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Current Genetics

, Volume 60, Issue 3, pp 193–200 | Cite as

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

  • Jenny Veide Vilg
  • Sita Dahal
  • Thomas Ljungdahl
  • Morten Grøtli
  • Markus J. Tamás
Research Article

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.

Keywords

Chemical biology Protein kinase Inhibitors Yeast Hog1 Elm1 

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

Notes

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.

Conflict of interest

The authors declare that they have no competing interests.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Jenny Veide Vilg
    • 1
    • 2
  • Sita Dahal
    • 1
  • Thomas Ljungdahl
    • 1
  • Morten Grøtli
    • 1
  • Markus J. Tamás
    • 1
  1. 1.Department of Chemistry and Molecular BiologyUniversity of GothenburgGöteborgSweden
  2. 2.Department of Chemical and Biological EngineeringChalmers University of TechnologyGöteborgSweden

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