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.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1452-1) contains supplementary material, which is available to authorized users.     

Activation of neutral-sphingomyelinase,MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester–induced apoptosis in C6 glioma cells

Background

Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells.

Results

C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869.

Conclusions

Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.     

Berberine chloride mediates its anti-leishmanial activity via differential regulation of the mitogen activated protein kinase pathway in macrophages

Background

A complex interplay between Leishmania and macrophages influences parasite survival and necessitates disruption of signaling molecules, eventually resulting in impairment of macrophage function. In this study, we demonstrate the immunomodulatory activity of Berberine chloride in Leishmania infected macrophages.

Principal Findings

The IC₅₀ of Berberine chloride, a quaternary isoquinoline alkaloid was tested in an amastigote macrophage model and its safety index measured by a cell viability assay. It eliminated intracellular amastigotes, the IC₅₀ being 2.8 fold lower than its IC₅₀ in promastigotes (7.10 µM vs. 2.54 µM) and showed a safety index >16. Levels of intracellular and extracellular nitric oxide (NO) as measured by flow cytometry and Griess assay respectively showed that Berberine chloride in Leishmania infected macrophages increased production of NO. Measurement of the mRNA expression of iNOS, IL-12 and IL-10 by RT-PCR along with levels of IL-12p40 and IL-10 by ELISA showed that in infected macrophages, Berberine chloride enhanced expression of iNOS and IL-12p40, concomitant with a downregulation of IL-10. The phosphorylation status of extracellular signal related kinase (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK) was studied by western blotting. In infected macrophages, Berberine chloride caused a time dependent activation of p38 MAPK along with deactivation of ERK1/2; addition of a p38 MAPK inhibitor SB203580 inhibited the increased generation of NO and IL-12p40 by Berberine chloride as also prevented its decrease of IL-10.

Conclusions

Berberine chloride modulated macrophage effector responses via the mitogen activated protein kinase (MAPK) pathway, highlighting the importance of MAPKs as an antiparasite target.     

Ethanol Impairs Intestinal Barrier Function in Humans through Mitogen Activated Protein Kinase Signaling: A Combined In Vivo and In Vitro Approach

Background

Ethanol-induced gut barrier disruption is associated with several gastrointestinal and liver disorders.

Aim

Since human data on effects of moderate ethanol consumption on intestinal barrier integrity and involved mechanisms are limited, the objectives of this study were to investigate effects of a single moderate ethanol dose on small and large intestinal permeability and to explore the role of mitogen activated protein kinase (MAPK) pathway as a primary signaling mechanism.

Methods

Intestinal permeability was assessed in 12 healthy volunteers after intraduodenal administration of either placebo or 20 g ethanol in a randomised cross-over trial. Localization of the tight junction (TJ) and gene expression, phosphorylation of the MAPK isoforms p38, ERK and JNK as indicative of activation were analyzed in duodenal biopsies. The role of MAPK was further examined in vitro using Caco-2 monolayers.

Results

Ethanol increased small and large intestinal permeability, paralleled by redistribution of ZO-1 and occludin, down-regulation of ZO-1 and up-regulation of myosin light chain kinase (MLCK) mRNA expression, and increased MAPK isoforms phosphorylation. In Caco-2 monolayers, ethanol increased permeability, induced redistribution of the junctional proteins and F-actin, and MAPK and MLCK activation, as indicated by phosphorylation of MAPK isoforms and myosin light chain (MLC), respectively, which could be reversed by pretreatment with either MAPK inhibitors or the anti-oxidant L-cysteine.

Conclusions

Administration of moderate ethanol dosage can increase both small and colon permeability. Furthermore, the data indicate a pivotal role for MAPK and its crosstalk with MLCK in ethanol-induced intestinal barrier disruption.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00928733","term_id":"NCT00928733"}}NCT00928733     

ERK1/2 and p38 MAPKs Are Complementarily Involved in Estradiol 17?-d-Glucuronide-Induced Cholestasis: Crosstalk with cPKC and PI3K

Objective

The endogenous, cholestatic metabolite estradiol 17ß-d-glucuronide (E₂17G) induces endocytic internalization of the canalicular transporters relevant to bile formation, Bsep and Mrp2. We evaluated here whether MAPKs are involved in this effect.

