PAR1b takes the stage in the morphogenetic and motogenetic activity of Helicobacter pylori CagA oncoprotein

Helicobacter pylori CagA oncoprotein is critically involved in gastric carcinogenesis. Upon delivery into gastric epithelial cells via type IV secretion, CagA induces an extremely elongated cell-shape known as the hummingbird phenotype, which is associated with massive changes in actin cytoskeleton and elevated motility. With the notion that the hummingbird phenotype reflects pathogenic/oncogenic activity of CagA, many studies have focused on the mechanism through which CagA induces the morphological change. Once delivered, CagA interacts with host proteins such as oncogenic phosphatase SHP2 and polarity-regulating kinase PAR1b. Whereas the essential role of the CagA-SHP2 interaction in inducing the hummingbird phenotype has been extensively investigated, involvement of the CagA-PAR1b interaction in the morphological change has remained uncertain. Recently, we found that the CagA-PAR1b interaction, which inhibits PAR1b kinase activity, influences the actin cytoskeletal system and potentiates the magnitude of the hummingbird phenotype. We also found that PAR1b inactivates a RhoA-specific GEF, GEF-H1, via phosphorylation and thereby inhibits cortical actin and stress fiber formation. Collectively, these findings indicate that CagA-mediated inhibition of PAR1b promotes RhoA-dependent actin-cytoskeletal rearrangement and thereby strengthens the hummingbird phenotype induced by CagA-stimulated SHP2 during infection with H. pylori cagA-positive strains.     

Helicobacter pylori stimulates epithelial cell migration via CagA-mediated perturbation of host cell signaling

Helicobacter pylori CagA is delivered into gastric epithelial cells, where undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif to interact with Src homology 2-containing protein tyrosine phosphatase-2 (SHP2) oncoprotein. CagA also binds to partitioning-defective 1 (PAR1) polarity-regulating kinase via the CagA multimerization (CM) sequence. To investigate pathophysiological role of CagA-SHP2 and/or CagA-PAR1 interaction in H. pylori infection, we generated H. pylori isogenic strains producing a phosphorylation-resistant CagA and a CagA without CM sequence. Infection studies revealed that deregulation of epithelial cell motility was more prominent in the wild-type strain than in the mutant strains. Thus, both CagA-SHP2 and CagA-PAR1 interactions are involved in the pathogenicity of cagA-positive H. pylori.     

Focal adhesion kinase is a substrate and downstream effector of SHP-2 complexed with Helicobacter pylori CagA

Infection with cagA-positive Helicobacter pylori (H. pylori) is associated with atrophic gastritis, peptic ulcer, and gastric adenocarcinoma. The cagA gene product CagA is translocated from H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation by Src family kinases (SFKs). Tyrosine-phosphorylated CagA binds and activates SHP-2 phosphatase and the C-terminal Src kinase (Csk) while inducing an elongated cell shape termed the "hummingbird phenotype." Here we show that CagA reduces the level of focal adhesion kinase (FAK) tyrosine phosphorylation in gastric epithelial cells. The decrease in phosphorylated FAK is due to SHP-2-mediated dephosphorylation of FAK at the activating phosphorylation sites, not due to Csk-dependent inhibition of SFKs, which phosphorylate FAK. Coexpression of constitutively active FAK with CagA inhibits induction of the hummingbird phenotype, whereas expression of dominant-negative FAK elicits an elongated cell shape characteristic of the hummingbird phenotype. These results indicate that inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. Impaired cell adhesion and increased motility by CagA may be involved in the development of gastric lesions associated with cagA-positive H. pylori infection.     

