A tumor-associated splice-isoform of MAP2K7 drives dedifferentiation in MBNL1-low cancers via JNK activation.

Master splicing regulator MBNL1 shapes large transcriptomic changes that drive cellular differentiation during development. Here we demonstrate that MBNL1 is a suppressor of tumor dedifferentiation. We surveyed MBNL1 expression in matched tumor/normal pairs across The Cancer Genome Atlas and found that MBNL1 was down-regulated in several common cancers. Down-regulation of MBNL1 predicted poor overall survival in breast, lung, and stomach adenocarcinomas and increased relapse and distant metastasis in triple-negative breast cancer. Down-regulation of MBNL1 led to increased tumorigenic and stem/progenitor-like properties in vitro and in vivo. A discrete set of alternative splicing events (ASEs) are shared between MBNL1-low cancers and embryonic stem cells including a MAP2K7∆exon2 splice variant that leads to increased stem/progenitor-like properties via JNK activation. Accordingly, JNK inhibition is capable of reversing MAP2K7∆exon2-driven tumor dedifferentiation in MBNL1-low cancer cells. Our work elucidates an alternative-splicing mechanism that drives tumor dedifferentiation and identifies biomarkers that predict enhanced susceptibility to JNK inhibition.

Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation.

Alternative splicing is prevalent among genes encoding signaling molecules; however, the functional consequence of differential isoform expression remains largely unknown. Here we demonstrate that, in response to T-cell activation, the Jun kinase (JNK) kinase MAP kinase kinase 7 (MKK7) is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a JNK-docking site within MKK7 that is disrupted in the larger isoform. Consistently, we show that skipping of MKK7 exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and up-regulation of TNF-α. Moreover, this splicing event is itself dependent on JNK signaling. Thus, MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. We further show that repression of MKK7 exon 2 is dependent on the presence of flanking sequences and the JNK-induced expression of the RNA-binding protein CELF2, which binds to these regulatory elements. Finally, we found that ∼25% of T-cell receptor-mediated alternative splicing events are dependent on JNK signaling. Strikingly, these JNK-dependent events are also significantly enriched for responsiveness to CELF2. Together, our data demonstrate a widespread role for the JNK-CELF2 axis in controlling splicing during T-cell activation, including a specific role in propagating JNK signaling.

MKK7 deficiency in mature neurons impairs parental behavior in mice.

c-Jun N-terminal kinases (JNKs) are constitutively activated in mammalian brains and are indispensable for their development and neural functions. MKK7 is an upstream activator of all JNKs. However, whether the common JNK signaling pathway regulates the brain's control of social behavior remains unclear. Here, we show that female mice in which Mkk7 is deleted specifically in mature neurons (Mkk7flox/flox Syn-Cre mice) give birth to a normal number of pups but fail to raise them due to a defect in pup retrieval. To explore the mechanism underlying this abnormality, we performed comprehensive behavioral tests. Mkk7flox/flox Syn-Cre mice showed normal locomotor functions and cognitive ability but exhibited depression-like behavior. cDNA microarray analysis of mutant brain revealed an altered gene expression pattern. Quantitative RT-PCR analysis demonstrated that mRNA expression levels of genes related to neural signaling pathways and a calcium channel were significantly different from controls. In addition, loss of neural MKK7 had unexpected regulatory effects on gene expression patterns in oligodendrocytes. These findings indicate that MKK7 has an important role in regulating the gene expression patterns responsible for promoting normal social behavior and staving off depression.

Mitogen Kinase Kinase (MKK7) Controls Cytokine Production In Vitro and In Vivo in Mice.

Mitogen kinase kinase 4 (MKK4) and mitogen kinase kinase 7 (MKK7) are members of the MAP2K family that can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, while MKK7 has been reported to activate only JNK in response to different stimuli. The stimuli, as well as the cell type determine which MAP2K member will mediate a given response. In various cell types, MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have also implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK4 and MKK7 contribute to innate immune responses in macrophages or during inflammation in vivo. To address this question and to elucidate the role of MKK4 and MKK7 in macrophage and in vivo, we developed MKK4- and MKK7-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for lipopolysaccharide (LPS)-induced cytokine production, M1 polarization, and migration, which appear to be a major contributor to the inflammatory response in vivo. Conversely, MKK4 plays a significant, but minor role in cytokine production in vivo.

