MAPK-Activated Protein Kinases: Servant or Partner?

Mitogen-activated protein kinase (MAPK)-activated protein kinases (MAPKAPKs) are defined by their exclusive activation by MAPKs. They can be activated by classical and atypical MAPKs that have been stimulated by mitogens and various stresses. Genetic deletions of MAPKAPKs and availability of highly specific small-molecule inhibitors have continuously increased our functional understanding of these kinases. MAPKAPKs cooperate in the regulation of gene expression at the level of transcription; RNA processing, export, and stability; and protein synthesis. The diversity of stimuli for MAPK activation, the crosstalk between the different MAPKs and MAPKAPKs, and the specific substrate pattern of MAPKAPKs orchestrate immediate-early and inflammatory responses in space and time and ensure proper control of cell growth, differentiation, and cell behavior. Hence, MAPKAPKs are promising targets for cancer therapy and treatments for conditions of acute and chronic inflammation, such as cytokine storms and rheumatoid arthritis.

Structural basis for the action of the drug trametinib at KSR-bound MEK.

The MAPK/ERK kinase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology1, and more recently in immunotherapy2,3 and ageing4. However, many MEK inhibitors are limited owing to on-target toxicities5-7 and drug resistance8-10. Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR (kinase suppressor of RAS) with various MEK inhibitors, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. In the bound complex, KSR remodels the prototypical allosteric pocket of the MEK inhibitor, thereby affecting binding and kinetics, including the drug-residence time. Moreover, trametinib binds KSR-MEK but disrupts the related RAF-MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these sub-complexes. On the basis of these insights, we created trametiglue, which limits adaptive resistance to MEK inhibition by enhancing interfacial binding. Our results reveal the plasticity of an interface pocket within MEK sub-complexes and have implications for the design of next-generation drugs that target the RAS pathway.

Rice calcium/calmodulin-dependent protein kinase directly phosphorylates a mitogen-activated protein kinase kinase to regulate abscisic acid responses.

Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment induces phosphorylation in the activation loop of OsMKK1, and the two phosphorylations, in the N-terminus and in the activation loop, are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in activating the MAPK cascade and ABA signaling.

Comprehensive analysis of MAPK gene family in Populus trichocarpa and physiological characterization of PtMAPK3-1 in response to MeJA induction.

Mitogen-activated protein kinases (MAPKs) play important roles in plant growth and development, as well as hormone and stress responses by signaling to eukaryotic cells, through MAPK cascade, the presence of various cues; thereby, regulating various responses. The MAPK cascade consists mainly of three gene families, MAPK, MAPKK, and MAPKKK, which activate downstream signaling pathways through sequential phosphorylation. Although the MAPK cascade gene family has been reported in several species, there is a lack of comprehensive analysis in poplar. We identified 21 MAPK genes, 11 MAPKK genes, and 104 MAPKKK genes in Populus trichocarpa. The phylogenetic classification was supported by conservative motif, gene structure and motif analysis. Whole genome duplication has an important role in the expansion of MAPK cascade genes. Analysis of promoter cis-elements and expression profiles indicates that MAPK cascade genes have important roles in plant growth and development, abiotic and biotic stresses, and phytohormone response. Expression profiling revealed a significant upregulation of PtMAPK3-1 expression in response to drought, salt and disease stresses. Poplar transiently overexpressing PtMAPK3-1 and treated with methyl jasmonic acid (MeJA) had higher catalase and peroxidase levels than non-overexpressing poplar. This work represents the first complete inventory of the MAPK cascade in P. trichocarpa, which reveals that PtMAPK3-1 is induced by the MeJA hormone and participates in the MeJA-induced enhancement of the antioxidant enzyme system.

PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and ß-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

Pseudokinases repurpose flexibility signatures associated with the protein kinase fold for noncatalytic roles.