Structural basis and functional consequence of Helicobacter pylori CagA multimerization in cells

Helicobacter pylori cagA-positive strains are associated with gastric adenocarcinoma. The cagA gene product CagA is delivered into gastric epithelial cells where it localizes to the plasma membrane and undergoes tyrosine phosphorylation at the EPIYA-repeat region, which contains the EPIYA-A segment, EPIYA-B segment, and Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D segment. In host cells, CagA specifically binds to and deregulates SHP-2 phosphatase via the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment, thereby inducing an elongated cell shape known as the hummingbird phenotype. In this study, we found that CagA multimerizes in cells in a manner independent of its tyrosine phosphorylation. Using a series of CagA mutants, we identified a conserved amino acid sequence motif (FPLXRXXXVXDLSKVG), which mediates CagA multimerization, within the EPIYA-C segment as well as in a sequence that located immediately downstream of the EPIYA-C or EPIYA-D segment. We also found that a phosphorylation-resistant CagA, which multimerizes but cannot bind SHP-2, inhibits the wild-type CagA-SHP-2 complex formation and abolishes induction of the hummingbird phenotype. Thus, SHP-2 binds to a preformed and tyrosinephosphorylated CagA multimer via its two Src homology 2 domains. These results, in turn, indicate that CagA multimerization is a prerequisite for CagA-SHP-2 interaction and subsequent deregulation of SHP-2. The present work raises the possibility that inhibition of CagA multimerization abolishes pathophysiological activities of CagA that promote gastric carcinogenesis.     

Attenuation of Helicobacter pylori CagA x SHP-2 signaling by interaction between CagA and C-terminal Src kinase

Helicobacter pylori (H. pylori) is a causative agent of gastric diseases ranging from gastritis to cancer. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like morphological change termed the "hummingbird phenotype." In this work, we demonstrate that CagA is also capable of interacting with C-terminal Src kinase (Csk). As is the case with SHP-2, Csk selectively binds tyrosine-phosphorylated CagA via its SH2 domain. Upon complex formation, CagA stimulates Csk, which in turn inactivates the Src family of protein-tyrosine kinases. Because Src family kinases are responsible for CagA phosphorylation, an essential prerequisite of CagA.SHP-2 complex formation and subsequent induction of the hummingbird phenotype, our results indicate that CagA-Csk interaction down-regulates CagA.SHP-2 signaling by both competitively inhibiting CagA.SHP-2 complex formation and reducing levels of CagA phosphorylation. We further demonstrate that CagA.SHP-2 signaling eventually induces apoptosis in AGS cells. Our results thus indicate that CagA-Csk interaction prevents excess cell damage caused by deregulated activation of SHP-2. Attenuation of CagA activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

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.     

Stability of Helicobacter pylori CagA oncoprotein in human gastric epithelial cells

Upon delivery into gastric epithelial cells, Helicobacter pylori cytotoxin-associated gene A (CagA) binds and deregulates cellular proteins such as Src homology 2 domain-containing protein tyrosine phosphatase 2 and partitioning-defective 1 (PAR1), thereby acting as an epigenetic oncoprotein that promotes early phases of gastric cancer development. To elucidate the spatial and temporal contribution of CagA to carcinogenesis, it is crucial to know the stability of CagA in host cells. Here we show that the biological half-life of CagA is about 200 min in gastric epithelial cells. Furthermore, deletion of the PAR1-binding sequence accelerates CagA degradation. Thus, CagA is a relatively short half-life protein whose stability may be modulated through complex formation with PAR1.     

Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells

Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor-like ('hummingbird') phenotype. In this study, using a cagA mutant complemented with wild-type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Anti-phosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA-green fluorescent protein (GFP) fusion proteins and two-dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C-terminus. Site-directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).     

Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.

The gastric pathogen Helicobacter pylori uses a type IV secretion system to inject the bacterial CagA protein into gastric epithelial cells. Within the host cell, CagA becomes phosphorylated on tyrosine residues and initiates cytoskeletal rearrangements. We demonstrate here that Src-like protein-tyrosine kinases mediate CagA phosphorylation in vitro and in vivo. First, the Src-specific tyrosine kinase inhibitor PP2 specifically blocks CagA phosphorylation and cytoskeletal rearrangements thereby inhibiting the CagA-induced hummingbird phenotype of gastric epithelial cells. Second, CagA is in vivo phosphorylated by transiently expressed c-Src. Third, recombinant c-Src and lysates derived from c-Src-expressing fibroblasts but not lysates derived from Src-, Yes-, and Fyn-deficient cells phosphorylated CagA in vitro. Fourth, a transfected CagA-GFP fusion protein is phosphorylated in vivo in Src-positive fibroblasts but not in Src-, Yes-, and Fyn-deficient cells. Because a CagA-GFP fusion protein mutated in an EPIYA motif is not efficiently phosphorylated in any of these fibroblast cells, the CagA EPIYA motif appears to constitute the major c-Src phosphorylation site conserved among CagA-positive Helicobacter strains.     

