title: Active kinase proteome screening reveals novel signal complexity in cardiomyopathy.
abstract: Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions.
authors.affiliation: Molecular Medicine Program, Ottawa Health Research Institute, Ottawa Hospital, Ottawa, Ontario K1H 8L6, Canada.
authors.forename: Pasan
authors.initials: P
authors.lastname: Fernando
authors.forename: Wen
authors.initials: W
authors.lastname: Deng
authors.forename: Beata
authors.initials: B
authors.lastname: Pekalska
authors.forename: Yves
authors.initials: Y
authors.lastname: DeRepentigny
authors.forename: Rashmi
authors.initials: R
authors.lastname: Kothary
authors.forename: John F
authors.initials: JF
authors.lastname: Kelly
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Megeney
mesh_terms: D000818:Animals; D009202:Cardiomyopathies / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D048669:MAP Kinase Kinase 6 / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis*; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D020543:Proteome; D048669:MAP Kinase Kinase 6