Helicobacter pylori CagA containing ITAM-like sequences localized to lipid rafts negatively regulates VacA-induced signaling in vivo.
BACKGROUND
Helicobacter pylori CagA is injected into the host cell and tyrosine-phosphorylated. We examined tyrosine-phosphorylation sites of CagA, as well as the function of CagA proteins in vivo and in vitro.


METHODS
After proteolytic digestion of CagA with lysyl endopeptidase, CagA tyrosine-phosphorylation sites were determined using quadropolar time-of-flight (Q-TOF) mass spectrometry analysis. Specific anti-pY CagA polyclonal and anti-CagA monoclonal antibodies were used to examine gastric mucosal biopsy specimens from H. pylori infected patients.


RESULTS
Mass spectrometry identified five crucial tyrosine-phosphorylation sites of CagA at Tyr893, Tyr912, Tyr965, Tyr999, and Tyr1033 within the five repeated EPIYA sequences of H. pylori (NCTC11637)-infected AGS cells. CagA protein also had an immuno-receptor tyrosine-based activation motif (ITAM)-like amino acid sequences in the 3' region of the cagA, EPIYATIx27EIYATI, which closely resembled the ITAM. CagA proteins: (i) were localized to the 1% TritonX-100 resistant membrane fraction (lipid rafts); (ii) formed a cluster of phosphorylated CagA protein complexes; (iii) associated with tyrosine-phosphorylated GIT1/Cat1 (G protein-coupled receptor kinase-interactor 1/Cool-associated tyrosine-phosphorylated 1), substrate molecules of receptor type protein-tyrosine phosphatase (RPTPzeta/beta), which is the receptor of VacA; and (iv) were involved in a delay and negative regulation of VacA-induced signal. Furthermore, immunohistochemical staining of gastric mucosal biopsy specimens provided strong evidence that tyrosine-phosphorylated CagA is found together with CagA at the luminal surface of gastric foveola in vivo.


CONCLUSIONS
These findings suggest an important role for CagA containing ITAM-like sequences in the pathogenesis of H. pylori-related disease.
Asahi
Momoyo
M
Faculty of Nursing and Social Welfare Science, Fukui Prefectural University, Fukui 910-1195, Japan.
Tanaka
Yukie
Y
Izumi
Toshimasa
T
Ito
Yoshiyuki
Y
Naiki
Hironobu
H
Kersulyte
Dangeruta
D
Tsujikawa
Kazutake
K
Saito
Masakazu
M
Sada
Kiyonao
K
Yanagi
Shigeru
S
Fujikawa
Akihiro
A
Noda
Masaharu
M
Itokawa
Yoshinori
Y
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D001665:Binding Sites; D002460:Cell Line; D005753:Gastric Mucosa / Q000382:microbiology; D005798:Genes, Bacterial; D016481:Helicobacter Infections / Q000382:microbiology; D016480:Helicobacter pylori / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000382:microbiology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C074689:VacA protein, Helicobacter pylori; C080866:cagA protein, Helicobacter pylori; D014443:Tyrosine