Discovery of Turn-On Fluorescent Probes for Detecting PDEδ Protein in Living Cells and Tumor Slices.

The first small-molecule fluorescent turn-on probes for detecting PDEδ protein were rationally designed, showing reasonable fluorescent properties and the fluorescent ability has been applied for visualization of the PDEδ protein in living cells and at tissue levels. The qPCR results showed that the mRNA expression of KRAS, PDEδ, AKT1, MAPK1, MEK7, RAF1, and mTOR were downregulated by probes 1-3 through PI3K/AKT/mTOR and MAPK signal pathways. The probes also can downregulate the protein level of pErk and tErk. Therefore, these small-molecule fluorescent probes are expected to be used in the screening of antipancreatic cancer drugs targeting the PDEδ protein, as well as in obtaining a better understanding of the pathological and physiological roles of PDEδ protein.

DeSUMOylation of MKK7 kinase by the SUMO2/3 protease SENP3 potentiates lipopolysaccharide-induced inflammatory signaling in macrophages.

Protein SUMOylation has been reported to play a role in innate immune response, but the enzymes, substrates, and consequences of the specific inflammatory signaling events are largely unknown. Reactive oxygen species (ROS) are abundantly produced during macrophage activation and required for Toll-like receptor 4 (TLR4)-mediated inflammatory signaling. Previously, we demonstrated that SENP3 is a redox-sensitive SUMO2/3 protease. To explore any links between reversible SUMOylation and ROS-related inflammatory signaling in macrophage activation, we generated mice with Senp3 conditional knock-out in myeloid cells. In bacterial lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models, we found that SENP3 deficiency markedly compromises the activation of TLR4 inflammatory signaling and the production of proinflammatory cytokines in macrophages exposed to LPS. Moreover, Senp3 conditional knock-out mice were significantly less susceptible to septic shock. Of note, SENP3 deficiency was associated with impairment in JNK phosphorylation. We found that MKK7, which selectively phosphorylates JNK, is a SENP3 substrate and that SENP3-mediated deSUMOylation of MKK7 may favor its binding to JNK. Importantly, ROS-dependent SENP3 accumulation and MKK7 deSUMOylation rapidly occurred after LPS stimulation. In conclusion, our findings indicate that SENP3 potentiates LPS-induced TLR4 signaling via deSUMOylation of MKK7 leading to enhancement in JNK phosphorylation and the downstream events. Therefore this work provides novel mechanistic insights into redox regulation of innate immune responses.

Inhibition of didscoidin domain receptor 1 reduces epithelial-mesenchymal transition and induce cell-cycle arrest and apoptosis in prostate cancer cell lines.

Didscoidin domain receptor 1 (DDR1) is involved in the progression of prostate cancer metastasis through stimulation of epithelial-mesenchymal transition (EMT). So DDR1 inhibition can be a helpful target for cancer metastasis prevention. So, we studied the effects of DDR1 inhibition on EMT as well as induction of cell-cycle arrest and apoptosis in prostate cancer cell lines. DDR1 expression was evaluated using reverse-transcription polymerase chain reaction and western blot analysis. The EMT-associated protein expression was determined using the western blot analysis and immunocytochemistry following treatment with various concentrations of DDR1 inhibitor. The activation of DDR1 and also downstream-signaling molecules Pyk2 and MKK7 were determined using western blot analysis. Cell survival and proliferation after DDR1 inhibition were evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, bromodeoxyuridine, and colony formation assays. Flow cytometry analysis was used to determine the effects of DDR1 inhibition on cell-cycle arrest and apoptosis using annexin V/propidium iodide-based flow cytometry. Results showed that the protein expression of N-cadherin and vimentin were decreased whereas protein expression of E-cadherin was increased after DDR1 inhibition. Results of our western blot analysis indicated that DDR1 inhibitor effectively downregulated P-DDR1, P-Pyk2, and P-MKK7 levels. This result also showed that DDR1 inhibition decreased cell survival and proliferation, induced G1 cell-cycle arrest, induced apoptosis by an increase in the Bax/Bcl-2 ratio and depletion of the mitochondrial membrane potential, and also by reactive oxygen species creation in prostate cancer cells. These data show that DDR1 inhibition can result in the EMT prevention via inhibition of Pyk2 and MKK7 signaling pathway and induces cell-cycle arrest and apoptosis in prostate cancer cell lines. Thus, this study identifies DDR1 as an important target for modulating EMT and induction of apoptosis in prostate cancer cells.

