title: Development of high-throughput phosphorylation profiling method for identification of Ser/Thr kinase specificity.
abstract: 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.
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
authors.forename: Eun-Mi
authors.initials: EM
authors.lastname: Kim
authors.forename: Jaehi
authors.initials: J
authors.lastname: Kim
authors.forename: Yun-Gon
authors.initials: YG
authors.lastname: Kim
authors.forename: Peter
authors.initials: P
authors.lastname: Lee
authors.forename: Dong-Sik
authors.initials: DS
authors.lastname: Shin
authors.forename: Mira
authors.initials: M
authors.lastname: Kim
authors.forename: Ji-Sook
authors.initials: JS
authors.lastname: Hahn
authors.forename: Yoon-Sik
authors.initials: YS
authors.lastname: Lee
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
mesh_terms: D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D051379:Mice; D019151:Peptide Library; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D019151:Peptide Library; D029701:Saccharomyces cerevisiae Proteins; D017346:Protein Serine-Threonine Kinases; C062391:YAK1 protein, S cerevisiae; D017868:Cyclic AMP-Dependent Protein Kinases