A phylogenetic study of the members of the MAPK and MEK families across Viridiplantae.

Protein phosphorylation is regulated by the activity of enzymes generically known as kinases. One of those kinases is Mitogen-Activated Protein Kinases (MAPK), which operate through a phosphorylation cascade conformed by members from three related protein kinase families namely MAPK kinase kinase (MEKK), MAPK kinase (MEK), and MAPK; these three acts hierarchically. Establishing the evolution of these proteins in the plant kingdom is an interesting but complicated task because the current MAPK, MAPKK, and MAPKKK subfamilies arose from duplications and subsequent sub-functionalization during the early stage of the emergence of Viridiplantae. Here, an in silico genomic analysis was performed on 18 different plant species, which resulted in the identification of 96 genes not previously annotated as components of the MAPK (70) and MEK (26) families. Interestingly, a deeper analysis of the sequences encoded by such genes revealed the existence of putative domains not previously described as signatures of MAPK and MEK kinases. Additionally, our analysis also suggests the presence of conserved activation motifs besides the canonical TEY and TDY domains, which characterize the MAPK family.

The evolutionarily conserved MAPK/Erk signaling promotes ancestral T-cell immunity in fish via c-Myc-mediated glycolysis.

The mitogen-activated protein kinase (MAPK) cascade is an ancient and evolutionarily conserved signaling pathway involved in numerous physiological processes. Despite great advances in understanding MAPK-mediated regulation of adaptive immune responses in mammals, its contribution to T-cell immunity in early vertebrates remains unclear. Herein, we used Nile tilapia (Oreochromis niloticus) to investigate the regulatory roles of MAPK/extracellular signal-regulated kinase (Erk) signaling in ancestral T-cell immunity of jawed fish. We found that Nile tilapia possesses an evolutionarily conserved MAPK/Erk axis that is activated through a classical three-tier kinase cascade, involving sequential phosphorylation of RAF proto-oncogene serine/threonine-protein kinase (Raf), MAPK/Erk kinase 1/2 (Mek1/2), and Erk1/2. In Nile tilapia, MAPK/Erk signaling participates in adaptive immune responses during bacterial infection. Upon T-cell activation, the MAPK/Erk axis is robustly activated, and MAPK/Erk blockade by specific inhibitors severely impairs T-cell activation. Furthermore, signals from MAPK/Erk were indispensable for primordial T cells to proliferate and exert their effector functions. Mechanistically, activation of the MAPK/Erk axis promoted glycolysis via induction of the transcriptional regulator proto-oncogene c-Myc (c-Myc), to ensure the proper activation and proliferation of fish T cells. Our results reveal the regulatory mechanisms of MAPK/Erk signaling in T-cell immunity in fish and highlight a close link between immune signals and metabolic programs. We propose that regulation of T-cell immunity by MAPK/Erk is a basic and sophisticated strategy that evolved before the emergence of the tetrapod lineage. These findings shed light on the evolution of the adaptive immune system.

StPBS2, a MAPK kinase gene, is involved in determining hyphal morphology, cell wall development, hypertonic stress reaction as well as the production of secondary metabolites in Northern Corn Leaf Blight pathogen Setosphaeria turcica.

Mitogen activated protein kinase kinase (MAPKK) is a crucial component in the MAPK signaling pathway. However, the functions of MAPKKs in foliar pathogens remain poorly understood. In the current study, a MAPKK gene designated as StPBS2 was cloned from Setosphaeria turcica and the functions of this gene were investigated by RNAi technology. Four independent StPBS2 gene silence transformants with different efficiencies were confirmed by real time PCR. Compared to the wild type strain (WT), these transformants showed decreased colony growth, shortened hyphae cell length, broadened cell width and an obvious reduction in conidium yield. Moreover, the cell wall of the transformants was thicker and they were also more sensitive to substances that interfere with cell wall biosynthesis than WT. Additionally, the transformants displayed higher sensitivity to hypertonic stress than WT and the sensitivity was associated with the level of silencing of StPBS2. They were also resistant to the fungicides iprodione, procymidone and fludioxonil, to which WT almost completely sensitive. The transformants produced more red secondary metabolites than WT and the production was enhanced with increasing silencing level and increased glucose content in PDA medium. Our results suggest that StPBS2 is involved in morphogenesis, condiogenesis, cell wall development, hypertonic stress reaction and resistance to fungicides, as well as in the biosynthesis of secondary metabolites in S. turcica.

