Science.gov

Sample records for active n-terminal kinase

  1. Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

    PubMed

    Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

    2015-12-01

    The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. Copyright © 2015 by the Genetics Society of America.

  2. Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development

    PubMed Central

    Park, Sung Yeon; Stultz, Brian G.; Hursh, Deborah A.

    2015-01-01

    The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. PMID:26500262

  3. SHP-1 inhibition by 4-hydroxynonenal activates Jun N-terminal kinase and glutamate cysteine ligase.

    PubMed

    Rinna, Alessandra; Forman, Henry Jay

    2008-07-01

    4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, is toxic at high concentrations, but at near-physiological concentrations it induces detoxifying enzymes. Previous data established that in human bronchial epithelial (HBE1) cells, both genes for glutamate cysteine ligase (GCL) are induced by HNE through the c-Jun N-terminal kinase (JNK) pathway. The protein-tyrosine phosphatase SH2 domain containing phosphatase-1 (SHP-1) is thought to play a role as a negative regulator of cell signaling, and has been implicated as such in the JNK pathway. In the present study, SHP-1 was demonstrated to contribute to HNE-induced-gclc expression via regulation of the JNK pathway in HBE1 cells. Treatment of HBE1 cells with HNE induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4), JNK, and c-Jun. HNE was able to inhibit protein tyrosine phosphatase activity of SHP-1 through increased degradation of the protein. Furthermore, transfection with small interference RNA SHP-1 showed an enhancement of JNK and c-Jun phosphorylation, but not of MKK4, leading to increased gclc expression. These results demonstrate that SHP-1 plays a role as a negative regulator of the JNK pathway and that HNE activated the JNK pathway by inhibiting SHP-1. Thus, SHP-1 acts as a sensor for HNE and is responsible for an important adaptive response to oxidative stress.

  4. c-Jun N-terminal kinase phosphorylation is a biomarker of plitidepsin activity.

    PubMed

    Muñoz-Alonso, María J; Álvarez, Enrique; Guillén-Navarro, María José; Pollán, Marina; Avilés, Pablo; Galmarini, Carlos M; Muñoz, Alberto

    2013-05-21

    Plitidepsin is an antitumor drug of marine origin currently in Phase III clinical trials in multiple myeloma. In cultured cells, plitidepsin induces cell cycle arrest or an acute apoptotic process in which sustained activation of c-Jun N-terminal kinase (JNK) plays a crucial role. With a view to optimizing clinical use of plitidepsin, we have therefore evaluated the possibility of using JNK activation as an in vivo biomarker of response. In this study, we show that administration of a single plitidepsin dose to mice xenografted with human cancer cells does indeed lead to increased phosphorylation of JNK in tumors at 4 to 12 h. By contrast, no changes were found in other in vitro plitidepsin targets such as the levels of phosphorylated-ERK, -p38MAPK or the protein p27KIP1. Interestingly, plitidepsin also increased JNK phosphorylation in spleens from xenografted mice showing similar kinetics to those seen in tumors, thereby suggesting that normal tissues might be useful for predicting drug activity. Furthermore, plitidepsin administration to rats at plasma concentrations comparable to those achievable in patients also increased JNK phosphorylation in peripheral mononuclear blood cells. These findings suggest that changes in JNK activity provide a reliable biomarker for plitidepsin activity and this could be useful for designing clinical trials and maximizing the efficacy of plitidepsin.

  5. c-Jun N-Terminal Kinase Phosphorylation Is a Biomarker of Plitidepsin Activity

    PubMed Central

    Muñoz-Alonso, María J.; Álvarez, Enrique; Guillén-Navarro, María José; Pollán, Marina; Avilés, Pablo; Galmarini, Carlos M.; Muñoz, Alberto

    2013-01-01

    Plitidepsin is an antitumor drug of marine origin currently in Phase III clinical trials in multiple myeloma. In cultured cells, plitidepsin induces cell cycle arrest or an acute apoptotic process in which sustained activation of c-Jun N-terminal kinase (JNK) plays a crucial role. With a view to optimizing clinical use of plitidepsin, we have therefore evaluated the possibility of using JNK activation as an in vivo biomarker of response. In this study, we show that administration of a single plitidepsin dose to mice xenografted with human cancer cells does indeed lead to increased phosphorylation of JNK in tumors at 4 to 12 h. By contrast, no changes were found in other in vitro plitidepsin targets such as the levels of phosphorylated-ERK, -p38MAPK or the protein p27KIP1. Interestingly, plitidepsin also increased JNK phosphorylation in spleens from xenografted mice showing similar kinetics to those seen in tumors, thereby suggesting that normal tissues might be useful for predicting drug activity. Furthermore, plitidepsin administration to rats at plasma concentrations comparable to those achievable in patients also increased JNK phosphorylation in peripheral mononuclear blood cells. These findings suggest that changes in JNK activity provide a reliable biomarker for plitidepsin activity and this could be useful for designing clinical trials and maximizing the efficacy of plitidepsin. PMID:23697951

  6. Role of c-Jun N-terminal kinase activation in blastema formation during planarian regeneration.

    PubMed

    Tasaki, Junichi; Shibata, Norito; Sakurai, Toshihide; Agata, Kiyokazu; Umesono, Yoshihiko

    2011-04-01

    The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts, which are the only mitotic somatic cells in adult planarians and are responsible for blastema formation after amputation. Little is known about the molecular mechanisms that drive blastema formation during planarian regeneration. Here we found that treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 blocked the entry of neoblasts into the M-phase of the cell cycle, while allowing neoblasts to successfully enter S-phase in the planarian Dugesia japonica. The rapid and efficient blockage of neoblast mitosis by treatment with the JNK inhibitor provided a method to assess whether temporally regulated cell cycle activation drives blastema formation during planarian regeneration. In the early phase of blastema formation, activated JNK was detected prominently in a mitotic region (the "postblastema") proximal to the blastema region. Furthermore, we demonstrated that undifferentiated mitotic neoblasts in the postblastema showed highly activated JNK at the single cell level. JNK inhibition by treatment with SP600125 during this period caused a severe defect of blastema formation, which accorded with a drastic decrease of mitotic neoblasts in regenerating animals. By contrast, these animals still retained many undifferentiated neoblasts near the amputation stump. These findings suggest that JNK signaling plays a crucial role in feeding into the blastema neoblasts for differentiation by regulating the G2/M transition in the cell cycle during planarian regeneration.

  7. c-Jun N-terminal kinase activity is required for efficient respiratory syncytial virus production.

    PubMed

    Caly, Leon; Li, Hong-Mei; Bogoyevitch, Marie A; Jans, David A

    2017-01-29

    Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly, leading to more deaths than influenza each year worldwide. With no RSV antiviral or efficacious vaccine currently available, improved understanding of the host-RSV interaction is urgently required. Here we examine the contribution to RSV infection of the host stress-regulated c-Jun N-terminal kinase (JNK), for the first time. Peak JNK1/2 phosphoactivation is observed at ∼24 h post-infection, correlating with the time of virus assembly. The release of infectious RSV virions from infected cells was significantly reduced by either JNK1/2 siRNA knockdown or treatment with the JNK-specific inhibitor, JNK-IN-VIII. High resolution microscopy confirmed RSV accumulation in the host cell cytoplasm. The results implicate JNK1/2 as a key host factor for RSV virus production, raising the possibility of agents targeting JNK activity as potential anti-RSV therapeutics.

  8. Activation of c-Jun N-Terminal Kinase (JNK) during Mitosis in Retinal Progenitor Cells

    PubMed Central

    Ribas, Vinicius Toledo; Gonçalves, Bruno Souza; Linden, Rafael; Chiarini, Luciana Barreto

    2012-01-01

    Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In this study, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosis of progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina. PMID:22496813

  9. Activation of c-Jun N-terminal kinase (JNK) during mitosis in retinal progenitor cells.

    PubMed

    Ribas, Vinicius Toledo; Gonçalves, Bruno Souza; Linden, Rafael; Chiarini, Luciana Barreto

    2012-01-01

    Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In this study, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosis of progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina.

  10. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    SciTech Connect

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G.

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  11. Activation by phosphorylation and purification of human c-Jun N-terminal Kinase (JNK) isoforms in milligram amounts

    PubMed Central

    Thévenin, Anastasia F.; Zony, Chati L.; Bahnson, Brian J.; Colman, Roberta F.

    2010-01-01

    c-Jun N-terminal kinases (JNKs) are part of the mitogen activated protein kinase (MAPK) signaling cascade. They are activated through dual phosphorylation of two residues in the activation loop, a threonine and a tyrosine, by MAP2 kinases (MKK4 and 7) in response to various extracellular stresses such as UV or osmotic shock, as well as by cytokines and growth factors. Only small amounts of phosphorylated, active JNKs have previously been produced because of difficulties in expressing these phosphorylated kinases in E. coli, which lack the appropriate upstream kinases. We have now established a novel activation and purification method that allows for reproducible production of milligram amounts of active, phosphorylated JNKs suitable for a variety of enzymatic, biophysical and structural characterizations. We utilize N-terminally His-tagged MKK4 that is coexpressed in E. coli with a constitutively active form of MEKK1. This phosphorylated, active His-MKK4 is purified by Ni-NTA chromatography and used to phosphorylate milligram amounts of three different isoforms of human JNKs (JNK1α1, JNK1α2 and JNK2α2) that had separately been expressed and purified from E. coli in their inactive forms. These in vitro activated JNKs are phosphorylated on both residues (T183, Y185) in their activation loops and are active towards their substrate, ATF2. PMID:20709173

  12. The N-terminal domains of cyclin-dependent kinase inhibitory proteins block the phosphorylation of cdk2/Cyclin E by the CDK-activating kinase.

    PubMed

    Rank, K B; Evans, D B; Sharma, S K

    2000-05-10

    It has been suggested that binding of p27 and p21 kinase inhibitory proteins (KIPs) to cyclin-dependent kinases (cdks) render them inaccessible to cdk-activating kinase (CAK), presumably by steric hindrance by the C-terminal residues. However, this common mechanism of inhibition is inconsistent with the known structural divergence in the p27 and p21 C-terminal domains. Therefore, we studied the direct binding of N-terminal minimal domain of p27 (amino acids 28-81) to cdk2/cyclin E. An unlabeled p27 minimal domain, mutated in the N-terminal LFG motif, was unable to compete with a labeled minimal domain for binding to cdk2/cyclin E. The p27 and its minimal domain inhibited CAK-mediated phosphorylation of cdk2/cyclin E. This inhibitory effect was significantly diminished with p27 minimal domain mutated in the LFG motif. A synthetic peptide, ACRRLFGPVDSE, from the N-terminal residues 17-28 of p21, was also a potent inhibitor of CAK-mediated cdk2/cyclin E phosphorylation. Taken together, these results show that anchoring of p27 or p21 KIPs to cyclin E via the N-terminal LFG-containing motif can block CAK access to its cdk2/cyclin E substrate.

  13. Sustained activation of c-Jun N-terminal and extracellular signal-regulated kinases in port-wine stain blood vessels.

