Objective: To construct the lentivirus carrying the mutated palmitoylation site of the linker for activation of T cells (LAT) and infect Jurkat cells with it to establish stable cell line, and to investigate the effect of LAT palmitoylation mutation on T cell signaling induced by CD59.
Methods: Negative control (neg-EGFP) and LAT-M-EGFP fusion protein gene vectors were respectively constructed and then packaged using lentivirus. Subsequently, Jurkat cells were infected with them to establish stable cell lines. Confocal laser scanning microscopy was used to observe the infection efficiency and the distribution of fusion proteins in Jurkat cells. CCK-8 assay was used to detect the change of cell proliferation activity after CD59 mAb supplementation. Flow cytometry was used to determine the apoptosis rate. Western blotting was used to examine the levels of phospholipase C-γ1 (PLC-γ1) and lymphocyte-specific protein tyrosine kinase (LCK).
Results: Confocal laser scanning microscopy revealed that LAT molecules of LAT-M group scattered on cell membrane, and there was no obvious clustered region after cross linkage with CD59 mAb. Compared with the negative control group, the cell proliferation activity of LAT-M group significantly decreased, and the quantity of middle-late apoptotic cells significantly increased; Western blotting showed that the expression levels of PLC-γ1 and LCK in LAT-M group was roughly the same with those in negative control group, and after CD59 mAb stimulation, there was no obvious change in LAT-M group, while the levels in negative control group were reduced.
Conclusion: LAT-M-EGFP fusion protein could not locate on lipid rafts of Jurkat cells infected with LAT palmitoylation mutation. In addition, the growth of the cells carrying the LAT-M-EGFP was inhibited. The palmitoylation mutation of LAT attenuated the signal transduction induced by glycosylphosphatidylinositol-anchored CD59 in T cells.