Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line
Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line
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DOI：
10.1093/intimm/11.6.943
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发表时间：
1999-06-01
影响因子：
4.4
通讯作者：
Samelson, LE
中科院分区：
文献类型：
--
作者：
Zhang, WG;Irvin, BJ;Samelson, LE
接头分子LAT （I细胞活化的连接物）是一种棕榈酰化的整体膜蛋白，定位于质膜中富含糖脂的微结构域。与TCR结合后，LAT在多个酪氨酸残基上被磷酸化，然后结合几个关键的信号分子。在这里，我们描述了一个缺乏LAT的细胞系的产生和特性，使用这个细胞系，我们证明了LAT是tcr介导的Ca2+动员和磷脂酶c - γ 1、Vav和SLP-76的最佳酪氨酸磷酸化所必需的，LAT也是Erk激活、CD69上调以及AP和nfat介导的基因转录所必需的。通过重组LAT突变体细胞系，我们也证明了LAT跨膜结构域和Cys26的棕榈酰化是LAT功能和TCR信号传递所必需的，而不是Cys29。这些研究为LAT分子的关键作用提供了进一步的证据，并证明了使用LAT缺陷细胞系分析LAT的结构和功能。
The adaptor molecule LAT (linker for activation of I cells) is a palmitoylated integral membrane protein that localizes to the glycolipid-enriched microdomains in the plasma membrane. Upon TCR engagement, LAT becomes phosphorylated on multiple tyrosine residues and then binds several critical signaling molecules. Here, we describe the generation and characterization of a LAT-deficient cell line, Using this cell line, we demonstrate that LAT is required for TCR-mediated Ca2+ mobilization and optimal tyrosine phosphorylation of phospholipase C-gamma 1,Vav and SLP-76, LAT is also required for Erk activation, CD69 up-regulation, and AP- and NFAT-mediated gene transcription. We also demonstrate, by reconstituting this cell line with LAT mutants, that the LAT transmembrane domain and palmitoylation at Cys26, but not Cys29, are required for LAT function and TCR signaling. These studies provide further evidence for the crucial role of the LAT molecule, and demonstrate the use of a LAT-deficient cell line for the analysis of LAT structure and function.