Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking
Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking
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DOI：
10.1073/pnas.1501373112
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发表时间：
2015-07-14
影响因子：
11.1
通讯作者：
Link, A. James
中科院分区：
文献类型：
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作者：
Futran, Alan S.;Kyin, Saw;Link, A. James
细胞外信号调节激酶（ERK）通过磷酸化其众多底物来协调细胞对一系列刺激的反应。这些底物之一，Capicua（Cic），是一种转录阻遏物，首先在果蝇中发现，并已涉及许多人类疾病。在这里，我们使用化学生物学方法来映射ERK和Cic的结合界面。将非经典氨基酸对叠氮苯丙氨酸（AzF）引入果蝇Cic的ERK结合区，利用光交联和串联质谱技术确定AzF在ERK上的结合位点。我们还鉴定了人Cic的ERK结合区，并表明它与ERK上的相同位点结合，尽管缺乏与果蝇Cic结合区的保守性。最后，我们使用AzF标记的ERK定位了参与人Cic与ERK结合的氨基酸。这些结果揭示了ERK-Cic相互作用的分子细节，并表明光交联方法是对现有的用于绘制激酶-底物结合界面的方法的补充。
Extracellular signal-regulated kinase (ERK) coordinates cellular responses to a range of stimuli by phosphorylating its numerous substrates. One of these substrates, Capicua (Cic), is a transcriptional repressor that was first identified in Drosophila and has been implicated in a number of human diseases. Here we use a chemical biology approach to map the binding interface of ERK and Cic. The noncanonical amino acid p-azidophenylalanine (AzF) was introduced into the ERK-binding region of Drosophila Cic, and photocrosslinking and tandem mass spectrometry were used to pinpoint its binding site on ERK. We also identified the ERK-binding region of human Cic and showed that it binds to the same site on ERK despite lacking conservation with the Drosophila Cic binding region. Finally, we mapped the amino acids involved in human Cic binding to ERK using AzF-labeled ERK. These results reveal the molecular details of the ERK-Cic interaction and demonstrate that the photocrosslinking approach is complementary to existing methods for mapping kinase-substrate binding interfaces.