The bilobal protein kinase-like fold in pseudokinases lack one or more catalytic residues, conserved in canonical protein kinases, and are considered enzymatically deficient. Tertiary structures of pseudokinases reveal that their loops topologically equivalent to activation segments of kinases adopt contracted configurations, which is typically extended in active conformation of kinases. Herein, anisotropic network model based normal mode analysis (NMA) was conducted on 51 active conformation structures of protein kinases and 26 crystal structures of pseudokinases. Our observations indicate that although backbone fluctuation profiles are similar for individual kinase-pseudokinase families, low intensity mean square fluctuations in pseudo-activation segment and other sub-structures impart rigidity to pseudokinases. Analyses of collective motions from functional modes reveal that pseudokinases, compared to active kinases, undergo distinct conformational transitions using the same structural fold. All-atom NMA of protein kinase-pseudokinase pairs from each family, sharing high amino acid sequence identities, yielded distinct community clusters, partitioned by residues exhibiting highly correlated fluctuations. It appears that atomic fluctuations from equivalent activation segments guide community membership and network topologies for respective kinase and pseudokinase. Our findings indicate that such adaptations in backbone and side-chain fluctuations render pseudokinases competent for catalysis-independent roles.

Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling.

Deregulation of the Ras-mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes. Kinase suppressor of Ras (KSR) is a MAPK scaffold that is subject to allosteric regulation through dimerization with RAF. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras-MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.

Quantifying ERK activity in response to inhibition of the BRAFV600E-MEK-ERK cascade using mathematical modelling.

BACKGROUND: Simultaneous inhibition of multiple components of the BRAF-MEK-ERK cascade (vertical inhibition) has become a standard of care for treating BRAF-mutant melanoma. However, the molecular mechanism of how vertical inhibition synergistically suppresses intracellular ERK activity, and consequently cell proliferation, are yet to be fully elucidated. METHODS: We develop a mechanistic mathematical model that describes how the mutant BRAF inhibitor, dabrafenib, and the MEK inhibitor, trametinib, affect BRAFV600E-MEK-ERK signalling. The model is based on a system of chemical reactions that describes cascade signalling dynamics. Using mass action kinetics, the chemical reactions are re-expressed as ordinary differential equations that are parameterised by in vitro data and solved numerically to obtain the temporal evolution of cascade component concentrations. RESULTS: The model provides a quantitative method to compute how dabrafenib and trametinib can be used in combination to synergistically inhibit ERK activity in BRAFV600E-mutant melanoma cells. The model elucidates molecular mechanisms of vertical inhibition of the BRAFV600E-MEK-ERK cascade and delineates how elevated BRAF concentrations generate drug resistance to dabrafenib and trametinib. The computational simulations further suggest that elevated ATP levels could be a factor in drug resistance to dabrafenib. CONCLUSIONS: The model can be used to systematically motivate which dabrafenib-trametinib dose combinations, for treating BRAFV600E-mutated melanoma, warrant experimental investigation.

Quantification of the Dynamic Phosphorylation Process of ERK Using Stable Isotope Dilution Selective Reaction Monitoring Mass Spectrometry.

Mitogen-activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three-tiered kinase module-MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase-that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine-glutamic acid-tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal-regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)-selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID-SRM-MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono-phosphorylatable mutants ERKT202A and ERKY204F indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers.

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

Comparative analysis of plant MKK gene family reveals novel expansion mechanism of the members and sheds new light on functional conservation.

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades play critical functions in almost every aspect of plant growth and development, which regulates many physiological and biochemical processes. As a middle nodal point of the MAPK cascades, although evolutionary analysis of MKK from individual plant families had some reports, their evolutionary history in entire plants is still not clear. RESULTS: To better understand the evolution and function of plant MKKs, we performed systematical molecular evolutionary analysis of the MAPKK gene family and also surveyed their gene organizations, sequence features and expression patterns in different subfamilies. Phylogenetic analysis showed that plant MAPKK fall into five different groups (Group A-E). Majority orthology groups seemed to be a single or low-copy genes in all plant species analyzed in Group B, C and D, whereas group A MKKs undergo several duplication events, generating multiple gene copies. Further analysis showed that these duplication events were on account of whole genome duplications (WGDs) in plants and the duplicate genes maybe have undergone functional divergence. We also found that group E MKKs had mutation with one change of serine or theronine might lead to inactivity originated through the ancient tandem duplicates in monocots. Moreover, we also identified MKK3 integrated NTF2 domain that might have gradually lost the cytoplasmic-nuclear trafficking activity, which suggests that they may involve with the gene function more and more sophistication in the evolutionary process. Moreover, expression analyses indicated that plant MKK genes play probable roles in UV-B signaling. CONCLUSION: In general, ancient gene and genome duplications are significantly conducive to the expansion of the plant MKK gene family. Our study reveals two distinct evolutionary patterns for plant MKK proteins and sheds new light on the functional evolution of this gene family.

