Veide Vilg Jenny, Dahal Sita, Ljungdahl Thomas, Grøtli Morten, Tamás Markus J
Department of Chemistry and Molecular Biology, University of Gothenburg, 405 30, Göteborg, Sweden.
Curr Genet. 2014 Aug;60(3):193-200. doi: 10.1007/s00294-014-0424-3. Epub 2014 Mar 19.
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.
抑制蛋白激酶活性的化学分子是评估蛋白激酶在活细胞中功能的重要工具。为了开发、测试和表征新型抑制剂，一种方便且可重复的激酶测定方法至关重要。在此，我们应用基于生物素化肽的方法来评估靶向酵母激酶Hog1、Elm1和Elm1-as的三磷酸腺苷竞争性抑制剂。肽底物包含13个氨基酸，涵盖磷酸化位点周围的共有序列。为了测试远端位点的缺失是否会影响抑制剂的效力，我们将基于肽的测定方法与使用全长蛋白作为底物的测定方法进行了比较。无论使用肽还是全长蛋白作为激酶底物，都获得了相似的抑制剂效率。因此，我们证明与蛋白质底物相比，先前使用的肽底物（Dinér等人，《公共科学图书馆·综合》6(5):e20012，2011）能给出准确的结果。我们还表明基于肽的方法适用于选择性测定和抑制剂筛选。生物素化肽底物的使用为蛋白激酶抑制剂的表征提供了一种简单可靠的测定方法。讨论了这种方法的实用性。
