[Impact of the palmitoylation of linker for activation of T cells on signal transduction pathway of CD59 in T cells]

Abstract

Objective: To transfect Jurkat cells with linker for activation of T cells (LAT)-EGFP or LAT-EGFP-M (two cysteines of which near the juxtamembrane region were substituted for alanine) by electroporation, and observe the change in T cell signal transduction after the cross linkage with anti-CD59 mAb.

Methods: By means of electroporation, Jurkat cells were transfected with LAT-EGFP or LAT-EGFP-M or PEGFP-N3 (control plasmid), and then subjected to cross linkage with anti-CD59 mAb. The locations of the fusion protein LAT-EGFP and LAT-EGFP-M were observed with confocal microscopy. The proliferation activity of the Jurkat cells was determined by MTT assay, and the content of interleukin 2 (IL-2) in cell supernatants was detected by ELISA. Western blotting was used to test the phosphorylation level of LAT in each group.

Results: After the cross linkage of anti-CD59 mAb, LAT-EGFP-M could not locate on lipid rafts just as LAT-EGFP did. The cell proliferation and IL-2 secretion of Jurkat cells transfected with LAT-EGFP-M decreased as compared with the ones transfected with LAT-EGFP, EGFP-N3 or the untransfected ones. The phosphorylation level of LAT-EGFP-M was much lower than that of LAT-EGFP.

Conclusion: The mutation of palmitoylation site of LAT-EGFP attenuated the signal transduction of CD59 in T cells.