Mitogen-activated Protein Kinase Subfamilies Research Articles

Deregulated JNK signaling enhances apoptosis during hyperthermia

Purpose c-Jun N-terminal kinases (JNKs) comprise a subfamily of mitogen-activated protein kinases (MAPKs). The JNK group is known to be activated by a variety of stimuli. However, the molecular mechanism underlying heat-induced JNK activation is largely unknown. The aim of this study was to clarify how JNK activity is stimulated by heat. Methods and materials The expression levels of various MAPK members in HeLa cells, with or without hyperthermia treatment, were evaluated via western blotting. The kinase activity of MAPK members was assessed through in vitro kinase assays. Cell death was assessed in the absence or presence of siRNAs targeting MAPK-related members. Results Hyperthermia decreased the levels of MAP3Ks, such as ASK1 and MLK3 which are JNK kinase kinase members, but not those of the downstream MAP2K/SEK1 and MAPK/JNK. Despite the reduced or transient phosphorylation of ASK1, MLK3, or SEK1, downstream JNK was phosphorylated in a temperature-dependent manner. In vitro kinase assays demonstrated that heat did not directly stimulate SEK1 or JNK. However, the expression levels of DUSP16, a JNK phosphatase, were decreased upon hyperthermia treatment. DUSP16 knockdown enhanced the heat-induced activation of ASK1–SEK1–JNK pathway and apoptosis. Conclusion JNK was activated in a temperature-dependent manner despite reduced or transient phosphorylation of the upstream MAP3K and MAP2K. Hyperthermia-induced degradation of DUSP16 may induce activation of the ASK1–SEK1–JNK pathway and subsequent apoptosis.

On the Therapeutic Potential of ERK4 in Triple-Negative Breast Cancer.

ERK3 and ERK4 define a distinct and understudied subfamily of mitogen-activated protein kinases (MAPKs). Little is known about the physiological roles of these atypical MAPKs and their association with human diseases. Interestingly, accumulating evidence points towards a role for ERK3 and ERK4 signaling in the initiation and progression of various types of cancer. Notably, a recent study reported that ERK4 is expressed in a subset of triple-negative breast cancer (TNBC) cell lines and that this expression is critical for AKT activation and for sustaining TNBC cell proliferation in vitro and tumor growth in mice. The authors also showed that depletion of ERK4 sensitizes TNBC cells to phosphatidylinositol-3-kinase (PI3K) inhibitors. They concluded that ERK4 is a promising therapeutic target for TNBC and has potential for combination therapy with PI3K inhibitors. Here, we raise concerns about the cellular models and experimental approaches used in this study, which compromise the conclusions on the oncogenic role of ERK4 in TNBC.

Evolution of mitogen-activated protein kinase family and their immune function in Apostichopus japonicus

The mitogen-activated protein kinase family plays an important role in cell differentiation, growth, proliferation, and survival. However, the current research on the mitogen-activated protein kinase (MAPK) family in invertebrates is limited to the individual gene, and the analysis has not been conducted at the family level. In the present study, echinoderm MAPK family was identified by genomic screening, and five members, including three ERK subfamily members, one c-Jun N-terminal kinase (JNK) subfamily, and one p38-MAPK member were detected. Phylogenetic analysis showed that three MAPK subfamilies were separated into three separated clusters, and ERK subfamily appeared earlier than the other two subfamilies. Synteny analysis revealed that the p38 subfamily might be derived from the continuous gene duplication events of MAPK14 subfamily in invertebrates, which displayed genome expansion via gene duplication in vertebrates. The role of MAPK family in echinoderm immune defense was determined by investigating the expression profiles of MAPKs in Vibrio splendidus-challenged Apostichopus japonicus and LPS-exposed coelomocytes. The result showed that five MAPK members displayed induced expression profiles both in vitro and in vivo, and the peak expression was detected at different time points. Our study provides new insights into the evolutionary history of the MAPK family and show the similar immune function among MAPK members.

