title: Kinase-Associated Phosphoisoform Assay: a novel candidate-based method to detect specific kinase-substrate phosphorylation interactions in vivo.
abstract: BACKGROUND
  Protein kinases are important components of signalling pathways, and kinomes have remarkably expanded in plants. Yet, our knowledge of kinase substrates in plants is scarce, partly because tools to analyse protein phosphorylation dynamically are limited. Here we describe Kinase-Associated Phosphoisoform Assay, a flexible experimental method for directed experiments to study specific kinase-substrate interactions in vivo. The concept is based on the differential phosphoisoform distribution of candidate substrates transiently expressed with or without co-expression of activated kinases. Phosphorylation status of epitope-tagged proteins is subsequently detected by high-resolution capillary isoelectric focusing coupled with nanofluidic immunoassay, which is capable of detecting subtle changes in isoform distribution.
  
  
  RESULTS
  The concept is validated by showing phosphorylation of the known mitogen-activated protein kinase (MAPK) substrate, ACS6, by MPK6. Next, we demonstrate that two transcription factors, WUS and AP2, both of which are shown to be master regulators of plant development by extensive genetic studies, exist in multiple isoforms in plant cells and are phosphorylated by activated MAPKs.
  
  
  CONCLUSIONS
  As plant development flexibly responds to environmental conditions, phosphorylation of developmental regulators by environmentally-activated kinases may participate in linking external cues to developmental regulation. As a counterpart of advances in unbiased screening methods to identify potential protein kinase substrates, such as phosphoproteomics and computational predictions, our results expand the candidate-based experimental toolkit for kinase research and provide an alternative in vivo approach to existing in vitro methodologies.
authors.affiliation: Department of Plant Cell Biology, Centre for Agricultural Research of the Hungarian Academy of Sciences, H-2462, Brunszvik u. 2, Martonvásár, Hungary.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Dory
authors.affiliation: Department of Pathogenetics, National Institute of Oncology, H-1122, Ráth György u. 7-9, Budapest, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Doleschall
authors.affiliation: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, H-1094, Tűzoltó u. 37-47, Budapest, Hungary.
authors.forename: Szilvia K
authors.initials: SK
authors.lastname: Nagy
authors.affiliation: Department of Plant Cell Biology, Centre for Agricultural Research of the Hungarian Academy of Sciences, H-2462, Brunszvik u. 2, Martonvásár, Hungary.
authors.forename: Helga
authors.initials: H
authors.lastname: Ambrus
authors.affiliation: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, H-1094, Tűzoltó u. 37-47, Budapest, Hungary.|Research Group for Technical Analytical Chemistry, Hungarian Academy of Sciences - Budapest University of Technology and Economics, H-1111, Szt. Gellért tér 4, Budapest, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Mészáros
authors.affiliation: Department of Plant Cell Biology, Centre for Agricultural Research of the Hungarian Academy of Sciences, H-2462, Brunszvik u. 2, Martonvásár, Hungary.
authors.forename: Beáta
authors.initials: B
authors.lastname: Barnabás
authors.affiliation: Department of Plant Cell Biology, Centre for Agricultural Research of the Hungarian Academy of Sciences, H-2462, Brunszvik u. 2, Martonvásár, Hungary. doczi.robert@agrar.mta.hu.
authors.forename: Róbert
authors.initials: R
authors.lastname: Dóczi
keywords: APETALA 2; Arabidopsis thaliana; Capillary isoelectric focusing; Nanofluidic immunoassay; Phosphorylation assay; Protein kinase; Protoplast transfection; Signal transduction; WUSCHEL