Design

ERK1/2, JNK1/2, and p38 MAPK activation was assessed by the increase in their phosphorylation status. Hepatocanalicular function was evaluated in isolated rat hepatocyte couplets (IRHCs) by quantifying the apical secretion of fluorescent Bsep and Mrp2 substrates, and in isolated, perfused rat livers (IPRLs), using taurocholate and 2,4-dinitrophenyl-S-glutathione, respectively. Protein kinase participation in E₂17G-induced secretory failure was assessed by co-administering selective inhibitors. Internalization of Bsep/Mrp2 was assessed by confocal microscopy and image analysis.

Results

E₂17G activated all kinds of MAPKs. The PI3K inhibitor wortmannin prevented ERK1/2 activation, whereas the cPKC inhibitor Gö6976 prevented p38 activation, suggesting that ERK1/2 and p38 are downstream of PI3K and cPKC, respectively. The p38 inhibitor SB203580 and the ERK1/2 inhibitor PD98059, but not the JNK1/2 inhibitor SP600125, partially prevented E₂17G-induced changes in transporter activity and localization in IRHCs. p38 and ERK1/2 co-inhibition resulted in additive protection, suggesting complementary involvement of these MAPKs. In IPRLs, E₂17G induced endocytosis of canalicular transporters and a rapid and sustained decrease in bile flow and biliary excretion of Bsep/Mrp2 substrates. p38 inhibition prevented this initial decay, and the internalization of Bsep/Mrp2. Contrarily, ERK1/2 inhibition accelerated the recovery of biliary secretion and the canalicular reinsertion of Bsep/Mrp2.

Conclusions

cPKC/p38 MAPK and PI3K/ERK1/2 signalling pathways participate complementarily in E₂17G-induced cholestasis, through internalization and sustained intracellular retention of canalicular transporters, respectively.     

An Inhibition of p38 Mitogen Activated Protein Kinase Delays the Platelet Storage Lesion

Background and Objectives

Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK). Another MAPK, extracellular signal-related kinase (ERK), is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors.

Materials and Methods

A single Trima apheresis platelet unit (n = 12) was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20–24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters.

Results

Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties.

Conclusion

Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.     

Effects of thioglycolic acid on parthenogenetic activation of Xenopus oocytes

Background

Existing in Permanent-wave solutions (PWS), thioglycolic acid (TGA) is widely used in hairdressing industry for its contribution to hair styling. However, the toxicity of TGA, especially its reproductive toxicity, gradually calls the attention of more and more researchers.

Method

In this work, xenopus oocytes were pretreated with different concentration of TGA, and then activated by calcium ionophore A23187. During culture, the oocytes activation rates were taken note at different time after adding calcium ionophore A23187. At the end of the culture period, the nuclear status was detected under confocal microscope. In addition, some other samples were collected for Western-Blotting analysis.

Result

TGA significantly inhibited the oocytes activation rate and pronuclear formation. It may be resulted from the inhibition of the degradation of p-ERK1, Mos and CyclinB2.

Conclusion

TGA inhibits in vitro parthenogenetic activation of xenopus oocytes with inhibited the degradation of proteins involved in mitogenic-activated protein kinase (MAPK) and maturation-promoting factor (MPF) pathways.     

Dexamethasone Inhibits Repair of Human Airway Epithelial Cells Mediated by Glucocorticoid-Induced Leucine Zipper (GILZ)

Background

Glucocorticoids (GCs) are a first-line treatment for asthma for their anti-inflammatory effects, but they also hinder the repair of airway epithelial injury. The anti-inflammatory protein GC-induced leucine zipper (GILZ) is reported to inhibit the activation of the mitogen-activated protein kinase (MAPK)-extracellular-signal-regulated kinase (ERK) signaling pathway, which promotes the repair of airway epithelial cells around the damaged areas. We investigated whether the inhibition of airway epithelial repair imposed by the GC dexamethasone (DEX) is mediated by GILZ.

