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Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response.

Churin Y, Al-Ghoul L, Kepp O, Meyer TF, Birchmeier W, Naumann M - J. Cell Biol. (2003)

Bottom Line: The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response.CagA could represent a bacterial adaptor protein that associates with phospholipase Cgamma but not Grb2-associated binder 1 or growth factor receptor-bound protein 2.The activation of the motogenic response in H. pylori-infected epithelial cells suggests that CagA could be involved in tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Experimental Internal Medicine, Medical Faculty, Otto-von-Guericke-University, Leipziger Strasse 44, 39120 Magdeburg, Germany.

ABSTRACT
Infection with the human microbial pathogen Helicobacter pylori is assumed to lead to invasive gastric cancer. We find that H. pylori activates the hepatocyte growth factor/scatter factor receptor c-Met, which is involved in invasive growth of tumor cells. The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response. CagA could represent a bacterial adaptor protein that associates with phospholipase Cgamma but not Grb2-associated binder 1 or growth factor receptor-bound protein 2. The H. pylori-induced motogenic response is suppressed and blocked by the inhibition of PLCgamma and of MAPK, respectively. Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling. The activation of the motogenic response in H. pylori-infected epithelial cells suggests that CagA could be involved in tumor progression.

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H. pylori–activated PLCγ interacts with CagA and is required for the motogenic response of AGS cells. (A) PLCγ interacts with CagA. CagA was immunoprecipitated (IP) from AGS cell lysates prepared at the indicated time points after infection. Immunoblot (IB) analysis of immunoprecipitates was performed using anti-PLCγ antibody. (B) H. pylori induces PLCγ phosphorylation. AGS cells were infected with H. pylori wild type or cagA and virB11 strains, and PLCγ immunoprecipitates were tested by Western blot analysis using antiphosphotyrosine antibody. (C) PLCγ inhibitor drastically reduces the motogenic response of AGS cells to H. pylori infection. AGS cells were pretreated with the PLCγ inhibitor U73122 and infected with H. pylori. Phase-contrast microscopy was performed at 4 h after infection. (D) CagA fails to interact with large adaptor protein Gab1. AGS cells were transiently transfected with the plasmid-expressed Flag-tagged Gab1 and infected with H. pylori. CagA immunoprecipitates were analyzed by immunoblotting with anti-Flag antibody.
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fig4: H. pylori–activated PLCγ interacts with CagA and is required for the motogenic response of AGS cells. (A) PLCγ interacts with CagA. CagA was immunoprecipitated (IP) from AGS cell lysates prepared at the indicated time points after infection. Immunoblot (IB) analysis of immunoprecipitates was performed using anti-PLCγ antibody. (B) H. pylori induces PLCγ phosphorylation. AGS cells were infected with H. pylori wild type or cagA and virB11 strains, and PLCγ immunoprecipitates were tested by Western blot analysis using antiphosphotyrosine antibody. (C) PLCγ inhibitor drastically reduces the motogenic response of AGS cells to H. pylori infection. AGS cells were pretreated with the PLCγ inhibitor U73122 and infected with H. pylori. Phase-contrast microscopy was performed at 4 h after infection. (D) CagA fails to interact with large adaptor protein Gab1. AGS cells were transiently transfected with the plasmid-expressed Flag-tagged Gab1 and infected with H. pylori. CagA immunoprecipitates were analyzed by immunoblotting with anti-Flag antibody.

