Components of the JNK-MAPK pathway play distinct roles in hepatocellular carcinoma.

PURPOSE: Mitogen-activated protein kinases (MAPK), specifically the c-Jun N-terminal kinase (JNK)-MAPK subfamily, play a crucial role in the development of various cancers, including hepatocellular carcinoma (HCC). However, the specific roles of JNK1/2 and their upstream regulators, MKK4/7, in HCC carcinogenesis remain unclear. METHODS: In this study, we performed differential expression analysis of JNK-MAPK components at both the transcriptome and protein levels using TCGA and HPA databases. We utilized Kaplan-Meier survival plots and receiver operating characteristic (ROC) curve analysis to evaluate the prognostic performance of a risk scoring model based on these components in the TCGA-HCC cohort. Additionally, we conducted immunoblotting, apoptosis analysis with FACS and soft agar assays to investigate the response of JNK-MAPK pathway components to various death stimuli (TRAIL, TNF-α, anisomycin, and etoposide) in HCC cell lines. RESULTS: JNK1/2 and MKK7 levels were significantly upregulated in HCC samples compared to paracarcinoma tissues, whereas MKK4 was downregulated. ROC analyses suggested that JNK2 and MKK7 may serve as suitable diagnostic genes for HCC, and high JNK2 expression correlated with significantly poorer overall survival. Knockdown of JNK1 enhanced TRAIL-induced apoptosis in hepatoma cells, while JNK2 knockdown reduced TNF-α/cycloheximide (CHX)-and anisomycin-induced apoptosis. Neither JNK1 nor JNK2 knockdown affected etoposide-induced apoptosis. Furthermore, MKK7 knockdown augmented TNF-α/CHX- and TRAIL-induced apoptosis and inhibited colony formation in hepatoma cells. CONCLUSION: Targeting MKK7, rather than JNK1/2 or MKK4, may be a promising therapeutic strategy to inhibit the JNK-MAPK pathway in HCC therapy.

Stress-Activated Protein Kinase JNK Modulates Depression-like Behaviors in Mice.

Stress is considered as a major cause of depression. C-Jun N-terminal kinase (JNK) is a member of the stress-induced mitogen activated protein (MAP) kinase family which is often activated through phosphorylation. Clinical studies and animal experiments have found that abnormal phosphorylation/activation of JNK exists in the occurrence of various psychiatric diseases. Recently, several studies linked JNK kinase activity to depression. However, whether excessive activation of JNK activity is directly responsible for the occurrence of depression and the underlying mechanisms remain unclear. Here, we constructed a conditional transgenic mouse which is specifically expressing MKK7-JNK1 (CAJNK1) in the central nervous system. CAJNK1 mice showed activation of JNK and lead to depression-like behavior in mice. Transcriptome analysis indicates reduced expression of synaptic-associated genes in CAJNK1 mice brains. Consistently, we found abnormal dendritic spine development and PSD95 downregulation in CAJNK1 hippocampal neurons. Our studies provide compelling evidence that activation of JNK as an intrinsic factor leading to depression-like behavior in mice provides direct clues for targeting the JNK activity as a potential therapeutic strategy for depression.

The schizophrenia risk gene Map2k7 regulates responding in a novel contingency-shifting rodent touchscreen gambling task.

In schizophrenia, subjects show reduced ability to evaluate and update risk/reward contingencies, showing correspondingly suboptimal performance in the Iowa gambling task. JNK signalling gene variants are associated with schizophrenia risk, and JNK modulates aspects of cognition. We therefore studied the performance of mice hemizygous for genetic deletion of the JNK activator MKK7 (Map2k7+/- mice) in a touchscreen version of the Iowa gambling task, additionally incorporating a novel contingency-switching stage. Map2k7+/- mice performed slightly better than wild-type (WT) littermates in acquisition and performance of the task. Although Map2k7+/- mice adapted well to subtle changes in risk/reward contingencies, they were profoundly impaired when the positions of 'best' and 'worst' choice selections were switched, and still avoided the previous 'worst' choice location weeks after the switch. This demonstrates a precise role for MKK7-JNK signalling in flexibility of risk/reward assessment and suggests that genetic variants affecting this molecular pathway may underlie impairment in this cognitive domain in schizophrenia. Importantly, this new contingency shift adaptation of the rodent touchscreen gambling task has translational utility for characterising these cognitive subprocesses in models of neuropsychiatric disorders.

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.

JunD, not c-Jun, is the AP-1 transcription factor required for Ras-induced lung cancer.

The AP-1 transcription factor c-Jun is required for Ras-driven tumorigenesis in many tissues and is considered as a classical proto-oncogene. To determine the requirement for c-Jun in a mouse model of K-RasG12D-induced lung adenocarcinoma, we inducibly deleted c-Jun in the adult lung. Surprisingly, we found that inactivation of c-Jun, or mutation of its JNK phosphorylation sites, actually increased lung tumor burden. Mechanistically, we found that protein levels of the Jun family member JunD were increased in the absence of c-Jun. In c-Jun-deficient cells, JunD phosphorylation was increased, and expression of a dominant-active JNKK2-JNK1 transgene further increased lung tumor formation. Strikingly, deletion of JunD completely abolished Ras-driven lung tumorigenesis. This work identifies JunD, not c-Jun, as the crucial substrate of JNK signaling and oncogene required for Ras-induced lung cancer.