Methods

We tested the effect of DEX on the expressions of GILZ mRNA and GILZ protein and the MAPK-ERK signaling pathway in human airway epithelial cells, via RT-PCR and Western blot. We further evaluated the role of GILZ in mediating the effect of DEX on the MAPK-ERK signaling pathway and in airway epithelium repair by utilizing small-interfering RNAs, MTT, CFSE labeling, wound-healing and cell migration assays.

Results

DEX increased GILZ mRNA and GILZ protein levels in a human airway epithelial cell line. Furthermore, DEX inhibited the phosphorylation of Raf-1, Mek1/2, Erk1/2 (components of the MAPK-ERK signaling pathway), proliferation and migration. However, the inhibitory effect of DEX was mitigated in cells when the GILZ gene was silenced.

Conclusions

The inhibition of epithelial injury repair by DEX is mediated in part by activation of GILZ, which suppressed activation of the MAPK-ERK signaling pathway, proliferation and migration. Our study implicates the involvement of DEX in this process, and furthers our understanding of the dual role of GCs.     

Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

Background

We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains.

Objective

The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of immunobiotics by evaluating the role of TLR2 and TLR negative regulators in the modulation of proinflammatory cytokine production and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways in PIE cells.

Results

Bifidobacteria longum BB536 and B. breve M-16V strains significantly downregulated levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and IL-6 in PIE cells challenged with heat-killed enterotoxigenic Escherichia coli. Moreover, BB536 and M-16V strains attenuated the proinflammatory response by modulating the NF-κB and MAPK pathways. In addition, our findings provide evidence for a key role for the ubiquitin-editing enzyme A20 in the anti-inflammatory effect of immunobiotic bifidobacteria in PIE cells.

Conclusions

We show new data regarding the mechanism involved in the anti-inflammatory effect of immunobiotics. Several strains with immunoregulatory capabilities used a common mechanism to induce tolerance in PIE cells. Immunoregulatory strains interacted with TLR2, upregulated the expression of A20 in PIE cells, and beneficially modulated the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of proinflammatory cytokines. We also show that the combination of TLR2 activation and A20 induction can be used as biomarkers to screen and select potential immunoregulatory bifidobacteria strains.     

Plk1 regulates MEK1/2 and proliferation in airway smooth muscle cells

Background

Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that has been implicated in the regulation of mitosis. In addition, the activation of mitogen-activated protein kinase (MAPK) is a key event in the early stage of the growth factor response. The role of Plk1 in MAPK phosphorylation in cells has not been investigated.

Methods

Immunoblot analysis was used to evaluate Plk1 and MAPK phosphorylation in cells upon stimulation with platelet-derived growth factor (PDGF). We also generated stable Plk1 knockdown (KD) cells to assess the role of Plk1 in MAPK activation and cell proliferation. Furthermore, we used a non-phosphorylatable Plk1 mutant to determine the function of Plk1 phosphorylation in these processes.

Results

Treatment with PDGF increased Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in human airway smooth muscle cells. Plk1 KD attenuated the PDGF-induced phosphorylation of MEK1/2 and ERK1/2 as well as cell proliferation. However, phosphorylation of Raf-1 and AKT upon stimulation with PDGF was not reduced in Plk1 KD cells. Furthermore, the expression of T210A Plk1 (alanine substitution at Thr-210) inhibited the PDGF-stimulated MEK1/2 phosphorylation, ERK1/2 phosphorylation and cell proliferation.

Conclusions

Together, these findings suggest that Plk1 is activated upon growth factor stimulation, which may control the activation of MEK1/2 and ERK1/2, and smooth muscle cell proliferation.     

A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

Background

RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation.

Methodology/Principal Findings

Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment.

Conclusions/Significance

A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.     

Candida albicans induces cyclo-oxygenase 2 expression and prostaglandin E2 production in synovial fibroblasts through an extracellular-regulated kinase 1/2 dependent pathway

Introduction

Synovial cells are potential sources of inflammatory mediators in bacterial-induced arthritis but their involvement in the inflammatory response to Candida albicans-induced septic arthritis is largely unknown.