Mentions: AGS cells grow on plastic as a nonpolarized monolayer. For effective migration, cells must establish an asymmetry in cell–substratum biophysical interactions permitting cellular protrusive and contractile motive forces to produce a net cell body translocation-polarized cell shape. Attachment of H. pylori to, and translocation of, CagA in the host cell could promote such asymmetry. PLCγ signaling is linked to cytoskeletal alterations and promotes cell migration by increasing the fraction of cells in a motility-permissive morphology (Wells et al., 1999). Therefore, we tested whether CagA could interact with PLCγ. AGS cells were infected with H. pylori and CagA was immunoprecipitated with anti-CagA antibody. Western blot analysis of immunoprecipitates, performed using antibody against PLCγ, showed the physical interaction of CagA and PLCγ (Fig. 4 A). PLCγ tyrosine phosphorylation, which is provoked by nearly all growth factor receptors, is necessary to achieve maximal enzymatic activity (Carpenter and Ji, 1999). Upon activation, PLCγ cleaves its membrane-bound substrate, phosphatidylinositol bisphophate (PIP2). PIP2 releases bound actin-modifying proteins such as gelsolin, profilin, and cofilin, which then interact with the submembrane actin cytoskeleton (Chen et al., 1996). We next tested whether H. pylori could stimulate tyrosine phosphorylation of PLCγ. H. pylori induced PLCγ phosphorylation, whereas both mutant strains cagA and virB11 failed to activate PLCγ (Fig. 4 B). Inhibition of the PLCγ signaling pathway blocks growth factor–induced cell motility (Kassis et al., 2001). In our work, we were able to suppress the motogenic response of AGS cells after H. pylori infection by using the pharmacological agent U73122 (Fig. 4 C). The motogenic response of AGS cells in the presence of U73122 was weak and resembled that after the infection of AGS cells with the H. pylori mutant strain cagA (Fig. 3 A). We have previously shown that wild-type H. pylori strains and the cagA mutant strain could activate Rho GTPases Rac1 and Cdc42 in AGS cells. Furthermore, Rac1 and Cdc42 were recruited to the site of bacterial attachment (Churin et al., 2001). Rho GTPases control polarity, protrusion, and adhesion during cell movement (Nobes and Hall, 1999). Thus, a weak motogenic response of AGS cells to the infection with the cagA mutant strain could be explained by activation of Rho GTPases that leads to the transient polarization of the host cells. Together, CagA–PLCγ physical interaction is necessary to produce the complete motogenic response of AGS cells after H. pylori infection.


Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response.

Churin Y, Al-Ghoul L, Kepp O, Meyer TF, Birchmeier W, Naumann M - J. Cell Biol. (2003)

H. pylori–activated PLCγ interacts with CagA and is required for the motogenic response of AGS cells. (A) PLCγ interacts with CagA. CagA was immunoprecipitated (IP) from AGS cell lysates prepared at the indicated time points after infection. Immunoblot (IB) analysis of immunoprecipitates was performed using anti-PLCγ antibody. (B) H. pylori induces PLCγ phosphorylation. AGS cells were infected with H. pylori wild type or cagA and virB11 strains, and PLCγ immunoprecipitates were tested by Western blot analysis using antiphosphotyrosine antibody. (C) PLCγ inhibitor drastically reduces the motogenic response of AGS cells to H. pylori infection. AGS cells were pretreated with the PLCγ inhibitor U73122 and infected with H. pylori. Phase-contrast microscopy was performed at 4 h after infection. (D) CagA fails to interact with large adaptor protein Gab1. AGS cells were transiently transfected with the plasmid-expressed Flag-tagged Gab1 and infected with H. pylori. CagA immunoprecipitates were analyzed by immunoblotting with anti-Flag antibody.
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Related In: Results  -  Collection