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.

An autophagy-related model of 4 key genes for predicting prognosis of patients with laryngeal cancer.

Autophagy, a major cause of cancer-related death, is correlated with the pathogenesis of various diseases including cancers. Our study aimed to develop an autophagy-related model for predicting prognosis of patients with laryngeal cancer.We analyzed the correlation between expression profiles of autophagy-related genes (ARGs) and clinical outcomes in 111 laryngeal cancer patients from The Cancer Genome Atlas (TCGA). Afterward, gene functional enrichment analyses of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to find the major biological attributes. Univariate Cox regression analyses and multivariate Cox regression analyses were performed to screen ARGs whose expression profiles were significantly associated with laryngeal cancer patients overall survival (OS). Furthermore, to provide the doctors and patients with a quantitative method to perform an individualized survival prediction, we constructed a prognostic nomogram.Thirty eight differentially expressed ARGs were screened out in laryngeal cancer patients through the TCGA database. Related functional enrichments may act as tumor-suppressive roles in the tumorigenesis of laryngeal cancer. Subsequently, 4 key prognostic ARGs (IKBKB, ST13, TSC2, and MAP2K7) were identified from all ARGs by the Cox regression model, which significantly correlated with OS in laryngeal cancer. Furthermore, the risk score was constructed, which significantly divided laryngeal cancer patients into high- and low-risk groups. Integrated with clinical characteristics, gender, N and the risk score are very likely associated with patients OS. A prognostic nomogram of ARGs was constructed using the Cox regression model.Our study could provide a valuable prognostic model for predicting the prognosis of laryngeal cancer patients and a new understanding of autophagy in laryngeal cancer.

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.

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.

Phosphatidic acid promotes the activation and plasma membrane localization of MKK7 and MKK9 in response to salt stress.

Phosphatidic acid (PA) is a lipid secondary messenger involved in intracellular signaling in eukaryotes. It has been confirmed that PA mediates salt stress signaling by promoting activation of Mitogen-activated Protein Kinase 6 (MPK6) which phosphorylates Na+/H+ antiporter SOS1. However, the MPK6-upstream kinases and their relationship to PA remain unclear. Here, we found that, among the six tested Arabidopsis Mitogen-activated Protein Kinase Kinases (MKKs), PA specifically bound to MKK7 and MKK9 which phosphorylate MPK6, and promoted the activation of MKK7/MKK9. Based on phenotypic and physiological analyses, we found that MKK7 and MKK9 positively regulate Arabidopsis salt tolerance and are functionally redundant. NaCl treatment can induce significant increase in MKK7/MKK9 activities, and this depends, in part, on the Phospholipase Dα1 (PLDα1). MKK7 and MKK9 also mediate the NaCl-induced activation of MPK6. Furthermore, PA or NaCl treatment could induce translocation of MKK7/MKK9 to the plasma membrane, whereas this translocation disappeared in pldα1. These results indicate that PA binds to MKK7 and MKK9, increases their kinase activity and plasma membrane localization during Arabidopsis response to salt stress. Together with the PA-MPK6-SOS1 pathway identified previously, this mechanism may maximize the signal transduction efficiency, providing novel insights into the link between lipid signaling and MAPK cascade.

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.

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.

Two novel MKKs (MKK4 and MKK7) from Ctenopharyngodon idella are involved in the intestinal immune response to bacterial muramyl dipeptide challenge.

Mitogen-activated protein kinase kinases (MKKs) are a class of evolutionarily conserved signalling intermediates of the MAPK signalling pathway that can be activated by a diverse range of pathogenic stimuli and are crucial for the regulation of host immune defence. In this study, two fish MKK genes (CiMKK4 and CiMKK7) were first identified and characterized from grass carp (Ctenopharyngodon idella). Similar to other reported MKKs, the present CiMKK4 and CiMKK7 contained a conserved serine/threonine protein kinase (S_TKc) domain and a canonical dual phosphorylation motif. Quantitative real-time PCR results showed that CiMKK4 and CiMKK7 were broadly transcribed in all selected tissues and developmental stages of grass carp. The mRNA expression levels of CiMKK4 and CiMKK7 in the intestine were significantly induced by bacterial muramyl dipeptide (MDP) challenge in a time-dependent manner (P < 0.01). Additionally, the stimulatory effects of bacterial MDP on CiMKK4 and CiMKK7 expression in the intestine were inhibited by the bioactive dipeptide ß-alanyl-l-histidine (carnosine) and alanyl-glutamine (Ala-Gln) (P < 0.05). Moreover, overexpression analysis revealed that CiMKK4 and CiMKK7 were localized throughout the entire cell and could significantly enhance AP-1 reporter gene activation in HEK293T cells. Taken together, these results provide the first experimental demonstration that CiMKK4 and CiMKK7 are involved in the intestinal immune response to MDP challenge in C. idella, which may provide new insight into the bacterial-induced intestinal inflammation of bony fishes.

