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Natural variant of the Helicobacter pylori CagA oncoprotein that lost the ability to interact with PAR1.

Hashi K, Murata-Kamiya N, Varon C, Mégraud F, Dominguez-Bello MG, Hatakeyama M - Cancer Sci. (2014)

Bottom Line: In the present study, we investigated the biological activity of v225d CagA, an Amerindian CagA of H. pylori isolated from a Venezuelan Piaroa Amerindian subject, because the variant CagA does not possess a canonical CM sequence.We found that v225d CagA interacts with SHP2 but not PAR1b.Furthermore, SHP2-binding activity of v225d CagA was much lower than that of CagA of H. pylori isolated from Western countries (Western CagA). v225d CagA also displayed a reduced ability to induce the hummingbird phenotype than that of Western CagA.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

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Mechanism of v225d CagA-SHP2 interaction. (a) Schematic view of v225d wild-type CagA and mutant CagA proteins. Phosphoresistant v225d CagA was constructed by mutating tyrosine residue of the EPIYA-D/CΔCM or EPIYA-pD/CΔCM segment to phenylalanine (v225d YF1 and v225d YF2, respectively). (b) AGS cells were transiently transfected with the indicated CagA-FLAG expression vectors. Cell lysates were immunoprecipitated with an anti-FLAG antibody and immunoblotted with the indicated antibodies. (c) Left panel: AGS cells were transfected with the indicated CagA expression vectors. Cell morphology was examined under a microscope at 24 h after transfection. Arrows indicate hummingbird cells induced by CagA. Bar, 100 μm. Right upper panel: the number of hummingbird cells at 24 h after transfection was counted. Error bars, ±SD (n = 3). *P < 0.01, Student's t-test. Right lower panel: total cell lysates (TCL) were also prepared and subjected to immunoblotting with the indicated antibodies. (d) Upper panel: AGS cells were transfected with an EGFP expression vector together with a CagA expression vector. At 24 h after transfection, the cell morphological change was examined under a fluorescence microscope (left). The length of cells was measured using images analyzed with the ImageJ software (right). Bar, 100 μm. Lower panel: the length of cells was calculated from images presented in the upper right panels. Red dots show cells that are longer than the maximum value of control cells. n = 200. *P < 0.01, Mann–Whitney U-test.
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fig04: Mechanism of v225d CagA-SHP2 interaction. (a) Schematic view of v225d wild-type CagA and mutant CagA proteins. Phosphoresistant v225d CagA was constructed by mutating tyrosine residue of the EPIYA-D/CΔCM or EPIYA-pD/CΔCM segment to phenylalanine (v225d YF1 and v225d YF2, respectively). (b) AGS cells were transiently transfected with the indicated CagA-FLAG expression vectors. Cell lysates were immunoprecipitated with an anti-FLAG antibody and immunoblotted with the indicated antibodies. (c) Left panel: AGS cells were transfected with the indicated CagA expression vectors. Cell morphology was examined under a microscope at 24 h after transfection. Arrows indicate hummingbird cells induced by CagA. Bar, 100 μm. Right upper panel: the number of hummingbird cells at 24 h after transfection was counted. Error bars, ±SD (n = 3). *P < 0.01, Student's t-test. Right lower panel: total cell lysates (TCL) were also prepared and subjected to immunoblotting with the indicated antibodies. (d) Upper panel: AGS cells were transfected with an EGFP expression vector together with a CagA expression vector. At 24 h after transfection, the cell morphological change was examined under a fluorescence microscope (left). The length of cells was measured using images analyzed with the ImageJ software (right). Bar, 100 μm. Lower panel: the length of cells was calculated from images presented in the upper right panels. Red dots show cells that are longer than the maximum value of control cells. n = 200. *P < 0.01, Mann–Whitney U-test.

Mentions: We wanted to gain insights into the mechanism of v225d CagA-SHP2 interaction. The finding that v225d CagA bound to SHP2 without PAR1b interaction raised the possibility that most of the v225d CagA-SHP2 interaction was mediated by cis-interaction of a single CagA rather than by trans-interaction of dimerized CagA. To investigate this idea, we generated two phosphoresistant v225d CagA mutants, v225d YF1 and v225d YF2, in which the tyrosine residue was replaced with phenylalanine in the EPIYA-D/CΔCM and EPIYA-pD/CΔCM segments, respectively (Fig.4a). AGS cells were transfected with a FLAG-tagged CagA expression vector and then total cell lysates prepared were immunoprecipitated with an anti-FLAG antibody. Because Western CagA with a greater number of EPIYA-C segments exhibits a better ability to bind to SHP2 than that with a lower number of EPIYA-C, ABCC CagA with two EPIYA-C segments was used as a control for the experiment as v225d CagA has EPIYA-D/CΔCM and EPIYA-pD/CΔCM segments (Fig.1b). Although SHP2 was co-immunoprecipitated with v225d CagA wild type (WT) and mutants (YF1 and YF2), the amount of SHP2 co-immunoprecipitated with v225d CagA was much smaller than that co-immunoprecipitated with ABCC CagA (Fig.4b). This result indicates that v225d CagA was capable of binding to SHP2 through both the EPIYA-D/CΔCM and EPIYA-pD/CΔCM segments. Also notably, the amount of SHP2 co-immuoprecipitated with v225d YF1 CagA was less than that co-immunoprecipitated with v225d YF2 CagA (Fig.4b).


