that stimulates SHP2. Accordingly, East Asian CagA and Western CagA exploit distinct mechanisms for SHP2 deregulation.

Most if not all gastric cancers are associated with chronic infection of the stomach mucosa with Helicobacter pylori cagA-positive strains(1-4). Approximately 10% of gastric cancers also harbour Epstein-Barr virus (EBV) in the cancer cells(5,6). Following delivery into gastric epithelial cells via type IV secretion(7,8), the cagA-encoded CagA protein undergoes tyrosine phosphorylation on the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs initially by Src family kinases (SFKs) and then by c-Abl(9,10). Tyrosine-phosphorylated CagA binds to the pro-oncogenic protein tyrosine phosphatase SHP2 and thereby deregulates the phosphatase activity(11,12), which has been considered to play an important role in gastric carcinogenesis(13). Here we show that the SHP2 homologue SHP1 interacts with CagA independently of the EPIYA motif. The interaction potentiates the phosphatase activity of SHP1 that dampens the oncogenic action of CagA by dephosphorylating the CagA EPIYA motifs. In vitro infection of gastric epithelial cells with EBV induces SHP1 promoter hypermethylation, which strengthens phosphorylation-dependent CagA action via epigenetic downregulation of SHP1 expression. Clinical specimens of EBV-positive gastric cancers also exhibit SHP1 hypermethylation with reduced SHP1 expression. The results reveal that SHP1 is the long-sought phosphatase that can antagonize CagA. Augmented H. pylori CagA activity, via SHP1 inhibition, might also contribute to the development of EBV-positive gastric cancer.

Infection with cagA-positive Helicobacter pylori is critically associated with the development of gastric cancer. The cagA-encoded CagA is delivered into gastric epithelial cells via type IV secretion, where it interacts with and thereby deregulates the pro-oncogenic phosphatase SHP2. East Asian CagA and Western CagA are two major CagA species produced by H. pylori circulating in East Asian countries and in the rest of the world, respectively. The SHP2 binding site of Western CagA, termed the EPIYA-C segment, variably duplicates and infection with H. pylori carrying Western CagA with multiple EPIYA-C segments is a distinct risk factor of gastric cancer. Here we show that duplication of EPIYA-C from one to two or more increases SHP2 binding of Western CagA by more than one hundredfold. Based on the decisive difference in SHP2 binding, Western CagA can be divided into two types: type I CagA carrying a single EPIYA-C segment and type II CagA carrying multiple EPIYA-C segments. Gastric epithelial cells expressing type II CagA acquire the ability to invade extracellular matrices, a malignant cellular trait associated with deregulated SHP2. A big leap in SHP2 binding activity may therefore provide molecular basis that makes type II Western CagA a distinct gastric cancer risk.

Chronic infection with Helicobacter pylori cagA-positive strains is associated with atrophic gastritis, peptic ulceration, and gastric carcinoma. The cagA gene product, CagA, is delivered into gastric epithelial cells via type IV secretion, where it undergoes tyrosine phosphorylation at the EPIYA motifs. Tyrosine-phosphorylated CagA binds and aberrantly activates the oncogenic tyrosine phosphatase SHP2, which mediates induction of elongated cell morphology (hummingbird phenotype) that reflects CagA virulence. CagA also binds and inhibits the polarity-regulating kinase partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) via the CagA multimerization (CM) sequence independently of tyrosine phosphorylation. Because PAR1 exists as a homodimer, two CagA proteins appear to be passively dimerized through complex formation with a PAR1 dimer in cells. Interestingly, a CagA mutant that lacks the CM sequence displays a reduced SHP2 binding activity and exhibits an attenuated ability to induce the hummingbird phenotype, indicating that the CagA-PAR1 interaction also influences the morphological transformation. Here we investigated the role of CagA dimerization in induction of the hummingbird phenotype with the use of a chemical dimerizer, coumermycin. We found that CagA dimerization markedly stabilizes the CagA-SHP2 complex and thereby potentiates SHP2 deregulation, causing an increase in the number of hummingbird cells. Protrusions of hummingbird cells induced by chemical dimerization of CagA are further elongated by simultaneous inhibition of PAR1. This study revealed a role of the CM sequence in amplifying the magnitude of SHP2 deregulation by CagA, which, in conjunction with the CM sequence-mediated inhibition of PAR1, evokes morphological transformation that reflects in vivo CagA virulence.