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Dramatic increase in SHP2 binding activity of Helicobacter pylori Western CagA by EPIYA-C duplication: its implications in gastric carcinogenesis.

ABSTRACT: 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.

SUBMITTER: Nagase L

PROVIDER: S-EPMC4623810 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Dramatic increase in SHP2 binding activity of Helicobacter pylori Western CagA by EPIYA-C duplication: its implications in gastric carcinogenesis.

Nagase Lisa L Hayashi Takeru T Senda Toshiya T Hatakeyama Masanori M

Scientific reports 20151028

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 EP ...[more]

PMID: 26507409

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Project description:The clinical outcome of Helicobacter pylori infections is determined by multiple host-pathogen interactions that may develop to chronic gastritis, and sometimes peptic ulcers or gastric cancer. Highly virulent strains encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-sequence motifs, called A, B and C in Western-type strains, by members of the oncogenic Src and Abl host kinases. Phosphorylated EPIYA-motifs mediate interactions of CagA with host signaling factors--in particular various SH2-domain containing human proteins--thereby hijacking multiple downstream signaling cascades. Observations of tyrosine-phosphorylated CagA are mainly based on the use of commercial phosphotyrosine antibodies, which originally were selected to detect phosphotyrosines in mammalian proteins. Systematic studies of phosphorylated EPIYA-motif detection by the different antibodies would be very useful, but are not yet available. To address this issue, we synthesized phospho- and non-phosphopeptides representing each predominant Western CagA EPIYA-motif, and determined the recognition patterns of seven different phosphotyrosine antibodies in Western blots, and also performed infection studies with diverse representative Western H. pylori strains. Our results show that a total of 9-11 amino acids containing the phosphorylated EPIYA-motifs are necessary and sufficient for specific detection by these antibodies, but revealed great variability in sequence recognition. Three of the antibodies recognized phosphorylated EPIYA-motifs A, B and C similarly well; whereas preferential binding to phosphorylated motif A and motifs A and C was found with two and one antibodies, respectively, and the seventh anti-phosphotyrosine antibody did not recognize any phosphorylated EPIYA-motif. Controls showed that none of the antibodies recognized the corresponding non-phospho CagA peptides, and that all of them recognized phosphotyrosines in mammalian proteins. These data are valuable in judicious application of commercial anti-phosphotyrosine antibodies and in characterization of CagA phosphorylation during infection and disease development.

Project description:Highly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming.This study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9-11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells.The results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.

Dataset Information

Dramatic increase in SHP2 binding activity of Helicobacter pylori Western CagA by EPIYA-C duplication: its implications in gastric carcinogenesis.

Publications

Dramatic increase in SHP2 binding activity of Helicobacter pylori Western CagA by EPIYA-C duplication: its implications in gastric carcinogenesis.

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