Department of Biomedicine, Biotechnology and Public Health (Immunology), Core Research Facility for Health Sciences, University of Cádiz and Puerto Real University Hospital Research Unit, Cádiz, Spain.
Servicio de Neumología-Alergia, Hospital Puerta del Mar, Cádiz, Spain.
Front Immunol. 2018 Feb 2;9:115. doi: 10.3389/fimmu.2018.00115. eCollection 2018.
The adaptor protein linker for activation of T cells (LAT) has an essential role transducing activatory intracellular signals coming from the TCR/CD3 complex. Previous reports have shown that upon T-cell activation, LAT interacts with the tyrosine kinase Lck, leading to the inhibition of its kinase activity. LAT-Lck interaction seemed to depend on a stretch of negatively charged amino acids in LAT. Here, we have substituted this segment of LAT between amino acids 113 and 126 with a non-charged segment and expressed the mutant LAT (LAT-NIL) in J.CaM2 cells in order to analyze TCR signaling. Substitution of this segment in LAT prevented the activation-induced interaction with Lck. Moreover, cells expressing this mutant form of LAT showed a statistically significant increase of proximal intracellular signals such as phosphorylation of LAT in tyrosine residues 171 and 191, and also enhanced ZAP70 phosphorylation approaching borderline statistical significance ( = 0.051). Nevertheless, downstream signals such as Ca influx or MAPK pathways were partially inhibited. Overall, our data reveal that LAT-Lck interaction constitutes a key element regulating proximal intracellular signals coming from the TCR/CD3 complex.
衔接蛋白激活 T 细胞（LAT）在传递 TCR/CD3 复合物传来的激活性细胞内信号中起着至关重要的作用。先前的报告表明，在 T 细胞激活后，LAT 与酪氨酸激酶 Lck 相互作用，导致其激酶活性受到抑制。LAT-Lck 相互作用似乎依赖于 LAT 中一段带负电荷的氨基酸。在这里，我们用不带电荷的片段替换了 LAT 中氨基酸 113 到 126 之间的这一段，并在 J.CaM2 细胞中表达了突变的 LAT（LAT-NIL），以分析 TCR 信号。LAT 中这一片段的替换阻止了激活诱导的与 Lck 的相互作用。此外，表达这种突变形式 LAT 的细胞显示出统计学上显著增加的近端细胞内信号，如酪氨酸残基 171 和 191 处的 LAT 磷酸化，以及接近统计学意义的 ZAP70 磷酸化（=0.051）。然而，下游信号，如 Ca2+内流或 MAPK 途径，部分受到抑制。总的来说，我们的数据表明，LAT-Lck 相互作用是调节 TCR/CD3 复合物传来的近端细胞内信号的关键因素。
