Influence of EPIYA-repeat polymorphism on the phosphorylation-dependent biological activity of Helicobacter pylori CagA.
OBJECTIVE
Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3'-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity.


METHODS
A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method.


RESULTS
CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA-SHP-2 interaction.


CONCLUSIONS
EPIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis.
Naito
Masanori
M
Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan.
Yamazaki
Takeshi
T
Tsutsumi
Ryouhei
R
Higashi
Hideaki
H
Onoe
Kazunori
K
Yamazaki
Shiho
S
Azuma
Takeshi
T
Hatakeyama
Masanori
M
D000942:Antigens, Bacterial / Q000145:classification / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D011110:Polymorphism, Genetic; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D047908:Intracellular Signaling Peptides and Proteins; C080866:cagA protein, Helicobacter pylori; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases