Study On Signal Transduction Of Transmembrane Co - Protein CBP And Its Palmitoylation Site Mutation In CD59 - Mediated T - Line Leukemia

Objective To explore the interactions between the transmembrane adaptor protein CBP and GPI anchored protein CD59 to the signal transduction pathway of T cells and study the mechanisms of CBP and CD59 in T lymphocytic leukemia, which can provide experimental basis in the treatment of cancer.Methods Using Jurkat cell line as the research object, CBP-EGFP and CBP-mutation-EGFP lentiviral vector were constructed for establishing stable cell lines through virus transfection. Using the CD59 monoclonal antibody were used to stimulate the cells in each group, we could observe the expression and localization of CBP and CD59 with immunofluorescence technique; the cell growth condition was observed by optical microscope; the complexity and ultrastructure of intracellular organelles were detected by electron microscope. Flow cytometry could check the basic parameters and apoptosis of Jurkat cells; cell proliferation was measured by CCK8 assay efficiency. Inhibition of tyrosine kinase CSK combined with CBP and LCK, FYN, PLCG1 and ZAP70 of TCR activation important signaling pathway were detected by Western Blot. Real time quantitative PCR could detect the expression of gene CBP and CD59, CSK, LCK, PI3 K, PLCG1 and GRB2.Results ① The results showed that CBP and CD59 molecules located on the cell membrane under confocal microscopy; after CD59 antibody cross-linking antibody stimulation, CBP-EGFP group showed CBP and CD59 molecules were ring or point cluster highlighted fluorescence distribution, cross overlap and distribution area. But in CBP-M-EGFP group, CBP and CD59 appeared the phenomenon of point cluster in some cells, but they do not overlap. ②Observed by the light microscope, the most cells were small cells, and shrinkage cells increased in CBP-EGFP; the morphology of CBP-M-EGFP group cells were different sizes, but with big cell. ③After transfection, autophagosome like mass was found in two groups under the organelle, but more in CBP-EGFP. At the same time, we found the nuclear cytoplasmic ratio decreased, nuclear small, irregular, organelles swelling. ④The real-time quantitative PCR showed that CBP gene expression was significantly up-regulated after transfection. The expression of CSK gene was up-regulated in CBP-EGFP, while lower in CBP-M-EGFP. CD59, CSK, LCK, PLCG1 and GRB2 were down-regulated in CBP-EGFP, but the result was opposite in CBP-M-EGFP. ⑤ Western Blot displayed the expression of CSK was increased in CBP-EGFP group and further increased after stimulated by CD59 monoclonal antibody. LCK, FYN, ZAP70 was decreased in the CBP group, and CBP-M-EGFP group was rising.⑥The CCK8 results showed that CBP could inhibit cell proliferation activity and efficiency, but the result was opposite when we mutanted palmitoylation sites. ⑦The results of apoptosis showed: The early apoptotic cells were the major in CBP-EGFP group, was about 40%, and the proportion of the late apoptosis was about 15%; early apoptotic cells accounted for 25% and the proportion of the late apoptotic cells was 12% in CBP-M-EGFP. After CD59 antibody cross-linking, CD59 helped CBP to inhibit cell viability, and increased the incidence of cell apoptosis. When the palmitoylation sites were mutated, CBP lost its inhibition effects.Conclusion Over expression of CBP can inhibit the proliferation of Jurkat cells. When the palmitoylation sites were mutated, the inhibitory effect decreased significantly. At the same time, the crosslinkage of CD59 antibody confirmed that CD59 can use the palmitoylation groups to phosphorylate CBP molecule to play biological effects, inhibited the activation and proliferation of T cells, cause the change of a series of related molecular pathways in the activation of TCR. This is a foundation to investigate the interaction between CBP and CD59 in the signal transduction pathway of T cells and tumor targeting therapy.