Kinase-Associated Phosphoisoform Assay: a novel candidate-based method to detect specific kinase-substrate phosphorylation interactions in vivo

BMC Plant Biology

Methodology Article

Szilvia K. Nagy¹  Tamás Mészáros²  Zoltán Doleschall³  Magdalena Dory⁴  Beáta Barnabás⁴  Róbert Dóczi⁴  Helga Ambrus⁴ 

[1] Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, H-1094, Tűzoltó u. 37-47, Budapest, Hungary;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;Department of Pathogenetics, National Institute of Oncology, H-1122, Ráth György u. 7-9, Budapest, Hungary;Department of Plant Cell Biology, Centre for Agricultural Research of the Hungarian Academy of Sciences, H-2462, Brunszvik u. 2, Martonvásár, Hungary;

关键词: Protein kinase;    Phosphorylation assay;    Signal transduction;    Protoplast transfection;    Capillary isoelectric focusing;    Nanofluidic immunoassay;    APETALA 2;    WUSCHEL;    Arabidopsis thaliana;   

DOI  :  10.1186/s12870-016-0894-1

 received in 2016-05-12, accepted in 2016-09-12,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundProtein 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.ResultsThe 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.ConclusionAs 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.

【 授权许可】

CC BY   
© The Author(s). 2016