    PubMed

    Tan, Wenbin; Chernova, Margarita; Gao, Lin; Sun, Victor; Liu, Huaxu; Jia, Wangcun; Langer, Stephanie; Wang, Gang; Mihm, Martin C; Nelson, J Stuart

    2014-11-01

    Port-wine stain (PWS) is a congenital, progressive vascular malformation but the pathogenesis remains incompletely understood. We sought to investigate the activation status of various kinases, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, AKT, phosphatidylinositol 3-kinase, P70 ribosomal S6 kinase, and phosphoinositide phospholipase C γ subunit, in PWS biopsy tissues. Immunohistochemistry was performed on 19 skin biopsy samples from 11 patients with PWS. c-Jun N-terminal kinase, extracellular signal-regulated kinase, and P70 ribosomal S6 kinase in pediatric and adult PWS blood vessels were consecutively activated. Activation of AKT and phosphatidylinositol 3-kinase was found in many adult hypertrophic PWS blood vessels but not in infants. Phosphoinositide phospholipase C γ subunit showed strong activation in nodular PWS blood vessels. Infantile PWS sample size was small. Our data suggest a subsequent activation profile of various kinases during different stages of PWS: (1) c-Jun N-terminal and extracellular signal-regulated kinases are firstly and consecutively activated in all PWS tissues, which may contribute to both the pathogenesis and progressive development of PWS; (2) AKT and phosphatidylinositol 3-kinase are subsequently activated, and are involved in the hypertrophic development of PWS blood vessels; and (3) phosphoinositide phospholipase C γ subunit is activated in the most advanced stage of PWS and may participate in nodular formation. Copyright © 2014 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  14. Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation

    PubMed Central

    Becker, Elena; Huynh-Do, Uyen; Holland, Sacha; Pawson, Tony; Daniel, Tom O.; Skolnik, Edward Y.

    2000-01-01

    The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62dok, RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs. PMID:10669731

  15. c-JUN N-terminal kinase (JNK) is activated and contributes to tumor cell proliferation in classical Hodgkin lymphoma.

    PubMed

    Leventaki, Vasiliki; Drakos, Elias; Karanikou, Maria; Psatha, Konstantina; Lin, Pei; Schlette, Ellen; Eliopoulos, Aris; Vassilakopoulos, Theodoros P; Papadaki, Helen; Patsouris, Efstratios; Medeiros, L Jeffrey; Rassidakis, George Z

    2014-03-01

    c-JUN N-terminal Kinase (JNK) is activated/phosphorylated by upstream MAPK kinases (MKK), and, in turn, phosphorylates and activates its major substrate c-JUN, a member of the activator protein-1 (AP-1) transcription factors. c-JUN is overexpressed and activated in Hodgkin and Reed Sternberg cells (HRS) of classical Hodgkin lymphoma (cHL), however, the mechanism of its activation remains unknown. JNK activation was immunohistochemically assessed in 60 cases of HL and in a control group of 151 B-cell non-Hodgkin lymphomas. The biologic effects of JNK activation in cultured HRS cells were investigated using colony formation, cell growth and viability assays and cell cycle analysis by flow cytometry. Western blotting was used to assess protein levels. p-JNK was expressed in 90% of HL, 83% of Burkitt lymphomas, 28% of mantle cell lymphomas, 23% of diffuse large B-cell lymphomas, 19% of follicular lymphomas, and 18% of extranodal marginal zone lymphomas of MALT type. None of the 48 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma and 18 cases of plasma cell myeloma showed JNK phosphorylation (P < 001, Kruskall-Wallis test). Pharmacological inhibition of JNK activity in cultured HRS cells resulted in a significant decrease of cell growth, which was associated with cell cycle arrest at the G2/M phase. The cell cycle effects were linked to deactivation of c-JUN and upregulation of its known target, the cyclin-dependent kinase inhibitor p21. JNK is highly activated in HRS cells, and may contribute to uncontrolled cell cycle progression and proliferation of tumor cells in cHL.

  16. Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.

    PubMed

    Pike, Brietta L; Tenis, Nora; Heierhorst, Jörg

    2004-09-17

    Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.

  17. Thrombin-induced apoptosis in neurons through activation of c-Jun-N-terminal kinase.

    PubMed

    Bao, Lei; Zu, Jie; He, Qianqian; Zhao, Hui; Zhou, Su; Ye, Xinchun; Yang, Xinxin; Zan, Kun; Zhang, Zuohui; Shi, Hongjuan; Cui, Guiyun

    2017-01-01

    Studies have shown that thrombin activation played a central role in cell injuries associated with intracerebral hemorrhage (ICH). Here, our study investigated the cytotoxicity of thrombin on neurons, and determined the involvement of JNK pathways in thrombin-induced neuronal apoptosis. Primary cultured neurons were treated with different doses of thrombin. Some neurons were given either SP600125 or vehicle. LDH release assay and flow cytometry were used to measure neuronal apoptosis caused by thrombin. The activation of JNK and capases-3 were measured by Western blot. Our results showed large doses of thrombin that increased the LDH release, the level of cleaved caspase-3 and apoptosis rate of neurons. JNK was activated by thrombin in a time-dependent manner. Administration of SP600125 protects neurons from thrombin-induced apoptosis. These data indicate that the activation of JNK is crucial for thrombin-induced neuronal apoptosis, and inhibition of JNK may be a potential therapeutic target for ICH.

  18. Vitexin 6, a novel lignan, induces autophagy and apoptosis by activating the Jun N-terminal kinase pathway.

    PubMed

    Zhou, Jun; Hu, Huiyong; Long, Jingpei; Wan, Fang; Li, Lili; Zhang, Suzhan; Shi, Yuenian E; Chen, Yiding

    2013-10-01

    Previous studies have reported that vitexins induce cytotoxic effects. In the present study, we investigate a new native lignan vitexin 6 (VB6) in vitro to determine the molecular mechanism underlying its cytotoxicity. We screened and cultured several tumor cell lines and subsequently analyzed VB6 cytotoxicity against 14 different tumor cell lines using a 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The expression of proteins that regulate apoptosis and autophagy was determined using western blot analysis. VB6 showed an excellent cytotoxic effect against various cancer cell lines in vitro. It induced apoptosis and autophagy of cancer cells. VB6-induced apoptosis showed a time-dependent and concentration-dependent relationship with cleaved poly (ADP-ribose) polymerase, cleaved caspase-3, Bax upregulation, and Bcl-2 downregulation. The levels of Beclin-1 and LC3-II, which are markers for cell autophagy, gradually increased after VB6 treatment. Jun N-terminal kinase (JNK) phosphorylation was increased after VB6 treatment, accompanied by upregulation of P-Bcl-2 and P-C-Jun expression. Cotreatment with a JNK inhibitor significantly decreased VB6-induced cell death and downregulated P-Bcl-2, and cleaved PARP and Beclin-1 expression. The new native lignan VB6 inhibits cancer cell proliferation by activating the JNK pathway. We believe that VB6 could be a valuable chemotherapeutic drug after further evaluation.

  19. c-Jun N-terminal kinase-3 (JNK3)/stress-activated protein kinase-beta (SAPKbeta) binds and phosphorylates the neuronal microtubule regulator SCG10.

    PubMed

    Neidhart, S; Antonsson, B; Gilliéron, C; Vilbois, F; Grenningloh, G; Arkinstall, S

    2001-11-16

    The neuronal growth-associated protein SCG10 is enriched in the growth cones of neurons where it destabilizes microtubules and thus contributes to the dynamic assembly and disassembly of microtubules. Since its microtubule-destabilizing activity is regulated by phosphorylation, SCG10 may link extracellular signals to rearrangements of the neuronal cytoskeleton. To identify signal transduction pathways that may lead to SCG10 phosphorylation, we tested a series of serine-threonine-directed protein kinases that phosphorylate SCG10 in vitro. We demonstrate that purified SCG10 can be phosphorylated by two subclasses of mitogen-activated protein (MAP) kinases, c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) and p38 MAP kinase. Moreover, SCG10 was found to bind tightly and specifically to JNK3/SAPKbeta. JNK3/SAPKbeta phosphorylation occurs at Ser-62 and Ser-73, residues that result in reduced microtubule-destabilizing activity for SCG10. Endogenous SCG10 also undergoes increased phosphorylation in sympathetic neurons at times of JNK3/SAPKbeta activation following deprivation from nerve growth factor. Together these observations indicate that activation of JNK/SAPKs provides a pathway for phosphorylation of SCG10 and control of growth cone microtubule formation following neuronal exposure to cellular stresses.

  20. Analysis of mitogen-activated protein kinase pathways used by interleukin 1 in tissues in vivo: activation of hepatic c-Jun N-terminal kinases 1 and 2, and mitogen-activated protein kinase kinases 4 and 7.

    PubMed Central

    Finch, A; Davis, W; Carter, W G; Saklatvala, J

    2001-01-01

    The effects of interleukin 1 (IL-1) are mediated by the activation of protein kinase signalling pathways, which have been well characterized in cultured cells. We have investigated the activation of these pathways in rabbit liver and other tissues after the systemic administration of IL-1alpha. In liver there was 30-40-fold activation of c-Jun N-terminal kinase (JNK) and 5-fold activation of both JNK kinases, mitogen-activated protein kinase (MAPK) kinase (MKK)4 and MKK7. IL-1alpha also caused 2-3-fold activation of p38 MAPK and degradation of the inhibitor of nuclear factor kappaB ('IkappaB'), although no activation of extracellular signal-regulated protein kinase (ERK) (p42/44 MAPK) was observed. The use of antibodies against specific JNK isoforms showed that, in liver, short (p46) JNK1 and long (p54) JNK2 are the predominant forms activated, with smaller amounts of long JNK1 and short JNK2. No active JNK3 was detected. A similar pattern of JNK activation was seen in lung, spleen, skeletal muscle and kidney. Significant JNK3 activity was detectable only in the brain, although little activation of the JNK pathway in response to IL-1alpha was observed in this tissue. This distribution of active JNK isoforms probably results from a different expression of JNKs within the tissues, rather than from a selective activation of isoforms. We conclude that IL-1alpha might activate a more restricted set of signalling pathways in tissues in vivo than it does in cultured cells, where ERK and JNK3 activation are often observed. Cultured cells might represent a 'repair' phenotype that undergoes a broader set of responses to the cytokine. PMID:11139391

  1. Glycogen Synthase Kinase 3β Is a Negative Regulator of Growth Factor-induced Activation of the c-Jun N-terminal Kinase*

    PubMed Central

    Liu, Shuying; Yu, Shuangxing; Hasegawa, Yutaka; LaPushin, Ruth; Xu, Hong-Ji; Woodgett, James R.; Mills, Gordon B.; Fang, Xianjun

    2016-01-01

    The c-Jun N-terminal kinase (JNK)/stress activated protein kinase is preferentially activated by stress stimuli. Growth factors, particularly ligands for G protein-coupled receptors, usually induce only modest JNK activation, although they may trigger marked activation of the related extracellular signal-regulated kinase. In the present study, we demonstrated that homozygous disruption of glycogen synthase kinase 3β (GSK-3β) dramatically sensitized mouse embryonic fibroblasts (MEFs) to JNK activation induced by lysophosphatidic acid (LPA) and sphingosine-1-phosphate, two prototype ligands for G protein-coupled receptors. To a lesser degree, a lack of GSK-3β also potentiated JNK activation in response to epidermal growth factor. In contrast, the absence of GSK-3β decreased UV light-induced JNK activation. The increased JNK activation induced by LPA in GSK-3β null MEFs was insufficient to trigger apoptotic cell death or growth inhibition. Instead, the increased JNK activation observed in GSK-3β−/− MEFs was associated with an increased proliferative response to LPA, which was reduced by the inhibition of JNK. Ectopic expression of GSK-3β in GSK-3β-negative MEFs restrained LPA-triggered JNK phosphorylation and induced a concomitant decrease in the mitogenic response to LPA compatible with GSK-3β through the inhibition of JNK activation, thus limiting LPA-induced cell proliferation. Mutation analysis indicated that GSK-3β kinase activity was required for GSK-3β to optimally inhibit LPA-stimulated JNK activation. Thus GSK-3β serves as a physiological switch to specifically repress JNK activation in response to LPA, sphingosine-1-phosphate, or the epidermal growth factor. These results reveal a novel role for GSK-3β in signal transduction and cellular responses to growth factors. PMID:15466414

  2. Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide

    NASA Technical Reports Server (NTRS)

    Ramachandran, Anup; Moellering, Douglas; Go, Young-Mi; Shiva, Sruti; Levonen, Anna-Liisa; Jo, Hanjoong; Patel, Rakesh P.; Parthasarathy, Sampath; Darley-Usmar, Victor M.