Genome-wide identification and analysis of MAPK and MAPKK gene family in Chinese jujube (Ziziphus jujuba Mill.).

BACKGROUND: Chinese jujube (Ziziphus jujuba Mill.) is one of the most important members in the Rhamnaceae family. The whole genome sequence and more than 30,000 proteins of Chinese jujube have been obtained in 2014. Mitogen-activated protein kinase cascades are universal signal transduction modules in plants, which is rapidly activated under various biotic and abiotic stresses. To date, there has been no comprehensive analysis of the MAPK and MAPKK gene family in Chinese jujube at the whole genome level. RESULTS: By performing a series of bioinformatics analysis, ten MAPK and five MAPKK genes were identified from the genome database of Chinese jujube, and then compared with the homologous genes from Arabidopsis. Phylogenetic analysis showed that ZjMAPKs was classified into four known groups, including A, B, C and D. ZjMAPKs contains five members of the TEY phosphorylation site and five members with the TDY motif. The ZjMAPKK family was subsequently divided into three groups, A, B and D. The gene structure, conserved motifs, functional annotation and chromosome distribution of ZjMAPKs and ZjMAPKKs were also predicted. ZjMAPKs and ZjMAPKKs were distributed on nine pseudo-chromosomes of Chinese jujube. Subsequently, expression analysis of ZjMAPK and ZjMAPKK genes using reverse transcription PCR and quantitative real-time PCR was carried out. The majority of ZjMAPK and ZjMAPKK genes were expressed in all tested organs/tissues with considerable differences in transcript levels indicating that they might be constitutively expressed. Moreover, ZjMKK5 was specific expressed in early development stage of jujube flower bud, indicating it plays some roles in reproductive organs development. The transcript expression of most ZjMAPK and ZjMAPKK genes was down-regulated in response to plant growth regulators, darkness treatment and phytoplasma infection. CONCLUSIONS: We identified ten ZjMAPK and five ZjMAPKK genes from the genome database of Chinese jujube, the research results shown that ZjMPKs and ZjMKKs have the different expression patterns, indicating that they might play different roles in response to various treatments. The results provide valuable information for the further elucidation of physiological functions and biological roles of jujube MAPKs and MAPKKs.

ghr-miR5272a-mediated regulation of GhMKK6 gene transcription contributes to the immune response in cotton.

Fusarium wilt is a major biotic stress affecting the productivity of cotton (Gossypium hirsutum). Although mitogen-activated protein kinase (MAPK) cascades play critical roles in plant disease resistance, their intricate regulation under fungal stress remains unclear, especially with regards to microRNA-mediated regulation of MAPK gene expression. In this study, we report that the MAPK kinase gene GhMKK6 and ghr-miR5272a work together in cotton resistance to Fusarium wilt. Silencing GhMKK6 in cotton decreased resistance to F. oxysporum by repressing the expression of known disease-resistance genes. Furthermore, although GhMKK6 played a positive role in disease resistance, excessive GhMKK6 activation caused an excessive hypersensitive response. ghr-miR5272a, a major regulator, prevents this excessive response by regulating GhMKK6 expression. ghr-miR5272a targets the GhMKK6 3'-untranslated region in cotton. Overexpressing miR5272a decreased the expression of GhMKK6 and disease-resistance genes, and increased sensitivity to F. oxysporum, yielding a similar phenotype to GhMKK6-silenced cotton. Overall, these results demonstrate that the ghr-miR5272a-mediated regulation of GhMKK6 expression contributes to the immune response in cotton, and reveal a new feedback loop mechanism in plant disease response.

Manipulation of mitogen-activated protein kinase kinase signaling in the Arabidopsis stomatal lineage reveals motifs that contribute to protein localization and signaling specificity.

When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four "stomatal" MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.

In silico docking studies of Lupeol with MAPK pathway proteins- Raf-1, MEK & ERK.

OBJECTIVE: Lupeol, A triterpenoid found in variety of plants is reported to have beneficial medicinal effects on several ailments. Lupeol is also found to show inhibitory effect on proliferation of breast cancer cells. Metastasis is considered to be a major cause for worldwide deaths related to cancer. Ras related MAPK Signaling Pathway is one of the crucial pathways leading to metastasis. Lupeols binding possibility with Ras is already reported. In present study, Interaction between with downstream proteins of Ras- MAPK pathway, Raf ,MEK ,ERK1/2 and their corresponding domains are studied using STRING Database and their structures are retrieved in PDB Format. Lupeols binding affinity with downstream proteins of these signaling proteins at their interacting domains are analyzed. Here in silico docking approach to identify binding sites of each of these proteins with Lupeol is used. FDA approved standard drug molecule CH5126766 was used as reference ligand. Lupeol shows potent binding at significant sites with extremely high affinity. Since it binds with all the proteins involved in the pathway with high efficiency it is an important compound which can be developed as a therapeutic molecule.

