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

Godl, K. et al. (2003) An efficient proteomics method to identify the cellular targets of protein kinase inhibitors. Proceedings of the National Academy of Sciences of the United States of America, 100(26), pp. 15434-15439. (doi: 10.1073/pnas.2535024100) (PMID:14668439) (PMCID:PMC307585)

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Abstract

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 p38α 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Cotten, Professor Matthew
Authors: Godl, K., Wissing, J., Kurtenbach, A., Habenberger, P., Blencke, S., Gutbrod, H., Salassidis, K., Stein-Gerlach, M., Missio, A., Cotten, M., and Daub, H.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
Journal Name:Proceedings of the National Academy of Sciences of the United States of America
Publisher:National Academy of Sciences
ISSN:0027-8424
ISSN (Online):1091-6490
Published Online:10 December 2003

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