Lck phosphorylates the activation loop tyrosine of the Itk kinase domain and activates Itk kinase activity
Lck phosphorylates the activation loop tyrosine of the Itk kinase domain and activates Itk kinase activity
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DOI：
10.1074/jbc.272.40.25401
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发表时间：
1997-10-03
影响因子：
4.8
通讯作者：
Berg, LJ
中科院分区：
文献类型：
--
作者：
Heyeck, SD;Wilcox, HM;Berg, LJ
Tec家族酪氨酸激酶Itk与T细胞受体（TCR）信号传导有关，但其确切的作用和激活机制仍不清楚。为了研究这些问题，我们研究了Itk对TCR刺激的生化反应。我们发现Itk在TCR交联后被酪氨酸磷酸化，并且这种磷酸化依赖于功能性Lck的存在。为了确定这种Lck依赖性是否是由Lck对Itk的直接磷酸化引起的，我们使用杆状病毒表达系统产生重组Itk和Lck，并在随后的生化分析中使用这些蛋白质。我们发现，在昆虫细胞中共表达时，Lck使Itk磷酸化，并且进一步地，Itk的这种磷酸化导致Itk体外激酶活性增加。Itk上Lck磷酸化的主要位点定位于Itk激酶结构域激活环中的保守酪氨酸（Tyr（511））。用苯丙氨酸取代该酪氨酸废除了昆虫细胞中的Itk激酶活性，表明该位点的磷酸化在调节Itk功能中起着关键作用。
The Tec family tyrosine kinase Itk has been implicated in T cell receptor (TCR) signaling, yet its precise role and mechanism of activation remain undefined. To investigate these issues, we examined the biochemical response of Itk to TCR stimulation. We found that Itk is tyrosine-phosphorylated after TCR cross-linking and that this phosphorylation depends on the presence of functional Lck. To determine if this Lck dependence results from direct phosphorylation of Itk by Lck, we generated recombinant Itk and Lck using a baculovirus expression system and used these proteins in subsequent biochemical analyses, We found that Lck phosphorylates Itk upon co-expression in insect cells and, further, that this phosphorylation of Itk results in increased Itk in vitro kinase activity. The major site of Lck phosphorylation on Itk was mapped to the conserved tyrosine (Tyr(511)) in the activation loop of the Itk kinase domain. Substitution of this tyrosine with phenylalanine abolishes Itk kinase activity in insect cells, indicating that phosphorylation at this site plays a critical role in regulating Itk function.