Comparison of laxative effects of fermented soybeans (Cheonggukjang) containing toxins and biogenic amines against loperamide-induced constipation mouse model.

BACKGROUND/OBJECTIVES Cheonggukjang is a traditional fermented soybean paste with significant health-promoting effects. On the other hand, there have been insufficient studies on the safety and efficacy of Cheonggukjang, which is produced using traditional methods containing toxins and biogenic amines (BAs). This study compared the laxative effect of Cheonggukjang, containing high or low levels of toxins and BAs (HTBC or LTBC) in a loperamide (Lop)-induced constipation mouse model.MATERIALS/METHODSTo induce constipation, Lop (5 mg/kg) was administered orally to ICR mice twice a day for 4 days, and the dose was increased to 8 mg/kg after a 3-day rest period. Cheonggukjang (500 mg/kg, HTBC, or LTBC respectively) was administered for four weeks before the Lop treatment.RESULTSThe number of stools, fecal weight, water contents, gastrointestinal transit, and histological alterations were recovered significantly in the HTBC or LTBC groups. HTBC and LTBC administration did not induce significant changes in body weight, dietary intake, and behavior. The opioid-receptor downstream signaling pathway in colon tissues was also evaluated. The c-Kit, stem cell kinase, and mitogen-activated protein kinases subfamilies, including extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinases, and p38, were all downregulated in the HTBC or LTBC-administered mice colon compared to the Lop group.CONCLUSIONThese results show that Cheonggukjang, containing high levels of toxins and BAs, have a similar laxative effect in a mouse model of Lop-induced constipation.

Stress Relief Techniques: p38 MAPK Determines the Balance of Cell Cycle and Apoptosis Pathways.

Protein signaling networks are formed from diverse and inter-connected cell signaling pathways converging into webs of function and regulation. These signaling pathways both receive and conduct molecular messages, often by a series of post-translation modifications such as phosphorylation or through protein–protein interactions via intrinsic motifs. The mitogen activated protein kinases (MAPKs) are components of kinase cascades that transmit signals through phosphorylation. There are several MAPK subfamilies, and one subfamily is the stress-activated protein kinases, which in mammals is the p38 family. The p38 enzymes mediate a variety of cellular outcomes including DNA repair, cell survival/cell fate decisions, and cell cycle arrest. The cell cycle is itself a signaling system that precisely controls DNA replication, chromosome segregation, and cellular division. Another indispensable cell function influenced by the p38 stress response is programmed cell death (apoptosis). As the regulators of cell survival, the BCL2 family of proteins and their dynamics are exquisitely sensitive to cell stress. The BCL2 family forms a protein–protein interaction network divided into anti-apoptotic and pro-apoptotic members, and the balance of binding between these two sides determines cell survival. Here, we discuss the intersections among the p38 MAPK, cell cycle, and apoptosis signaling pathways.

Identification, Characterization, and Expression Analysis Reveal Diverse Regulated Roles of Three MAPK Genes in Chlamys farreri Under Heat Stress.

Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in all eukaryotic organisms, participating growth and development, as well as stress response. In the present study, three MAPK genes were successfully identified from the genome of Chlamys farreri, respectively, named CfERK1/2, CfJNK, and Cfp38, and only one copy of ERK, JNK, and p38 were detected. Domain analysis indicated that CfMAPKs possessed the typical domains, including S_TKc, Pkinase, and PKc_like domain. Phylogenetic analysis showed that three CfMAPKs of MAPK subfamilies exists in the common ancestor of vertebrates and invertebrates. All CfMAPKs specifically expressed during larval development and in adult tissues, and the expression level of CfERK1/2 and Cfp38 was apparently higher than that of CfJNK. Under heat stress, the expression of CfERK1/2 and Cfp38 were significantly downregulated and then upregulated in four tissues, while the expression of CfJNK increased in all tissues; these different expression patterns suggested a different molecular mechanism of CfMAPKs for bivalves to adapt to temperature changes. The diversity of CfMAPKs and their specific expression patterns provide valuable information for better understanding of the functions of MAPK cascades in bivalves.

A phylogenetic study of the members of the MAPK and MEK families across Viridiplantae.

Protein phosphorylation is regulated by the activity of enzymes generically known as kinases. One of those kinases is Mitogen-Activated Protein Kinases (MAPK), which operate through a phosphorylation cascade conformed by members from three related protein kinase families namely MAPK kinase kinase (MEKK), MAPK kinase (MEK), and MAPK; these three acts hierarchically. Establishing the evolution of these proteins in the plant kingdom is an interesting but complicated task because the current MAPK, MAPKK, and MAPKKK subfamilies arose from duplications and subsequent sub-functionalization during the early stage of the emergence of Viridiplantae. Here, an in silico genomic analysis was performed on 18 different plant species, which resulted in the identification of 96 genes not previously annotated as components of the MAPK (70) and MEK (26) families. Interestingly, a deeper analysis of the sequences encoded by such genes revealed the existence of putative domains not previously described as signatures of MAPK and MEK kinases. Additionally, our analysis also suggests the presence of conserved activation motifs besides the canonical TEY and TDY domains, which characterize the MAPK family.

The MAPK Pathway-Based Drug Therapeutic Targets in Pituitary Adenomas.

Mitogen-activated protein kinases (MAPKs) include ERK, p38, and JNK MAPK subfamilies, which are crucial regulators of cellular physiology, cell pathology, and many diseases including cancers. For the MAPK signaling system in pituitary adenomas (PAs), the activation of ERK signaling is generally thought to promote cell proliferation and growth; whereas the activations of p38 and JNK signaling are generally thought to promote cell apoptosis. The role of MAPK in treatment of PAs is demonstrated through the effects of currently used medications such as somatostatin analogs such as SOM230 and OCT, dopamine agonists such as cabergoline and bromocriptine, and retinoic acid which inhibit the MAPK pathway. Further, there are potential novel therapies based on putative molecular targets of the MAPK pathway, including 18beta-glycyrrhetinic acid (GA), dopamine-somatostatin chimeric compound (BIM-23A760), ursolic acid (UA), fulvestrant, Raf kinase inhibitory protein (RKIP), epidermal growth factor pathway substrate number 8 (Eps8), transmembrane protein with EGF-like and two follistatin-like domains (TMEFF2), cold inducible RNA-binding protein (CIRP), miR-16, and mammaliansterile-20-like kinase (MST4). The combined use of ERK inhibitor (e.g., SOM230, OCT, or dopamine) plus p38 activator (e.g., cabergoline, bromocriptine, and fulvestrant) and/or JNK activator (e.g., UA), or the development of single drug (e.g., BIM-23A760) to target both ERK and p38 or JNK pathways, might produce better anti-tumor effects on PAs. This article reviews the advances in understanding the role of MAPK signaling in pituitary tumorigenesis, and the MAPK pathway-based potential therapeutic drugs for PAs.

Commonly Used Anesthesia/Euthanasia Methods for Brain Collection Differentially Impact MAPK Activity in Male and Female C57BL/6 Mice.

The mitogen-activated protein kinases (MAPKs) are a family of protein kinases that regulate crucial neuronal functions such as neuronal differentiation, proliferation, and apoptosis through phosphorylation of subsequent protein kinases. The three classical MAPK subfamilies, extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 kinase have been linked to various neurological disorders often in conjunction with activation of a wide range of G protein-coupled receptors and receptor tyrosine kinases. Many studies investigating MAPK function in these disorders rely on histochemistry or immunoblotting that require brain isolation following euthanasia. Here, we evaluated to what degree different modes of anesthesia/euthanasia impact MAPK activity in adult male and female C57BL/6 mice. Mice were decapitated following ketamine/xylazine or isoflurane anesthesia, carbon dioxide asphyxiation, or without anesthesia. We selectively chose five brain regions (the prefrontal cortex, the dorsal hippocampus, the dorsal striatum, the nucleus accumbens, and the amygdala) that are heavily implicated in neuropsychiatric disorders. We found that relative to carbon dioxide asphyxiation, the other methods displayed significantly stronger ERK1/2 phosphorylation in select brain regions of male and female mice, with no pronounced sex difference. A similar, yet, less pronounced trend was observed for JNK activity, whereas the choice of euthanasia method did not differentially impact p38 phosphorylation. Our study results reveal how small differences in experimental design may impact whether one will be able to detect drug- or disease-related changes in MAPK activity. These findings are timely in a period where experimental rigor is emphasized to increase reproducibility of research.

Correlation of P38 Mitogen-Activated Protein Kinase Expression to Clinical Stage in Nasopharyngeal Carcinoma.

BACKGROUND:Nasopharyngeal carcinoma (NPC) is uncommon and usually diagnosed at the advanced stage. A subfamily of mitogen-activated protein kinase which is called p38 mitogen-activated protein kinase (MAPK) involved in response to stress, and plays an important role in cell regulation. There is a suggestion that p38 mitogen-activated protein kinase could be a potential biomarker to determine the clinical stage of nasopharyngeal carcinoma.AIM:The aim of this study is for observing and analysing the correlation of p38 mitogen-activated protein kinase expression in regards to nasopharyngeal carcinoma patient’s clinical stage.METHODS:This study involved 126 nasopharyngeal carcinoma patients admitted to Haji Adam Malik General Hospital.RESULTS:The result of this study indicates that nasopharyngeal carcinoma mostly found in the age group 41-60 years, male, non-keratinizing squamous cell carcinoma, and stage IV group. In immunohistochemistry evaluation, most of p38 mitogen-activated protein kinase overexpressed in non-keratinizing squamous cell carcinoma, T3-T4, N2-N3 and clinical stage III-IV. Spearman’s test for categorical correlation yield p-value of < 0.001.CONCLUSION:In conclusion, there is a significant correlation between p38 mitogen-activated protein kinase expression and the clinical stage of nasopharyngeal carcinoma.

Triptolide induces mitochondrial apoptosis through modulating dual specificity phosphatase 1/mitogen-activated protein kinases cascade in osteosarcoma cells.

Due to chemoresistance and metastasis, the overall prognosis of osteosarcoma (OS) has not improved over the last two decades. Exploring novel therapeutic agents that can circumvent theses malignant phenotypes of OS would be essential to improve the survival of OS patients. Triptolide is a unique diterpene triepoxide that possesses potent antitumor activities.However, the effects and mechanism of triptolide on OS cells remain unknown. The effects of triptolide on viability, apoptosis, cell cycle distribution and migratory ability of OS cells were measured using MTT, flow cytometry and wound healing and transwell invasion assays. And an OS tumor xenograft mouse model was produced to further study the in vivo antitumor effects of triptolide. The expression of DUSP1 at the protein and mRNA level in OS cells was detected by western blot and qPCR. We report that triptolide exhibits multidimensional antitumor activities in OS cells, including the induction of apoptosis and G1 phase accumulation, inhibition of cell viability, migration, and invasion. We further demonstrate that triptolide inhibits the expression of dual-specificity protein phosphatase1 (DUSP1) through inhibiting its promoter activity, which causes sustained activation of three subfamilies of mitogen-activated protein kinase (MAPK). And the modulation of DUSP1/MAPK cascade is associated with the apoptosis of OS cells, since the ectopic expression of DUSP1 or the inhibition of MAPK using specific inhibitors can counteract triptolide-induced apoptosis. In addition, triptolide enhances doxorubicin-induced apoptosis. In summary, our study suggests that DUSP1 is an important cellular target of triptolide, and triptolide may be a promising treatment option for OS as a single agent or combined with other chemotherapeutics.

Metal oxide nanoparticles interact with immune cells and activate different cellular responses.

Besides cell death, nanoparticles (Nps) can induce other cellular responses such as inflammation. The potential immune response mediated by the exposure of human lymphoid cells to metal oxide Nps (moNps) was characterized using four different moNps (CeO2, TiO2, Al2O3, and ZnO) to study the three most relevant mitogen-activated protein kinase subfamilies and the nuclear factor kappa-light-chain-enhancer of the activated B-cell inhibitor, IκBα, as well as the expression of several genes by immune cells incubated with these Nps. The moNps activated different signaling pathways and altered the gene expression in human lymphocyte cells. The ZnO Nps were the most active and the release of Zn2+ ions was the main mechanism of toxicity. CeO2 Nps induced the smallest changes in gene expression and in the IκBα protein. The effects of the particles were strongly dependent on the type and concentration of the Nps and on the cell activation status prior to Np exposure.

The Diversification of Evolutionarily Conserved MAPK Cascades Correlates with the Evolution of Fungal Species and Development of Lifestyles.

The fungal kingdom displays an extraordinary diversity of lifestyles, developmental processes, and ecological niches. The MAPK (mitogen-activated protein kinase) cascade consists of interlinked MAPKKK, MAPKK, and MAPK, and collectively such cascades play pivotal roles in cellular regulation in fungi. However, the mechanism by which evolutionarily conserved MAPK cascades regulate diverse output responses in fungi remains unknown. Here we identified the full complement of MAPK cascade components from 231 fungal species encompassing 9 fungal phyla. Using the largest data set to date, we found that MAPK family members could have two ancestors, while MAPKK and MAPKKK family members could have only one ancestor. The current MAPK, MAPKK, and MAPKKK subfamilies resulted from duplications and subsequent subfunctionalization during the emergence of the fungal kingdom. However, the gene structure diversification and gene expansion and loss have resulted in significant diversity in fungal MAPK cascades, correlating with the evolution of fungal species and lifestyles. In particular, a distinct evolutionary trajectory of MAPK cascades was identified in single-celled fungi in the Saccharomycetes. All MAPK, MAPKK, and MAPKKK subfamilies expanded in the Saccharomycetes; genes encoding MAPK cascade components have a similar exon–intron structure in this class that differs from those in other fungi.

The NADase-Negative Variant of the Streptococcus pyogenes Toxin NAD⁺ Glycohydrolase Induces JNK1-Mediated Programmed Cellular Necrosis.

ABSTRACTVirulence factors are often multifunctional and contribute to pathogenesis through synergistic mechanisms. For the human pathogen Streptococcus pyogenes, two factors that act synergistically are the S. pyogenes NAD+ glycohydrolase (SPN) and streptolysin O (SLO). Through distinct mechanisms, SLO forms pores in host cell membranes and translocates SPN into the host cell cytosol. Two natural variants of SPN exist, one that exhibits NADase activity and one that lacks this function, and both versions are translocated and act in concert with SLO to cause an accelerated death response in epithelial cells. While NADase+ SPN is known to trigger a metabolic form of necrosis through the depletion of NAD+, the mechanism by which NADase− SPN induces cell death was unknown. In the studies described here, we examined the pathway of NADase− cell death through analysis of activation patterns of mitogen-activated protein kinases (MAPKs). S. pyogenes infection resulted in activation of members of three MAPK subfamilies (p38, ERK, and JNK). However, only JNK was activated in an SLO-specific manner. NADase− SPN induced necrosis in HeLa epithelial cells associated with depolarization of mitochondrial membranes, activation of NF-κB, and the generation of reactive oxygen species. Remarkably, RNA interference (RNAi) silencing of JNK protected cells from NADase−-SPN-mediated necrosis, suggesting that NADase− SPN triggers a form of programmed necrosis dependent on JNK signaling. Taken together, these data demonstrate that SPN acts with SLO to elicit necrosis through two different mechanisms depending on its NADase activity, i.e., metabolic (NADase+) or programmed (NADase−), leading to distinct inflammatory profiles.

Expression of ERK1 and ERK2 in prostate cancer

Prostate cancer may emerge as result of dysregulated balance between cell proliferation and death rates, increased angiogenesis and chronic. These processes are regulated by numerous signaling proteins, including the mitogen-activated protein kinases (MAPKs). JNK, p38 and extracellular signal-regulated kinase (ERK) are the three major sub-families of MAPKs. The pro-oncogenic effects of ERK isoforms (ERK1 and ERK2) lie in their aberrant activation through phosphorylation by any mutation along the pathway of receptor tyrosine kinase (RTK)-Ras-Raf-MEK-ERK1/2. Once activated, ERKs phosphorylate cytoskeletal proteins, kinases, and transcription factors. Active ERK proteins induce strong proliferative and anti-apoptotic effects. Our group has tested variations in expression, activation and localization of ERKs in human prostate. Differential ERK1/2 expression and phosphorylation status may be linked to the progression of prostate cancer. The major striking observation is that ERKs are expressed in tumors with higher proportion than normal prostate. We believe that this is an important notion because the status (expression, localization, phosphorylation and the ERK1/ERK2 ratio) of ERK in the prostate may be developed into an important prognostic marker that predicts patient responce to the anti-cancer treatment.

Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus

Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK) family members to primate torpor were compared in six organs of control (aroused) versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs), c-jun NH2-terminal kinases (JNKs), MAPK kinase (MEK), and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27). The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor.

Inhibition of protein kinase (PK) Cδ attenuates methamphetamine-induced dopaminergic toxicity via upregulation of phosphorylation of tyrosine hydroxylase at Ser40 by modulation of protein phosphatase 2A and PKA.

Recently, we proposed that inhibition of protein kinase (PK) Cδ may be a useful target for protection against methamphetamine (MA)-induced dopaminergic toxicity. We demonstrated that treatment with MA resulted in a significant decrease in phosphorylation of tyrosine hydroxylase (TH) at Ser(40) in the striatum, but not in the phosphorylation of TH at Ser(31) . In the present study, treatment with rottlerin (1.5 or 3.0 μg, i.c.v, once a day for 5 days), a PKCδ inhibitor, or a PKCδ antisense oligonucleotide (ASO; 2.5 μg/μl, i.c.v., 3 times) significantly attenuated MA-induced reductions in the phosphorylation of TH at Ser(40) and in the expression of PKA in the striatum of mice. This attenuation was significantly counteracted by H89 (10 or 30 ng, i.c.v., 1 h after the last MA administration), a PKA inhibitor. Treatment with rottlerin or ASO significantly attenuated the MA-induced increase in protein phosphatase (PP) 2A activity. FTY720 (1 or 5 mg/kg, i.p., 1 h after the last MA administration), a PP2A activator, significantly reversed the recovery in TH phosphorylation mediated by inhibition of PKCδ after MA treatment. Both H89 and FTY720 counteracted the recovery of MA-induced behavioural impairments induced by PKCδ inhibition. The effects, mediated by rottlerin or ASO in MA-treated wild-type mice were comparable with those in MA-treated PKCδ(-/-) mice. However, neither inhibition of the mitogen-activated protein kinase subfamily (extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38) nor inhibition of calcium calmodulin kinase II significantly altered PKCδ inhibition-mediated attenuation of MA-induced impairment of TH phosphorylation. The results suggest that genetic or pharmacological inhibition of PKCδ requires modulation of PKA expression and/or PP2A activity to attenuate the impairment of TH phosphorylation at Ser(40) and behavioural activity induced by MA.

Selective mitogen activated protein kinase activity sensors through the application of directionally programmable D domain motifs.