CagA phosphorylation-dependent MMP-9 expression in gastric epithelial cells

Background: Infection of cagA‐positive Helicobacter pylori is associated with increased expression of MMPs in gastric epithelial cells. The role of phosphorylated CagA in the induction of MMP‐9, a protease‐degrading basement membrane, in gastric epithelial cells has not been clearly defined yet. The aim of this study is to analyze whether the presence of CagA and its phosphorylation status play a role in increased expression of MMP‐9 in gastric epithelial cells. Materials and Methods: Induction of MMP‐9 secretion was analyzed in gastric epithelial AGS cells harboring CagA with or without EPIYA motif, which is injected by H. pylori or ectopically expressed. In addition, signaling pathways involved in the CagA‐dependent MMP‐9 production have been studied. Results: The 147C strain of H. pylori expressing tyrosine‐phosphorylated CagA (EPIYA present) induced higher MMP‐9 secretion by AGS cells than the 147A strain expressing non‐tyrosine‐phosphorylated CagA (EPIYA absent). In addition, in bacteria‐free CagA‐inducible AGS cells, expression of wild‐type CagA induced more MMP‐9 secretion than phosphorylation‐resistant CagA. Inhibition of CagA phosphorylation by the Src family kinase inhibitor PP1 downregulated CagA‐mediated MMP‐9 secretion. Knockdown of SHP‐2 phosphatase dramatically reduced MMP‐9 secretion. ERK inhibitors, PD98059 and U0126, and NF‐κB pathway inhibitors, sulfasalazine and N‐acetyl‐l ‐cysteine, also inhibited MMP‐9 expression. Conclusion: These results support a model whereby the EPIYA motif of CagA is phosphorylated by Src family kinases in gastric epithelial cells, which initiates activation of SHP‐2. In addition, they suggest that the resultant activation of ERK pathway along with CagA‐dependent NF‐κB activation is critical for the induction of MMP‐9 secretion.     

Role of Abl and Src family kinases in actin-cytoskeletal rearrangements induced by the Helicobacter pylori CagA protein

The clinical outcome of infections with Helicobacter pylori is determined by a complex interplay of host-pathogen interactions, and persistent infection with this pathogen is the major cause of developing chronic gastritis, peptic ulcers and gastric cancer. Highly virulent strains encode a so-called type IV secretion system which translocates the CagA effector protein into gastric epithelial target cells. Injected CagA becomes tyrosine-phosphorylated on EPIYA sequence motifs by Src and Abl family kinase members. CagA then binds to and activates/inactivates various signalling proteins in a phosphorylation-dependent and phosphorylation-independent manner. In this way injected CagA can act as a master key that evolved during evolution the ability to highjack multiple downstream signalling cascades. Here we review our knowledge on the tyrosine phosphorylation motifs in CagA, the recent advances in the interaction of CagA with Src and Abl tyrosine kinases and their role in signalling events leading to changes of the phosphorylation status of actin-binding proteins cortactin, ezrin and vinculin followed by actin-cytoskeletal rearrangements, cell scattering and elongation. Detailed investigation of these pathways will help to yield novel insights and to elucidate the mechanisms of H. pylori-induced pathogenesis.     

