Helicobacter pylori CagA Induces Ras-independent Morphogenetic Response through SHP-2 Recruitment and Activation

Abstract

The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wildtype or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA (siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.

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

“…This in turn leads to the downregulation of FAK kinase activity, which is critically involved in the induction of the hummingbird phenotype by CagA. Consistent with the function of SHP-2 in potentiating the Erk microtubule-associated protein (MAP) kinase pathway by both Ras-dependent and Ras-independent mechanisms, CagA also elicits sustained Erk activation, which has an important function in G1 to S phase cell-cycle progression, in gastric epithelial cells (Higashi et al, 2004).…”

Helicobacter pylori CagA interacts with E-cadherin and deregulates the β-catenin signal that promotes intestinal transdifferentiation in gastric epithelial cells

“…Immunoprecipitation and immunoblotting were performed as described previously (Higashi et al, 2004).…”

“…The cagA gene product CagA is delivered into H. pylori-attached gastric epithelial cells via the type IV secretion system (Segal et al, 1999;Odenbreit et al, 2000). Upon tyrosine phosphorylation by Src family kinases, CagA binds and activates SHP-2 tyrosine phosphatase to induce an elongated cell shape termed the 'hummingbird phenotype' (Higashi et al, 2002(Higashi et al, , 2004. CagA also interacts with and deregulates a number of cellular proteins such as Grb2, c-Met and ZO-1 in a phosphorylation-independent manner (Mimuro et al, 2002;Amieva et al, 2003;Churin et al, 2003).…”

Isolation of a distinct class of gain-of-function SHP-2 mutants with oncogenic RAS-like transforming activity from solid tumors

“…Mammalian expression vectors for SHP-2 and HRAS V12 were described previously (Higashi et al, 2004;Tsutsumi et al, 2006). cDNAs encoding mutant SHP-2 proteins were made from PTPN11 cDNA by site-directed mutagenesis and were cloned into pSP65SRa vector.…”