    2002-01-01

    Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy-1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steady-state concentrations (0.1-0.5 nmol x min(-1) x 10(6) cells) of H2O2 generated intracellularly activate c-Jun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both c-Jun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.

  3. Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide

    NASA Technical Reports Server (NTRS)

    Ramachandran, Anup; Moellering, Douglas; Go, Young-Mi; Shiva, Sruti; Levonen, Anna-Liisa; Jo, Hanjoong; Patel, Rakesh P.; Parthasarathy, Sampath; Darley-Usmar, Victor M.

    2002-01-01

    Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy-1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steady-state concentrations (0.1-0.5 nmol x min(-1) x 10(6) cells) of H2O2 generated intracellularly activate c-Jun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both c-Jun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.

  4. Pigment epithelium-derived factor (PEDF) suppresses IL-1β-mediated c-Jun N-terminal kinase (JNK) activation to improve hepatocyte insulin signaling.

    PubMed

    Gattu, Arijeet K; Birkenfeld, Andreas L; Iwakiri, Yasuko; Jay, Steven; Saltzman, Mark; Doll, Jennifer; Protiva, Petr; Samuel, Varman T; Crawford, Susan E; Chung, Chuhan

    2014-04-01

    Pigment epithelium-derived factor (PEDF) is an antiinflammatory protein that circulates at high levels in the metabolic syndrome. Metabolic studies of PEDF knockout (KO) mice were conducted to investigate the relationship between PEDF, inflammatory markers, and metabolic homeostasis. Male PEDF KO mice demonstrated a phenotype consisting of increased adiposity, glucose intolerance, and elevated serum levels of metabolites associated with the metabolic syndrome. Genome expression analysis revealed an increase in IL-1β signaling in the livers of PEDF KO mice that was accompanied by impaired IRS and Akt signaling. In human hepatocytes, PEDF blocked the effects of an IL-1β challenge by suppressing activation of the inflammatory mediator c-Jun N-terminal kinase while restoring Akt signaling. RNA interference of PEDF in human hepatocytes was permissive for c-Jun N-terminal kinase activation and decreased Akt signaling. A metabolomics profile identified elevated circulating levels of tricarboxyclic acid cycle intermediates including succinate, an inducer of IL-1β, in PEDF KO mice. Succinate-dependent IL-1β expression was blocked by PEDF in PEDF KO, but not wild-type hepatocytes. In vivo, PEDF restoration reduced hyperglycemia and improved hepatic insulin signaling in PEDF KO mice. These findings identify elevated PEDF as a homeostatic mechanism in the human metabolic syndrome.

  5. Low concentrations of paraquat induces early activation of extracellular signal-regulated kinase 1/2, protein kinase B, and c-Jun N-terminal kinase 1/2 pathways: role of c-Jun N-terminal kinase in paraquat-induced cell death.

    PubMed

    Niso-Santano, Mireia; Morán, José M; García-Rubio, Lourdes; Gómez-Martín, Ana; González-Polo, Rosa A; Soler, Germán; Fuentes, José M

    2006-08-01

    Paraquat is a herbicide with a potential risk to induce parkinsonism due to its demonstrated neurotoxicity and its strong structural similarity to 1-methyl-4-phenylpyridinium (MPP(+)), a well-known neurotoxin which causes a clinical syndrome similar to Parkinson's disease (PD). However, at present very little is known about the signaling pathways activated by paraquat in any cell system. In this study, we have investigated the effect of paraquat on extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and protein kinase B (PKB) activation in E18 cells. Low concentrations of paraquat stimulated very early increases in ERK1/2, JNK1/2, and PKB phosphorylation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited early paraquat-induced increases in PKB phosphorylation. Furthermore, early paraquat-mediated increases in ERK1/2 activation were sensitive to the mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK1/2 responses were blocked by the JNK1/2 inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Pretreatment with wortmannin, LY 294002, or PD 98059 had no effect on paraquat cell death in E18 cells. In contrast, SP 600125 significantly decreased paraquat-induced cell death in E18 cells. In conclusion, we have shown that low concentrations of paraquat stimulate robust very early increases in ERK1/2, JNK1/2, and PKB phosphorylation in E18 cells. Furthermore, the data presented clearly suggest that inhibition of the JNK1/2 pathway protects E18 cells from paraquat-induced cell death and support the fact that inhibition of early activation of JNK1/2 can constitute a potential strategy in PD treatment.

  6. Frabin, a novel FGD1-related actin filament-binding protein capable of changing cell shape and activating c-Jun N-terminal kinase.

    PubMed

    Obaishi, H; Nakanishi, H; Mandai, K; Satoh, K; Satoh, A; Takahashi, K; Miyahara, M; Nishioka, H; Takaishi, K; Takai, Y

    1998-07-24

    We purified from rat brain a novel F-actin-binding protein with a Mr of about 105,000 (p105), which was estimated by SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a rat brain cDNA library and characterized it. p105 was a protein of 766 amino acids and showed a calculated Mr of 86,449. p105 consisted of one F-actin-binding domain at the N-terminal region, one Dbl homology domain and one pleckstrin homology domain at the middle region, and one cysteine-rich domain at the C-terminal region. This domain organization of p105 was similar to that of FGD1, which has been determined to be the genetic locus responsible for faciogenital dysplasia or Aarskog-Scott syndrome. We therefore named p105 frabin (FGD1-related F-actin-binding protein). Frabin bound along the sides of F-actin and showed F-actin-cross-linking activity. Overexpression of frabin in Swiss 3T3 cells and COS7 cells induced cell shape change and c-Jun N-terminal kinase activation, respectively, as described for FGD1. Because FGD1 has been shown to serve as a GDP/GTP exchange protein for Cdc42 small G protein, it is likely that frabin is a direct linker between Cdc42 and the actin cytoskeleton.

  7. Neuronal angiotensin II type 1 receptor upregulation in heart failure: activation of activator protein 1 and Jun N-terminal kinase.

    PubMed

    Liu, Dongmei; Gao, Lie; Roy, Shyamal K; Cornish, Kurtis G; Zucker, Irving H

    2006-10-27

    Chronic heart failure (CHF) is a leading cause of mortality in developed countries. Angiotensin II (Ang II) plays an important role in the development and progression of CHF. Many of the important functions of Ang II are mediated by the Ang II type 1 receptor (AT(1)R), including the increase in sympathetic nerve activity in CHF. However, the central regulation of the AT(1)R in the setting of CHF is not well understood. This study investigated the AT(1)R in the rostral ventrolateral medulla (RVLM) of rabbits with CHF, its downstream pathway, and its gene regulation by the transcription factor activator protein 1 (AP-1). Studies were performed in 5 groups of rabbits: sham (n=5), pacing-induced (3 to 4 weeks) CHF (n=5), CHF with intracerebroventricular (ICV) losartan treatment (n=5), normal with ICV Ang II treatment (n=5), and normal with ICV Ang II plus losartan treatment (n=5). AT(1)R mRNA and protein expressions, plasma Ang II, and AP-1-DNA binding activity were significantly higher in RVLM of CHF compared with Sham rabbits (240.4+/-30.2%, P<0.01; 206.6+/-25.8%, P<0.01; 280+/-36.5%, P<0.05; 207+/-16.4%, P<0.01, respectively). Analysis of the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) pathway showed that phosphorylated c-Jun proteins, phosphorylated JNK proteins, and JNK activity increased significantly in RVLM of CHF compared with sham (262.9+/-48.1%, 213.8+/-27.7%, 148.2+/-10.1% of control, respectively). Importantly, ICV losartan in CHF rabbits attenuated these increases. ICV Ang II in normal rabbits simulated the molecular changes seen in CHF. This effect was blocked by concomitant ICV losartan. In addition, Ang II-induced AT(1)R expression was blocked by losartan and a JNK inhibitor, but not by extracellular signal-regulated kinase or p38 MAP kinase inhibitors in a neuronal cell culture. These data suggest that central Ang II activates the AT(1)R, SAPK/JNK pathway. AP-1 may further regulate gene expression in RVLM in the CHF state.

  8. TRPM7 Activates m-Calpain by Stress-Dependent Stimulation of p38 MAPK and c-Jun N-Terminal Kinase

    PubMed Central

    Su, Li-Ting; Chen, Hsiang-Chin; González-Pagán, Omayra; Overton, Jeffrey D.; Xie, Jia; Yue, Lixia; Runnels, Loren W.

    2010-01-01

    Summary TRPM7 is a Ca2+ and Mg2+ permeant ion channel in possession of its own kinase domain. In a previous study we showed that overexpression of the channel-kinase in HEK-293 cells produced cell rounding and loss of adhesion which was dependent upon the Ca2+-dependent protease m-calpain. The TRPM7-elicited change in cell morphology was channel-dependent and occurred without any significant increase in cytosolic Ca2+. Here we demonstrate that overexpression of TRPM7 increased levels of cellular reactive oxygen species (ROS) and nitric oxide (NO), causing the activation of p38 MAP kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). Application of inhibitors of p38 MAPK and JNK blocked TRPM7-induced cell rounding and activation of m-calpain, without affecting the phosphorylation state of the protease. Overexpression of TRPM7 increased intracellular Mg2+; however, when the concentrations of either external Ca2+ or Mg2+ was increased to favor permeation of one divalent cation over the other, a similar increase in cell rounding and calpain activity was detected, indicating that TRPM7-mediated activation of m-calpain is not dependent on the nature of the divalent conducted by the channel. Application of inhibitors of nitric oxide synthase and mitochondrial-derived ROS reduced TRPM7-induced increases in nitric oxide and ROS production, blocked the change in cell morphology, and reduced cellular calpain activity. Collectively, our data reveal that excessive TRPM7 channel activity causes oxidative and nitrosative stress, producing cell rounding mediated by p38 MAPK/JNK dependent activation of m-calpain. PMID:20070945

  9. MST kinases monitor actin cytoskeletal integrity and signal via c-Jun N-terminal kinase stress-activated kinase to regulate p21Waf1/Cip1 stability.

    PubMed

    Densham, Ruth M; O'Neill, Eric; Munro, June; König, Ireen; Anderson, Kurt; Kolch, Walter; Olson, Michael F

    2009-12-01

    As well as providing a structural framework, the actin cytoskeleton plays integral roles in cell death, survival, and proliferation. The disruption of the actin cytoskeleton results in the activation of the c-Jun N-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway; however, the sensor of actin integrity that couples to the JNK pathway has not been characterized in mammalian cells. We now report that the mammalian Ste20-like (MST) kinases mediate the activation of the JNK pathway in response to the disruption of the actin cytoskeleton. One consequence of actin disruption is the JNK-mediated stabilization of p21(Waf1/Cip1) (p21) via the phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that, in addition to being components of the Salvador-Warts-Hippo tumor suppressor network and binding partners of c-Raf and the RASSF1A tumor suppressor, MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.

  10. Activation of c-Jun N-Terminal Kinase 1 by UV Irradiation Is Inhibited by Wortmannin without Affecting c-jun Expression

    PubMed Central

    Fritz, G.; Kaina, B.

    1999-01-01

    Activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases is an early response of cells upon exposure to DNA-damaging agents. JNK-mediated phosphorylation of c-Jun is currently understood to stimulate the transactivating potency of AP-1 (e.g., c-Jun/c-Fos; c-Jun/ATF-2), thereby increasing the expression of AP-1 target genes. Here we show that stimulation of JNK1 activity is not a general early response of cells exposed to genotoxic agents. Treatment of NIH 3T3 cells with UV light (UV-C) as well as with methyl methanesulfonate (MMS) caused activation of JNK1 and an increase in c-Jun protein and AP-1 binding activity, whereas antineoplastic drugs such as mafosfamide, mitomycin C, N-hydroxyethyl-N-chloroethylnitrosourea, and treosulfan did not elicit this response. The phosphatidylinositol 3-kinase inhibitor wortmannin specifically blocked the UV-stimulated activation of JNK1 but did not affect UV-driven activation of extracellular regulated kinase 2 (ERK2). To investigate the significance of JNK1 for transactivation of c-jun, we analyzed the effect of UV irradiation on c-jun expression under conditions of wortmannin-mediated inhibition of UV-induced stimulation of JNK1. Neither the UV-induced increase in c-jun mRNA, c-Jun protein, and AP-1 binding nor the activation of the collagenase and c-jun promoters was affected by wortmannin. In contrast, the mitogen-activated protein kinase/ERK kinase inhibitor PD98056, which blocked ERK2 but not JNK1 activation by UV irradiation, impaired UV-driven c-Jun protein induction and AP-1 binding. Based on the data, we suggest that JNK1 stimulation is not essential for transactivation of c-jun after UV exposure, whereas activation of ERK2 is required for UV-induced signaling leading to elevated c-jun expression. PMID:10022864

  11. Catalytic Activity of cGMP-Dependent Protein Kinase Type I in Intact Cells Is Independent of N-Terminal Autophosphorylation

    PubMed Central

    Vallur, Raghavan; Kalbacher, Hubert; Feil, Robert

    2014-01-01

    Although cGMP-dependent protein kinase type I (cGKI) is an important mediator of cGMP signaling and upcoming drug target, its in vivo-biochemistry is not well understood. Many studies showed that purified cGKI autophosphorylates multiple sites at its N-terminus. Autophosphorylation might be involved in kinase activation, but it is unclear whether this happens also in intact cells. To study cGKI autophosphorylation in vitro and in vivo, we have generated phospho-specific antisera against major in vitro-autophosphorylation sites of the cGKI isoforms, cGKIα and cGKIβ. These antisera detected specifically and with high sensitivity phospho-cGKIα (Thr58), phospho-cGKIα (Thr84), or phospho-cGKIβ (Thr56/Ser63/Ser79). Using these antisera, we show that ATP-induced autophosphorylation of cGKI in purified preparations and cell extracts did neither require nor induce an enzyme conformation capable of substrate heterophosphorylation; it was even inhibited by pre-incubation with cGMP. Interestingly, phospho-cGKI species were not detectable in intact murine cells and tissues, both under basal conditions and after induction of cGKI catalytic activity. We conclude that N-terminal phosphorylation, although readily induced in vitro, is not required for the catalytic activity of cGKIα and cGKIβ in vivo. These results will also inform screening strategies to identify novel cGKI modulators. PMID:24897423

  12. NELL-1-dependent mineralisation of Saos-2 human osteosarcoma cells is mediated via c-Jun N-terminal kinase pathway activation.

    PubMed

    Chen, Feng; Walder, Ben; James, Aaron W; Soofer, Donnalisa E; Soo, Chia; Ting, Kang; Zhang, Xinli

    2012-10-01

    NELL-1 is a novel osteoinductive growth factor that has shown promising results for the regeneration of bone. Moreover, NELL-1 has been used successfully in bone regeneration in the axial, appendicular and calvarial skeleton of both small and large animal models. Despite increasing evidence of NELL-1 efficacy and future usefulness as an alternative to traditional bone graft substitutes, much has yet to be understood regarding the mechanisms of action of this novel protein. The activation of the mitogen-activated protein kinase (MAPK) pathway has been well studied in the setting of growth factor-mediated changes in osteogenic differentiation. In this study, we provide evidence of the involvement of MAPK signalling pathways in NELL-1-induced terminal osteogenic differentiation of Saos-2 human osteosarcoma cells. Activation of extracellular signal-regulated kinase (ERK1/2), P38 and c-Jun N-terminal kinase (JNK) pathways were screened with MAPK signalling protein array after recombinant human (rh)NELL-1 treatment. Next, the mineralisation and intracellular phosphate levels after rhNELL-1 stimulation were assessed in the presence or absence of specific MAPK inhibitors. Results showed that rhNELL-1 predominantly increased JNK pathway activation. Moreover, the specific JNK inhibitor SP600125 blocked rhNELL-1-induced mineralisation and intracellular phosphate accumulation, whereas ERK1/2 and P38 inhibitors showed no effect. Thus, activation of the JNK pathway is necessary to mediate terminal osteogenic differentiation of Saos-2 osteosarcoma cells by rhNELL-1. Future studies will extend these in vitro mechanisms to the in vivo effects of NELL-1 in dealing with orthopaedic defects caused by skeletal malignancies or other aetiologies.

  13. Critical role of c-Jun N-terminal protein kinase activation in troglitazone-induced apoptosis of human HepG2 hepatoma cells.

    PubMed

    Bae, Myung-Ae; Song, Byoung J

    2003-02-01

    The peroxisome proliferator-activated receptor agonist troglitazone (TRO) was used for treatment of non-insulin-dependent diabetes until its removal from the market because of its severe hepatotoxicity. However, the mechanism for its hepatotoxicity is still poorly understood. In this study, we investigated whether TRO caused cell death by altering signaling pathways associated with cell damage and survival in human hepatoma cells. Our data reveal that TRO caused time- and concentration-dependent apoptosis of HepG2 and Chang liver human hepatoma cells, as evidenced by DNA fragmentation and staining with Hoechst 33342. In contrast, 50 or 100 microM rosiglitazone, a structural analog of TRO, did not cause apoptosis in these hepatoma cells. TRO activated both c-Jun N-terminal protein kinase (JNK) and p38 kinase about 5-fold between 0.5 and 8 h before they returned to control levels at 16 h in HepG2 cells. In contrast, TRO failed to activate the extracellular signal-regulated kinase. Furthermore, TRO increased the levels of proapoptotic proteins, Bad, Bax, release of cytochrome c, and cleavage of Bid in a time-dependent manner. The antiapoptotic Bcl-2 protein level decreased in hepatoma cells treated with TRO. Pretreatment of hepatoma cells with a selective JNK inhibitor, anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), significantly reduced the rate of TRO-induced cell death, whereas 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), an inhibitor of p38 kinase, had little effect on apoptosis. Pretreatment with SP600125 also prevented JNK activation and c-Jun phosphorylation. In addition, rosiglitazone, which is not as toxic to hepatoma cells as TRO, did not stimulate JNK activity. Transfection of cDNA for the dominant-negative mutant JNK-KR (Lys-->Arg) or SEK1-KR (Lys-->Arg), an immediate upstream kinase of JNK, significantly reduced TRO-induced JNK activation and cell death rate. Furthermore, SP600125 pretreatment effectively prevented the

  14. Blockade of the translocation and activation of c-Jun N-terminal kinase 3 (JNK3) attenuates dopaminergic neuronal damage in mouse model of Parkinson's disease.

    PubMed

    Pan, Jing; Xiao, Qin; Sheng, Cheng-Yu; Hong, Zhen; Yang, Hong-Qi; Wang, Gang; Ding, Jian-Qing; Chen, Sheng-Di

    2009-06-01

    Increasing evidence suggests that c-Jun N-terminal kinase (JNK) is an important kinase mediating neuronal death in Parkinson's disease (PD) model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). JNK3, the only neural-specific isoform, may play an important role in mediating the neurotoxic effects of MPTP in dopaminergic neuronal injury. To analyze the variation in JNK3 activation, the levels of phospho-JNK3 were measured at the various time points of occurrence of MPTP-induced lesions. In our study, we observed that during MPTP intoxication, two peaks of JNK3 activation appeared at 8 and 24h. To further define the mechanism of JNK3 activation and translocation, the antioxidant N-acetylcysteine (NAC), the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX) were administered to the mice 30 min after each of the four MPTP injections. The results revealed that NAC clearly inhibited JNK3 activation during the early intoxication, whereas ketamine preferably attenuated JNK3 activation during the latter intoxication. DNQX had no significant effects on JNK3 activation during intoxication. Consequently, reactive oxygen species (ROS) and the NMDA receptor were closely associated with JNK3 activation following MPTP intoxication. NAC and ketamine exerted a preventive effect against MPTP-induced loss of tyrosine hydroxylase-positive neurons and suppressed the nuclear translocation of JNK3, suggesting that NAC and ketamine can prevent MPTP-induced dopaminergic neuronal death by suppressing JNK3 activation.

  15. Transcriptional Activity of Neural Retina Leucine Zipper (Nrl) Is Regulated by c-Jun N-Terminal Kinase and Tip60 during Retina Development

    PubMed Central

    Kim, Jung-Woong; Jang, Sang-Min; Kim, Chul-Hong; An, Joo-Hee

    2012-01-01

    Neural retina leucine zipper (Nrl), a key basic motif leucine zipper (bZIP) transcription factor, modulates rod photoreceptor differentiation by activating rod-specific target genes. In searching for factors that might couple with Nrl to modulate its transcriptional activity through posttranslational modification, we observed the novel interactions of Nrl with c-Jun N-terminal kinase 1 (JNK1) and HIV Tat-interacting protein 60 (Tip60). JNK1 directly interacted with and phosphorylated Nrl at serine 50, which enhanced Nrl transcriptional activity on the rhodopsin and Ppp2r5c promoters. Use of an inactive JNK1 mutant or treatment with a JNK inhibitor (SP600125) significantly reduced JNK1-mediated phosphorylation and transcriptional activity of Nrl in cultured retinal explants. We also found that Nrl activated rhodopsin and Ppp2r5c transcription by recruiting Tip60 to promote histone H3/H4 acetylation. The binding affinity of phospho-Nrl for Tip60 was significantly greater than that of the unphosphorylated Nrl. Thus, the histone acetyltransferase-containing Tip60 behaved as a coactivator in the Nrl-dependent transcriptional regulation of the rhodopsin and Ppp2r5c genes in the developing mouse retina. A transcriptional network of interactive proteins, including Nrl, JNK1, and Tip60, may be required to precisely control spatiotemporal photoreceptor-specific gene expression during retinal development. PMID:22354990

  16. Transcriptional activity of neural retina leucine zipper (Nrl) is regulated by c-Jun N-terminal kinase and Tip60 during retina development.

    PubMed

    Kim, Jung-Woong; Jang, Sang-Min; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

    2012-05-01

    Neural retina leucine zipper (Nrl), a key basic motif leucine zipper (bZIP) transcription factor, modulates rod photoreceptor differentiation by activating rod-specific target genes. In searching for factors that might couple with Nrl to modulate its transcriptional activity through posttranslational modification, we observed the novel interactions of Nrl with c-Jun N-terminal kinase 1 (JNK1) and HIV Tat-interacting protein 60 (Tip60). JNK1 directly interacted with and phosphorylated Nrl at serine 50, which enhanced Nrl transcriptional activity on the rhodopsin and Ppp2r5c promoters. Use of an inactive JNK1 mutant or treatment with a JNK inhibitor (SP600125) significantly reduced JNK1-mediated phosphorylation and transcriptional activity of Nrl in cultured retinal explants. We also found that Nrl activated rhodopsin and Ppp2r5c transcription by recruiting Tip60 to promote histone H3/H4 acetylation. The binding affinity of phospho-Nrl for Tip60 was significantly greater than that of the unphosphorylated Nrl. Thus, the histone acetyltransferase-containing Tip60 behaved as a coactivator in the Nrl-dependent transcriptional regulation of the rhodopsin and Ppp2r5c genes in the developing mouse retina. A transcriptional network of interactive proteins, including Nrl, JNK1, and Tip60, may be required to precisely control spatiotemporal photoreceptor-specific gene expression during retinal development.