MKK7 transcription positively or negatively regulated by SP1 and KLF5 depends on HDAC4 activity in glioma.

JNK activity has been implicated in the malignant proliferation, invasion and drug-resistance of glioma cells (GCs), but the molecular mechanisms underlying JNK activation are currently unknown. Here, we reported that MKK7, not MKK4, directly activates JNK in GCs and exerts oncogenic effects on tumor formation. Notably, MKK7 expression in glioma tissues was closely correlated with the grade of the glioma and JNK/c-Jun activation. Mechanistically, MKK7 transcription critically depends on the complexes formed by HDAC4 and the transcriptional factors SP1 and Krüppel-like factor-5 (KLF5), wherein HDAC4 directly deacetylates both SP1 and KLF5 and synergistically upregulates MKK7 transcription through two SP1 sites located on its promoter. In contrast, the increases in acetylated-SP1 and acetylated-KLF5 after HDAC4 inhibition switched to transcriptionally suppress MKK7. Selective inhibition of HDAC4 by LMK235, siRNAs or blockage of SP1 and KLF5 by the ectopic dominant-negative SP1 greatly reduced the malignant capacity of GCs. Furthermore, suppression of both MKK7 expression and JNK/c-Jun activities was involved in the tumor-growth inhibitory effects induced by LMK235 in U87-xenograft mice. Interestingly, HDAC4 is highly expressed in glioma tissues, and the rate of HDAC4 nuclear import is closely correlated with glioma grade, as well as with MKK7 expression. Collectively, these findings demonstrated that highly expressed MKK7 contributes to JNK/c-Jun signaling-mediated glioma formation. MKK7 transcription, regulated by SP1 and KLF5, critically depends on HDAC4 activity, and inhibition of HDAC4 presents a potential strategy for suppressing the oncogenic roles of MKK7/JNK/c-Jun signaling in GCs.

JNK signalling mediates aspects of maternal immune activation: importance of maternal genotype in relation to schizophrenia risk.

BACKGROUND: Important insight into the mechanisms through which gene-environmental interactions cause schizophrenia can be achieved through preclinical studies combining prenatal immune stimuli with disease-related genetic risk modifications. Accumulating evidence associates JNK signalling molecules, including MKK7/MAP2K7, with genetic risk. We tested the hypothesis that Map2k7 gene haploinsufficiency in mice would alter the prenatal immune response to the viral mimetic polyriboinosinic-polyribocytidylic acid (polyI:C), specifically investigating the impact of maternal versus foetal genetic variants. METHODS: PolyI:C was administered to dams (E12.5), and cytokine/chemokine levels were measured 6 h later, in maternal plasma, placenta and embryonic brain. RESULTS: PolyI:C dramatically elevated maternal plasma levels of most cytokines/chemokines. Induction of IL-1ß, IL-2, IL-10, IL-12, TNF-α and CXCL3 was enhanced, while CCL5 was suppressed, in Map2k7 hemizygous (Hz) dams relative to controls. Maternal polyI:C administration also increased embryonic brain chemokines, influenced by both maternal and embryonic genotype: CCL5 and CXCL10 levels were higher in embryonic brains from Map2k7 dams versus control dams; for CCL5, this was more pronounced in Map2k7 Hz embryos. Placental CXCL10 and CXCL12 levels were also elevated by polyI:C, the former enhanced and the latter suppressed, in placentae from maternal Map2k7 Hzs relative to control dams receiving polyI:C. CONCLUSIONS: The results demonstrate JNK signalling as a mediator of MIA effects on the foetus. Since both elevated CXCL10 and supressed CXCL12 compromise developing GABAergic interneurons, the results support maternal immune challenge contributing to schizophrenia-associated neurodevelopmental abnormalities. The influence of Map2k7 on cytokine/chemokine induction converges the genetic and environmental aspects of schizophrenia, and the overt influence of maternal genotype offers an intriguing new insight into modulation of embryonic neurodevelopment by genetic risk.

