title: Global analysis of human nonreceptor tyrosine kinase specificity using high-density peptide microarrays.
abstract: Protein kinases phosphorylate substrates in the context of specific phosphorylation site sequence motifs. The knowledge of the specific sequences that are recognized by kinases is useful for mapping sites of phosphorylation in protein substrates and facilitates the generation of model substrates to monitor kinase activity. Here, we have adapted a positional scanning peptide library method to a microarray format that is suitable for the rapid determination of phosphorylation site motifs for tyrosine kinases. Peptide mixtures were immobilized on glass slides through a layer of a tyrosine-free Y33F mutant avidin to facilitate the analysis of phosphorylation by radiolabel assay. A microarray analysis provided qualitatively similar results in comparison with the solution phase peptide library "macroarray" method. However, much smaller quantities of kinases were required to phosphorylate peptides on the microarrays, which thus enabled a proteome scale analysis of kinase specificity. We illustrated this capability by microarray profiling more than 80% of the human nonreceptor tyrosine kinases (NRTKs). Microarray results were used to generate a universal NRTK substrate set of 11 consensus peptides for in vitro kinase assays. Several substrates were highly specific for their cognate kinases, which should facilitate their incorporation into kinase-selective biosensors.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Deng
authors.forename: Nilda L
authors.initials: NL
authors.lastname: Alicea-Velázquez
authors.forename: Ludovic
authors.initials: L
authors.lastname: Bannwarth
authors.forename: Soili I
authors.initials: SI
authors.lastname: Lehtonen
authors.forename: Titus J
authors.initials: TJ
authors.lastname: Boggon
authors.forename: Heung-Chin
authors.initials: HC
authors.lastname: Cheng
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Turk
mesh_terms: D006801:Humans; D040081:Protein Array Analysis; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011505:Protein-Tyrosine Kinases
keywords: drug discovery; enzyme specificity; kinase inhibitors; nonreceptor tyrosine kinases; peptide libraries; peptide microarrays; protein kinases