Association of Grb2 Gads, and phospholipase C-γ1 with phosphorylated LAT tyrosine residues -: Effect of LAT tyrosine mutations on T cell antigen receptor-mediated signaling
Association of Grb2 Gads, and phospholipase C-γ1 with phosphorylated LAT tyrosine residues -: Effect of LAT tyrosine mutations on T cell antigen receptor-mediated signaling
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DOI：
10.1074/jbc.m000404200
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发表时间：
2000-07-28
影响因子：
4.8
通讯作者：
Samelson, LE
中科院分区：
文献类型：
--
作者：
Zhang, WG;Trible, RP;Samelson, LE
T细胞活化连接物(LAT)是T细胞抗原受体(TCR)介导的信号转导和胸腺细胞发育所必需的关键适配分子。当T细胞激活时，LAT在酪氨酸残基上高度磷酸化，Grb2、Gads和磷脂酶C(PLC)-Gamma 1通过Src同源-2结构域与LAT结合。在LAT缺乏突变的Jurkat细胞中，TCR的参与不能诱导ERK的激活、钙离子通量以及AP-1和NF-AT的激活。我们通过在LAT缺陷细胞中表达带有酪氨酸到苯丙氨酸突变的LAT突变体，定位了LAT中负责与这些特定信号分子相互作用的酪氨酸残基。我们的结果表明，三个末端的酪氨酸，Tyr(171)，Tyr(191)和Tyr(226)负责Grb2结合；Tyr171和Tyr(191)是Gads结合所必需的，但不是Tyr(226)。仅Tyr(132)的突变就取消了PLC-Gamma 1结合。3个酪氨酸末端的突变也取消了PLC-Gamma 1的结合，提示PLC-Gamma 1可能有多个结合部位。Tyr(132)突变影响了钙离子的流动，并阻断了Erk和NF-AT的激活。由于Grb2结合不受该突变的影响，这些结果强烈表明PLC-Gamma激活调节这些细胞中的RAS激活。单个Grb2结合位点的突变没有功能影响，但其中两个或三个结合位点的突变也会影响ERK。和核因子-AT的激活。
The linker for activation of T cells (LAT) is a critical adaptor molecule required for T cell antigen receptor (TCR)-mediated signaling and thymocyte development. Upon T cell activation, LAT becomes highly phosphorylated on tyrosine residues, and Grb2, Gads, and phospholipase C (PLC)-gamma 1 bind LAT via Src homology-2 domains. In LAT-deficient mutant Jurkat cells, TCR engagement fails to induce ERK activation, Ca2+ flux, and activation of AP-1 and NF-AT. We mapped the tyrosine residues in LAT responsible for interaction with these specific signaling molecules by expressing LAT mutants with tyrosine to phenylalanine mutations in LAT-deficient cells. Our results showed that three distal tyrosines, Tyr(171), Tyr(191), and Tyr(226), are responsible for Grb2-binding; Tyr171, and Tyr(191), but not Tyr(226) necessary for Gads binding. Mutation of Tyr(132) alone abolished PLC-gamma 1 binding. Mutation of all three distal tyrosines also abolished PLC-gamma 1 binding, suggesting there might be multiple binding sites for PLC-gamma 1. Mutation of Tyr(132) affected calcium flux and blocked Erk and NF-AT activation. Since Grb2 binding is not affected by this mutation, these results strongly suggest that PLC-gamma activation regulates Ras activation in these cells. Mutation of individual Grb2 binding sites had no functional effect, but mutation of two or three of these sites, in combination, also affected Erk. and NF-AT activation.