Mitogen-Activated Protein Kinase Activity and Reporter Gene Assays in Plants
Mitogen-activated protein (MAP) kinase pathways are conserved in eukaryotes and transmit a plethora of stimuli. MAP kinases (MAPKs) are part of signalling modules that consist of three to four tiers of protein kinases in a phosphorylation cascade. MAPKs a
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roduction Mitogen-activated protein (MAP) kinase signal transduction modules mediate the transfer of information from extracellular signals to cellular responses. These signalling pathways are ubiquitously found from yeast to animal and plant cells throughout eukaryotes.
N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_5, © Springer Science+Business Media, LLC 2011
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R. Dóczi et al.
MAP kinases (MAPK) cascades are composed of distinct combinations of three to four families of protein kinases: MAP kinase kinase kinase kinase (MAP4K), MAP kinase kinase kinase (MAP3K/MEKK), MAP kinase kinase (MAP2K/MEK/MKK), and MAP kinase (MAPK/MPK), which are activated in a sequential phosphorylation cascade (1, 2). The activated MAPK then phosphorylates various downstream components, including transcription factors and other kinases thereby modulating their activity, stability, or localisation. In Arabidopsis thaliana, genes encoding 20 MPKs, 10 MKKs, and 60–80 MAP3Ks and 10 MAP4Ks were identified based on the annotation of the genome sequence (3, 4). In plants, MAPK signalling pathways play important regulatory roles in several processes such as biotic and abiotic stress, as well as hormone and developmental signalling (1, 5). MAPK activities are rapidly and in most cases transiently switched on in response to specific signals, and therefore appropriate time-course experiments that typically span 0, 1, 2, 5, 10, 20, 30, and 60 min are used. Detection of MAPK activity status under various conditions is an important part of functional MAPK analysis. MAPK activity in plants is most commonly determined by measuring the phosphotransferase activity by use of radiolabelled ATP and measuring the amount of incorporated 32PO43− into a MAPK artificial substrate such as myelin basic protein (MBP) (e.g. (6–8)). To this end, MAPKs can be immunoprecipitated from crude protein extracts (immunocomplex kinase assay) or the reaction can be performed after separation of protein samples by polyacrylamide gel electrophoresis (PAGE) where the MBP substrate is incorporated into the gel (in gel kinase assay) (7). The third approach to detect MAPK activities is the use of antibodies specific to the phosphorylated form of MAPKs (9). Currently, only antibodies against the phosphorylated form of animal MAPKs are commercially available. The threonin, glutamate, tyrosine (TEY) motif that gets phosphorylated during the activation of MAPKs by MEKs is conserved and present in a number of plant MAPKs, but the surrounding amino acids are not fully conserved. Therefore, the phospho-ERK (animal extracellular (signal-)regulated kinase; one of the prominent models for MAPKs) antibodies, which are widely used against phospho-MAPKs, only weakly recognise plant MAPKs, and thus can only be used with variable success. Protoplasts are plant cells of which cell walls have been enzymatically removed. Protoplasts are responsive to external stimuli such as hormones, s
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