Natural variant of the Helicobacter pylori CagA oncoprotein that lost the ability to interact with PAR1.

Hashi K, Murata-Kamiya N, Varon C, Mégraud F, Dominguez-Bello MG, Hatakeyama M - Cancer Sci. (2014)

Mechanism of v225d CagA-SHP2 interaction. (a) Schematic view of v225d wild-type CagA and mutant CagA proteins. Phosphoresistant v225d CagA was constructed by mutating tyrosine residue of the EPIYA-D/CΔCM or EPIYA-pD/CΔCM segment to phenylalanine (v225d YF1 and v225d YF2, respectively). (b) AGS cells were transiently transfected with the indicated CagA-FLAG expression vectors. Cell lysates were immunoprecipitated with an anti-FLAG antibody and immunoblotted with the indicated antibodies. (c) Left panel: AGS cells were transfected with the indicated CagA expression vectors. Cell morphology was examined under a microscope at 24 h after transfection. Arrows indicate hummingbird cells induced by CagA. Bar, 100 μm. Right upper panel: the number of hummingbird cells at 24 h after transfection was counted. Error bars, ±SD (n = 3). *P < 0.01, Student's t-test. Right lower panel: total cell lysates (TCL) were also prepared and subjected to immunoblotting with the indicated antibodies. (d) Upper panel: AGS cells were transfected with an EGFP expression vector together with a CagA expression vector. At 24 h after transfection, the cell morphological change was examined under a fluorescence microscope (left). The length of cells was measured using images analyzed with the ImageJ software (right). Bar, 100 μm. Lower panel: the length of cells was calculated from images presented in the upper right panels. Red dots show cells that are longer than the maximum value of control cells. n = 200. *P < 0.01, Mann–Whitney U-test.
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fig04: Mechanism of v225d CagA-SHP2 interaction. (a) Schematic view of v225d wild-type CagA and mutant CagA proteins. Phosphoresistant v225d CagA was constructed by mutating tyrosine residue of the EPIYA-D/CΔCM or EPIYA-pD/CΔCM segment to phenylalanine (v225d YF1 and v225d YF2, respectively). (b) AGS cells were transiently transfected with the indicated CagA-FLAG expression vectors. Cell lysates were immunoprecipitated with an anti-FLAG antibody and immunoblotted with the indicated antibodies. (c) Left panel: AGS cells were transfected with the indicated CagA expression vectors. Cell morphology was examined under a microscope at 24 h after transfection. Arrows indicate hummingbird cells induced by CagA. Bar, 100 μm. Right upper panel: the number of hummingbird cells at 24 h after transfection was counted. Error bars, ±SD (n = 3). *P < 0.01, Student's t-test. Right lower panel: total cell lysates (TCL) were also prepared and subjected to immunoblotting with the indicated antibodies. (d) Upper panel: AGS cells were transfected with an EGFP expression vector together with a CagA expression vector. At 24 h after transfection, the cell morphological change was examined under a fluorescence microscope (left). The length of cells was measured using images analyzed with the ImageJ software (right). Bar, 100 μm. Lower panel: the length of cells was calculated from images presented in the upper right panels. Red dots show cells that are longer than the maximum value of control cells. n = 200. *P < 0.01, Mann–Whitney U-test.
Mentions: We wanted to gain insights into the mechanism of v225d CagA-SHP2 interaction. The finding that v225d CagA bound to SHP2 without PAR1b interaction raised the possibility that most of the v225d CagA-SHP2 interaction was mediated by cis-interaction of a single CagA rather than by trans-interaction of dimerized CagA. To investigate this idea, we generated two phosphoresistant v225d CagA mutants, v225d YF1 and v225d YF2, in which the tyrosine residue was replaced with phenylalanine in the EPIYA-D/CΔCM and EPIYA-pD/CΔCM segments, respectively (Fig.4a). AGS cells were transfected with a FLAG-tagged CagA expression vector and then total cell lysates prepared were immunoprecipitated with an anti-FLAG antibody. Because Western CagA with a greater number of EPIYA-C segments exhibits a better ability to bind to SHP2 than that with a lower number of EPIYA-C, ABCC CagA with two EPIYA-C segments was used as a control for the experiment as v225d CagA has EPIYA-D/CΔCM and EPIYA-pD/CΔCM segments (Fig.1b). Although SHP2 was co-immunoprecipitated with v225d CagA wild type (WT) and mutants (YF1 and YF2), the amount of SHP2 co-immunoprecipitated with v225d CagA was much smaller than that co-immunoprecipitated with ABCC CagA (Fig.4b). This result indicates that v225d CagA was capable of binding to SHP2 through both the EPIYA-D/CΔCM and EPIYA-pD/CΔCM segments. Also notably, the amount of SHP2 co-immuoprecipitated with v225d YF1 CagA was less than that co-immunoprecipitated with v225d YF2 CagA (Fig.4b).

Bottom Line: In the present study, we investigated the biological activity of v225d CagA, an Amerindian CagA of H. pylori isolated from a Venezuelan Piaroa Amerindian subject, because the variant CagA does not possess a canonical CM sequence.We found that v225d CagA interacts with SHP2 but not PAR1b.Furthermore, SHP2-binding activity of v225d CagA was much lower than that of CagA of H. pylori isolated from Western countries (Western CagA). v225d CagA also displayed a reduced ability to induce the hummingbird phenotype than that of Western CagA.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus