High-throughput identification of potential Arabidopsis MAP kinases substrates

T. Feilner, C. Hultschig, J. Lee, S. Meyer, R.G.H. Immink, A. Koenig, A. Possling, H. Seitz, A. Beveridge, D. Scheel, D.J. Cahill, H. Lehrach, J. Kreutzberger, B. Kersten

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    201 Citations (Scopus)

    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 to identify potential substrates for Arabidopsis thaliana mitogen-activated protein kinases 3 (MPK3) and 6 (MPK6), which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomics 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 MPKs 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 (ACS-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 allows to shortlist candidate substrates of MAP kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance
    Original languageEnglish
    Pages (from-to)1558-1568
    JournalMolecular and Cellular Proteomics
    Volume4
    Issue number10
    DOIs
    Publication statusPublished - 2005

    Keywords

    • unnatural nucleotide specificity
    • antibody microarray technology
    • transcriptional regulation
    • fluorescence detection
    • innate immunity
    • phosphorylation
    • expression
    • stress
    • chips
    • scale

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