title: High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates.
abstract: Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.
authors: Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics (MPI-MG), Ihnestr. 73, 14195 Berlin, Germany.
authors: Tanja
authors: T
authors: Feilner
authors: Claus
authors: C
authors: Hultschig
authors: Justin
authors: J
authors: Lee
authors: Svenja
authors: S
authors: Meyer
authors: Richard G H
authors: RG
authors: Immink
authors: Andrea
authors: A
authors: Koenig
authors: Alexandra
authors: A
authors: Possling
authors: Harald
authors: H
authors: Seitz
authors: Allan
authors: A
authors: Beveridge
authors: Dierk
authors: D
authors: Scheel
authors: Dolores J
authors: DJ
authors: Cahill
authors: Hans
authors: H
authors: Lehrach
authors: Jürgen
authors: J
authors: Kreutzberger
authors: Birgit
authors: B
authors: Kersten
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D019295:Computational Biology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015723:Gene Library; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D040081:Protein Array Analysis; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases