Application of a peptide-based assay to characterize inhibitors targeting protein kinases from yeast
Artikel i vetenskaplig tidskrift, 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.

Elm1

Yeast

Chemical biology

Protein kinase

Inhibitors

Hog1

Författare

Jenny Veide Vilg

Göteborgs universitet

S. Dahal

Göteborgs universitet

T. Ljungdahl

Göteborgs universitet

Morten Grøtli

Göteborgs universitet

Markus J. Tamás

Göteborgs universitet

Current Genetics

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

Vol. 60 193-200

Ämneskategorier

Biokemi och molekylärbiologi

DOI

10.1007/s00294-014-0424-3