Bio-orthogonal Affinity Purification of Direct Kinase Substrates

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• 作者：Jasmina J. Allen ; Scott E. Lazerwith ; and Kevan M. Shokat
• 刊名：Journal of the American Chemical Society
• 出版年：2005
• 出版时间：April 20, 2005
• 年：2005
• 卷：127
• 期：15
• 页码：5288 - 5289
• 全文大小：87K
• 年卷期：v.127,no.15(April 20, 2005)
• ISSN：1520-5126

文摘

Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signaling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. Herein, we describe a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second nonenzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the alkylated phosphorothioate epitope recognize these labeled substrates, but not alkylation products of other cellular nucleophiles. This strategy is demonstrated with Cdk1/cyclinB substrates using ELISA, western blotting, and immunoprecipitation in the context of whole cell lysates.