Methods

Primary cultures of rat synovial fibroblasts were infected with C. albicans (ATCC90028). Immunocytochemistry, western blotting, and RT-PCR were performed to assess cyclo-oxygenase 2 induction. Phosphorylation of extracellular-regulated kinase (ERK1/2) following infection in the absence or presence of U0126 was assessed by western blotting whilst prostaglandin E2 production was measured by ELISA. Nuclear factor κB (NFκB) translocation was evaluated by an electrophoretic mobility shift assay.

Results

Infection of synovial fibroblasts with C. albicans resulted in cyclo-oxygenase 2 expression and prostaglandin E2 production. Cyclo-oxygenase 2 expression and prostaglandin E2 production was dependent upon extracellular-regulated kinase 1/2 phosphorylation, associated with activation of NFκB and significantly elevated in the presence of laminarin, an inhibitor of dectin-1 activity. Synovial fibroblasts adjacent to C. albicans hyphae aggregates appeared to be the major contributors to the increased levels of cyclo-oxygenase 2 and phosphorylated extracellular-regulated kinase 1/2.

Conclusions

C. albicans infection of synovial fibroblasts in vitro results in upregulation of cyclo-oxygenase 2 and prostaglandin E2 by mechanisms that may involve activation of extracellular-regulated kinase 1/2 and are associated with NFκB activation.     

Docking of PRAK/MK5 to the Atypical MAPKs ERK3 and ERK4 Defines a Novel MAPK Interaction Motif

