Abstract
Chemical molecules that inhibit protein kinase activity are important tools to assess the functions of protein kinases in living cells. To develop, test and characterize novel inhibitors, a convenient and reproducible kinase assay is of importance. Here, we applied a biotinylated peptide-based method to assess adenosine triphosphate-competitive inhibitors that target the yeast kinases Hog1, Elm1 and Elm1-as. The peptide substrates contained 13 amino acids, encompassing the consensus sequence surrounding the phosphorylation site. To test whether the lack of distal sites affects inhibitor efficacy, we compared the peptide-based assay with an assay using full-length protein as substrate. Similar inhibitor efficiencies were obtained irrespective of whether peptide or full-length protein was used as kinase substrates. Thus, we demonstrate that the peptide substrates used previously (Dinér et al. in PLoS One 6(5):e20012, 2011) give accurate results compared with protein substrates. We also show that the peptide-based method is suitable for selectivity assays and for inhibitor screening. The use of biotinylated peptide substrates provides a simple and reliable assay for protein kinase inhibitor characterization. The utility of this approach is discussed.
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Abbreviations
- IC50 :
-
Half maximal inhibitory concentration
- ATP:
-
Adenosine triphosphate
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- MAPK:
-
Mitogen-activated protein kinase
- GST:
-
Glutathione-S-transferase
- ASKA:
-
Analog-sensitive kinase allele
- as mutant:
-
Analog-sensitive mutant
- DMSO:
-
Dimethyl sulfoxide
- HEPES:
-
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
- TD buffer:
-
Tris–DTT buffer
- DTT:
-
Dithiothreitol
- BPTIP:
-
4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)-N-isopropylpyridin-2-amine
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Acknowledgments
We thank Francesc Posas (Barcelona), Martin Schmidt (Pittsburgh) and Karin Elbing (Lund) for providing plasmids and purified protein. This work was supported by the University of Gothenburg Chemical Biology Platform and the Signhild Engkvist and Olle Engkvist Byggmästare foundations.
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The authors declare that they have no competing interests.
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Communicated by S. Hohmann.
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Veide Vilg, J., Dahal, S., Ljungdahl, T. et al. Application of a peptide-based assay to characterize inhibitors targeting protein kinases from yeast. Curr Genet 60, 193–200 (2014). https://doi.org/10.1007/s00294-014-0424-3
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DOI: https://doi.org/10.1007/s00294-014-0424-3