Bacterial effector NleL promotes enterohemorrhagic E. coli-induced attaching and effacing lesions by ubiquitylating and inactivating JNK.

As a major diarrheagenic human pathogen, enterohemorrhagic Escherichia coli (EHEC) produce attaching and effacing (A/E) lesions, characterized by the formation of actin pedestals, on mammalian cells. A bacterial T3SS effector NleL from EHEC O157:H7 was recently shown to be a HECT-like E3 ligase in vitro, but its biological functions and host targets remain elusive. Here, we report that NleL is required to effectively promote EHEC-induced A/E lesions and bacterial infection. Furthermore, human c-Jun NH2-terminal kinases (JNKs) were identified as primary substrates of NleL. NleL-induced JNK ubiquitylation, particularly mono-ubiquitylation at the Lys 68 residue of JNK, impairs JNK's interaction with an upstream kinase MKK7, thus disrupting JNK phosphorylation and activation. This subsequently suppresses the transcriptional activity of activator protein-1 (AP-1), which modulates the formation of the EHEC-induced actin pedestals. Moreover, JNK knockdown or inhibition in host cells complements NleL deficiency in EHEC infection. Thus, we demonstrate that the effector protein NleL enhances the ability of EHEC to infect host cells by targeting host JNK, and elucidate an inhibitory role of ubiquitylation in regulating JNK phosphorylation.

The MKK7 p.Glu116Lys Rare Variant Serves as a Predictor for Lung Cancer Risk and Prognosis in Chinese.

Accumulated evidence indicates that rare variants exert a vital role on predisposition and progression of human diseases, which provides neoteric insights into disease etiology. In the current study, based on three independently retrospective studies of 5,016 lung cancer patients and 5,181 controls, we analyzed the associations between five rare polymorphisms (i.e., p.Glu116Lys, p.Asn118Ser, p.Arg138Cys, p.Ala195Thr and p.Leu259Phe) in MKK7 and lung cancer risk and prognosis. To decipher the precise mechanisms of MKK7 rare variants on lung cancer, a series of biological experiments was further performed. We found that the MKK7 p.Glu116Lys rare polymorphism was significantly associated with lung cancer risk, progression and prognosis. Compared with Glu/Glu common genotype, the 116Lys rare variants (Lys/Glu/+ Lys/Lys) presented an adverse effect on lung cancer susceptibility (odds ratio [OR] = 3.29, 95% confidence interval [CI] = 2.70-4.01). These rare variants strengthened patients' clinical progression that patients with 116Lys variants had a significantly higher metastasis rate and advanced N, M stages at diagnosis. In addition, the patients with 116Lys variants also contributed to worse cancer prognosis than those carriers with Glu/Glu genotype (hazard ratio [HR] = 1.53, 95% CI = 1.32-1.78). Functional experiments further verified that the MKK7 p.116Lys variants altered the expression of several cancer-related genes and thus affected lung cancer cells proliferation, tumor growth and metastasis in vivo and in vitro. Taken together, our findings proposed that the MKK7 p.Glu116Lys rare polymorphism incurred a pernicious impact on lung cancer risk and prognosis through modulating expressions of a serial of cancer-related genes.

Dissection of MAPK signaling specificity through protein engineering in a developmental context.

BACKGROUND: Mitogen-activated protein kinases (MAPK) signaling affects many processes, some of which have different outcomes in the same cell. In Arabidopsis, activation of a MAPK cascade consisting of YODA, MKK4/5 and MPK3/6 inhibits early stages of stomatal developmental, but the ability to halt stomatal progression is lost at the later stage when guard mother cells (GMCs) transition to guard cells (GCs). Rather than downregulating cascade components, stomatal precursors must have a mechanism to prevent late stage inhibition because the same MKKs and MPKs mediate other physiological responses. RESULTS: We artificially activated the MAPK cascade using MKK7, another MKK that can modulate stomatal development, and found that inhibition of stomatal development is still possible in GMCs. This suggests that MKK4/5, but not MKK7, are specifically prevented from inhibiting stomatal development. To identify regions of MKKs responsible for cell-type specific regulation, we used a domain swap approach with MKK7 and a battery of in vitro and in vivo kinase assays. We found that N-terminal regions of MKK5 and MKK7 establish specific signal-to-output connections like they do in other organisms, but they do so in combination with previously undescribed modules in the C-terminus. One of these modules encoding the GMC-specific regulation of MKK5, when swapped with sequences from the equivalent region of MKK7, allows MKK5 to mediate robust inhibition of late stomatal development. CONCLUSIONS: Because MKK structure is conserved across species, the identification of new MKK specificity modules and signaling rules furthers our understanding of how eukaryotes create specificity in complex biological systems.