A novel MKK gene (AjMKK3/6) in the sea cucumber Apostichopus japonicus: Identification, characterization and its response to pathogenic challenge.

The mitogen-activated protein kinase kinases (MKKs) are key components of MAP kinase (MAPK) cascades and function as redox-regulated signaling factors in pathological and physiological processes. In this study, we identified a novel MKK3/6 gene in the sea cucumber Apostichopus japonicus (designated as AjMKK3/6) by transcriptome database mining and rapid amplification of cDNA ends (RACE) approaches. Sequence analysis and protein structure prediction showed that AjMKK3/6 is highly conserved as compared to those from other invertebrate and vertebrate species. Molecular phylogeny result revealed that AjMKK3/6 exhibited a closest relationship with that from Strongylocentrotus purpuratus. Quantitative real-time PCR was employed to determine the expression profiles of AjMKK3/6 in healthy adult A. japonicus tissues and in coelomocytes after Vibrio splendidus infection in vivo, respectively. As results shown, AjMKK3/6 was ubiquitously expressed in all examined tissues of healthy adult A. japonicus with a relative expression level from high to low as body wall > tube feet > coelomocytes > respiratory tree > intestine > longitudinal muscle. Significant expression changes of AjMKK3/6 in coelomocytes were observed at 12 h- and 72 h-after V. splendidus infection, respectively. In general, the current study will enrich our knowledge of characterizations and immno-functions of MKK3/6 in sea cucumbers.

The crystal structure of an inactive dimer of PDZ-binding kinase.

The overexpression of PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) has been associated with hematologic tumors, breast cancer and various other cancers. However, the three-dimensional structure of PBK has not been solved. In this study, we determined the crystal structure of human PBK, which has two phospho-mimicking mutations T9E and T198E. The structural data indicated that PBK may assemble into an inactive dimer in alkaline conditions. Analytical size-exclusion chromatography and analytical ultracentrifugation confirmed that PBK exists in a conformational transition between dimers and monomers at different pH conditions. Co-IP and kinase assays suggested that the active state of PBK is a monomer and does not form a dimer even under alkaline conditions. These results showed that the conformational transition of PBK is important for its kinase activity regulation. Collectively, our observations may provide a novel starting point for structure-based functional studies.

Isolation and expression analysis of an MAPKK gene from Fenneropenaeus chinensis in response to white spot syndrome virus infection.

Mitogen-activated kinase kinase (MAPKK) is an important gene involved in the host-virus interaction process. To obtain a better understanding of MAPKK in the interaction process between the Chinese shrimp Fenneropenaeus chinensis and white spot syndrome virus (WSSV), we cloned the sequence of an MAPKK cDNA from F. chinensis (FcMAPKK) and investigated the effect of FcMAPKK on WSSV infection. The results showed that the FcMAPKK gene contained a 1227 bp open reading frame (ORF), which encoded a highly conserved protein with a serine/threonine protein kinase catalytic (S_TKc) domain. The deduced amino acid sequence of FcMAPKK shared identities between 11.9 and 92.6% with MAPKKs from vertebrate, invertebrate, plant and fungus species. The FcMAPKK was expressed in all the examined tissues in the normal F. chinensis. FcMAPKK expression level was highest in the hepatopancreas where it was approximately 2.6-fold the expression level in the gill, and lowest in the muscle where it was approximately 0.3-fold the expression level in the hepatopancreas. The FcMAPKK expression levels in the muscle, gill, and hepatopancreas were all changed post WSSV challenge. The FcMAPKK expression was significantly (P < 0.01) up-regulated in the muscle of F. chinensis at 48 h post WSSV infection. The WSSV began to replicate quickly in the normal F. chinensis at 48 h post infection, while the WSSV replication in the U0126-treated F. chinensis could be significantly (P < 0.05) inhibited. The results suggested that FcMAPKK might be involved in the WSSV infection process, and hijacking of FcMAPKK might be required for WSSV replication in F. chinensis.