Integration analysis of MKK and MAPK family members highlights potential MAPK signaling modules in cotton.

Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development, as well as their biotic and abiotic stress responses. As a nodal point of the MAPK cascade, the MKK gene family has not been systematically studied in cotton. Here, we identified 11 putative MKK genes in the Gossypium raimondii genome. Phylogenetic analysis showed that the MKKs were supported by architectures of conserved protein motifs. Expression patterns of MKKs under hormone treatments or abiotic stresses revealed their diverse functions in stress responses. Based on a yeast two hybrid, a total of 63 interactive pairs of MKKs and MAPKs were identified in cotton. Among these, 40 interactive pairs were newly identified compared to that reported previously in Arabidopsis. Integration analysis of the interaction network and expression patterns of MKK and MAPK family members revealed 13 potential MAPK signaling modules that are involved in the complicated cross-talk between hormones and abiotic stresses. Taken together, our data enhance the understanding of the evolution and function of MAPK cascades in cotton, and lay the foundation for the improvement of various defense responses that use MAPK signaling modules in the future.

Genome-wide exploration of the molecular evolution and regulatory network of mitogen-activated protein kinase cascades upon multiple stresses in Brachypodium distachyon.

BACKGROUND: Brachypodium distachyon is emerging as a widely recognized model plant that has very close relations with several economically important Poaceae species. MAPK cascade is known to be an evolutionarily conserved signaling module involved in multiple stresses. Although the gene sequences of MAPK and MAPKK family have been fully identified in B. distachyon, the information related to the upstream MAPKKK gene family especially the regulatory network among MAPKs, MAPKKs and MAPKKKs upon multiple stresses remains to be understood. RESULTS: In this study, we have identified MAPKKKs which belong to the biggest gene family of MAPK cascade kinases. We have systematically investigated the evolution of whole MAPK cascade kinase gene family in terms of gene structures, protein structural organization, chromosomal localization, orthologs construction and gene duplication analysis. Our results showed that most BdMAPK cascade kinases were located at the low-CpG-density region, and the clustered members in each group shared similar structures of the genes and proteins. Synteny analysis showed that 62 or 21 pairs of duplicated orthologs were present between B. distachyon and Oryza sativa, or between B. distachyon and Arabidopsis thaliana respectively. Gene expression data revealed that BdMAPK cascade kinases were rapidly regulated by stresses and phytohormones. Importantly, we have constructed a regulation network based on co-expression patterns of the expression profiles upon multiple stresses performed in this study. CONCLUSIONS: BdMAPK cascade kinases were involved in the signaling pathways of multiple stresses in B. distachyon. The network of co-expression regulation showed the most of duplicated BdMAPK cascade kinase gene orthologs demonstrated their convergent function, whereas few of them developed divergent function in the evolutionary process. The molecular evolution analysis of identified MAPK family genes and the constructed MAPK cascade regulation network under multiple stresses provide valuable information for further investigation of the functions of BdMAPK cascade kinase genes.

ZmMKK1, a novel group A mitogen-activated protein kinase kinase gene in maize, conferred chilling stress tolerance and was involved in pathogen defense in transgenic tobacco.

As an important intracellular signaling module, the mitogen-activated protein kinase (MAPK) cascades have been previously implicated in signal transduction during plants responsing to various environmental stresses as well as pathogen attack. The mitogen-activated protein kinase kinase acts as the convergent point of MAPK cascades during a variety of stress signaling. In this study, a novel MAPKK gene, ZmMKK1, in maize (Zea mays L.) belonging to group A MAPKK was isolated and functionally characterized. ZmMKK1 was mainly localized in the cytoplasm and its constitutive kinase-active form ZmMKK1DD was localized in both cytoplasm and nucleus. QRT-PCR analysis uncovered that ZmMKK1 expression was triggered by abiotic and biotic stresses and exogenous signaling molecules. Moreover, hydrogen peroxide (H2O2) and Ca(2+) mediated 12°C-induced up-regulated expressing of ZmMKK1 at mRNA level. Ectopic expression of ZmMKK1 in tobacco (Nicotiana tabacum) conferred tolerance to chilling stress by higher antioxidant enzyme activities, more accumulation of osmoregulatory substances and more significantly up-expression of ROS-related and stress-responsive genes compared with empty vector control plants. Furthermore, ZmMKK1 played differential functions in biotrophic versus necrotrophic pathogen-induced responses. These results suggested ZmMKK1 played a crucial role in chilling stress and pathogen defense in plants.