Role of p-MKK7 in myricetin-induced protection against intestinal ischemia/reperfusion injury.

Intestinal ischemia reperfusion (I/R) may cause inflammation-, oxidative stress-, and apoptosis-related tissue injuries and facilitate bacterial infection, leading to multiple organ failure. Myricetin, a flavonoid, is found to have diverse biological effects including anti-inflammatory, anti-oxidative, and anti-bacterial effects. Based on our pre-experiment, we proposed that myricetin pretreatment (25, 50mg/kg) could ameliorate intestinal I/R injury and myricetin-induced modulation on MKK7/JNK signal pathway might play a key role in the amelioration. The present study was designed to verify the proposal by using both rat intestinal I/R model in vivo and hypoxia/reoxygenation (H/R)-injured intestinal epithelial cell line (IEC-6 cells) model in vitro. The results confirmed our proposal. Myricetin selectively ameliorated I/R- and H/R-induced injuries in vivo and in vitro respectively without significantly affecting the corresponding normal controls. Myricetin significantly alleviated I/R-induced rat intestinal injury by reducing the generation of pro-inflammatory cytokines including TNF-α, IL-1ß, and IL-6 and by reducing MPO activity. Myricetin significantly reduced oxidative stress through decreasing MDA level and increasing the levels of SOD and GSH in the intestinal tissues compared with I/R control rats. Myricetin significantly decreased apoptosis by selectively down-regulating the expression of p-MKK7 and p-JNK without affecting MKK7 and JNK, inhibiting Bax, caspase-3 protein expression, and up-regulating Bcl-2 protein expression in I/R-injured jejunum of rats. In vitro study indicated that MKK7 siRNA transfection significantly decreased both MKK7 and p-MKK7 and other apoptosis-related proteins, partially simulating myricetin-induced anti-apoptotic effects. MKK7 siRNA transfection+myricetin could not further decrease MKK7, p-MKK7, and other apoptosis-related proteins, suggesting that inhibition of MKK7/JNK pathway plays a key role in myricetin-induced protection against intestinal I/R. MKK7 overexpression by cDNA transfection abrogated myricetin-reduced apoptosis-related protein expression, confirming that the MKK7/JNK signal pathway is the key target for myricetin-induced amelioration. The present study indicated that pretreatment of myricetin induced selective protection against intestinal I/R injury without significantly affecting corresponding normal controls and p-MKK7 was the key target, suggesting that myricetin is worth further translational studies.

Genetic variants of JNK and p38α pathways and risk of non-small cell lung cancer in an Eastern Chinese population.

The JNK and p38α pathways play an important role in carcinogenesis. Therefore, we hypothesize that single nucleotide polymorphisms (SNPs) of genes involved in these pathways are associated with risk of lung cancer. We first selected and genotyped 11 independent SNPs of the JNK and p38α pathway-related genes in a discovery set of 1,002 non-small cell lung cancer (NSCLC) cases and 1,025 cancer-free controls of Eastern Chinese. Then, we validated those significant SNPs in a replication set of 1,333 NSCLC cases and 1,339 cancer-free controls of Eastern Chinese. Multifactor dimensionality reduction (MDR) and classification and regression tree (CART) analyses were used to identify interactions between significant SNPs and other covariates. In both discovery and replication as well as their pooled analysis, carriers of GADD45G rs8252T variant genotypes had a significantly lower risk of NSCLC (adjusted OR = 0.81 and 0.79, 95% CI = 0.72-0.92 and 0.64-0.99 and p = 0.001 and 0.040 for dominant and recessive genetic models, respectively) and carriers of MAP2K7 rs3679T variant genotypes had an increased risk of NSCLC (adjusted OR = 1.19 and 1.29, 95% CI = 1.05-1.34 and 1.09-1.54 and p = 0.005 and 0.004 for dominant and recessive genetic models, respectively). Furthermore, rs8252 variant CT/TT carriers showed significantly higher levels of GADD45G mRNA expression than CC carriers in the target tissues. We observed some evidence of interactions between rs8252 genotypes and sex in NSCLC risk. These results indicate that GADD45G rs8252 and MAP2K7 rs3679 SNPs may be susceptibility biomarkers for NSCLC in Eastern Chinese populations.

