Proteome-Wide Discovery of Unknown ATP-Binding Proteins and Kinase Inhibitor Target Proteins Using an ATP Probe

ATP-binding proteins, including protein kinases, play essential roles in many biological and pathological processes and thus these proteins are attractive as drug targets. Acyl-ATP probes have been developed as efficient probes for kinase enrichment, and these probes have also been used to enrich ot...

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Veröffentlicht in:Journal of proteome research 2014-12, Vol.13 (12), p.5461-5470
Hauptverfasser: Adachi, Jun, Kishida, Marina, Watanabe, Shio, Hashimoto, Yuuki, Fukamizu, Kazuna, Tomonaga, Takeshi
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Sprache:eng
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Zusammenfassung:ATP-binding proteins, including protein kinases, play essential roles in many biological and pathological processes and thus these proteins are attractive as drug targets. Acyl-ATP probes have been developed as efficient probes for kinase enrichment, and these probes have also been used to enrich other ATP-binding proteins. However, a robust method to identify ATP-binding proteins with systematic elimination of nonspecific binding proteins has yet to be established. Here, we describe an ATP competition assay that permitted establishment of a rigorous ATP-binding protein list with virtual elimination of nonspecific proteins. A total of 539 ATP-binding protein candidates were identified, including 178 novel candidates. In informatics analysis, ribosomal proteins were overrepresented in the list of novel candidates. We also found multiple ATP-competitive sites for several kinases, including epidermal growth factor receptor, serine/threonine-protein kinase PRP4 homologue, cyclin-dependent kinase 12, eukaryotic elongation factor 2 kinase, ribosomal protein S6 kinase alpha-1, and SRSF protein kinase 1. Using our cataloged ATP-binding protein list, a selectivity profiling method that covers the kinome and ATPome was established to identify off-target binding sites of ATP-competitive kinase inhibitors, staurosporine and crizotinib.
ISSN:1535-3893
1535-3907
DOI:10.1021/pr500845u