Identification of mitogen-activated protein kinase kinase gene family and MKK-MAPK interaction network in maize.

Plant mitogen-activated protein kinases (MAPK) are involved in important processes, including stress signaling and development. MAPK kinases (MAPKK, MKK) have been investigated in several plant species including Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, and Brachypodium distachyon. In the present study, nine putative maize MKK genes have been identified. Analysis of the conserved protein motifs, exon-intron junctions and intron phase has revealed high levels of conservation within the phylogenetic groups. Next, we defined four new ZmMKK-ZmMPK interactions using yeast two-hybrid. Finally, we examined the biological functions of the ZmMKK4 gene. Overexpression of ZmMKK4 in Arabidopsis conferred tolerance to oxidative stress by increased germination rate and early seedling growth compared with WT plants. Taken together, we provide a comprehensive bioinformatics analysis of the MKK gene family in maize genome and our data provide an important foundation for further functional study of MAPK and MKK families in maize.

Detection and quantification of anthrax lethal factor in serum by mass spectrometry.

The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.

Ancient signals: comparative genomics of plant MAPK and MAPKK gene families.

MAPK signal transduction modules play crucial roles in regulating many biological processes in plants, and their components are encoded by highly conserved genes. The recent availability of genome sequences for rice and poplar now makes it possible to examine how well the previously described Arabidopsis MAPK and MAPKK gene family structures represent the broader evolutionary situation in plants, and analysis of gene expression data for MPK and MKK genes in all three species allows further refinement of those families, based on functionality. The Arabidopsis MAPK nomenclature appears sufficiently robust to allow it to be usefully extended to other well-characterized plant systems.

LmxPK4, a mitogen-activated protein kinase kinase homologue of Leishmania mexicana with a potential role in parasite differentiation.

Members of the mitogen-activated protein (MAP) kinase cascade are important for the establishment of a Leishmania mexicana infection and are involved in flagellar length control, although the underlying molecular mechanisms remain to be elucidated. This study reports the cloning and characterization of LmxPK4, a MAP kinase kinase homologue of L. mexicana displaying putative plant-like regulatory phosphorylation sites. The recombinant protein has autophosphorylating activity and phosphorylates myelin basic protein. An LmxPK4 gene deletion mutant showed a proliferation defect after infection of macrophages and no or delayed lesion development in mice. Irrespective of the onset of lesion development parasites showed an early and homogeneous lesion development in re-infection experiments. This is indicative for a compensation of the null mutant phenotype. Additionally, this phenotype could be reverted by reintroduction of the wild-type gene into the deletion background. Mutants expressing loss-of-function or N-terminally truncated versions of LmxPK4 retained the null mutant phenotype. LmxPK4 is stage-specifically expressed in promastigotes and during differentiation to amastigotes, but is not detectable in amastigotes isolated from the mammalian host. Moreover, its in vitro kinase activity increases with temperature rise up to 40 degrees C. Our results suggest that LmxPK4 is involved in the differentiation process and affects virulence of Leishmania mexicana.

Mitogen-activated protein kinases and their role in regulation of cellular processes.

Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes connecting cell-surface receptors to critical regulatory targets within cells. The three major MAPK cascades are known, the extracellular signal-regulated protein kinase (ERK) cascade, c-Jun amino-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) cascade and p38-MAPK cascade. This paper is focused on characterization of these MAPK cascades in terms of their distribution and biological role in some pathological processes (apoptosis, hypertrophy) with a special orientation on the role of MAPKs in cardiovascular system during ischemia/reperfusion.