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.

中文翻译：

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通过高通量特异性筛选揭示的 Src 家族激酶 Lck 底物偏好的微调

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。我们开发了一种通过将肽库的细菌表面展示与下一代测序相结合来分析酪氨酸激酶特异性的方法。利用这一点，我们发现酪氨酸激酶 ZAP-70 对 T 细胞信号传导至关重要，它通过在其催化域内编码的静电选择机制区分底物（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源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，我们发现密切相关的 Src 家族激酶 Lck 和 c-Src 之间的底物静电识别存在差异。这种差异可能反映了 Lck 在 T 细胞信号传导中与 ZAP-70 协同作用的专业化。这些结果表明直接识别激酶活性位点在微调特异性中的重要性。