CagA is a significant oncogenic factor injected into host cells by Helicobacter pylori, which is divided into two subtypes: East Asian type (CagAE), characterized by the EPIYA-D motif, and western type (CagAW), harboring the EPIYA-C motif. CagAE has been reported to have higher carcinogenicity than CagAW, although the underlying reason is not fully understood. SHIP2 is an intracellular phosphatase that can be recruited by CagA to perturb the homeostasis of intracellular signaling pathways. In this study, we found that SHIP2 contributes to the higher oncogenicity of CagAE. Co-Immunoprecipitation and Pull-down assays showed that CagAE bind more SHIP2 than CagAW. Immunofluorescence staining showed that a higher amount of SHIP2 recruited by CagAE to the plasma membrane catalyzes the conversion of PI(3,4,5)P3 into PI(3,4)P2. This alteration causes higher activation of Akt signaling, which results in enhanced IL-8 secretion, migration, and invasion of the infected cells. SPR analysis showed that this stronger interaction between CagAE and SHIP2 stems from the higher affinity between the EPIYA-D motif of CagAE and the SH2 domain of SHIP2. Structural analysis revealed the crucial role of the Phe residue at the Y + 5 position in EPIYA-D. After mutating Phe of CagAE into Asp (the corresponding residue in the EPIYA-C motif) or Ala, the activation of downstream Akt signaling was reduced and the malignant transformation of infected cells was alleviated. These findings revealed that CagAE hijacks SHIP2 through its EPIYA-D motif to enhance its carcinogenicity, which provides a better understanding of the higher oncogenic risk of H. pylori CagAE.
CagA, one of the key virulence factors of Helicobacter pylori , plays a significant role in H. pylori -associated gastric cancer by actively participating in neoplastic transformation. Among CagA variants, East Asian type CagA (CagA E ) bearing the EPIYA-D motif exhibits a higher risk than the Western-type (CagA W ) with EPIYA-C motifs. In this study, we investigated the interactions of CagA E and CagA W and host intracellular Grb2 and found a pronouncedly greater recruitment of Grb2 by CagA E compared to CagA W . Our findings revealed the phosphorylated tyrosine in the EPIYA motif is very important for the binding of CagA to Grb2. Phe at Y + 5 position in EPIYA-D, which interacts with Trp 121 in SH2 domain of Grb2, established the higher affinity than the interaction of EPIYA-C and Grb2-SH2. The substitute of Phe to Asp/Ala in CagA E EPIYA-D reduced the recruitment of Grb2 by CagA E , and neutralized the stronger induced malignant characteristics of the recipient cells. These findings provide additional insights into the distinct regulatory mechanisms employed by CagA E and CagA W , contributing to a better understanding of the higher oncogenic risk associated with H. pylori CagA E .
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