Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates
Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates
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DOI：
10.1073/pnas.0708966105
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发表时间：
2008-02-05
影响因子：
11.1
通讯作者：
Shokat, Kevan M.
中科院分区：
文献类型：
--
作者：
Blethrow, Justin D.;Glavy, Joseph S.;Shokat, Kevan M.
我们描述了一种快速鉴定蛋白激酶底物的方法。CDK1被设计为接受一种ATP类似物，使其能够用生物正交磷酸盐类似物标签唯一地标记其底物。开发了一种高度特异的共价捕获和释放方法，用于快速纯化来自标记底物蛋白的标记多肽。应用这种方法发现CDK1-Cyclin B底物，得到了>；70底物和磷酸化位点的鉴定。已知许多这些位点在体内被磷酸化，但大多数蛋白质还没有被鉴定为CDK1-Cyclin B底物。这种方法有可能扩大我们对信号网络中的激酶-底物连接的理解。
We describe a method for rapid identification of protein kinase substrates. Cdk1 was engineered to accept an ATP analog that allows it to uniquely label its substrates with a bio-orthogonal phosphate analog tag. A highly specific, covalent capture-and-release methodology was developed for rapid purification of tagged peptides derived from labeled substrate proteins. Application of this approach to the discovery of Cdk1-cyclin B substrates yielded identification of >70 substrates and phosphorylation sites. Many of these sites are known to be phosphorylated in vivo, but most of the proteins have not been characterized as Cdk1-cyclin B substrates. This approach has the potential to expand our understanding of kinase-substrate connections in signaling networks.