ERK3 and ERK4 are atypical MAPKs in which the canonical TXY motif within the activation loop of the classical MAPKs is replaced by SEG. Both ERK3 and ERK4 bind, translocate, and activate the MAPK-activated protein kinase (MK) 5. The classical MAPKs ERK1/2 and p38 interact with downstream MKs (RSK1–3 and MK2–3, respectively) through conserved clusters of acidic amino acids, which constitute the common docking (CD) domain. In contrast to the classical MAPKs, the interaction between ERK3/4 and MK5 is strictly dependent on phosphorylation of the SEG motif of these kinases. Here we report that the conserved CD domain is dispensable for the interaction of ERK3 and ERK4 with MK5. Using peptide overlay assays, we have defined a novel MK5 interaction motif (FRIEDE) within both ERK4 and ERK3 that is essential for binding to the C-terminal region of MK5. This motif is located within the L16 extension lying C-terminal to the CD domain in ERK3 and ERK4 and a single isoleucine to lysine substitution in FRIEDE totally abrogates binding, activation, and translocation of MK5 by both ERK3 and ERK4. These findings are the first to demonstrate binding of a physiological substrate via this region of the L16 loop in a MAPK. Furthermore, the link between activation loop phosphorylation and accessibility of the FRIEDE interaction motif suggests a switch mechanism for these atypical MAPKs in which the phosphorylation status of the activation loop regulates the ability of both ERK3 and ERK4 to bind to a downstream effector.Mitogen-activated protein kinase (MAPK)² phosphorylation cascades play important roles in the regulation of diverse cellular functions such as cell proliferation, differentiation, migration, and apoptosis (1, 2). A characteristic and conserved feature of this family of signaling pathways is their organization into modules comprising a sequential three-tiered kinase cascade. This contains a MAPK kinase kinase, a MEK, and the MAPK itself. Four such MAPK signaling modules have been described in mammals: ERK1 and ERK2, the c-Jun N-terminal kinases 1–3, the p38 kinases (p38α/β/γ/δ), and ERK5 (3–7). The MAPK kinase kinases phosphorylate and activate the MEKs, which in turn activate the MAPKs by dual phosphorylation on both the threonine and the tyrosine residue of a highly conserved TXY motif in the kinase activation loop. MAPKs are Ser/Thr kinases, which phosphorylate a wide range of substrates with the minimal consensus sequence (S/T)P (2).ERK4 and its close relative ERK3 are regarded as atypical members of the MAPK family. In contrast to the classical MAPKs, ERK3 and ERK4 harbor an SEG motif in the activation loop and thus lack a second phosphoacceptor site. In addition, protein kinases all possess a conserved APE motif located just C-terminal to the phosphoacceptor sites within subdomain VIII, in which the conserved glutamate is important for maintaining the stability of the kinase domain. In both ERK3 and ERK4, this motif is substituted by SPR, and ERK3 and ERK4 are the only two protein kinases in the human genome with an arginine residue in this position (8). Although they display significant sequence homology (44% identity) with ERK1 and ERK2 within their kinase domains, both ERK3 and ERK4 have unique C-terminal extensions, which account for the large differences in size observed between ERK1/2 (∼360 amino acids) and ERK3/ERK4 (721/587 amino acids). Whereas classical MAPKs have been highly conserved throughout evolution, with examples found in both unicellular and multicellular organisms, ERK3 and ERK4 are only present in vertebrates. Finally, in contrast to many of the classical MAPKs, the regulation, substrate specificity, and physiological functions of ERK3 and ERK4 are poorly understood. Although ERK3 and ERK4 are very similar to each other, there are significant differences between them. For instance, whereas ERK4, like most classical MAPKs, is a stable protein, ERK3 is highly unstable and subject to rapid proteosomal degradation. Thus, ERK3 activity may be regulated at the level of cellular abundance, and taken together these features indicate that ERK3 and ERK4 may perform specialized functions and enjoy different modes of regulation when compared with classical MAPKs (9–11).Despite the striking differences between ERK3 and ERK4 and the classical MAPKs, they do share one property with the ERK1/2, p38, and ERK5, namely the ability to interact with a group of downstream Ser/Thr protein kinases, termed MAPK-activated protein kinases (MAPKAPKs or MKs) (12, 13). In the case of ERK3 and ERK4, both proteins interact with, translocate, and activate the MK5 protein kinase. Several studies have drawn attention to the role of specific docking interactions that contribute to both substrate selectivity and regulation in MAPK pathways (14–17). These interactions involve docking domains, which specifically recognize small peptide docking motifs (D motifs) located on functional MAPK partner proteins including downstream substrates, scaffold proteins, as well as positive and negative regulators. The docking domains, although located within the kinase domains, are distinct from the active site. Similarly the D motifs, which these docking domains recognize, are also distinct from the phosphoacceptor sites within protein substrates (18). There are several classes of D motifs. The motifs found in MAPKAP kinases including MK5 have the consensus sequence LX_1–2(K/R)_2–5 where X is any amino acid (12). The corresponding docking domains within the MAPKs have also been characterized (16, 19, 20). The common docking (CD) domain is a cluster of negatively charged amino acids located in the L16 extension directly C-terminal to the kinase domain in the MAPK primary structure. A second domain termed ED (Glu-Asp) also contributes to binding specificity. This latter site is located near the CD domain in the MAPK tertiary structure. Whereas the CD domain is considered commonly important for all docking interactions, the ED site is thought to be important for the determination of specificity (16). Other residues and regions distinct from the ED and CD domains have also been shown to be important for docking.(21–25).This work has so far been largely confined to analysis of the classical MAPKs, and much less is known about the nature of substrate or regulatory docking interactions for the atypical MAPKs. We and others (9, 11, 26) have recently reported that the region encompassing residues 326–340 within both ERK3 and ERK4 is required for their ability to interact with and activate MK5. Furthermore, a truncated mutant of MK5, which lacks the 50 C-terminal residues (MK5 1–423), was unable to bind to ERK4 despite the fact that it retains its D domain. Finally, in contrast to conventional MAPKs, the interaction between ERK3 and ERK4 and MK5 requires activation loop phosphorylation of ERK3 and ERK4 (27, 28). Taken together these observations suggest that the mechanism by which the atypical MAPKs recognize and bind to at least one important class of effector kinases may be distinct to that found in the classical MAPKs such as ERK1/2 and p38.Here we demonstrate that two separate C-terminal regions, encompassing residues 383–393 and 460–465, respectively, are necessary for MK5 to interact with both ERK3 and ERK4. These regions are distinct from the D motif previously identified within MK5, suggesting that binding to ERK3 and ERK4 may be mediated by a different mechanism to that seen in the classical MAPKs. In support of this, the conserved CD domains within ERK3 and ERK4 are shown to be completely dispensable for MK5 interaction. Using peptide overlay assays, we have defined a minimal MK5 interaction motif FRIEDE in ERK4. Furthermore, we demonstrate that a single point mutation (ERK3 I334K or ERK4 I330K) within this FRIEDE motif is sufficient to disrupt the binding of both ERK3 and ERK4 to MK5 and consequently their ability to both translocate and activate MK5. The FRIEDE motif is located within the L16 extension C-terminal to the CD domain in both ERK3 and ERK4. Interestingly, molecular modeling of the corresponding region in ERK2 suggests that it undergoes a significant conformational change as a result of activation loop phosphorylation, making this part of the L16 extension more accessible (29). We propose that the FRIEDE motif represents a novel MAPK interaction motif, the function of which is linked to activation loop phosphorylation and MAPK activation.     

Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia

Background

T cells play a dominant role in the pathogenesis of asthma. Costimulation of T cells is necessary to fully activate them. An inducible costimulator (ICOS) of T cells is predominantly expressed on Th2 cells. Therefore, interference of signaling pathways precipitated by ICOS may present new therapeutic options for Th2 dominated diseases such as asthma. However, these signaling pathways are poorly characterized in vitro and in vivo.

Methods

Human primary CD4⁺ T cells from blood were activated by beads with defined combinations of surface receptor stimulating antibodies and costimulatory receptor ligands. Real-time RT-PCR was used for measuring the production of cytokines from activated T cells. Activation of mitogen activated protein kinase (MAPK) signaling pathways leading to cytokine synthesis were investigated by western blot analysis and by specific inhibitors. The effect of inhibitors in vivo was tested in a murine asthma model of late phase eosinophilia. Lung inflammation was assessed by differential cell count of the bronchoalveolar lavage, determination of serum IgE and lung histology.

Results

We showed in vitro that ICOS and CD28 are stimulatory members of an expanding family of co-receptors, whereas PD1 ligands failed to co-stimulate T cells. ICOS and CD28 activated different MAPK signaling cascades necessary for cytokine activation. By means of specific inhibitors we showed that p38 and ERK act downstream of CD28 and that ERK and JNK act downstream of ICOS leading to the induction of various T cell derived cytokines. Using a murine asthma model of late phase eosinophilia, we demonstrated that the ERK inhibitor U0126 and the JNK inhibitor SP600125 inhibited lung inflammation in vivo. This inhibition correlated with the inhibition of Th2 cytokines in the BAL fluid. Despite acting on different signaling cascades, we could not detect synergistic action of any combination of MAPK inhibitors. In contrast, we found that the p38 inhibitor SB203580 antagonizes the action of the ERK inhibitor U0126 in vitro and in vivo.

Conclusion

These results demonstrate that the MAPKs ERK and JNK may be suitable targets for anti-inflammatory therapy of asthma, whereas inhibition of p38 seems to be an unlikely target.     

A Non-Redundant Role for Drosophila Mkk4 and Hemipterous/Mkk7 in TAK1-Mediated Activation of JNK

Background

The JNK pathway is a mitogen-activated protein (MAP) kinase pathway involved in the regulation of numerous physiological processes during development and in response to environmental stress. JNK activity is controlled by two MAPK kinases (MAPKK), Mkk4 and Mkk7. Mkk7 plays a prominent role upon Tumor Necrosis Factor (TNF) stimulation. Eiger, the unique TNF-superfamily ligand in Drosophila, potently activates JNK signaling through the activation of the MAPKKK Tak1.

Methodology/Principal Findings

In a dominant suppressor screen for new components of the Eiger/JNK-pathway in Drosophila, we have identified an allelic series of the Mkk4 gene. Our genetic and biochemical results demonstrate that Mkk4 is dispensable for normal development and host resistance to systemic bacterial infection but plays a non-redundant role as a MAPKK acting in parallel to Hemipterous/Mkk7 in dTAK1-mediated JNK activation upon Eiger and Imd pathway activation.

Conclusions/Significance

In contrast to mammals, it seems that in Drosophila both MAPKKs, Hep/Mkk7 and Mkk4, are required to induce JNK upon TNF or pro-inflammatory stimulation.