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 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.