  17. Tricetin Induces Apoptosis of Human Leukemic HL-60 Cells through a Reactive Oxygen Species-Mediated c-Jun N-Terminal Kinase Activation Pathway

    PubMed Central

    Chien, Ming-Hsien; Chow, Jyh-Ming; Lee, Wei-Jiunn; Chen, Hui-Yu; Tan, Peng; Wen, Yu-Ching; Lin, Yung-Wei; Hsiao, Pei-Ching

    2017-01-01

    Tricetin is a dietary flavonoid with cytostatic properties and antimetastatic activities in various solid tumors. The anticancer effect of tricetin in nonsolid tumors remains unclear. Herein, the molecular mechanisms by which tricetin exerts its anticancer effects on acute myeloid leukemia (AML) cells were investigated. Results showed that tricetin inhibited cell viability in various types of AML cell lines. Tricetin induced morphological features of apoptosis such as chromatin condensation and phosphatidylserine (PS) externalization, and significantly activated proapoptotic signaling including caspase-8, -9, and -3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in HL-60 AML cells. Of note, tricetin-induced cell growth inhibition was dramatically reversed by a pan caspase and caspase-8- and -9-specific inhibitors, suggesting that this compound mainly acts through a caspase-dependent pathway. Moreover, treatment of HL-60 cells with tricetin induced sustained activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), and inhibition of ERK and JNK by their specific inhibitors respectively promoted and abolished tricetin-induced cell apoptosis. Dichlorofluorescein (DCF) staining showed that intracellular reactive oxygen species (ROS) levels were higher in tricetin-treated HL-60 cells compared to the control group. Moreover, an ROS scavenger, N-acetylcysteine (NAC), reversed tricetin-induced JNK activation and subsequent cell apoptosis. In conclusion, our results indicated that tricetin induced cell death of leukemic HL-60 cells through induction of intracellular oxidative stress following activation of a JNK-mediated apoptosis pathway. A combination of tricetin and an ERK inhibitor may be a better strategy to enhance the anticancer activities of tricetin in AML. PMID:28758971

  18. Jun N-terminal kinase signaling makes a face

    PubMed Central

    Hursh, Deborah A.; Stultz, Brian G.; Park, Sung Yeon

    2016-01-01

    ABSTRACT decapentaplegic (dpp), the Drosophila ortholog of BMP 2/4, directs ventral adult head morphogenesis through expression in the peripodial epithelium of the eye-antennal disc. This dpp expressing domain exerts effects both on the peripodial epithelium, and the underlying disc proper epithelium. We have uncovered a role for the Jun N-terminal kinase (JNK) pathway in dpp-mediated ventral head development. JNK activity is required for dpp's action on the disc proper, but in the absence of dpp expression, excessive JNK activity is produced, leading to specific loss of maxillary palps. In this review we outline our hypotheses on how dpp acts by both short range and longer range mechanisms to direct head morphogenesis and speculate on the dual role of JNK signaling in this process. Finally, we describe the regulatory control of dpp expression in the eye-antennal disc, and pose the problem of how the various expression domains of a secreted protein can be targeted to their specific functions. PMID:27384866

  19. Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation.

    PubMed

    Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

    2016-01-01

    Rice (Oryza sativa) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin E2, RHE: rice hull extract, ROS: reactive oxygen species, TNF

  20. Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation

    PubMed Central

    Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

    2016-01-01

    Background: Rice (Oryza sativa) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. Materials and Methods: In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Results: We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. Conclusion: This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. SUMMARY Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin

  1. Downregulation of cellular c-Jun N-terminal protein kinase and NF-κB activation by berberine may result in inhibition of herpes simplex virus replication.

    PubMed

    Song, Siwei; Qiu, Min; Chu, Ying; Chen, Deyan; Wang, Xiaohui; Su, Airong; Wu, Zhiwei

    2014-09-01

    Berberine is a quaternary ammonium salt from the protoberberine group of isoquinoline alkaloids. Some reports show that berberine exhibits anti-inflammatory, antitumor, and antiviral properties by modulating multiple cellular signaling pathways, including p53, nuclear factor κB (NF-κB), and mitogen-activated protein kinase. In the present study, we investigated the antiviral effect of berberine against herpes simplex virus (HSV) infection. Current antiherpes medicines such as acyclovir can lessen the recurring activation when used early at infection but are unable to prevent or cure infections where treatment has selected for resistant mutants. In searching for new antiviral agents against herpesvirus infection, we found that berberine reduced viral RNA transcription, protein synthesis, and virus titers in a dose-dependent manner. To elucidate the mechanism of its antiviral activity, the effect of berberine on the individual steps of viral replication cycle of HSV was investigated via time-of-drug addition assay. We found that berberine acted at the early stage of HSV replication cycle, between viral attachment/entry and genomic DNA replication, probably at the immediate-early gene expression stage. We further demonstrated that berberine significantly reduced HSV-induced NF-κB activation, as well as IκB-α degradation and p65 nuclear translocation. Moreover, we found that berberine also depressed HSV-induced c-Jun N-terminal kinase (JNK) phosphorylation but had little effect on p38 phosphorylation. Our results suggest that the berberine inhibition of HSV infection may be mediated through modulating cellular JNK and NF-κB pathways.

  2. Downregulation of Cellular c-Jun N-Terminal Protein Kinase and NF-κB Activation by Berberine May Result in Inhibition of Herpes Simplex Virus Replication

    PubMed Central

    Song, Siwei; Qiu, Min; Chu, Ying; Chen, Deyan; Wang, Xiaohui; Su, Airong

    2014-01-01

    Berberine is a quaternary ammonium salt from the protoberberine group of isoquinoline alkaloids. Some reports show that berberine exhibits anti-inflammatory, antitumor, and antiviral properties by modulating multiple cellular signaling pathways, including p53, nuclear factor κB (NF-κB), and mitogen-activated protein kinase. In the present study, we investigated the antiviral effect of berberine against herpes simplex virus (HSV) infection. Current antiherpes medicines such as acyclovir can lessen the recurring activation when used early at infection but are unable to prevent or cure infections where treatment has selected for resistant mutants. In searching for new antiviral agents against herpesvirus infection, we found that berberine reduced viral RNA transcription, protein synthesis, and virus titers in a dose-dependent manner. To elucidate the mechanism of its antiviral activity, the effect of berberine on the individual steps of viral replication cycle of HSV was investigated via time-of-drug addition assay. We found that berberine acted at the early stage of HSV replication cycle, between viral attachment/entry and genomic DNA replication, probably at the immediate-early gene expression stage. We further demonstrated that berberine significantly reduced HSV-induced NF-κB activation, as well as IκB-α degradation and p65 nuclear translocation. Moreover, we found that berberine also depressed HSV-induced c-Jun N-terminal kinase (JNK) phosphorylation but had little effect on p38 phosphorylation. Our results suggest that the berberine inhibition of HSV infection may be mediated through modulating cellular JNK and NF-κB pathways. PMID:24913175

  3. C-Jun N-terminal Kinase and Apoptotic Signaling in Prostate Cancer

    DTIC Science & Technology

    2002-01-01

    hydrogen peroxide (H20 2) to induce JNK activation varied in different cell types. Pyrrolidine dithiocarbamate (PDTC), a presumed antioxidant (13,14...Down-regulation of the c-Jun N-terminal kinase (JNK) phosphatase M3/6 and activation of JNK by hydrogen peroxide and pyrrolidine dithiocarbamate...and Tan, T.-H. (2001) Down-regulation of the c-Jun N-terminal kinase (JNK) phosphatase M3/6 and activation of JNK by hydrogen peroxide and pyrrolidine

  4. The c-Jun N-terminal kinase pathway of a vector insect is activated by virus capsid protein and promotes viral replication

    PubMed Central

    Wang, Wei; Zhao, Wan; Li, Jing; Luo, Lan; Kang, Le; Cui, Feng

    2017-01-01

    No evidence has shown whether insect-borne viruses manipulate the c-Jun N-terminal kinase (JNK) signaling pathway of vector insects. Using a system comprising the plant virus Rice stripe virus (RSV) and its vector insect, the small brown planthopper, we have studied the response of the vector insect’s JNK pathway to plant virus infection. We found that RSV increased the level of Tumor Necrosis Factor-α and decreased the level of G protein Pathway Suppressor 2 (GPS2) in the insect vector. The virus capsid protein competitively bound GPS2 to release it from inhibiting the JNK activation machinery. We confirmed that JNK activation promoted RSV replication in the vector, whereas JNK inhibition caused a significant reduction in virus production and thus delayed the disease incidence of plants. These findings suggest that inhibition of insect vector JNK may be a useful strategy for controling the transmission of plant viruses. DOI: http://dx.doi.org/10.7554/eLife.26591.001 PMID:28716183

  5. Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase.

    PubMed

    Du, J; Suzuki, H; Nagase, F; Akhand, A A; Yokoyama, T; Miyata, T; Kurokawa, K; Nakashima, I

    2000-03-01

    Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.

  6. Low Dose Acetaminophen Induces Reversible Mitochondrial Dysfunction Associated with Transient c-Jun N-Terminal Kinase Activation in Mouse Liver.

    PubMed

    Hu, Jiangting; Ramshesh, Venkat K; McGill, Mitchell R; Jaeschke, Hartmut; Lemasters, John J

    2016-03-01

    Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and c-jun N-terminal kinase (JNK) activation. Because the safe limit of APAP dosing is controversial, our aim was to evaluate the role of the mitochondrial permeability transition (MPT) and JNK in mitochondrial dysfunction after APAP dosing considered nontoxic by criteria of serum alanine aminotransferase (ALT) release and histological necrosis in vivo. C57BL/6 mice were given APAP with and without the MPT inhibitor, N-methyl-4-isoleucine cyclosporin (NIM811), or the JNK inhibitor, SP600125. Fat droplet formation, cell viability, and mitochondrial function in vivo were monitored by intravital multiphoton microscopy. Serum ALT, liver histology, total JNK, and activated phospho(p)JNK were also assessed. High APAP (300 mg/kg) caused ALT release, necrosis, irreversible mitochondrial dysfunction, and hepatocellular death. By contrast, lower APAP (150 mg/kg) caused reversible mitochondrial dysfunction and fat droplet formation in hepatocytes without ALT release or necrosis. Mitochondrial protein N-acetyl-p-benzoquinone imine adducts correlated with early JNK activation, but irreversible mitochondrial depolarization and necrosis at high dose were associated with sustained JNK activation and translocation to mitochondria. NIM811 prevented cell death and/or mitochondrial depolarization after both high and low dose APAP. After low dose, SP600125 decreased mitochondrial depolarization. In conclusion, low dose APAP produces reversible MPT-dependent mitochondrial dysfunction and steatosis in hepatocytes without causing ALT release or necrosis, whereas high dose leads to irreversible mitochondrial dysfunction and cell death associated with sustained JNK activation. Thus, nontoxic APAP has the potential to cause transient mitochondrial dysfunction that may synergize with other stresses to promote liver damage and steatosis. © The Author 2015. Published by Oxford University Press on

  7. Low Dose Acetaminophen Induces Reversible Mitochondrial Dysfunction Associated with Transient c-Jun N-Terminal Kinase Activation in Mouse Liver

    PubMed Central

    Hu, Jiangting; Ramshesh, Venkat K.; McGill, Mitchell R.; Jaeschke, Hartmut; Lemasters, John J.