Genome-wide identification of MAPK, MAPKK, and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber.

BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade consists of three types of reversibly phosphorylated kinases, namely, MAPK, MAPK kinase (MAPKK/MEK), and MAPK kinase kinase (MAPKKK/MEKK), playing important roles in plant growth, development, and defense response. The MAPK cascade genes have been investigated in detail in model plants, including Arabidopsis, rice, and tomato, but poorly characterized in cucumber (Cucumis sativus L.), a major popular vegetable in Cucurbitaceae crops, which is highly susceptible to environmental stress and pathogen attack. RESULTS: A genome-wide analysis revealed the presence of at least 14 MAPKs, 6 MAPKKs, and 59 MAPKKKs in the cucumber genome. Phylogenetic analyses classified all the CsMAPK and CsMAPKK genes into four groups, whereas the CsMAPKKK genes were grouped into the MEKK, RAF, and ZIK subfamilies. The expansion of these three gene families was mainly contributed by segmental duplication events. Furthermore, the ratios of non-synonymous substitution rates (Ka) and synonymous substitution rates (Ks) implied that the duplicated gene pairs had experienced strong purifying selection. Real-time PCR analysis demonstrated that some MAPK, MAPKK and MAPKKK genes are preferentially expressed in specific organs or tissues. Moreover, the expression levels of most of these genes significantly changed under heat, cold, drought, and Pseudoperonospora cubensis treatments. Exposure to abscisic acid and jasmonic acid markedly affected the expression levels of these genes, thereby implying that they may play important roles in the plant hormone network. CONCLUSION: A comprehensive genome-wide analysis of gene structure, chromosomal distribution, and evolutionary relationship of MAPK cascade genes in cucumber are present here. Further expression analysis revealed that these genes were involved in important signaling pathways for biotic and abiotic stress responses in cucumber, as well as the response to plant hormones. Our first systematic description of the MAPK, MAPKK, and MAPKKK families in cucumber will help to elucidate their biological roles in plant.

Characterization, expression patterns and functional analysis of the MAPK and MAPKK genes in watermelon (Citrullus lanatus).

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades, which consist of three functionally associated protein kinases, namely MEKKs, MKKs and MPKs, are universal signaling modules in all eukaryotes and have been shown to play critical roles in many physiological and biochemical processes in plants. However, little or nothing is known about the MPK and MKK families in watermelon. RESULTS: In the present study, we performed a systematic characterization of the ClMPK and ClMKK families including the identification and nomenclature, chromosomal localization, phylogenetic relationships, ClMPK-ClMKK interactions, expression patterns in different tissues and in response to abiotic and biotic stress and transient expression-based functional analysis for their roles in disease resistance. Genome-wide survey identified fifteen ClMPK and six ClMKK genes in watermelon genome and phylogenetic analysis revealed that both of the ClMPK and ClMKK families can be classified into four distinct groups. Yeast two-hybrid assays demonstrated significant interactions between members of the ClMPK and ClMKK families, defining putative ClMKK2-1/ClMKK6-ClMPK4-1/ClMPK4-2/ClMPK13 and ClMKK5-ClMPK6 cascades. Most of the members in the ClMPK and ClMKK families showed differential expression patterns in different tissues and in response to abiotic (e.g. drought, salt, cold and heat treatments) and biotic (e.g. infection of Fusarium oxysporum f. sp. niveum) stresses. Transient expression of ClMPK1, ClMPK4-2 and ClMPK7 in Nicotiana benthamiana resulted in enhanced resistance to Botrytis cinerea and upregulated expression of defense genes while transient expression of ClMPK6 and ClMKK2-2 led to increased susceptibility to B. cinerea. Furthermore, transient expression of ClMPK7 also led to hypersensitive response (HR)-like cell death and significant accumulation of H2O2 in N. benthamiana. CONCLUSION: We identified fifteen ClMPK and six ClMKK genes from watermelon and analyzed their phylogenetic relationships, expression patterns and protein-protein interactions and functions in disease resistance. Our results demonstrate that ClMPK1, ClMPK4-2 and ClMPK7 positively but ClMPK6 and ClMKK2-2 negatively regulate the resistance to B. cinerea when transiently expressed in N. benthamiana and that ClMPK7 functions as a regulator of HR-like cell death through modulating the generation of H2O2.