An efficient proteomics method to identify the cellular targets of protein kinase inhibitors

被引:294
作者
Godl, K [1 ]
Wissing, J [1 ]
Kurtenbach, A [1 ]
Habenberger, P [1 ]
Blencke, S [1 ]
Gutbrod, H [1 ]
Salassidis, K [1 ]
Stein-Gerlach, M [1 ]
Missio, A [1 ]
Cotten, M [1 ]
Daub, H [1 ]
机构
[1] Axxima Pharmaceut AG, D-81377 Munich, Germany
关键词
D O I
10.1073/pnas.2535024100
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38alpha whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action.
引用
收藏
页码:15434 / 15439
页数:6
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