Identification of new substrates of the protein-tyrosine phosphatase PTP1B by Bayesian integration of proteome evidence

There is growing evidence that tyrosine phosphatases display an intrinsic enzymatic preference for the sequence context flanking the target phosphotyrosines. On the other hand, substrate selection in vivo is decisively guided by the enzyme-substrate connectivity in the protein interaction network. We describe here a system wide strategy to infer physiological substrates of protein-tyrosine phosphatases. Here we integrate, by a Bayesian model, proteome wide evidence about in vitro substrate preference, as determined by a novel high-density peptide chip technology, and "closeness" in the protein interaction network. This allows to rank candidate substrates of the human PTP1B phosphatase. Ultimately a variety of in vitro and in vivo approaches were used to verify the prediction that the tyrosine phosphorylation levels of five high-ranking substrates, PLC-γ1, Gab1, SHP2, EGFR, and SHP1, are indeed specifically modulated by PTP1B. In addition, we demonstrate that the PTP1B-mediated dephosphorylation of Gab1 negatively affects its EGF-induced association with the phosphatase SHP2. The dissociation of this signaling complex is accompanied by a decrease of ERK MAP kinase phosphorylation and activation.     

c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs

The human pathogen Helicobacter pylori colonizes the mucous layer of the stomach. During parasitic infection, freely swimming bacteria adhere to the gastric epithelial cells and trigger intracellular signalling pathways. This process requires the translocation of the effector protein CagA into the host cell through a specialized type IV secretion system encoded in the cag pathogenicity island. Following transfer, CagA is phosphorylated on tyrosine residues by a host cell kinase. Here, we describe how the tyrosine phosphorylation of CagA is restricted to a previously identified repeated sequence called D1. This sequence is located in the C-terminal half of the protein and contains the five-amino-acid motif EPIYA, which is amplified by duplications in a large fraction of clinical isolates. Tyrosine phosphorylation of CagA is essential for the activation process that leads to dramatic changes in the morphology of cells growing in culture. In addition, we observed that two members of the src kinases family, c-Src and Lyn, account for most of the CagA-specific kinase activity in host cell lysates. Thus, CagA translocation followed by tyrosine phosphorylation at the EPIYA motifs promotes a growth factor-like response with intense cytoskeletal rearrangements, cell elongation effects and increased cellular motility.     

Grb2 is a key mediator of helicobacter pylori CagA protein activities

CagA delivered from Helicobacter pylori into gastric epithelial cells undergoes tyrosine phosphorylation and induces host cell morphological changes. Here we show that CagA can interact with Grb2 both in vitro and in vivo, which results in the activation of the Ras/MEK/ERK pathway and leads to cell scattering as well as proliferation. Importantly, this ability of CagA is independent from the tyrosine phosphorylation, which occurs within the five repeated EPIYA sequences (PY region) of CagA. However, the PY region appears to be indispensable for the Grb2 binding and induction of the cellular responses. Thus, intracellular CagA via its binding to Grb2 may act as a transducer for stimulating growth factor-like downstream signals which lead to cell morphological changes and proliferation, the causes of H. pylori-induced gastric hyperplasia.     

Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool

The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection.     

The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation

The gastric pathogen Helicobacter pylori translocates the CagA protein into epithelial cells by a type IV secretion process. Translocated CagA is tyrosine phosphorylated (CagA(P-Tyr)) on specific EPIYA sequence repeats by Src family tyrosine kinases. Phos phorylation of CagA induces the dephosphorylation of as yet unidentified cellular proteins, rearrangements of the host cell actin cytoskeleton and cell scattering. We show here that CagA(P-Tyr) inhibits the catalytic activity of c-Src in vivo and in vitro. c-Src inactivation leads to tyrosine dephosphorylation of the actin binding protein cortactin. Concomitantly, cortactin is specifically redistributed to actin-rich cellular protrusions. c-Src inactivation and cortactin dephosphorylation are required for rearrangements of the actin cytoskeleton. Moreover, CagA(P-Tyr)-mediated c-Src inhibition downregulates further CagA phosphorylation through a negative feedback loop. This is the first report of a bacterial virulence factor that inhibits signalling of a eukaryotic tyrosine kinase and on a role of c-Src inactivation in host cell cytoskeletal rearrangements.     

A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.     

Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein (SPRED1), a tyrosine-protein phosphatase non-receptor type 11 (SHP2) substrate in the Ras/extracellular signal-regulated kinase (ERK) pathway

SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway.