ATP-Mediated Kinome Selectivity: The Missing Link in Understanding the Contribution of Individual JAK Kinase Isoforms to Cellular Signaling

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• 作者：Atli Thorarensen ; Mary Ellen Banker ; Andrew Fensome ; Jean-Baptiste Telliez ; Brian Juba ; Fabien Vincent ; Robert M. Czerwinski ; Agustin Casimiro-Garcia
• 刊名：ACS Chemical Biology
• 出版年：2014
• 出版时间：July 18, 2014
• 年：2014
• 卷：9
• 期：7
• 页码：1552-1558
• 全文大小：393K
• ISSN：1554-8937

文摘

Kinases constitute an important class of therapeutic targets being explored both by academia and the pharmaceutical industry. The major focus of this effort was directed toward the identification of ATP competitive inhibitors. Although it has long been recognized that the intracellular concentration of ATP is very different from the concentrations utilized in biochemical enzyme assays, little thought has been devoted to incorporating this discrepancy into our understanding of translation from enzyme inhibition to cellular function. Significant work has been dedicated to the discovery of JAK kinase inhibitors; however, a disconnect between enzyme and cellular function is prominently displayed in the literature for this class of inhibitors. Herein, we demonstrate utilizing the four JAK family members that the difference in the ATP K_m of each individual kinase has a significant impact on the enzyme to cell inhibition translation. We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either 1 mM ATP or K_m ATP for the four isoforms as well as in primary cell assays. This data set provided the opportunity to examine individual kinase contributions to the heterodimeric kinase complexes mediating cellular signaling. In contrast to a recent study, we demonstrate that for IL-15 cytokine signaling it is sufficient to inhibit either JAK1 or JAK3 to fully inhibit downstream STAT5 phosphorylation. This additional data thus provides a critical piece of information explaining why JAK1 has incorrectly been thought to have a dominant role over JAK3. Beyond enabling a deeper understanding of JAK signaling, conducting similar analyses for other kinases by taking into account potency at high ATP rather than K_m ATP may provide crucial insights into a compound鈥檚 activity and selectivity in cellular contexts.