Mutation of tyrosines 492/493 in the kinase domain of ZAP-70 affects multiple T-cell receptor signaling pathways
Mutation of tyrosines 492/493 in the kinase domain of ZAP-70 affects multiple T-cell receptor signaling pathways
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DOI：
10.1074/jbc.271.51.32644
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发表时间：
1996-12-20
影响因子：
4.8
通讯作者：
Acuto, O
中科院分区：
文献类型：
--
作者：
Mege, D;DiBartolo, V;Acuto, O
蛋白酪氨酸激酶ZAP-70与Src激酶p56（lck）一起参与控制T细胞抗原受体（TCR）信号级联的早期步骤。为了帮助进一步阐明ZAP-70调节这些初始事件的机制，我们使用了显性负突变体方法。我们在Jurkat T细胞系中过表达ZAP-70，其激酶结构域的推定调节环中的Tyr-492和Tyr-493突变。该突变体通过干扰细胞内钙增加和Ras调节的细胞外信号调节激酶的活化来抑制TCR诱导的活化T细胞的核因子的活化。此外，发现TCR诱导的pp36 - 38磷酸化（被认为在这些途径的上游起作用）减少。相反，野生型ZAP-70的过表达诱导活化T细胞的核因子的组成性活化。本文研究的ZAP-70突变体在与TCR ζ链结合时可以在酪氨酸上磷酸化，并且能够结合p56（lck）。该结果表明Tyr-492和Tyr-493不负责Src同源结构域α介导的p56（lck）与ZAP-70的缔合。我们的数据与其中募集至TCR允许ZAP-70自磷酸化并结合至p56（lck）的模型最一致，p56（lck）进而磷酸化Tyr-492和/或Tyr-493，随后上调ZAP-70激酶活性。然后，ZAP-70将能够有效地控制其底物的磷酸化并导致基因激活。
The protein-tyrosine kinase ZAP-70 is implicated, together with the Src kinase p56(lck), in controlling the early steps of the T-cell antigen receptor (TCR) signaling cascade. To help elucidate further the mechanism by which ZAP-70 regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited TCR induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp36-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wildtype ZAP-70 induced constitutive activation of nuclear factor of activated T cells. The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR zeta chain and was able to bind p56(lck). This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain a-mediated association of p56(lck) with ZAP-70. Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56(lck), which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation.