The kinase p38α MAPK (p38α) plays a pivotal role in many biological processes. p38α is activated by canonical upstream kinases that phosphorylate the activation region. The purpose of our study was to determine whether such activation may depend on redox-sensing cysteines within p38α. p38α was activated and formed a disulfide-bound heterodimer with MAP2K3 (MKK3) in rat cardiomyocytes and isolated hearts exposed to H2O2 This disulfide heterodimer was sensitive to reduction by mercaptoethanol and was enhanced by the thioredoxin-reductase inhibitor auranofin. We predicted that Cys-119 or Cys-162 of p38α, close to the known MKK3 docking domain, were relevant for these redox characteristics. The C119S mutation decreased whereas the C162S mutation increased the dimer formation, suggesting that these two Cys residues act as vicinal thiols, consistent with C119S/C162S being incapable of sensing H2O2 Similarly, disulfide heterodimer formation was abolished in H9C2 cells expressing both MKK3 and p38α C119S/C162S and subjected to simulated ischemia and reperfusion. However, the p38α C119S/C162S mutants did not exhibit appreciable alteration in activating dual phosphorylation. In contrast, the anti-inflammatory agent 10-nitro-oleic acid (NO2-OA), a component of the Mediterranean diet, reduced p38α activation and covalently modified Cys-119/Cys-162, probably obstructing MKK3 access. Moreover, NO2-OA reduced the dephosphorylation of p38α by hematopoietic tyrosine phosphatase (HePTP). Furthermore, steric obstruction of Cys-119/Cys-162 by NO2-OA pretreatment in Langendorff-perfused murine hearts prevented the p38-MKK3 disulfide dimer formation and attenuated H2O2-induced contractile dysfunction. Our findings suggest that cysteine residues within p38α act as redox sensors that can dynamically regulate the association between p38 and MKK3.

Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat.

Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition.

Zinc chloride is known to be effective in combatting hepatitis A virus (HAV) infection, and zinc ions seem to be especially involved in Toll-like receptor (TLR) signaling pathways. In the present study, we examined this involvement in human hepatoma cell lines using a human TLR signaling target RT-PCR array. We also observed that zinc chloride inhibited mitogen-activated protein kinase kinase 3 (MAP2K3) expression, which could downregulate HAV replication in human hepatocytes. It is possible that zinc chloride may inhibit HAV replication in association with its inhibition of MAP2K3. In that regard, this study set out to determine whether MAP2K3 could be considered a modulating factor in the development of the HAV pathogen-associated molecular pattern (PAMP) and its triggering of interferon-β production. Because MAP2K3 seems to play a role in antiviral immunity against HAV infection, it is a promising target for drug development. The inhibition of MAP2K3 may also prevent HAV patients from developing a severe hepatitis A infection.

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

Mitogen-activated protein kinase phosphatase 3 (MKP-3) is a negative regulator of extracellular signal-related kinase signaling. Our laboratory recently demonstrated that MKP-3 plays an important role in obesity-related hyperglycemia by promoting hepatic glucose output. This study shows that MKP-3 deficiency attenuates body weight gain induced by a high-fat diet (HFD) and protects mice from developing obesity-related hepatosteatosis. Triglyceride (TG) contents are dramatically decreased in the liver of MKP-3(-/-) mice fed an HFD compared with wild-type (WT) controls. The absence of MKP-3 also reduces adiposity, possibly by repressing adipocyte differentiation. In addition, MKP-3(-/-) mice display increased energy expenditure, enhanced peripheral glucose disposal, and improved systemic insulin sensitivity. We performed global phosphoproteomic studies to search for downstream mediators of MKP-3 action in liver lipid metabolism. Our results revealed that MKP-3 deficiency increases the phosphorylation of histone deacetylase (HDAC) 1 on serine 393 by 3.3-fold and HDAC2 on serine 394 by 2.33-fold. Activities of HDAC1 and 2 are increased in the livers of MKP-3(-/-) mice fed an HFD. Reduction of HDAC1/2 activities is sufficient to restore TG content of MKP-3(-/-) primary hepatocytes to a level similar to that in WT cells.

