Phosphorylation of Tyrosine 972 of the Helicobacter Pylori CagA Protein Is Essential for Induction of a Scattering Phenotype in Gastric Epithelial Cells

Mol Microbiol. 2001 Nov;42(3):631-44. doi: 10.1046/j.1365-2958.2001.02649.x.

Abstract

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / microbiology
  • Antigens, Bacterial*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Epithelial Cells / metabolism
  • Epithelial Cells / microbiology
  • Gastric Mucosa / metabolism
  • Gastric Mucosa / microbiology*
  • Helicobacter Infections / microbiology
  • Helicobacter pylori / classification*
  • Helicobacter pylori / genetics
  • Helicobacter pylori / pathogenicity*
  • Humans
  • Immunoblotting
  • Microscopy, Confocal
  • Phenotype
  • Phosphorylation
  • Stomach Neoplasms / microbiology
  • Tumor Cells, Cultured
  • Tyrosine / metabolism*
  • Virulence

Substances

  • Antigens, Bacterial
  • Bacterial Proteins
  • cagA protein, Helicobacter pylori
  • Tyrosine