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fig4: H. pylori–activated PLCγ interacts with CagA and is required for the motogenic response of AGS cells. (A) PLCγ interacts with CagA. CagA was immunoprecipitated (IP) from AGS cell lysates prepared at the indicated time points after infection. Immunoblot (IB) analysis of immunoprecipitates was performed using anti-PLCγ antibody. (B) H. pylori induces PLCγ phosphorylation. AGS cells were infected with H. pylori wild type or cagA and virB11 strains, and PLCγ immunoprecipitates were tested by Western blot analysis using antiphosphotyrosine antibody. (C) PLCγ inhibitor drastically reduces the motogenic response of AGS cells to H. pylori infection. AGS cells were pretreated with the PLCγ inhibitor U73122 and infected with H. pylori. Phase-contrast microscopy was performed at 4 h after infection. (D) CagA fails to interact with large adaptor protein Gab1. AGS cells were transiently transfected with the plasmid-expressed Flag-tagged Gab1 and infected with H. pylori. CagA immunoprecipitates were analyzed by immunoblotting with anti-Flag antibody.
Mentions: AGS cells grow on plastic as a nonpolarized monolayer. For effective migration, cells must establish an asymmetry in cell–substratum biophysical interactions permitting cellular protrusive and contractile motive forces to produce a net cell body translocation-polarized cell shape. Attachment of H. pylori to, and translocation of, CagA in the host cell could promote such asymmetry. PLCγ signaling is linked to cytoskeletal alterations and promotes cell migration by increasing the fraction of cells in a motility-permissive morphology (Wells et al., 1999). Therefore, we tested whether CagA could interact with PLCγ. AGS cells were infected with H. pylori and CagA was immunoprecipitated with anti-CagA antibody. Western blot analysis of immunoprecipitates, performed using antibody against PLCγ, showed the physical interaction of CagA and PLCγ (Fig. 4 A). PLCγ tyrosine phosphorylation, which is provoked by nearly all growth factor receptors, is necessary to achieve maximal enzymatic activity (Carpenter and Ji, 1999). Upon activation, PLCγ cleaves its membrane-bound substrate, phosphatidylinositol bisphophate (PIP2). PIP2 releases bound actin-modifying proteins such as gelsolin, profilin, and cofilin, which then interact with the submembrane actin cytoskeleton (Chen et al., 1996). We next tested whether H. pylori could stimulate tyrosine phosphorylation of PLCγ. H. pylori induced PLCγ phosphorylation, whereas both mutant strains cagA and virB11 failed to activate PLCγ (Fig. 4 B). Inhibition of the PLCγ signaling pathway blocks growth factor–induced cell motility (Kassis et al., 2001). In our work, we were able to suppress the motogenic response of AGS cells after H. pylori infection by using the pharmacological agent U73122 (Fig. 4 C). The motogenic response of AGS cells in the presence of U73122 was weak and resembled that after the infection of AGS cells with the H. pylori mutant strain cagA (Fig. 3 A). We have previously shown that wild-type H. pylori strains and the cagA mutant strain could activate Rho GTPases Rac1 and Cdc42 in AGS cells. Furthermore, Rac1 and Cdc42 were recruited to the site of bacterial attachment (Churin et al., 2001). Rho GTPases control polarity, protrusion, and adhesion during cell movement (Nobes and Hall, 1999). Thus, a weak motogenic response of AGS cells to the infection with the cagA mutant strain could be explained by activation of Rho GTPases that leads to the transient polarization of the host cells. Together, CagA–PLCγ physical interaction is necessary to produce the complete motogenic response of AGS cells after H. pylori infection.

Bottom Line: The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response.CagA could represent a bacterial adaptor protein that associates with phospholipase Cgamma but not Grb2-associated binder 1 or growth factor receptor-bound protein 2.The activation of the motogenic response in H. pylori-infected epithelial cells suggests that CagA could be involved in tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Experimental Internal Medicine, Medical Faculty, Otto-von-Guericke-University, Leipziger Strasse 44, 39120 Magdeburg, Germany.

ABSTRACT
Infection with the human microbial pathogen Helicobacter pylori is assumed to lead to invasive gastric cancer. We find that H. pylori activates the hepatocyte growth factor/scatter factor receptor c-Met, which is involved in invasive growth of tumor cells. The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response. CagA could represent a bacterial adaptor protein that associates with phospholipase Cgamma but not Grb2-associated binder 1 or growth factor receptor-bound protein 2. The H. pylori-induced motogenic response is suppressed and blocked by the inhibition of PLCgamma and of MAPK, respectively. Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling. The activation of the motogenic response in H. pylori-infected epithelial cells suggests that CagA could be involved in tumor progression.

Show MeSH
Related in: MedlinePlus