Protein-protein interaction is one of the crucial ways to decipher the functions of proteins and to understand their role in complex pathways at cellular level. Such a protein-protein interaction network in many crop plants remains poorly defined owing largely to the involvement of high costs, requirement for state of the art laboratory, time and labour intensive techniques. Here, we employed computational docking using ZDOCK and RDOCK programmes to identify interaction network between members of Oryza sativa mitogen activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK). The 3-dimentional (3-D) structures of five MAPKKs and eleven MAPKs were determined by homology modelling and were further used as input for docking studies. With the help of the results obtained from ZDOCK and RDOCK programmes, top six possible interacting MAPK proteins were predicted for each MAPKK. In order to assess the reliability of the computational prediction, yeast two-hybrid (Y2H) analyses were performed using rice MAPKKs and MAPKs. A direct comparison of Y2H assay and computational prediction of protein interaction was made. With the exception of one, all the other MAPKK-MAPK pairs identified by Y2H screens were among the top predictions by computational dockings. Although, not all the predicted interacting partners could show interaction in Y2H, yet, the harmony between the two approaches suggests that the computational predictions in the present work are reliable. Moreover, the present Y2H analyses per se provide interaction network among MAPKKs and MAPKs which would shed more light on MAPK signalling network in rice.

Hypoxia, or low oxygen tension, is a hallmark of the tumor microenvironment. The hypoxia-inducible factor-1α (HIF-1α) subunit plays a critical role in the adaptive cellular response of hypoxic tumor cells to low oxygen tension by activating gene-expression programs that control cancer cell metabolism, angiogenesis, and therapy resistance. Phosphorylation is involved in the stabilization and regulation of HIF-1α transcriptional activity. HIF-1α is activated by several factors, including the mitogen-activated protein kinase (MAPK) superfamily. MAPK phosphatase 3 (MKP-3) is a cytoplasmic dual-specificity phosphatase specific for extracellular signal-regulated kinase 1/2 (Erk1/2). Recent evidence indicates that hypoxia increases the endogenous levels of both MKP-3 mRNA and protein. However, its role in the response of cells to hypoxia is poorly understood. Herein, we demonstrated that small-interfering RNA (siRNA)-mediated knockdown of MKP-3 enhanced HIF-1α (not HIF-2α) levels. Conversely, MKP-3 overexpression suppressed HIF-1α (not HIF-2α) levels, as well as the expression levels of hypoxia-responsive genes (LDHA, CA9, GLUT-1, and VEGF), in hypoxic colon cancer cells. These findings indicated that MKP-3, induced by HIF-1α in hypoxia, negatively regulates HIF-1α protein levels and hypoxia-responsive genes. However, we also found that long-term hypoxia (>12 h) induced proteasomal degradation of MKP-3 in a lactic acid-dependent manner. Taken together, MKP-3 expression is modulated by the hypoxic conditions prevailing in colon cancer, and plays a role in cellular adaptation to tumor hypoxia and tumor progression. Thus, MKP-3 may serve as a potential therapeutic target for colon cancer treatment.

The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are poorly understood in crops. Here, microscopic, phenotypic, proteomic, and biochemical analyses revealed that roots of independent transcription activator-like effector nuclease (TALEN)-based knockout lines of barley (Hordeum vulgare L.) MAPK 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of extracellular hyphae was considerably deregulated, mycelia development was less efficient, and number of appressoria-like structures and their penetration potential were substantially reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild-type (WT), but ROS production was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with elevated abundance of catalase (CAT) and ascorbate peroxidase (APX). Moreover, differential proteomic analysis revealed downregulation of several defense-related proteins in WT, and the upregulation of pathogenesis-related protein 1 (PR-1) and cysteine proteases in HvMPK3 KO lines. Proteins involved in suberin formation, such as peroxidases, lipid transfer proteins (LTPs), and the GDSL esterase/lipase (containing "GDSL" aminosequence motif) were differentially regulated in HvMPK3 KO lines after F. graminearum inoculation. Consistent with proteomic analysis, microscopic observations showed enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to barley root resistance against Fusarium root rot.

