Cellular reprogramming through mitogen-activated protein kinases

Abstract

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Keywords: MAPK substrates; chemical defense; metabolome; phosphoproteome; phosphorylation.

Figure 1

MAMP (microbe-associated molecular pattern)-induced cellular signaling. (A) Schematic representation of the MAMP-induced cellular signaling pathway in plant cells, using flg22 peptide as an example of a classical MAMP. See introduction text for description. Highlighted in red are the events generated by in vivo activation of the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, by transgenic expression of a constitutively active MAPK kinase (MKK5-CA). (B) Western blot analysis for detecting activated forms of MAPKs in Arabidopsis protoplasts after flg22 treatment or transfection of c-myc-tagged MKK5-CA construct. The identities of the MAPK bands are indicated on the right. Protoplast transfection and western blotting to identify specific activated MAPKs was performed as described (Ranf et al., ; Eschen-Lippold et al., ; Lassowskat et al., 2014). Abbreviations used: FLS2, flg22 receptor; rbohD, NADPH oxidase responsible for flg22-induced reactive oxygen species (ROS) production; CDPKs, calcium-dependent protein kinases; CBL, calcineurin-B-like protein; CIPK, CBL-interacting protein kinase; CaM, calmodulin.

Figure 2

Protein interaction network based on 538 putative MPK3/MPK6 substrates and downstream phosphoproteins. The protein network was plotted with STRING 10.0 (high confidence score 0.9, confidence view). Each node represents a protein labeled by its protein name abbreviation or the Arabidopsis locus identifier. Protein nodes without edges are masked and not displayed. The main protein clusters relevant for the four presented Key Concepts are color-coded. Red = (Key Concept 1: Transcriptional, post-transcriptional, translational, and post-translational control); orange = (Key Concept 2: Cellular re-compartmentalization); green (Key Concept 3: Chemical defense); and light blue = (Key Concept 4: Interplay with downstream cellular signaling). Additionally, dark blue = proteins for energy or precursor supply that support processes of one of the other key concepts.