Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3 Cells
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Amino Acid Motifs
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Amino Acid Sequence
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Animals
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Antigens, Bacterial*
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Bacterial Proteins / chemistry*
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Bacterial Proteins / metabolism
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Binding Sites
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Cells, Cultured
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Cytoskeleton / metabolism
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Enzyme Inhibitors / pharmacology
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Escherichia coli / metabolism
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Fibroblasts / metabolism
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Green Fluorescent Proteins
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Helicobacter pylori / enzymology*
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Humans
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Luminescent Proteins / metabolism
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Mice
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Molecular Sequence Data
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Phenotype
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Phosphorylation
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Protein Binding
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Proto-Oncogene Proteins / physiology
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Proto-Oncogene Proteins c-fyn
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Proto-Oncogene Proteins c-yes
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Proto-Oncogene Proteins pp60(c-src) / metabolism*
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Proto-Oncogene Proteins pp60(c-src) / physiology*
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Transfection
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src-Family Kinases*
Substances
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Antigens, Bacterial
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Bacterial Proteins
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Enzyme Inhibitors
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Luminescent Proteins
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Proto-Oncogene Proteins
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Recombinant Fusion Proteins
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Recombinant Proteins
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cagA protein, Helicobacter pylori
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Green Fluorescent Proteins
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FYN protein, human
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Fyn protein, mouse
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Proto-Oncogene Proteins c-fyn
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Proto-Oncogene Proteins c-yes
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Proto-Oncogene Proteins pp60(c-src)
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Yes1 protein, mouse
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src-Family Kinases