Src Is the Kinase of the Helicobacter pylori CagA Protein in Vitro and in Vivo

Abstract

The gastric pathogen Helicobacter pylori uses a type IV secretion system to inject the bacterial CagA protein into gastric epithelial cells. Within the host cell, CagA becomes phosphorylated on tyrosine residues and initiates cytoskeletal rearrangements. We demonstrate here that Src-like protein-tyrosine kinases mediate CagA phosphorylation in vitro and in vivo. First, the Src-specific tyrosine kinase inhibitor PP2 specifically blocks CagA phosphorylation and cytoskeletal rearrangements thereby inhibiting the CagA-induced hummingbird phenotype of gastric epithelial cells. Second, CagA is in vivo phosphorylated by transiently expressed c-Src. Third, recombinant c-Src and lysates derived from c-Src-expressing fibroblasts but not lysates derived from Src-, Yes-, and Fyn-deficient cells phosphorylated CagA in vitro. Fourth, a transfected CagA-GFP fusion protein is phosphorylated in vivo in Src-positive fibroblasts but not in Src-, Yes-, and Fyn-deficient cells. Because a CagA-GFP fusion protein mutated in an EPIYA motif is not efficiently phosphorylated in any of these fibroblast cells, the CagA EPIYA motif appears to constitute the major c-Src phosphorylation site conserved among CagA-positive Helicobacter strains.

The versatile bacterial type IV secretion systems

“…In parallel, studies by several laboratories are generating extensive information about the cellular consequences of CagA translocation. On transfer, CagA localizes on the inner surface of the plasma membrane where it interacts with and is phosphorylated by the Src family of protein tyrosine kinases, such as c-Src [84][85][86][87][88][89][90][91] . The CagA phosphorylation sites have been mapped to the so-called EPIYA motifs that share homology with c-Src consensus phosphorylation sites and are present in variable numbers in the carboxy-terminal half of the protein 89,91 .…”

“…4; Table 3). For example, on phosphorylation, CagA P-Tyr inactivates c-Src kinase activity, which results in dephosphorylation of another c-Src substrate, CORTACTIN 91 . Dephosphorylated cortactin relocalizes in the cell and is thought to have enhanced ACTIN cross-linking activity, causing actin rearrangements associated with the hummingbird phenotype.…”

Linking epithelial polarity and carcinogenesis by multitasking Helicobacter pylori virulence factor CagA

“…CagL-integrin interaction triggers type IV injection of CagA into gastric epithelial cells. Within the host cells, CagA localizes to the inner surface of the plasma membrane, where it undergoes tyrosine phosphorylation by Src family kinases or Abl kinase (Selbach et al, 2002;Stein et al, 2002;Poppe et al, 2007;Tammer et al, 2007). The tyrosine phosphorylation site of CagA is characterized by the Glu-Pro-IleTyr-Ala (EPIYA) motif, which is present in multiple numbers in the carboxy-terminal polymorphic region (EPIYA-repeat region) of the protein (Higashi et al, 2002a, b).…”

Phosphorylation of Helicobacter pylori CagA by c-Abl leads to cell motility

“…Translocated CagA induces cellular processes, which lead to stimulation of cell dissemination followed by cell motility, and invasive growth in gastric epithelial cells (Segal et al, 1999;Bagnoli et al, 2005). The molecular mechanisms leading to the CagAdependent dissemination of cell colonies are only partially known, but it became apparently clear that tyrosine phosphorylation of CagA is a crucial step in the epithelial cell migration (Selbach et al, 2002;Stein et al, 2002). It was further hypothesized that phosphorylated CagA might function as an adapter protein to recruit eukaryotic proteins in a multi-protein and -enzyme complex, which includes zonula occludens-1 (ZO-1) (Amieva et al, 2003), Src homology 2 domain tyrosine phosphatase (SHP-2) (Higashi et al, 2002), Grb2 (Mimuro et al, 2002), c-Met and phospholipase C-g (PLC-g) (Churin et al, 2003) and that it mediates signals leading to cell motility.…”