Mitogen-activated Protein Kinase Phosphatase 3 (MKP-3) has been involved in the negative regulation of insulin signaling. The absence of MKP-3 is also associated with reduced adiposity, increased energy expenditure and improved insulin sensitivity. The MKP-3 is known as the main Erk1/2 phosphatase and FoxO1 activator, which has repercussions on the gluconeogenesis pathway and hyperglycemia in obese mice. Recently, we showed that MKP-3 overexpression decreases FoxO1 phosphorylation in the hypothalamus of lean mice. However, the hypothalamic interaction between MKP-3 and FoxO1 during obesity was not investigated yet. Here, the MKP-3 expression and the effects on food intake and energy expenditure, were investigated in high-fat diet-induced obese mice. The results indicate that obesity in mice increased the MKP-3 protein content in the hypothalamus. This hypothalamic upregulation led to an increase of food intake, adiposity, and body weight. Furthermore, the obese mice with increased MKP-3 showed an insulin signaling impairment with reduction of insulin-induced FoxO1 and Erk1/2 phosphorylation in the hypothalamus. Moreover, a bioinformatics analysis of data demonstrated that hypothalamic MKP-3 mRNA levels were positively correlated with body weight and negatively correlated to oxygen consumption (VO2) in BXD mice. Taken together, our study reports that obesity is associated with increased protein levels of hypothalamic MKP-3, which is related to the reduction of FoxO1 and Erk1/2 phosphorylation in the hypothalamus as well as to an increase in body weight and a reduction in energy expenditure.

microRNA 21 (miR-21) has been demonstrated to be significantly elevated in many types of cancers, including the hepatocellular carcinoma (HCC). In the present study, we investigated the role of miR-21 in HCC by identifying its novel targets, as well as its underlying molecular mechanism.

Cerebral cavernous malformation 2 (CCM2) functions as an adaptor protein implicated in various biological processes. By interacting with the mitogen-activated protein kinase MEKK3, CCM2 either mediates the activation of MEKK3 signaling in response to osmotic stress or negatively regulates MEKK3 signaling, which is important for normal cardiovascular development. However, the molecular basis governing CCM2-MEKK3 interaction is largely unknown. Here we report the crystal structure of the CCM2 C-terminal part (CCM2ct) containing both the five-helix domain (CCM2cts) and the following C-terminal tail. The end of the C-terminal tail forms an isolated helix, which interacts intramolecularly with CCM2cts. By biochemical studies we identified the N-terminal amphiphilic helix of MEKK3 (MEKK3-nhelix) as the essential structural element for CCM2ct binding. We further determined the crystal structure of CCM2cts-MEKK3-nhelix complex, in which MEKK3-nhelix binds to the same site of CCM2cts for CCM2ct intramolecular interaction. These findings build a structural framework for understanding CCM2ct-MEKK3 molecular recognition.

