Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP

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• 作者：Isabelle Becher ; Mikhail M. Savitski ; Maria F盲lth Savitski ; Carsten Hopf ; Marcus Bantscheff ; Gerard Drewes
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：March 15, 2013
• 年：2013
• 卷：8
• 期：3
• 页码：599-607
• 全文大小：625K
• 年卷期：v.8,no.3(March 15, 2013)
• ISSN：1554-8937

文摘

Most kinase inhibitor drugs target the binding site of the nucleotide cosubstrate ATP. The high intracellular concentration of ATP can strongly affect inhibitor potency and selectivity depending on the affinity of the target kinase for ATP. Here we used a defined chemoproteomics system based on competition-binding assays in cell extracts from Jurkat and SK-MEL-28 cells with immobilized ATP mimetics (kinobeads). This system enabled us to assess the affinities of more than 200 kinases for the cellular nucleotide cofactors ATP, ADP, and GTP and the effects of the divalent metal ions Mg²⁺ and Mn²⁺. The affinity values determined in this system were largely consistent across the two cell lines, indicating no major dependence on kinase expression levels. Kinase-ATP affinities range from low micromolar to millimolar, which has profound consequences for the prediction of cellular effects from inhibitor selectivity profiles. Only a small number of kinases including CK2, MEK, and BRAF exhibited affinity for GTP. This extensive and consistent data set of kinase-nucleotide affinities, determined for native enzymes under defined experimental conditions, will represent a useful resource for kinase drug discovery.