Accurate and quantitative methods for measuring the dynamic fluctuations of protein kinase activities are critically needed as diagnostic tools and for the evaluation of kinase-targeted inhibitors, which represent a major therapeutic development area in the treatment of cancer and other diseases. In particular, rapid and economical methods that utilize simple instrumentation and provide quantitative data in a high throughput format will have the most impact on basic research in systems biology and medicine. There are over 500 protein kinases in the human kinome. Among these, the mitogen activated protein (MAP) kinases are recognized to be central players in key cellular signaling events and are associated with essential processes including growth, proliferation, differentiation, migration, and apoptosis. The major challenge with MAP kinase sensor development is achieving high selectivity since these kinases rely acutely on secondary interactions distal to the phosphorylation site to impart substrate specificity. Herein we describe the development and application of selective sensors for three MAP kinase subfamilies, ERK1/2, p38α/β, and JNK1/2/3. The new sensors are based on a modular design, which includes a sensing element that exploits a sulfonamido-oxine (Sox) fluorophore for reporting phosphorylation, a recognition and specificity element based on reported docking domain motifs and a variable linker, which can be engineered to optimize the intermodule distance and relative orientation. Following rigorous validation, the capabilities of the new sensors are exemplified through the quantitative analysis of the target MAP kinases in breast cancer progression in a cell culture model, which reveals a strong correlation between p38α/β activity and increased tumorgenicity.

Map2k4δ — Identification and functional characterization of a novel Map2k4 splice variant

Mitogen-activated protein kinase kinase 4 (Map2k4) is a dual specificity serin/threonine protein kinase that is unique among all MAP2Ks in activating two different subfamilies of mitogen-activated protein kinases, the c-Jun N-terminal kinases (JNKs) and p38 kinases. Map2k4 is essential during embryogenesis and involved in a variety of physiological and pathological processes. However, studies on its role in cancer development revealed partially conflicting data. In the present study, we report the identification of a novel splice variant of Map2k4, Map2k4δ, with an additional exon in front of the substrate binding D-domain. Map2k4δ is expressed together with Map2k4 in various tissues from rat, mouse and human. In PC12 cells, both splice variants control cell cycle progression and basal apoptosis by using different signaling pathways. If expression and activation of Map2k4 and Map2k4δ are at a certain, cell type-specific equilibrium, an appropriate cell growth is ensured. Overexpression of one kinase disrupts the intricate balance and either results in a highly proliferative or pro-apoptotic phenotype, partially reflecting the discrepancies in the literature on Map2k4 and its role in tumor development. Our findings contribute to the understanding of previous studies and point out that Map2k4 has not always a definite function, but rather triggers a cellular reaction in concert with other modulators.

Enhancement of (−)-epigallocatechin-3-gallate and theaflavin-3-3′-digallate induced apoptosis by ascorbic acid in human lung adenocarcinoma SPC-A-1 cells and esophageal carcinoma Eca-109 cells via MAPK pathways

Tea polyphenols (−)-epigallocatechin-3-gallate (EGCG) and theaflavin-3-3′-digallate (TF3) are two prospective compounds in cancer prevention and treatment. Ascorbic acid (Vc) is essential to a healthy diet as well as being a highly effective antioxidant. In this work, the effects of the combination of EGCG or TF3 with Vc on the apoptosis and caspases-3/9 activities in human lung adenocarcinoma SPC-A-1 cells and esophageal carcinoma Eca-109 cells were determined. Furthermore, the role of mitogen-activated protein kinases (MAPK) pathways in the apoptosis induced by TF3 or EGCG together with Vc were studied using three MAPK inhibitors (ERK inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580). Our results showed that Vc could enhance the EGCG and TF3 induced apoptosis in SPC-A-1 and Eca-109 cells, and this effect involved the activation of caspase-3 and 9. EGCG, TF3 and Vc could activate MAPK pathways respectively, and each compound activated different MAPK subfamilies in different cells. This may explain the enhancement of EGCG and TF3 induced apoptosis by Vc in SPC-A-1 and Eca-109 cells, and will ultimately aid the design of more effective anti-cancer treatments.