Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland, 20892-4256, USA.
INSERM, U1068, CNRS UMR7258, Aix-Marseille Université UM105, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, 13009, Marseille, France.
Sci Rep. 2018 Mar 28;8(1):5336. doi: 10.1038/s41598-018-23549-2.
Cish, participates within a multi-molecular E3 ubiquitin ligase complex, which ubiquitinates target proteins. It has an inhibitory effect on T cell activation mediated by PLC-γ1 regulation, and it functions as a potent checkpoint in CD8 T cell tumor immunotherapy. To study the structural and functional relationships between Cish and PLC-γ1 during CD8 T cell activation, we tested mutants of the Cish-SH2 (R107K) and D/BC (L222Q, C226Q) domains. We confirmed that Cish-SH2-specific binding was essential for PLC-γ1 ubiquitination and degradation. This domain was essential for the Cish-mediated inhibition of Ca release upon TCR stimulation. No effect on inhibition of cytokine release was observed with SH2 or D/BC mutants, although the absence of Cish led to an increased release of IFN-γ and TNF-α. Using imaging we showed that Cish was expressed mostly in the cytoplasm and we did not see any Cish clustering at the plasma membrane upon stimulation. We conclude that the Cish-SH2 domain is essential for PLC-γ1 regulation in TCR-stimulated CD8 T cells.
Cish 参与了一个多分子 E3 泛素连接酶复合物，该复合物可泛素化靶蛋白。它通过调节 PLC-γ1 对 T 细胞激活具有抑制作用，并且在 CD8 T 细胞肿瘤免疫治疗中作为一个有效的检查点。为了研究 CD8 T 细胞激活过程中 Cish 和 PLC-γ1 之间的结构和功能关系，我们测试了 Cish-SH2（R107K）和 D/BC（L222Q、C226Q）结构域的突变体。我们证实了 Cish-SH2 特异性结合对于 PLC-γ1 的泛素化和降解是必需的。该结构域对于 Cish 介导的 TCR 刺激后 Ca2+释放的抑制是必需的。尽管缺乏 Cish 会导致 IFN-γ 和 TNF-α的释放增加，但 SH2 或 D/BC 突变体对抑制细胞因子释放没有影响。通过成像，我们表明 Cish 主要在细胞质中表达，并且在刺激时我们没有看到任何 Cish 在质膜上聚集。我们得出结论，Cish-SH2 结构域对于 TCR 刺激的 CD8 T 细胞中 PLC-γ1 的调节是必需的。