Mitogen-activated protein kinase kinase 3 (MKK3) is a dual threonine/tyrosine protein kinase that regulates inflammation, proliferation and apoptosis through specific phosphorylation and activation of the p38 mitogen-activated protein kinase. However, the role of MKK3 beyond p38-signaling remains elusive. Recently, we reported a protein-protein interaction (PPI) network of cancer-associated genes, termed OncoPPi, as a resource for the scientific community to generate new biological models. Analysis of the OncoPPi connectivity identified MKK3 as one of the major hub proteins in the network. Here, we show that MKK3 interacts with a large number of proteins critical for cell growth and metabolism, including the major oncogenic driver MYC. Multiple complementary approaches were used to demonstrate the direct interaction of MKK3 with MYC in vitro and in vivo. Computational modeling and experimental studies mapped the interaction interface to the MYC helix-loop-helix domain and a novel 15-residue MYC-binding motif in MKK3 (MBM). The MBM in MKK3 is distinct from the known binding sites for p38 or upstream kinases. Functionally, MKK3 stabilized MYC protein, enhanced its transcriptional activity and increased expression of MYC-regulated genes. The defined MBM peptide mimicked the MKK3 effect in promoting MYC activity. Together, the exploration of OncoPPi led to a new biological model in which MKK3 operates by two distinct mechanisms in cellular regulation through its phosphorylation of p38 and its activation of MYC through PPI.

To identify signaling pathways activated by oxycodone self-administration (SA), Sprague-Dawley rats self-administered oxycodone for 20 days using short-(ShA, 3 h) and long-access (LgA, 9 h) paradigms. Animals were euthanized 2 h after SA cessation and dorsal striata were used in post-mortem molecular analyses. LgA rats escalated their oxycodone intake and separated into lower (LgA-L) or higher (LgA-H) oxycodone takers. LgA-H rats showed increased striatal protein phosphorylation of ERK1/2 and MSK1/2. Histone H3, phosphorylated at serine 10 and acetylated at lysine 14 (H3S10pK14Ac), a MSK1/2 target, showed increased abundance only in LgA-H rats. RT-qPCR analyses revealed increased AMPA receptor subunits, GluA2 and GluA3 mRNAs, in the LgA-H rats. GluA3, but not GluA2, mRNA expression correlated positively with changes in pMSK1/2 and H3S10pK14Ac. These findings suggest that escalated oxycodone SA results in MSK1/2-dependent histone phosphorylation and increases in striatal gene expression. These observations offer potential avenues for interventions against oxycodone addiction.

The objective of the present study was to investigate if MAPK can be activated by a non-receptor agonist KCl, which depolarizes membrane to increase intracellular Ca(2+) and contracts cerebral arteries. Rabbit basilar arteries were used in isometric tension and western blot analysis studies. KCl produced a concentration-dependent contraction and an elevation of phospho-MAPK, which can be abolished by nicardipine, a voltage-dependent Ca(2+) channel blocker, and by PD98059 or U0126, MAPK kinase inhibitors. Thus, MAPK can be activated by the elevation of intracellular Ca(2+), independent of the activation of either G-protein coupled receptors or receptor tyrosine kinase. KCl which not only depolarizes membrane potentials, opens voltage-dependent Ca(2+), and increases intracellular Ca(2+), but also, probably by elevation of intracellular Ca(2+), triggers the activation of MAPK which seems responsible for a predominant part of the contraction of KCl in the rabbit basilar arteries.

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Multiple reports have elucidated that microRNAs are promising biomarkers for AD diagnosis and treatment. Herein, the effect of miR-191-5p on microglial cell injury and the underlying mechanism were explored. APP/PS1 transgenic mice were utilized to establish mouse model of AD. Amyloid-β protein 1-42 (Aβ1-42)-treated microglia were applied to establish in vitro cell model of AD. MiR-191-5p expression in hippocampus and microglia was measured by reverse transcription quantitative polymerase chain reaction. The viability and apoptosis of microglia were evaluated by Cell Counting Kit-8 assays and flow cytometry analyses, respectively. The binding relationship between miR-191-5p and its downstream target mitogen-activated protein kinase kinase kinase 12 (Map3k12) was determined by luciferase reporter assays. Pathological degeneration of hippocampus was tested using hematoxylin-eosin staining and Nissl staining. Aβ expression in hippocampus was examined via immunohistochemistry. In this study, miR-191-5p was downregulated in Aβ1-42-stimulated microglia and hippocampal tissues of APP/PS1 mice. MiR-191-5p overexpression facilitated cell viability and inhibited apoptosis rate of Aβ1-42-treated microglia. Mechanically, miR-191-5p targeted Map3k12 3'-untranslated region to downregulate Map3k12 expression. MiR-191-5p inhibited Aβ1-42-induced microglial cell injury and inactivated the MAPK signaling by downregulating Map3k12. Overall, miR-191-5p alleviated Aβ1-42-induced microglia cell injury by targeting Map3k12 to inhibit the MAPK signaling pathway in microglia.

