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 mitogenactivated 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.

MAPK cascades and major abiotic stresses

“…In a highthroughput phosphorylation approach, 48 potential substrates of MPK3 and 39 of MPK6 were identified (Feilner et al 2005). However, in vivo characterization will be required to confirm the potential interaction between these MAP kinases and their corresponding partners.…”

Mitogen-Activated Protein Kinases and Reactive Oxygen Species Signaling in Plants

“…Congruent with these studies, expression of constitutively active Arabidopsis MKK4 or MKK5, the orthologs of tobacco MEK2 (and activators of MPK3/6), resulted in generation of H 2 O 2 and cell death (Ren et al, 2002); protein kinase inhibitors blocked elicitininduced cell death, oxidative burst, and expression of defense genes in tobacco (Sasabe et al, 2000). Considering that two thioredoxins as well as an ascorbate oxidase-like protein were recently identified as potential substrates for MPK3 and MPK6 (Feilner et al, 2005), other scenarios for regulating ROS levels via MAPKs are conceivable. Manipulation of the ratio of ascorbate to dehydroascorbate or reduced to oxidized thioredoxin has an enormous impact.…”

MYB75 Phosphorylation by MPK4 Is Required for Light-Induced Anthocyanin Accumulation in Arabidopsis

“…However, phosphorylation-mediated activation of MAPKs is generally transient, as is their interaction with downstream substrate proteins. This makes it difficult to identify MAPK substrates by genome wide proteomic or yeast two-hybrid approaches (Feilner et al, 2005). To circumvent this difficulty, we exploited a recently described, constitutively active MPK4 form (CA-MPK4), which results from two mutations (D198G/E202A) and retains its substrate specificity and physiological functions (Berriri et al, 2012).…”