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首页> 外文期刊>Biochemistry >Activation of Zap-70 tyrosine kinase due to a structural rearrangement induced by tyrosine phosphorylation and/or ITAM binding.
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Activation of Zap-70 tyrosine kinase due to a structural rearrangement induced by tyrosine phosphorylation and/or ITAM binding.

机译：ZAP-70酪氨酸激酶的激活由于酪氨酸磷酸化和/或ITAM结合诱导的结构重排。

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摘要

The protein tyrosine kinase ZAP-70 is implicated in the early steps of the T-cell antigen receptor (TCR) signaling. Binding of ZAP-70 to the phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain through its two src-homology 2 (SH2) domains results in its activation coupled to phosphorylation on multiple tyrosine residues, mediated by Src kinases including Lck as well as by autophosphorylation. The mechanism of ZAP-70 activation following receptor binding is still not completely understood. Here we investigated the effect of intramolecular interactions and autophosphorylation by following the kinetics of recombinant ZAP-70 activation in a spectrophotometric substrate phosphorylation assay. Under these conditions, we observed a lag phase of several minutes before full ZAP-70 activation, which was not observed using a truncated form lacking the first 254 residues, suggesting that it might be due to an intramolecular interaction involving the interdomain A and SH2 region. Accordingly, the lag phase could be reproduced by testing the truncated form in the presence of recombinant SH2 domains and was abolished by the addition of diphosphorylated ITAM peptide. Preincubation with ATP or phosphorylation by Lck also abolished the lag phase and resulted in a more active enzyme. The same results were obtained using a ZAP-70 mutant lacking the interdomain B tyrosines. These findings are consistent with a mechanism in which ZAP-70 phosphorylation/autophosphorylation on tyrosine(s) other than 292, 315, and 319, as well as engagement of the SH2 domains by the phosphorylated TCR, can induce a conformational change leading to accelerated enzyme kinetics and higher catalytic efficiency.
机译：蛋白酪氨酸激酶ZAP-70涉及T细胞抗原受体（TCR）信号传导的早期步骤。 ZAP-70通过其两种SRC-同源性2（SH2）结构域的TCR Zeta链的磷酸化免疫感染酪氨酸酪氨酸的活化基序（ITAM）导致其激活与多个酪氨酸残基上的磷酸化，由SRC激酶介导，包括LCK以及自动磷酸化。 ZAP-70活化后的机制仍未完全理解。在这里，我们通过在分光光度法基质磷酸化测定中进行重组ZAP-70活化的动力学来研究分子内相互作用和自磷酸化的影响。在这些条件下，我们观察到在全ZAP-70激活前几分钟的滞后阶段，这未使用缺少前254个残基的截短形式观察，这表明它可能是由于涉及跨域A和SH2区域的分子内相互作用。因此，可以通过在重组SH2结构域的存在下测试截短的形式来再现滞后阶段，并通过加入二磷酸化的ITAM肽来消除。通过LCK与ATP或磷酸化的预孵育也废除了滞后阶段并导致更活跃的酶。使用缺乏杂交B酪氨酸的ZAP-70突变体获得相同的结果。这些发现是一致的一种机制，其中ZAP-70磷酸化/自磷酸化在292,315和319之外的酪氨酸，以及SH2域通过磷酸化的TCR接合，可以诱导导致加速的构象变化酶动力学及催化效率较高。

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