Background: CagA undergoes tyrosine phosphorylation in gastric epithelial cell lines, such as the AGS cell line. Interestingly, it has also been demonstrated that CagA is phosphorylated in non-gastric cell lines, such as COS-7 cells. The following experimental approach supports the notion that CagA undergoes tyrosine phosphorylation in the COS-7 cell line:
- Experimental Approach: Analysis of tyrosine phosphorylation of CagA transiently expressed in COS-7 cells using anti-phosphotyrosine antibody after immunoprecipitation (Higashi et al., 2002)
- Experiment: HA-tagged wild-type CagA and a phosphorylation-resistant CagA mutant (generated by substituting the tyrosine residues within the EPIYA motifs with phenylalanine) were transiently expressed in COS-7 cells, a monkey kidney fibroblast-like cell line. Following transfection, CagA proteins were immunoprecipitated and their phosphorylation status was assessed by immunoblotting using anti-phosphotyrosine antibodies.
- Materials: COS-7 cell line, HA-tagged CagA wild-type construct, HA-tagged phosphorylation-resistant CagA mutant construct
- Methods: Molecular cloning, transfection, cell culture, immunoprecipitation, SDS-PAGE, immunoblotting (Western blot).
- Observation: CagA transiently expressed in COS-7 cells was successfully phosphorylated on tyrosine residues but no phosphorylation was detected on mutated CagA. This finding demonstrated that phosphorylation of CagA is not restricted to gastric epithelial cells and can occur in different cellular contexts.
- Reference: Higashi et al., 2002. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science. 295(5555), 683-686. PMID-11743164; Full Text: Science, Science (Download PDF)
- Conclusion: These results indicate that tyrosine phosphorylation of CagA is a host cell-independent process and does not require specialized factors unique to gastric epithelial cells. Instead, it suggests that general host cell kinases are sufficient to phosphorylate CagA upon expression.