    2016-01-01

    Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and c-jun N-terminal kinase (JNK) activation. Because the safe limit of APAP dosing is controversial, our aim was to evaluate the role of the mitochondrial permeability transition (MPT) and JNK in mitochondrial dysfunction after APAP dosing considered nontoxic by criteria of serum alanine aminotransferase (ALT) release and histological necrosis in vivo. C57BL/6 mice were given APAP with and without the MPT inhibitor, N-methyl-4-isoleucine cyclosporin (NIM811), or the JNK inhibitor, SP600125. Fat droplet formation, cell viability, and mitochondrial function in vivo were monitored by intravital multiphoton microscopy. Serum ALT, liver histology, total JNK, and activated phospho(p)JNK were also assessed. High APAP (300 mg/kg) caused ALT release, necrosis, irreversible mitochondrial dysfunction, and hepatocellular death. By contrast, lower APAP (150 mg/kg) caused reversible mitochondrial dysfunction and fat droplet formation in hepatocytes without ALT release or necrosis. Mitochondrial protein N-acetyl-p-benzoquinone imine adducts correlated with early JNK activation, but irreversible mitochondrial depolarization and necrosis at high dose were associated with sustained JNK activation and translocation to mitochondria. NIM811 prevented cell death and/or mitochondrial depolarization after both high and low dose APAP. After low dose, SP600125 decreased mitochondrial depolarization. In conclusion, low dose APAP produces reversible MPT-dependent mitochondrial dysfunction and steatosis in hepatocytes without causing ALT release or necrosis, whereas high dose leads to irreversible mitochondrial dysfunction and cell death associated with sustained JNK activation. Thus, nontoxic APAP has the potential to cause transient mitochondrial dysfunction that may synergize with other stresses to promote liver damage and steatosis. PMID:26721299

  8. Inhibition of c-Jun N-terminal kinase activation reverses Alzheimer disease phenotypes in APPswe/PS1dE9 mice.

    PubMed

    Zhou, Qiong; Wang, Man; Du, Ying; Zhang, Wei; Bai, Miao; Zhang, Zhuo; Li, Zhuyi; Miao, Jianting

    2015-04-01

    Growing evidence indicates that the activation of c-Jun N-terminal kinase (JNK) is implicated in the multiple major pathological features of Alzheimer disease (AD). However, whether specific inhibition of JNK activation could prevent disease progression in adult transgenic AD models at moderate stage remains unknown. Here we first investigated the potential disease-modifying therapeutic effect of systemic administration of SP600125, a small-molecule JNK-specific inhibitor, in middle-aged APPswe/PS1dE9 mice. Using behavioral, histological, and biochemical methods, outcomes of SP600125 treatment on neuropathology and cognitive deficits were studied in APPswe/PS1dE9 mice. Compared with vehicle-treated APPswe/PS1dE9 mice, chronic treatment of SP600125 for 12 weeks potently inhibited JNK activation, which resulted in a marked improvement of behavioral measures of cognitive deficits and a dramatic reduction in amyloid plaque burden, β-amyloid production, tau hyperphosphorylation, inflammatory responses, and synaptic loss in these transgenic animals. In particular, we found that SP600125 treatment strongly promoted nonamyloidogenic amyloid precursor protein (APP) processing and inhibited amyloidogenic APP processing via regulating APP-cleavage secretase expression (ie, ADAM10, BACE1, and PS1) in APPswe/PS1dE9 mice. Our findings demonstrate that chronic SP600125 treatment is powerfully effective in slowing down disease progression by markedly reducing multiple pathological features and ameliorating cognitive deficits associated with AD. This study highlights the concept that active JNK actually contributes to the development of the disease, and provides critical preclinical evidence that specific inhibition of JNK activation by SP600125 treatment may be a novel promising disease-modifying therapeutic strategy for the treatment of AD. © 2015 American Neurological Association.

  9. Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway.

    PubMed

    Kamio, Koichiro; Usuki, Jiro; Azuma, Arata; Matsuda, Kuniko; Ishii, Takeo; Inomata, Minoru; Hayashi, Hiroki; Kokuho, Nariaki; Fujita, Kazue; Saito, Yoshinobu; Miya, Toshimichi; Gemma, Akihiko

    2015-06-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested

  10. Spinal astrocytic c-Jun N-terminal kinase (JNK) activation as counteracting mechanism to the amitriptyline analgesic efficacy in painful peripheral neuropathies.

    PubMed

    Sanna, Maria Domenica; Ghelardini, Carla; Galeotti, Nicoletta

    2017-03-05

    Several drugs and agents are currently used for the treatment of neuropathic pain. Among them amitriptyline, a tricyclic antidepressant drug, represent a first line treatment. Despite its well-documented clinical efficacy, amitriptyline is ineffective in some animal models of neuropathic pain. The aim of this study was to investigate into amitriptyline poor efficacy in neuropathic pain and to determine the role of c-Jun N-terminal kinase (JNK) activation as counteracting mechanism to the analgesic effects of this drug. Experiments were performed in mice with painful peripheral neuropathies due to the antiretroviral agent 2,3-dideoxycytidine (ddC), and with the partial sciatic nerve injury produced in the spared nerve injury model (SNI). In mice subjected to SNI and antiretroviral treatment, amitriptyline did not attenuate mechanical allodynia and thermal hyperalgesia. Conversely, intrathecal injection of the JNK inhibitor SP600125 prevented SNI and ddC-induced nociceptive behavior and, its inactive dose co-administrated with amitriptyline induced an antinociceptive effect. Western blotting analysis showed an upregulation of p-JNK in the lumbar spinal cord of SNI and ddC-exposed mice, that was further enhanced after amitriptyline administration. Additionally, amitriptyline further promoted astrocyte activation in neuropathic mice, as illustrated by the increased expression of glial fibrillary acidic protein (GFAP), that was attenuated by intrathecal injection of the JNK inhibitor. These data indicate astrocyte JNK activation as counteracting pathway to amitriptyline analgesic response. Targeting the JNK pathway in spinal astroglia may present an efficient way to improve the analgesic efficacy of amitriptyline in the neuropathic pain treatment.

  11. Hydrogen-Rich Saline Attenuates Lipopolysaccharide-Induced Heart Dysfunction by Restoring Fatty Acid Oxidation in Rats by Mitigating C-Jun N-Terminal Kinase Activation.

    PubMed

    Tao, Bingdong; Liu, Lidan; Wang, Ni; Tong, Dongyi; Wang, Wei; Zhang, Jin

    2015-12-01

    Sepsis is common in intensive care units (ICU) and is associated with high mortality. Cardiac dysfunction complicating sepsis is one of the most important causes of this mortality. This dysfunction is due to myocardial inflammation and reduced production of energy by the heart. A number of studies have shown that hydrogen-rich saline (HRS) has a beneficial effect on sepsis. Therefore, we tested whether HRS prevents cardiac dysfunction by increasing cardiac energy. Four groups of rats received intraperitoneal injections of one of the following solutions: normal saline (NS), HRS, lipopolysaccharide (LPS), and LPS plus HRS. Cardiac function was measured by echocardiography 8 h after the injections. Gene and protein expression related to fatty acid oxidation (FAO) were measured by quantitative polymerase chain reaction (PCR) and Western blot analysis. The injection of LPS compromised heart function through decreased fractional shortening (FS) and increased left ventricular diameter (LVD). The addition of HRS increased FS, palmitate triphosphate, and the ratio of phosphocreatinine (PCr) to adenosine triphosphate (ATP) as well as decreasing LVD. The LPS challenge reduced the expression of genes related to FAO, including perioxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), perioxisome proliferator-activated receptor alpha (PPARα), Estrogen-related receptor alpha (ERRα), and their downstream targets, in mRNA and protein level, which were attenuated by HRS. However, HRS had little effect on glucose metabolism. Furthermore, HRS inhibited c-Jun N-terminal kinase (JNK) activation in the rat heart. Inhibition of JNK by HRS showed beneficial effects on LPS-challenged rats, at least in part, by restoring cardiac FAO.

  12. Heroin Activates ATF3 and CytC via c-Jun N-Terminal Kinase Pathways to Mediate Neuronal Apoptosis

    PubMed Central

    Pu, Hongwei; Wang, Xuemei; Su, Liping; Ma, Chuang; Zhang, Yan; Zhang, Liping; Chen, Xiao; Li, Xiujuan; Wang, Hua; Liu, Xiaoshan; Zhang, Jianlong

    2015-01-01

    Background Drug abuse and addiction has become a major public health problem that impacts all societies. The use of heroin may cause spongiform leukoencephalopathy (SLE). Material/Methods Cerebellar granule cells were derived from 7-day-old Sprague-Dawley rat pups. Neurons were dissociated from freshly dissected cerebella by mechanical disruption in the presence of 0.125% trypsin and DNaseI and then seeded at a density of 4×106 cells/ml in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 ham’s containing 10% fetal bovine serum and Arc-C(sigma) at concentrations to inhibit glial cell growth inoculated into 6-well plates and a small dish. Results We found that heroin induces the apoptosis of primary cultured cerebellar granule cells (CGCS) and that the c-Jun N-terminal kinase (JNK) pathway was activated under heroin treatment and stimulated obvious increases in the levels of C-jun, Cytc, and ATF3mRNA. CYTC and ATF3 were identified as candidate targets of the JNK/c-Jun pathway in this process because the specificity inhibitors SP600125 of JNK/C-jun pathways reduced the levels of C-jun, Cytc, and ATF3mRNA. The results suggested that SP600125 of JNK/C-jun can inhibit heroin-induced apoptosis of neurons. Conclusions The present study analyzes our understanding of the critical role of the JNK pathway in the process of neuronal apoptosis induced by heroin, and suggests a new and effective strategy to treat SLE. PMID:25848832

  13. Cell Volume Decrease as a Link between Azaspiracid-Induced Cytotoxicity and c-Jun-N-Terminal Kinase Activation in Cultured Neurons

    PubMed Central

    Vale, Carmen; Nicolaou, Kyriacos C.; Frederick, Michael O.; Vieytes, Mercedes R.; Botana, Luis M.

    2010-01-01

    Azaspiracids (AZAs) are a group of marine toxins recently described that currently includes 20 members. Not much is known about their mechanism of action, although the predominant analog in nature, AZA-1 targets several organs in vivo, including the central nervous system, and exhibits high neurotoxicity in vitro. AZA distribution is increasing globally with mussels being most widely implicated in AZA-related food poisoning events, with human poisoning by AZAs emerging as an increasing worldwide problem in recent years. We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Several targets for AZA-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2–3 days in vitro; however, it did not modify membrane potential in mature neurons. Furthermore, in immature cells, AZA-1 decreased the membrane depolarization evoked by exposure of the neurons to 50mM K+. Preincubation of the neurons with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), 4-acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid (SITS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), amiloride, or ouabain before addition of AZA-1 decreased the AZA-1-induced neurotoxicity and the increase in phosphorylated c-Jun-N-terminal kinase (JNK) caused by the toxin, indicating that disruption in ion fluxes was involved in the neurotoxic effect of AZA-1. Furthermore, short exposures of cultured neurons to AZA-1 caused a significant decrease in neuronal volume that was reverted by preincubation of the neurons with DIDS or amiloride before addition of the toxin. The results presented here indicate that the JNK activation induced by AZA-1 is secondary to the decrease in cellular volume elicited by the toxin. PMID:19815690

  14. Extracellular Signal-Regulated Protein Kinase, c-Jun N-terminal Protein Kinase, and Calcineurin Regulate Transient Receptor Potential M3 (TRPM3) Induced Activation of AP-1.

    PubMed

    Lesch, Andrea; Rössler, Oliver G; Thiel, Gerald

    2017-01-23

    Stimulation of transient receptor potential M3 (TRPM3) cation channels with pregnenolone sulfate induces an influx of Ca(2+) ions into the cells and a rise in the intracellular Ca(2+) concentration, leading to the activation of the activator protein-1 (AP-1) transcription factor. Here, we show that expression of a constitutively active mutant of the Ca(2+) /calmodulin-dependent protein phosphatase calcineurin attenuated pregnenolone sulfate-induced AP-1 activation in TRPM3-expressing cells. Likewise, expression of the regulatory B subunit of calcineurin reduced AP-1 activity in the cells following stimulation of TRPM3 channels. MAP kinase phosphatase-1 has been shown to attenuate TRPM3-mediated AP-1 activation. Here, we show that pregnenolone sulfate-induced stimulation of TRPM3 triggers the phosphorylation and activation of the MAP kinase extracellular signal-regulated protein kinase (ERK1/2). Pharmacological and genetic experiments revealed that stimulation of ERK1/2 is essential for the activation of AP-1 in cells expressing stimulated TRPM3 channels. ERK1/2 is required for the activation of the transcription factor c-Jun, a key component of the AP-1 transcription factor, and regulates c-Fos promoter activity. In addition, we identified c-Jun N-terminal protein kinase (JNK1/2) as a second signal transducer of activated TRPM3 channels. Together, the data show that calcineurin and the protein kinases ERK1/2 and JNK1/2 are important regulators within the signaling cascade connecting TRPM3 channel stimulation with increased AP-1-regulated transcription. This article is protected by copyright. All rights reserved.

  15. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    SciTech Connect

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Xie, Yuchao; Farhood, Anwar; Vinken, Mathieu; Jaeschke, Hartmut

    2013-12-15

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB

  16. Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

    PubMed

    Garg, Aditya; Zhao, Angela; Erickson, Sarah L; Mukherjee, Subhajit; Lau, Aik Jiang; Alston, Laurie; Chang, Thomas K H; Mani, Sridhar; Hirota, Simon A

    2016-10-01

    The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 μM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD. Copyright © 2016 by The American Society for Pharmacology and

  17. Oxidative Unfolding of the Rubredoxin Domain and the Natively Disordered N-terminal Region Regulate the Catalytic Activity of Mycobacterium tuberculosis Protein Kinase G.

    PubMed

    Wittwer, Matthias; Luo, Qi; Kaila, Ville R I; Dames, Sonja A

    2016-12-30

    Mycobacterium tuberculosis escapes killing in human macrophages by secreting protein kinase G (PknG). PknG intercepts host signaling to prevent fusion of the phagosome engulfing the mycobacteria with the lysosome and, thus, their degradation. The N-terminal NORS (no regulatory secondary structure) region of PknG (approximately residues 1-75) has been shown to play a role in PknG regulation by (auto)phosphorylation, whereas the following rubredoxin-like metal-binding motif (RD, residues ∼74-147) has been shown to interact tightly with the subsequent catalytic domain (approximately residues 148-420) to mediate its redox regulation. Deletions or mutations in NORS or the redox-sensitive RD significantly decrease PknG survival function. Based on combined NMR spectroscopy, in vitro kinase assay, and molecular dynamics simulation data, we provide novel insights into the regulatory roles of the N-terminal regions. The NORS region is indeed natively disordered and rather dynamic. Consistent with most earlier data, autophosphorylation occurs in our assays only when the NORS region is present and, thus, in the NORS region. Phosphorylation of it results only in local conformational changes and does not induce interactions with the subsequent RD. Although the reduced, metal-bound RD makes tight interactions with the following catalytic domain in the published crystal structures, it can also fold in its absence. Our data further suggest that oxidation-induced unfolding of the RD regulates substrate access to the catalytic domain and, thereby, PknG function under different redox conditions, e.g. when exposed to increased levels of reactive oxidative species in host macrophages.

  18. Regulation of Yersinia Protein Kinase A (YpkA) Kinase Activity by Multisite Autophosphorylation and Identification of an N-terminal Substrate-binding Domain in YpkA*

    PubMed Central

    Pha, Khavong; Wright, Matthew E.; Barr, Tasha M.; Eigenheer, Richard A.; Navarro, Lorena

    2014-01-01

    The serine/threonine protein kinase YpkA is an essential virulence factor produced by pathogenic Yersinia species. YpkA is delivered into host mammalian cells via a type III secretion system and localizes to the inner side of the plasma membrane. We have previously shown that YpkA binds to and phosphorylates the α subunit of the heterotrimeric G protein complex, Gαq, resulting in inhibition of Gαq signaling. To identify residues in YpkA involved in substrate binding activity we generated GFP-YpkA N-terminal deletion mutants and performed coimmunoprecipitation experiments. We located a substrate-binding domain on amino acids 40–49 of YpkA, which lies within the previously identified membrane localization domain on YpkA. Deletion of amino acids 40–49 on YpkA interfered with substrate binding, substrate phosphorylation and substrate inhibition. Autophosphorylation regulates the kinase activity of YpkA. To dissect the mechanism by which YpkA transmits signals, we performed nano liquid chromatography coupled to tandem mass spectrometry to map in vivo phosphorylation sites. Multiple serine phosphorylation sites were identified in the secretion/translocation region, kinase domain, and C-terminal region of YpkA. Using site-directed mutagenesis we generated multiple YpkA constructs harboring specific serine to alanine point mutations. Our results demonstrate that multiple autophosphorylation sites within the N terminus regulate YpkA kinase activation, whereas mutation of serine to alanine within the C terminus of YpkA had no effect on kinase activity. YpkA autophosphorylation on multiple sites may be a strategy used by pathogenic Yersinia to prevent inactivation of this important virulence protein by host proteins. PMID:25086045

  19. Alpha 1-Antichymotrypsin, an Inflammatory Protein Overexpressed in the Brains of Patients with Alzheimer's Disease, Induces Tau Hyperphosphorylation through c-Jun N-Terminal Kinase Activation

    PubMed Central

    Tyagi, Ethika; Fiorelli, Tina; Norden, Michelle

    2013-01-01

    The association of inflammatory proteins with neuritic plaques in the brains of Alzheimer's disease (AD) patients has led to the hypothesis that inflammation plays a pivotal role in the development of pathology in AD. Earlier studies have shown that alpha 1-antichymotrypsin (ACT) enhances amyloid beta fibrillization and accelerated plaque formation in APP transgenic mice. Later studies from our laboratory have shown that purified ACT induces tau hyperphosphorylation and degeneration in neurons. In order to understand the mechanisms by which inflammatory proteins enhance tau hyperphosphorylation, we injected interleukin-1β (IL-1β) intracerebroventricularly into mice expressing human ACT, human tau, or both transgenes. It was found that the hyperphosphorylation of tau in ACT and ACT/htau mice after IL-1β injection correlated with increased phosphorylation of c-Jun N-terminal kinase (JNK). We verified the involvement of JNK in ACT-induced tau phosphorylation by utilizing JNK inhibitors in cultured primary neurons treated with ACT, and we found that the inhibitor showed complete prevention of ACT-induced tau phosphorylation. These results indicate that JNK is one of the major kinases involved in the ACT-mediated tau hyperphosphorylation and suggest that inhibitors of this kinase may protect against inflammation-induced tau hyperphosphorylation and neurodegeneration associated with AD. PMID:24175110

  20. Effects of curcumin on apoptosis and oxidoinflammatory regulation in a rat model of acetic acid-induced colitis: the roles of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase.

    PubMed

    Topcu-Tarladacalisir, Yeter; Akpolat, Meryem; Uz, Yesim Hulya; Kizilay, Gulnur; Sapmaz-Metin, Melike; Cerkezkayabekir, Aysegul; Omurlu, Imran Kurt

    2013-04-01

    The present study evaluated the effects of curcumin on epithelial cell apoptosis, the immunoreactivity of the phospho-c-Jun N-terminal kinase (JNK) and phospho-p38 mitogen-activated protein kinases (MAPKs) in inflamed colon mucosa, and oxidative stress in a rat model of ulcerative colitis induced by acetic acid. Rats were randomly divided into three groups: control, acetic acid, and acetic acid+curcumin. Curcumin (100 mg/kg per day, intragastrically) was administered 10 days before the induction of colitis and was continued for two additional days. Acetic acid-induced colitis caused a significant increase in the macroscopic and microscopic tissue ranking scores as well as an elevation in colonic myeloperoxidase (MPO) activity, malondialdehyde (MDA) levels, and the number of apoptotic epithelial cells in colon tissue compared to controls. In the rat colon, immunoreactivity of phospho-p38 MAPK was increased, whereas the phospho-JNK activity was decreased following the induction of colitis. Curcumin treatment was associated with amelioration of macroscopic and microscopic colitis sores, decreased MPO activity, and decreased MDA levels in acetic acid-induced colitis. Furthermore, oral curcumin supplementation clearly prevented programmed cell death and restored immunreactivity of MAPKs in the colons of colitic rats. The results of this study suggest that oral curcumin treatment decreases colon injury and is associated with decreased inflammatory reactions, lipid peroxidation, apoptotic cell death, and modulating p38- and JNK-MAPK pathways.

  1. The catalytic subunit of Dictyostelium cAMP-dependent protein kinase -- role of the N-terminal domain and of the C-terminal residues in catalytic activity and stability.

    PubMed

    Etchebehere, L C; Van Bemmelen, M X; Anjard, C; Traincard, F; Assemat, K; Reymond, C; Véron, M

    1997-09-15

    The C subunit of Dictyostelium cAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI. Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence-comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.

  2. Retinoic acids acting through retinoid receptors protect hippocampal neurons from oxygen-glucose deprivation-mediated cell death by inhibition of c-jun-N-terminal kinase and p38 mitogen-activated protein kinase.

    PubMed

    Shinozaki, Y; Sato, Y; Koizumi, S; Ohno, Y; Nagao, T; Inoue, K

    2007-06-15

    Retinoic acids (RAs), including all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), play fundamental roles in a variety of physiological events in vertebrates, through their specific nuclear receptors: retinoic acid receptor (RAR) and retinoid X receptor (RXR). Despite the physiological importance of RA, their functional significance under pathological conditions is not well understood. We examined the effect of ATRA on oxygen/glucose-deprivation/reperfusion (OGD/Rep)-induced neuronal damage in cultured rat hippocampal slices, and found that ATRA significantly reduced neuronal death. The cytoprotective effect of ATRA was observed not only in cornu ammonis (CA) 1 but also in CA2 and dentate gyrus (DG), and was attenuated by selective antagonists for RAR or RXR. By contrast, in the CA3 region, no protective effects of ATRA were observed. The OGD/Rep also increased phosphorylated forms of c-jun-N-terminal kinase (P-JNK) and p38 (P-p38) in hippocampus, and specific inhibitors for these kinases protected neurons. ATRA prevented the increases in P-JNK and P-p38 after OGD/Rep, as well as the decrease in NeuN and its shrinkage, all of which were inhibited by antagonists for RAR or RXR. These findings suggest that the ATRA signaling via retinoid receptors results in the inhibition of JNK and p38 activation, leading to the protection of neurons against OGD/Rep-induced damage in the rat hippocampus.