Rare variant of MAP2K7 is associated with increased risk of COPD in southern and eastern Chinese.

BACKGROUND AND OBJECTIVE: A wide range of common loci have been extensively screened and evaluated for their associations with various complex diseases; however, the relevance of rare variants causing missense substitutions in the protein-coding genes in human diseases is still poorly understood. METHODS: In this study, we conducted a two-stage retrospective study of a total of 1791 patients with COPD and 1940 controls in southern and eastern Chinese to test relevancies of five rare variants (i.e. p.Glu116Lys, p.Asn118Ser, p.Arg138Cys, p.Ala195Thr and p.Leu259Phe) of human mitogen-activated protein kinase kinase 7 (MAP2K7) to COPD susceptibility. The effects of these loci on lung function were further estimated. RESULTS: The p.Glu116Lys rare variant had significant associations with COPD risk. Compared to individuals with Glu/Glu wild-genotype, those with 116Lys rare variants (Lys/Glu+Lys/Lys) had an increased risk of COPD (OR = 3.83, 95% CI: 2.64-5.56; P = 1.45 × 10-12 ). Meanwhile, the carriers with 116Lys rare variants (Lys/Glu+Lys/Lys) had lower pre-forced expiratory volume in 1 s (pre-FEV1 : 1.74 ± 0.70 vs 2.00 ± 0.68; P = 3.97 × 10-5 ) and lower pre-FEV1 to pre-forced vital capacity ratio (pre-FEV1 /FVC: 0.68 ± 0.14 vs 0.75 ± 0.12; P = 2.40 × 10-10 ) than those with Glu/Glu genotype. However, for other rare variants, no significant association with either COPD risk or lung function was observed. CONCLUSION: Our data strongly suggest that the p.Glu116Lys rare variant in MAP2K7 predisposes its carriers to develop COPD, which would provide a useful genetic biomarker for COPD susceptibility in Chinese.

Hepatitis B Virus X Protein Modulates Apoptosis in NRK-52E Cells and Activates Fas/FasL Through the MLK3-MKK7-JNK3 Signaling Pathway.

BACKGROUND/AIMS: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. METHODS: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. RESULTS: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. CONCLUSIONS: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway.

A crucial role of Cys218 in configuring an unprecedented auto-inhibition form of MAP2K7.

Mitogen-activated protein kinase kinase 7 (MAP2K7) is an indispensable kinase of the c-Jun N-terminal kinase signal cascade and is rigorously regulated via phosphorylation. To investigate the regulatory mechanism of the inactive non-phosphorylated state of MAP2K7, the crystal structures of the wild-type and C218S mutant were solved. The wild-type apo-structure revealed an unprecedented auto-inhibition form that occluded the ATP site. This closed form was configured by the n-σ* interaction of Cys218, a non-conserved residue among the MAP2K family kinases, with Gly145 in the glycine-rich loop. The interaction was unaltered in the presence of an ATP analog, whereas the C218S mutation precluded the closed configuration. These structural insights are potentially valuable for drug discovery of highly selective MAP2K7 inhibitors.

Identification and characterization of MKK7 as an upstream activator of JNK in Litopenaeus vannamei.

