Single-Well Monitoring of Protein-Protein Interaction and Phosphorylation-Dephosphorylation Events
Single-Well Monitoring of Protein-Protein Interaction and Phosphorylation-Dephosphorylation Events
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DOI：
10.1021/bi100253p
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发表时间：
2010-04-20
期刊：
影响因子：
2.9
通讯作者：
Dahan, Sophie
中科院分区：
文献类型：
--
作者：
Arcand, Mathieu;Roby, Philippe;Dahan, Sophie
我们将氧通道分析与两种不同的化学发光珠结合起来，同时检测通常单独监测的蛋白质磷酸化和相互作用事件。这种新方法在ERK1/2 MAP激酶途径中进行了测试。它首次被用于直接监测map激酶ERK2在磷酸化后与MEK1的解离，并评估map激酶磷酸酶（mapkinase phosphatase， MKP）的选择性和作用机制。此外，MEK1和ERK2被一种ATP竞争者和一种变构MEK1抑制剂探测，它们产生了不同的磷酸化相互作用模式。同时监测蛋白质相互作用和底物磷酸化可以为酶活性和小分子作用提供重要的机制见解。
We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.