Unit of Cytokine Signaling, Department of Immunology, Institut Pasteur, Paris, France.
PLoS One. 2010 Nov 30;5(11):e15114. doi: 10.1371/journal.pone.0015114.
One of the earliest activation events following stimulation of the T cell receptor (TCR) is the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3-associated complex by the Src family kinase Lck. There is accumulating evidence that a large pool of Lck is constitutively active in T cells but how the TCR is connected to Lck and to the downstream signaling cascade remains elusive.
METHODOLOGY/PRINCIPAL FINDINGS: We have analyzed the phosphorylation state of Lck and Fyn and TCR signaling in human naïve CD4+ T cells and in the transformed T cell line, Hut-78. The latter has been shown to be similar to primary T cells in TCR-inducible phosphorylations and can be highly knocked down by RNA interference. In both T cell types, basal phosphorylation of Lck and Fyn on their activatory tyrosine was observed, although this was much less pronounced in Hut-78 cells. TCR stimulation led to the co-precipitation of Lck with the transmembrane adaptor protein LAT (linker for activation of T cells), Erk-mediated phosphorylation of Lck and no detectable dephosphorylation of Lck inhibitory tyrosine. Strikingly, upon LAT knockdown in Hut-78 cells, we found that LAT promoted TCR-induced phosphorylation of Lck and Fyn activatory tyrosines, TCRζ chain phosphorylation and Zap-70 activation. Notably, LAT regulated these events at low strength of TCR engagement.
CONCLUSIONS/SIGNIFICANCE: Our results indicate for the first time that LAT promotes TCR signal initiation and suggest that this adaptor may contribute to maintain active Lck in proximity of their substrates.
T 细胞受体（TCR）受到刺激后最早的激活事件之一是Src 家族激酶 Lck 对 CD3 相关复合物中免疫受体酪氨酸基激活基序（ITAMs）的磷酸化。越来越多的证据表明，T 细胞中有大量的 Lck 处于持续激活状态，但 TCR 如何与 Lck 以及下游信号级联连接仍然难以捉摸。
方法/主要发现：我们分析了人幼稚 CD4+T 细胞和转化的 T 细胞系 Hut-78 中 Lck 和 Fyn 的磷酸化状态和 TCR 信号。后者已被证明在 TCR 诱导的磷酸化方面与原代 T 细胞相似，并且可以通过 RNA 干扰高度敲低。在两种 T 细胞类型中，都观察到 Lck 和 Fyn 在其激活酪氨酸上的基础磷酸化，尽管在 Hut-78 细胞中这种磷酸化程度要低得多。TCR 刺激导致 Lck 与跨膜衔接蛋白 LAT（T 细胞激活的衔接蛋白）共沉淀，Erk 介导的 Lck 磷酸化和 Lck 抑制性酪氨酸的可检测去磷酸化。引人注目的是，在 Hut-78 细胞中敲低 LAT 后，我们发现 LAT 促进了 TCR 诱导的 Lck 和 Fyn 激活酪氨酸、TCRζ 链磷酸化和 Zap-70 激活。值得注意的是，LAT 在 TCR 结合强度较低时调节这些事件。
结论/意义：我们的研究结果首次表明 LAT 促进了 TCR 信号的起始，并表明该衔接蛋白可能有助于维持其底物附近活性的 Lck。
