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 , EPIYATI x(27) EIYATI, 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. Conclusion. These findings suggest an important role for CagA containing ITAM-like sequences in the pathogenesis of H. pylori- related disease.