Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen.
Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen.
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DOI：
10.7554/elife.35190
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发表时间：
2018-03-16
期刊：
影响因子：
7.7
通讯作者：
Kuriyan J
中科院分区：
文献类型：
--
作者：
Shah NH;Löbel M;Weiss A;Kuriyan J
酪氨酸激酶的特异性主要归因于由非催化结构域决定的定位效应。我们开发了一种方法，通过结合细菌表面展示肽库与下一代测序来分析酪氨酸激酶的特异性。利用这一点，我们表明对于T细胞信号传导至关重要的酪氨酸激酶ZAP-70通过其催化结构域中编码的静电选择机制来区分底物（Shah等人，2016年）。在这里，我们扩展了这个高通量平台，以分析任何酪氨酸激酶结构域对来自人类酪氨酸磷酸化位点的数千种肽的内在特异性。使用这种方法，我们发现了密切相关的Src家族激酶Lck和c-Src之间的静电识别基板的差异。这种差异可能反映了Lck的特化，以与ZAP-70在T细胞信号传导中协同作用。这些结果表明，在微调特异性的激酶活性位点的直接识别的重要性。
The specificity of tyrosine kinases is attributed predominantly to localization effects dictated by non-catalytic domains. We developed a method to profile the specificities of tyrosine kinases by combining bacterial surface-display of peptide libraries with next-generation sequencing. Using this, we showed that the tyrosine kinase ZAP-70, which is critical for T cell signaling, discriminates substrates through an electrostatic selection mechanism encoded within its catalytic domain (Shah et al., 2016). Here, we expand this high-throughput platform to analyze the intrinsic specificity of any tyrosine kinase domain against thousands of peptides derived from human tyrosine phosphorylation sites. Using this approach, we find a difference in the electrostatic recognition of substrates between the closely related Src-family kinases Lck and c-Src. This divergence likely reflects the specialization of Lck to act in concert with ZAP-70 in T cell signaling. These results point to the importance of direct recognition at the kinase active site in fine-tuning specificity.