Application of a peptide-based assay to characterize inhibitors targeting protein kinases from yeast

作者：Vilg Jenny Veide; Dahal Sita; Ljungdahl Thomas; Grotli Morten; Tamas Markus J^*

来源：Current Genetics, 2014, 60(3): 193-200.

DOI：10.1007/s00294-014-0424-3

• Chemical biology
• Protein kinase
• Inhibitors
• Yeast
• Hog1
• Elm1

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 (Din,r 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.

• 出版日期2014-8