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Development of high‐throughput phosphorylation profiling method for identification of Ser/Thr kinase specificity
Author(s) -
Kim EunMi,
Kim Jaehi,
Kim YunGon,
Lee Peter,
Shin DongSik,
Kim Mira,
Hahn JiSook,
Lee YoonSik,
Kim ByungGee
Publication year - 2011
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.1312
Subject(s) - phosphorylation , profiling (computer programming) , kinase , computational biology , chemistry , microbiology and biotechnology , biology , biochemistry , computer science , operating system
Identification of substrate specificity of kinases is crucial to understand the roles of the kinases in cellular signal transduction pathways. Here, we present an approach applicable for the discovery of substrate specificity of Ser/Thr kinases. The method, which is named as the ‘high‐throughput phosphorylation profiling (HTPP)’ method was developed on the basis of a fully randomized one‐bead one‐compound (OBOC) combinatorial ladder type peptide library and MALDI‐TOF MS. The OBOC ladder peptide library was constructed by the ‘split and pool’ method on a HiCore resin. The peptide library sequence was Ac‐Ala‐X‐X‐X‐Ser‐X‐X‐Ala‐BEBE‐PLL resin. The substrate specificity of murine PKA (cAMP‐dependent protein kinase A) and yeast Yak1 kinase was identified using this method. On the basis of the result, we identified Ifh1, which is a co‐activator for the transcription of ribosomal protein genes, as a novel substrate of Yak1 kinase. The putative Yak1‐dependent phosphorylation site of Ifh1 was verified by in vitro kinase assay. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.