The following experimental approaches support the notion that “Membrane localization of CagA is independent of tyrosine phosphorylation”.
- Immunofluorescence analysis of subcellular localization of phosphorylation-resistant CagA, transiently expressed in AGS cell line:
- Experiment: A phosphorylation-resistant mutant of CagA was generated by substituting tyrosine (Y) residue of EPIYA motifs with phenylalanine (F), thereby abolishing the potential for tyrosine phosphorylation. This mutant CagA was expressed in AGS cells, and its subcellular localization was analyzed using immunofluorescence microscopy.
- Materials: AGS cell line, HA-tagged CagA wild-type construct, HA-tagged phosphorylation-resistant CagA mutant construct
- Experimental methods: Molecular cloning (mutant creation), Transfection, Cell culture, Immunocytochemistry, Confocal microscopy
- Observation: Similar to the wild-type CagA, the phosphorylation-deficient mutant localized to the plasma membrane of AGS cells after transfection. However, in contrast to the wild-type protein, the mutant CagA failed to induce the characteristic hummingbird phenotype, a hallmark of CagA-mediated morphological transformation.
- Conclusion: These results demonstrate that membrane localization of CagA is independent of tyrosine phosphorylation. Nevertheless, tyrosine phosphorylation is essential for the biological activity of CagA, particularly for the induction of morphological changes such as the hummingbird phenotype.
- 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)