Insulin plays an important role in the control of hepatic glucose production. Insulin resistant states are commonly associated with excessive hepatic glucose production, which contributes to both fasting hyperglycaemia and exaggerated postprandial hyperglycaemia. In this regard, increased activity of phosphatases may contribute to the dysregulation of gluconeogenesis. Mitogen-activated protein kinase phosphatase-3 (MKP-3) is a key protein involved in the control of gluconeogenesis. MKP-3-mediated dephosphorylation activates FoxO1 (a member of the forkhead family of transcription factors) and subsequently promotes its nuclear translocation and binding to the promoters of gluconeogenic genes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). In this study, we investigated the effects of exercise training on the expression of MKP-3 and its interaction with FoxO1 in the livers of obese animals. We found that exercised obese mice had a lower expression of MKP-3 and FoxO1/MKP-3 association in the liver. Further, the exercise training decreased FoxO1 phosphorylation and protein levels of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and gluconeogenic enzymes (PEPCK and G6Pase). These molecular results were accompanied by physiological changes, including increased insulin sensitivity and reduced hyperglycaemia, which were not caused by reductions in total body mass. Similar results were also observed with oligonucleotide antisense (ASO) treatment. However, our results showed that only exercise training could reduce an obesity-induced increase in HNF-4α protein levels while ASO treatment alone had no effect. These findings could explain, at least in part, why additive effects of exercise training treatment and ASO treatment were not observed. Finally, the suppressive effects of exercise training on MKP-3 protein levels appear to be related, at least in part, to the reduced phosphorylation of Extracellular signal-regulated kinases (ERK) in the livers of obese mice.

The mitogen-activation protein kinase ERK2 is tightly regulated by multiple phosphatases, including those of the kinase interaction motif (KIM) PTP family (STEP, PTPSL and HePTP). Here, we use small angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) to show that the ERK2:STEP complex is compact and that residues outside the canonical KIM motif of STEP contribute to ERK2 binding. Furthermore, we analyzed the interaction of PTPSL with ERK2 showing that residues outside of the canonical KIM motif also contribute to ERK2 binding. The integration of this work with previous studies provides a quantitative and structural map of how the members of a single family of regulators, the KIM-PTPs, differentially interact with their corresponding MAPKs, ERK2 and p38α.

The growing demands for gene therapy have encouraged development of safe and efficient lentiviral vector (LV) preparation. While much progress has been made in this field, it remains to be explored how to boost its production from producer cells. This paper reports that transient co-expression of RelA or several mitogen-activated protein kinase kinase kinases (MAP3Ks) with packaging constructs can potently enhance LV production in HEK293T producer cells. Adding in transfection a small amount of effector plasmid is sufficient to achieve 3- to 4-fold enhancement, which can further be augmented by co-expression of IκB kinase 2 or HIV Tat. It is also shown that expression of RelA or MAP3K1 can increase LV production in HEK293T/17SF cells grown in suspension. These results indicate that stimulation of intracellular signaling pathways in producer cells represents a powerful means for enhancing LV production.

Increased proliferation, inflammation, and endothelial microparticle (EMP) generation in the pulmonary vasculature lead to endothelial dysfunction in pulmonary hypertension (PH). Interestingly, MK2, a downstream of p38MAPK, is a central regulator of inflammation, proliferation, and EMP generation in cardiovascular diseases. However, the role of MK2 in pulmonary endothelial dysfunction remains unexplored.