Application of a peptide-based assay to characterize inhibitors targeting protein kinases from yeast
Journal article, 2014

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. © 2014 Springer-Verlag Berlin Heidelberg.

Chemical biology

Inhibitors

Hog1

Yeast

Protein kinase

Elm1

Author

Jenny Veide Vilg

Applied Chemistry

S. Dahal

University of Gothenburg

T. Ljungdahl

University of Gothenburg

Morten Grötli

University of Gothenburg

Markus J. Tamás

University of Gothenburg

Current Genetics

0172-8083 (ISSN) 1432-0983 (eISSN)

Vol. 60 3 193-200

Subject Categories

Biochemistry and Molecular Biology

DOI

10.1007/s00294-014-0424-3

More information

Latest update

7/24/2019