Mitogen-activated protein kinase kinase 7 (MKK7) is a key signal transduction regulator in c-Jun N-terminal kinase (JNK) signaling pathway, which is involved in a wide range of physiological and pathological processes. In this study, we described the molecular cloning of a new member of MKK7 group from Litopenaeus vannamei named as LvMKK7. The full-length cDNA of LvMKK7 was 3093 bp in length, with an open reading frame (ORF) of 1440bp encoding a putative protein of 479 amino acids. LvMKK7 contained a conserved kinase domain of 261 amino acids in which there was a characteristic S-K-A-K-T motif as a potential target site of phosphorylation by MKKK. Moreover, subcellular localization showed LvMKK7 was located in both the cytoplasm and the nucleus of Drosophila S2 cells. Real-time PCR indicated that LvMKK7 was universally expressed in all tested tissues and its expression in hepatopancreas was responsive to the challenge of LPS, Poly (I:C), Vibrio parahaemolyticus, Staphhylococcus aureus and white spot syndrome virus (WSSV). In addition, co-immunoprecipitation assay demonstrated that LvJNK was phosphorylated and activated by LvMKK7, which suggested LvMKK7 was the upper regulator of LvJNK. Furthermore, RNAi-mediated knockdown of LvMKK7 enhanced the sensitivity of shrimps to V. parahaemolyticus infection. Overall, our results suggested that LvMKK7 may play important roles in the shrimp innate immunity.

MKK7 confers different activities to viral infection of Singapore grouper iridovirus (SGIV) and nervous necrosis virus (NNV) in grouper.

Mitogen-activated protein kinase 7 (MKK7) is one of the major stress-activated protein kinase (SAPK)-activating kinases in response to environmental or physiological stimuli. Here a MKK7 named as Ec-MKK7 was identified from orange-spotted grouper, Epinephelus coioides. The full-length cDNA of Ec-MKK7 was 1853 bp, with an open reading frame (ORF) of 1272 bp encoding a putative protein of 423 amino acids. A characteristic S-K-A-K-T motif was contained in the domain of dual-specificity protein kinase, mitogen-activated protein kinase kinase 7 (PKc_MKK7). Intracellular localization showed that Ec-MKK7 was localized in both the cytoplasm and the nucleus of grouper spleen (GS) and/or grouper brain (EAGB) cells. Moreover, Ec-MKK7 was universally expressed in all examined tissues and showed expression modulation to challenges of lipopolysacchride (LPS), Singapore grouper iridovirus (SGIV) and polyriboinosinic polyribocytidylic acid (poly I:C) in vivo. A gene targeting strategy over-expressing Ec-MKK7 was performed to examine the activities of MKK7 to viral infection in vitro. Our data showed that Ec-MKK7 was involved in the evasion and replication of SGIV but played an antiviral role to the infection of nervous necrosis virus (NNV). All results demonstrated that Ec-MKK7 could play important roles in grouper innate immunity and show distinct functions on virus infection.

The structural pathway of interleukin 1 (IL-1) initiated signaling reveals mechanisms of oncogenic mutations and SNPs in inflammation and cancer.

Interleukin-1 (IL-1) is a large cytokine family closely related to innate immunity and inflammation. IL-1 proteins are key players in signaling pathways such as apoptosis, TLR, MAPK, NLR and NF-κB. The IL-1 pathway is also associated with cancer, and chronic inflammation increases the risk of tumor development via oncogenic mutations. Here we illustrate that the structures of interfaces between proteins in this pathway bearing the mutations may reveal how. Proteins are frequently regulated via their interactions, which can turn them ON or OFF. We show that oncogenic mutations are significantly at or adjoining interface regions, and can abolish (or enhance) the protein-protein interaction, making the protein constitutively active (or inactive, if it is a repressor). We combine known structures of protein-protein complexes and those that we have predicted for the IL-1 pathway, and integrate them with literature information. In the reconstructed pathway there are 104 interactions between proteins whose three dimensional structures are experimentally identified; only 15 have experimentally-determined structures of the interacting complexes. By predicting the protein-protein complexes throughout the pathway via the PRISM algorithm, the structural coverage increases from 15% to 71%. In silico mutagenesis and comparison of the predicted binding energies reveal the mechanisms of how oncogenic and single nucleotide polymorphism (SNP) mutations can abrogate the interactions or increase the binding affinity of the mutant to the native partner. Computational mapping of mutations on the interface of the predicted complexes may constitute a powerful strategy to explain the mechanisms of activation/inhibition. It can also help explain how an oncogenic mutation or SNP works.