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Phosphorylation of Runx protein controls helper CD4+ T cell versus cytotoxic CD8+ T cell lineage choice
作者信息Chihiro Ogawa, Kazuki Okuyama, Satoshi Kojo, Kohei Nishino, Hiroaki Machiyama, Aditya K Padhi, Hirotaka Takahashi, Takashi Ebihara, Mari Tenno, Qin Zhizhen, Sawako Muroi, Kosei Ito, Tatsuya Sawasaki, Kam Y J Zhang, Hai-Hui Xue, Tadashi Yokosuka, Hidetaka Kosako, Ichiro Taniuchi
摘要
MHC-I- and MHC-II-selected CD4+CD8+ precursor thymocytes differentiate into cytotoxic CD8+ and helper CD4+ lineage T cells, during which suppression of Cd4 and Thpok genes by Runx-dependent-silencers in those genes is crucial to segregate the two lineages. However, how TCR signals are linked to cytotoxic-lineage-specific Cd4-Thpok silencing remains unclear. Here we show that the terminal Y residue within the evolutionarily conserved C-terminal WRPY motif in Runx1, which is essential for interacting with TLE co-repressor proteins, was phosphorylated more in CD4-CD8+ thymocytes than in CD4+CD8- thymocytes, inducing an interaction with TLE co-repressors for cytotoxic-lineage specific Cd4-Thpok silencing. Non-receptor tyrosine kinases Lck and Zap70 interacted with Runx in the cytoplasm more in MHC-I-signaled CD4-CD8+ thymocytes than in CD4+CD8- thymocytes. Collectively, these findings reveal that differential phosphorylation states at the terminal tyrosine residue in Runx connect MHC restriction with the helper versus cytotoxic T cell lineage choice.
实验方法
产品清单
| 名称 | 品牌 | 货号 |
|---|---|---|
| pX330质粒 | Addgene | #42230 |
| BD FACSCanto II流式细胞仪 | BD Biosciences | -- |
| BD FACSAria III流式细胞分选仪 | BD Biosciences | -- |
| RNeasy Mini试剂盒 | Qiagen | -- |
| NEBNext RNA文库制备试剂盒（用于Illumina） | NEB | -- |
| Illumina HiSeq 2500测序仪 | Illumina | -- |
| EasySep细胞分离系统（含EasySep Mouse Streptavidin RapidSpheres分离试剂盒） | STEMCELL Technologies | -- |
| QuantStudio 3实时PCR系统 | Applied Biosystems | -- |
| XL2000型超声波细胞破碎仪 | MICROSON | -- |
| NEBNext ChIP-seq文库制备Master Mix试剂盒（用于Illumina） | NEB | -- |
| Illumina HiSeq 1500测序仪 | Illumina | -- |
| Covaris S220超声波破碎仪 | Covaris | -- |
| Picoruptor 2超声波破碎仪 | Diagnode | -- |
| 384孔Opti板 | PerkinElmer | -- |
| AlphaScreen IgG（蛋白A）检测试剂盒 | Perkin Elmer | -- |
| EnVision多功能酶标仪 | -- | -- |
| EASY-nLC 1200超高效液相色谱仪 | Thermo Fisher Scientific | -- |
| Q Exactive Plus质谱仪 | Thermo Fisher Scientific | -- |
| 纳升电喷雾离子源 | Thermo Fisher Scientific | -- |
| C18反相色谱柱 | Nikkyo Technos | -- |
| Amersham Imager 680成像仪 | Cytiva | -- |
| Cytospin 4细胞离心涂片机 | Thermo Fisher Scientific | -- |
| BZ-X810显微镜 | Keyence | -- |
| ECLIPSE Ti-TIRF显微镜 | Nikon | -- |
| sCMOS相机 | Hamamatsu Photonics | ORCA flash 4.0 |