A role for GPI-CD59 in promoting T-cell signal transduction via LAT

Abstract

Cluster of differentiation 59 (CD59) is a glycosylphosphatidylinositol-anchored protein. Cross-linking of CD59 with specific monoclonal antibodies can cause a series of intracellular signal transduction events. However, the underlying molecular mechanisms are poorly understood. Linker for activation of T-cells (LAT) is a crucial adaptor protein in T-cell signaling, and its phosphorylation and palmitoylation are essential for its localization and function. In a previous study by the present authors, it was demonstrated that CD59 may be responsible for LAT palmitoylation, thereby regulating T-cell signal transduction. The present study detected the co-localization of LAT and CD59 in lipid rafts by transfecting Jurkat cells with lentivirus vectors carrying the LAT-enhanced green fluorescent protein fusion protein. In addition, LAT and CD59 were shown to have a synergistic effect on the proliferation of Jurkat cells. The results also indicated that CD59 may transfer the palmitate group from phosphatidylinositol to LAT to form LAT palmitate, which then localizes to lipid rafts to regulate T-cell activation. The results of the present study provided novel insights into the role of CD59 in T-cell signal transduction.

Keywords: T-cell; cluster of differentiation 59; glycosylphosphatidylinositol; linker for activation of T-cells; signal transduction.

Figure 1.

Transfection efficiency of neg-EGFP and LAT-EGFP lentivirus vectors. Successfully transfected cells with enhanced green fluorescence protein. (A) Jurkat cells. (B) Jurkat cells transfected with neg-EGFP. (C) Jurkat cells. (D) Jurkat cells transfected with LAT-EGFP. EGFP, enhanced green fluorescence protein; LAT, linker for activation of T-cells; neg, negative.

Figure 2.

Cellular localization of CD59 and LAT. (A) Neg group. Jurkat cells transfected with neg-EGFP lentiviruses showed green fluorescence corresponding to EGFP in the cytoplasm. CD59 molecules were stained red on the cytomembrane of the neg group cells. The merged images showed green and red fluorescence in neg cells corresponding to EGFP and CD59, respectively. (B) Neg + CD59Ab group. Neg cells were stimulated with CD59Ab. Green fluorescence corresponding to EGFP was expressed in the cytoplasm. CD59 red fluorescence was higher following stimulation with CD59Ab, as compared with the neg group. The merged images showed green and red fluorescence corresponding to EGFP and CD59, respectively, in neg cells following CD59Ab stimulation. (C) LAT group. Jurkat cells transfected with LAT-EGFP lentiviruses showed green fluorescence corresponding to LAT on the cell surface. CD59 was stained red on the cytomembrane of LAT group cells. The merged images showed colocalization of LAT and CD59 molecules to lipid rafts. (D) LAT + CD59Ab group. LAT group cells were stimulated with CD59Ab. Green fluorescence on the cell surface corresponding to LAT was higher in the LAT + CD59Ab group cells compared with the LAT group cells. Similarly, CD59 staining was higher in the LAT + CD59Ab group cells compared with the LAT group cells. The merged images showed that LAT and CD59 molecules were colocalized to lipid rafts in LAT cells stimulated with CD59Ab. CD59Ab, cluster of differentiation 59 antibody; LAT, linker for activation of T-cells; EGFP, enhanced green fluorescent protein.

Figure 3.

Cell proliferation was assessed using the CCK-8 assay. The neg, neg + CD59Ab, LAT and LAT + CD59Ab cells were treated with CCK-8 for 4 h. Data are expressed as the mean ± standard deviation. *P<0.01 vs. the neg group; ^#P<0.01 vs. the LAT group. CCK-8, cell counting kit-8; neg, negative; CD59Ab, cluster of differentiation 59 antibody; LAT, linker for activation of T-cells.

Figure 4.

Effect of CD59 on the apoptosis of neg and LAT cells. The apoptosis of (A) Jurkat cells transfected with neg-EGFP lentivirus, (B) neg cells treated with the CD59 monoclonal antibody for 2 h, (C) neg cells treated with the CD59 monoclonal antibody for 6 h, (D) Jurkat cells transfected with LAT-EGFP lentivirus, (E) LAT cells treated with the CD59 monoclonal antibody for 2 h and (F) LAT cells treated with the CD59 monoclonal antibody for 6 h was assessed by flow cytometry. (G) Statistical analysis of the percentages of early apoptotic cells in the different groups. *P<0.05 vs. the neg group. (H) Statistical analysis of the percentages of late apoptotic cells in the different groups. *P<0.05 vs. the neg group. ^▲P<0.05 vs. the LAT group. CD59Ab, cluster of differentiation 59 antibody; LAT, linker for activation of T-cells; EGFP, enhanced green fluorescent protein; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin.

Figure 5.

Western blotting was performed to detect the expression of PLCG1, PI3K, ZAP70, Fyn and Lck in the neg, neg + CD59Ab, LAT and LAT + CD59Ab groups. CD59Ab, cluster of differentiation 59 antibody; LAT, linker for activation of T-cells; PLCG1, phospholipase C-γ1; PI3K, phosphoinositide 3-kinase; ZAP70, zeta-chain-associated protein kinase 70; Lck, lymphocyte-specific protein tyrosine kinase.