DOI: 10.1126/science.1067147. SHP-2 Tyrosine Phosphatase as an Intracellular Target of <i>Helicobacter pylori</i> CagA Protein

SHP-2 Tyrosine Phosphatase as an Intracellular Target of Helicobacter pylori CagA Protein

10.1126/science.1067147

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

Helicobacter pylori CagA protein is associated with severe gastritis and gastric carcinoma. CagA is injected from the attached Helicobacter pylori into host cells and undergoes tyrosine phosphorylation. Wild-type but not phosphorylation-resistant CagA induced a growth factor–like response in gastric epithelial cells. Furthermore, CagA formed a physical complex with the SRC homology 2 domain (SH2)–containing tyrosine phosphatase SHP-2 in a phosphorylation-dependent manner and stimulated the phosphatase activity. Disruption of the CagA–SHP-2 complex abolished the CagA-dependent cellular response. Conversely, the CagA effect on cells was reproduced by constitutively active SHP-2. Thus, upon translocation, CagA perturbs cellular functions by deregulating SHP-2.

Bibliography

Higashi, H., Tsutsumi, R., Muto, S., Sugiyama, T., Azuma, T., Asaka, M., & Hatakeyama, M. (2002). SHP-2 Tyrosine Phosphatase as an Intracellular Target of Helicobacter pylori CagA Protein. Science, 295(5555), 683â686.

Authors 7

Hideaki Higashi (first)
Ryouhei Tsutsumi (additional)
Syuichi Muto (additional)
Toshiro Sugiyama (additional)
Takeshi Azuma (additional)
Masahiro Asaka (additional)
Masanori Hatakeyama (additional)

References 34 Referenced 809

10.1126/science.284.5418.1328
Blaser M. J., et al., Cancer Res. 55, 2111 (1995). / Cancer Res. by Blaser M. J. (1995)
10.1093/jnci/87.23.1777
10.1136/gut.40.3.297
10.1073/pnas.96.25.14559
10.1084/jem.191.4.593
10.1073/pnas.97.3.1263
10.1126/science.287.5457.1497
10.1046/j.1462-5822.2000.00043.x
Thirty μg of plasmids were transfected into AGS cells (1.8 x 10 6 cells) using Lipofectamine 2000 reagent (Invitrogen Carlsbad CA). COS-7 cells (1.5 × 10 6 cells) were transfected with 20 μg of plasmids using the calcium phosphate method. Cells were harvested at 36 hours after transfection and lysed in lysis buffer (Tris-HCl at pH 7.5 100 mM NaCl 5 mM EDTA and 1% Brij-35) containing 2 mM of Na 3 VO 4 2 mM of phenylmethylsulfonyl fluoride (PMSF) 10 μg/ml of leupeptin 10 μg/ml of trypsin inhibitor and 10 μg/ml of aprotinin. The lysates were treated with appropriate antibodies and the immune complexes were trapped on protein A– or G Sepharose beads. The immunoprecipitates were subjected to SDS-polyacrylamide gel (SDS-PAGE).
Antibodies used were anti-HA antibodies Y-11 (Santa Cruz Santa Cruz CA) and 12CA5 antibody to Myc (antibody 9E10) antibody to SHP-2 (antibody C-18) (Santa Cruz) and antibody to phosphotyrosine (antibody 4G10) (Upstate Biotechnology Lake Placid NY).
10.1073/pnas.90.12.5791
AGS cells transfected with CagA-HA or SHP-2-Myc were fixed with 3% paraformaldehyde. Cells were then treated with antibody to HA (Y-11) or antibody to Myc (9E10). Primary antibodies were localized by Alexa Fluor 546-conjugated anti-rabbit or Alexa Fluor 488-conjugated anti-mouse antibody (Molecular Probes Eugene OR). Images were acquired using a confocal microscope system (Fluoview Olympus Tokyo Japan).
10.1126/science.8096088
R. M. Jr. Freeman
10.1073/pnas.89.3.1123
10.1073/pnas.90.6.2197
10.1091/mbc.11.8.2565
H. Higashi et al. unpublished data.
10.1016/0092-8674(93)90404-E
SHP-2ΔPD-Myc consists of amino acid residues 1 to 192 of SHP-2-Myc. SHP-2ΔSH2-Myc lacks amino acid sequences between 33 and 191 of SHP-2-Myc. Myr-SHP-2ΔSH2-Myc was made by replacing the NH 2 -terminal 32 amino acids of SHP-2ΔSH2-Myc with the myristoylation signal sequence of v-Src (28). The DNAs were inserted into pSP65SRα vector.
10.1016/S0021-9258(20)80562-6
10.1074/jbc.270.7.2897
10.1016/S0021-9258(17)37036-9
10.1016/S0021-9258(17)37504-X
10.1016/S0092-8674(00)80938-1
SHP-2 phosphatase activity was measured with the use of p -nitrophenyl phosphate ( p NPP) as a substrate. SHP-2 immunoprecipitates were incubated in phosphatase assay buffer (100 mM sodium acetate at pH 5.0 and 1.6 mM dithiothreitol) containing 10 mM p NPP at 30°C for 1 hour. Absorbance of the reaction mixture was measured at 410 nm.
10.1016/S0960-9822(00)00831-9
10.1126/science.7694365
10.1093/emboj/16.9.2352
10.1128/MCB.19.4.3125
10.1128/MCB.19.4.3205
10.1074/jbc.273.33.21125
We thank R. A. Weinberg for reading of the manuscript. We also thank T. Matozaki for human SHP-2 cDNA. Supported by a Grant-in Aid for Scientific Research on Priority Areas from the Ministry of Education Culture Sports Science and Technology of Japan; a Research Grant from the Human Frontier Science Program Organization; and a Research Grant from the Nippon Boehringer Ingelheim Co. Ltd.

Dates

Type	When
Created	23 years, 1 month ago (July 27, 2002, 5:47 a.m.)
Deposited	1 year, 7 months ago (Jan. 9, 2024, 6:07 p.m.)
Indexed	1 week ago (Aug. 23, 2025, 9:48 p.m.)
Issued	23 years, 7 months ago (Jan. 25, 2002)
Published	23 years, 7 months ago (Jan. 25, 2002)
Published Print	23 years, 7 months ago (Jan. 25, 2002)

Funders 0

None

BibTeX

@article{Higashi_2002, title={SHP-2 Tyrosine Phosphatase as an Intracellular Target of Helicobacter pylori CagA Protein}, volume={295}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.1067147}, DOI={10.1126/science.1067147}, number={5555}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Higashi, Hideaki and Tsutsumi, Ryouhei and Muto, Syuichi and Sugiyama, Toshiro and Azuma, Takeshi and Asaka, Masahiro and Hatakeyama, Masanori}, year={2002}, month=jan, pages={683–686} }

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