Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein isessential for induction of a scattering phenotype in gastric epithelial cells

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 ph osphorylated. This process is associated with characteristic cytoskeletal r earrangements, resulting in a scatter factor-like ('hummingbird') phenotype . In this study, using a cagA mutant complemented with wild-type cagA and t ransiently expressing CagA in AGS cells, we have demonstrated that transloc ated CagA is necessary for rearrangements of the actin cytoskeleton to occu r. Antiphosphotyrosine immunoblotting studies and treatment of infected cel ls with phosphotyrosine kinase inhibitors suggested that not only transloca tion but also phosphorylation of CagA is important in this process. Transie nt expression of CagA-green fluorescent protein (GFP) fusion proteins and t wo-dimensional gel electrophoresis of CagA protein species demonstrated tyr osine phosphorylation in the C-terminus. Site-directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellul ar phenotype. We have also demonstrated that translocation and phosphorylat ion of CagA is necessary but not sufficient for induction of the hummingbir d phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).