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.

Covalent Docking Identifies a Potent and Selective MKK7 Inhibitor.

The c-Jun NH2-terminal kinase (JNK) signaling pathway is central to the cell response to stress, inflammatory signals, and toxins. While selective inhibitors are known for JNKs and for various upstream MAP3Ks, no selective inhibitor is reported for MKK7--one of two direct MAP2Ks that activate JNK. Here, using covalent virtual screening, we identify selective MKK7 covalent inhibitors. We optimized these compounds to low-micromolar inhibitors of JNK phosphorylation in cells. The crystal structure of a lead compound bound to MKK7 demonstrated that the binding mode was correctly predicted by docking. We asserted the selectivity of our inhibitors on a proteomic level and against a panel of 76 kinases, and validated an on-target effect using knockout cell lines. Lastly, we show that the inhibitors block activation of primary mouse B cells by lipopolysaccharide. These MKK7 tool compounds will enable better investigation of JNK signaling and may serve as starting points for therapeutics.

Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin.

Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/ß3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.

Truncation- and motif-based pan-cancer analysis reveals tumor-suppressing kinases.

A major challenge in cancer genomics is identifying "driver" mutations from the many neutral "passenger" mutations within a given tumor. To identify driver mutations that would otherwise be lost within mutational noise, we filtered genomic data by motifs that are critical for kinase activity. In the first step of our screen, we used data from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas to identify kinases with truncation mutations occurring within or before the kinase domain. The top 30 tumor-suppressing kinases were aligned, and hotspots for loss-of-function (LOF) mutations were identified on the basis of amino acid conservation and mutational frequency. The functional consequences of new LOF mutations were biochemically validated, and the top 15 hotspot LOF residues were used in a pan-cancer analysis to define the tumor-suppressing kinome. A ranked list revealed MAP2K7, an essential mediator of the c-Jun N-terminal kinase (JNK) pathway, as a candidate tumor suppressor in gastric cancer, despite its mutational frequency falling within the mutational noise for this cancer type. The majority of mutations in MAP2K7 abolished its catalytic activity, and reactivation of the JNK pathway in gastric cancer cells harboring LOF mutations in MAP2K7 or the downstream kinase JNK suppressed clonogenicity and growth in soft agar, demonstrating the functional relevance of inactivating the JNK pathway in gastric cancer. Together, our data highlight a broadly applicable strategy to identify functional cancer driver mutations and define the JNK pathway as tumor-suppressive in gastric cancer.

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.

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.

Involvement of mitogen activated kinase kinase 7 intracellular signalling pathway in Sunitinib-induced cardiotoxicity.

The tyrosine kinase inhibitor Sunitinib is used to treat cancer and is linked to severe adverse cardiovascular events. Mitogen activated kinase kinase 7 (MKK7) is involved in the development of cardiac injury and is a component of the c-Jun N-terminal kinase (JNK) signal transduction pathway. Apoptosis signal-regulating kinase 1 (ASK1) is the upstream activator of MKK7 and is specifically inhibited by 2,7-dihydro-2,7-dioxo-3H-naphtho[1,2,3-de]quinoline-1-carboxylic acid ethyl ester (NQDI-1). This study investigates the role of ASK1, MKK7 and JNK during Sunitinib-induced cardiotoxicity. Infarct size were measured in isolated male Sprague-Dawley rat Langendorff perfused hearts treated for 125 min with Sunitinib in the presence and absence of NQDI-1. Left ventricular cardiac tissue samples were analysed by qRT-PCR for MKK7 mRNA expression and cardiotoxicity associated microRNAs (miR-1, miR-27a, miR-133a and miR-133b) or Western blot analysis to measure ASK1/MKK7/JNK phosphorylation. Administration of Sunitinib (1 µM) during Langendorff perfusion resulted in increased infarct size, increased miR-133a expression, and decreased phosphorylation of the ASK1/MKK7/JNK pathway compared to control. Co-administration of NQDI-1 (2.5 µM) attenuated the increased Sunitinib-induced infarct size, reversed miR-133a expression and restored phosphorylated levels of ASK1/MKK7/JNK. These findings suggest that the ASK1/MKK7/JNK intracellular signalling pathway is important in Sunitinib-induced cardiotoxicity. The anti-cancer properties of Sunitinib were also assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Sunitinib significantly decreased the cell viability of human acute myeloid leukemia 60 cell line (HL60). The combination of Sunitinib (1 nM-10 µM) with NQDI-1 (2.5 µM) enhanced the cancer-fighting properties of Sunitinib. Investigations into the ASK1/MKK7/JNK transduction pathway could lead to development of cardioprotective adjunct therapy, which could prevent Sunitinib-induced cardiac injury.