Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP

James S. Duncan, Timothy A.J. Haystead, David W. Litchfield

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

8 Scopus citations

Abstract

Protein kinase inhibitors have emerged as indispensable tools for the elucidation of the biological functions of specific signal transduction pathways and as promising candidates for molecular-targeted therapy. However, because many protein kinase inhibitors are ATP-competitive inhibitors targeting the catalytic site of specific protein kinases, the large number of protein kinases that are encoded within eukaryotic genomes and the existence of many other cellular proteins that bind ATP result in the prospect of off-target effects for many of these compounds. Many of the potential off-target effects remain unrecognized because protein kinase inhibitors are often developed and tested primarily on the basis of in vitro assays using purified components. To overcome this limitation, we describe a systematic approach to characterize ATP-competitive protein kinase inhibitors employing ATP-sepharose to capture the purine-binding proteome from cell extracts. Protein kinase inhibitors can be used in competition experiments to prevent binding of specific cellular proteins to ATP-sepharose or to elute bound proteins from ATP-sepharose. Collectively, these strategies can enable validation of interactions between a specific protein kinase and an inhibitor in complex mixtures and can yield the identification of inhibitor targets.

Original languageEnglish
Title of host publicationKinase Inhibitors
Subtitle of host publicationMethods and Protocols
EditorsBernhard Kuster
Pages119-134
Number of pages16
Volume795
DOIs
StatePublished - 2012

Publication series

NameMethods in Molecular Biology
Volume795
ISSN (Print)1064-3745

Keywords

  • 2D Gel electrophoresis
  • ATP-sepharose
  • Chemoproteomics
  • Mass spectrometry
  • Protein kinase CK2
  • Protein kinase inhibitors

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