  3. Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: evidence for the role of c-Jun N-terminal kinase activation.

    PubMed

    Cheng, Hairong; Hong, Bo; Zhou, Lanlan; Allen, Joshua E; Tai, Guihua; Humphreys, Robin; Dicker, David T; Liu, Yingqiu Y; El-Deiry, Wafik S

    2012-09-01

    The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53-/-) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.

  4. c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways link capacitation with apoptosis and seminal plasma proteins protect sperm by interfering with both routes†.

    PubMed

    Luna, Carolina; Mendoza, Noelia; Casao, Adriana; Pérez-Pé, Rosaura; Cebrián-Pérez, José A; Muiño-Blanco, Teresa

    2017-04-01

    The mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38) signaling cascades are involved in triggering apoptosis in somatic cells. Given that spermatozoa are able to undergo apoptosis, we tested the hypothesis that these pathways might be functional in ram spermatozoa as two signal transduction mechanisms that contribute to the modulation of capacitation and apoptosis. Indirect immunofluorescence and western blot analysis evidenced the presence of JNK and p38 in ram spermatozoa. To verify the involvement of these enzymes in sperm physiology, we determined the effect of specific inhibitors of JNK or p38 on in vitro capacitation induced with either cAMP-elevating agents or epidermal growth factor (EGF). Both inhibitions reduced the EGF-induced capacitation with a decrease in the chlortetracycline capacitated-sperm pattern, protein tyrosine phosphorylation, phosphatidylserine externalization, caspase-3 and -7 activation, and the proportion of DNA-damaged spermatozoa. No significant changes were found in the high-cAMP capacitated samples. The addition of 3.4 mg/ml seminal plasma proteins (SPPs) to the EGF-containing samples, either alone or together with each inhibitor, resulted in a decreased proportion of capacitated sperm pattern, protein tyrosine phosphorylation, loss of plasma membrane integrity, and apoptotic alterations. Furthermore, SPPs significantly reduced the phosphorylation level of JNK and p38 MAPK (active forms). These findings show a relationship between capacitation and apoptosis, and represent a step forward in the knowledge of the SPP protective mechanism in spermatozoa. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.

  5. Ketamine inhibits tumor necrosis factor-{alpha} and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

    SciTech Connect

    Wu, G.-J.; Chen, T.-L.; Ueng, Y.-F.; Chen, R.-M.

    2008-04-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 {mu}M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 {mu}M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-{alpha} and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-{alpha} and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 {mu}M) significantly inhibited LPS-induced TNF-{alpha} and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-{alpha} and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-{alpha} and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms

  6. Ketamine inhibits tumor necrosis factor-alpha and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation.

    PubMed

    Wu, Gone-Jhe; Chen, Ta-Liang; Ueng, Yune-Fang; Chen, Ruei-Ming

    2008-04-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 microM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 microM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-alpha and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-alpha and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 microM) significantly inhibited LPS-induced TNF-alpha and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-alpha and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-alpha and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through

  7. Exercise training improves basal blood glucose metabolism with no changes of cytosolic inhibitor B kinase or c-Jun N-terminal kinase activation in skeletal muscle of Otsuka Long-Evans Tokushima fatty rats.

    PubMed

    Lee, Hojun; Chang, Hyukki; Park, Jae-Young; Kim, Si-Young; Choi, Kyung-Mook; Song, Wook

    2011-07-01

    Redox-sensitive stress kinases and heat shock protein 72 (Hsp72) have been considered to be associated with the development of type 2 diabetes in skeletal muscle. However, the effect of exercise training on skeletal muscle of type 2 diabetic models is largely unknown. The purpose of this study was to investigate the effect of 12 weeks of exercise training on gastrocnemius of type 2 diabetic rats, by examining the activation of c-Jun N-terminal kinase (JNK), the nuclear factor B (NF-B) pathway and Hsp72. Total hydroperoxide and 4-hydroxynoneal, as oxidative stress markers, were also examined. Otsuka Long-Evans Tokushima fatty (OLEFT) rats were randomly divided into an exercise training group (Ex-OLETF, n = 8) and a sedentary group (Sed-OLETF, n = 8), while Long-Evans Tokushima Otsuka (LETO) rats were used as a control group (Con-LETO, n = 5). The Ex-OLETF rats were trained on a treadmill five times a week for 12 weeks. The levels of hydroperoxide and 4-hydroxynoneal in both Ex-OLETF and Sed-OLETF were significantly higher compared with Con-LETO, but there was no difference between Ex-OLETF and Sed-OLETF. Levels of inhibitor B kinase, JNK activation and p65 nuclear translocation followed a similar pattern to that observed in oxidative stress markers. The level of Hsp72 in Ex-OLETF was increased by exercise training, but it did not reach the level observed in Con-LETO. The NF-B DNA binding activity in Sed-OLETF was significantly higher compared with Con-LETO. Although it was not statistically significant, exercise training in Ex-OLETF showed a trend to reduce the activation of NF-B DNA binding activity compared with Sed-OLETF (P = 0.104). Our findings indicate that exercise training improves basal glucose metabolism without a change in stress kinases, and that nuclear regulation of NF-B activity in diabetic muscle could be regulated independently of the cytosolic pathway. Our study also suggests a possibility that exercise-induced Hsp72 serves as a protective

  8. A novel splice variant of mouse interleukin-1-receptor-associated kinase-1 (IRAK-1) activates nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK).

    PubMed Central

    Yanagisawa, Ken; Tago, Kenji; Hayakawa, Morisada; Ohki, Motomichi; Iwahana, Hiroyuki; Tominaga, Shin-Ichi

    2003-01-01

    Interleukin-1 (IL-1)-receptor-associated kinase (IRAK) is an indispensable signalling molecule for host-defence responses initiated by a variety of ligands that bind to members of the Toll/IL-1 receptor family. Here we report a novel splice variant of mouse IRAK-1, IRAK-1-S, which is generated by utilizing a new splicing acceptor site within exon 12. IRAK-1-S cDNA is shorter than the originally reported IRAK-1 (IRAK-1-W) cDNA by 271 nucleotides, and the subsequent frameshift causes a premature termination of translation after 23 amino acids, which are unique to the IRAK-1-S protein. To elucidate the physiological function of IRAK-1-S, we overexpressed it in 293T cells and studied the effects on the IL-1 signalling cascade. As it lacks the C-terminal region of IRAK-1-W that has been reported to contain the TRAF6 (tumour necrosis factor receptor-associated factor 6) binding domain, IRAK-1-S was unable to bind TRAF6 protein, which is a proposed downstream signalling molecule. However, IRAK-1-S overexpressed in 293T cells induced constitutive activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) independent of stimulation by IL-1, as did IRAK-1-W. To clarify the mechanism of NF-kappaB activation by IRAK-1-S in the absence of binding to TRAF6, we demonstrated that IRAK-1-S binds to IRAK-1-W through its death domain; the findings suggested that overexpressed IRAK-1-S may bind endogenous IRAK-1-W and activate TRAF6 through IRAK-1-W. These results also indicate that this novel variant may play roles in the activation of NF-kappaB and JNK by IL-1 and other ligands whose signal transduction is dependent on IRAK-1 under physiological conditions. PMID:12418963

  9. Receptor-interacting protein kinase 2 promotes triple-negative breast cancer cell migration and invasion via activation of nuclear factor-kappaB and c-Jun N-terminal kinase pathways

    PubMed Central

    2014-01-01

    Introduction Metastasis is the main cause of breast cancer morbidity and mortality. Processes that allow for tumor cell migration and invasion are important therapeutic targets. Here we demonstrate that receptor-interacting protein kinase 2 (RIP2), a kinase known to be involved in inflammatory processes, also has novel roles in cancer cell migration and invasion. Methods A total of six breast cancer expression databases, including The Cancer Genome Atlas, were assessed for RIP2 expression among various clinical subtypes and its role as a prognostic biomarker. mRNA fluorescence in situ hybridization (FISH) for RIP2 was performed on 17 stage III breast cancers to determine if there was a correlation between RIP2 expression and lymph node involvement. RNA-interference was used to knock-down RIP2 expression in MDA-MB-231, Htb126, SUM149PT, MCF7, T47D, and HCC1428 cells. Cell migration and invasion were measured in vitro by scratch/wound healing and transwell migration assays. A xenograft mouse model was used to assess tumor growth and chemosensitivity to docetaxel in vivo in MDA-MB-231 cells with and without RIP2 small hairpin RNA knockdown. Western blot and immunofluorescence imaging were used to evaluate protein expressions. Results Interrogation of expression databases showed that RIP2 expression is significantly over-expressed in triple-negative breast cancers (TNBC: estrogen-receptor (ER) negative, progesterone-receptor (PR) negative, Her2/neu- (Her2) negative), compared to other clinical subtypes. High RIP2 expression correlates with worse progression-free survival using a combined breast cancer expression array dataset consisting of 946 patients. Multivariate analysis shows RIP2 as an independent prognostic biomarker. Knock-down of RIP2 significantly decreases migration in both scratch/wound healing and transwell migration assays in MDA-MB-231, Htb126, SUM149PT, MCF7, and T47D cells and is correlated with decreased Nuclear Factor-kappaB and c-Jun N-terminal

  10. Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling★

    PubMed Central

    Ma, Yinghuan; Bao, Yongxin; Li, Chenghao; Jiao, Fubin; Xin, Hongjie; Yuan, Zhengwei

    2012-01-01

    Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway. PMID:25337099

  11. Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

    PubMed

    Wang, Xiao-Hu; Hong, Xin; Zhu, Lei; Wang, Yun-Tao; Bao, Jun-Ping; Liu, Lei; Wang, Feng; Wu, Xiao-Tao

    2015-04-01

    Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

  12. The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase.

    PubMed

    Beltman, J; McCormick, F; Cook, S J

    1996-10-25

    The role of protein kinase C (PKC) in inflammation, mitogenesis, and differentiation has been deduced in part through the use of a variety of PKC inhibitors. Two widely used inhibitors are the structurally related compounds GF109203X and Ro-31-8220, both of which potently inhibit PKC activity and are believed to be highly selective. While using GF109203X and Ro-31-8220 to address the role of PKC in immediate early gene expression, we observed striking differential effects by each of these two compounds. Growth factors induce the expression of the immediate early gene products MAP kinase phosphatase-1 (MKP-1), c-Fos and c-Jun. Ro-31-8220 inhibits growth factor-stimulated expression of MKP-1 and c-Fos but strongly stimulated c-Jun expression, even in the absence of growth factors. GF109203X displays none of these properties. These data suggest that Ro-31-8220 may have other pharmacological actions in addition to PKC inhibition. Indeed, Ro-31-8220 strongly stimulates the stress-activated protein kinase, JNK1. Furthermore, Ro-31-8220 apparently activates JNK in a PKC-independent manner. Neither the down-regulation of PKC by phorbol esters nor the inhibition of PKC by GF109203X affected the ability of Ro-31-8220 to activate JNK1. These data suggest that, in addition to potently inhibiting PKC, Ro-31-8220 exhibits novel pharmacological properties which are independent of its ability to inhibit PKC.

  13. Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways.

    PubMed

    Duan, Wen-Jun; Li, Qi-Sheng; Xia, Ming-Yu; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2011-01-01

    Our previous research demonstrated that hepatic-protectant silibinin induced autophagy in human fibro-sarcoma HT1080 cells through reactive oxygen species (ROS) pathway. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced autophagy and reversed silibinin's growth-inhibitory effect; besides, PFT-α decreased the activation of caspase-3, a crucial executor of apoptosis. Silibinin upregulated expression of p53/phosphorylated-p53 (p-p53) in a time-dependent manner. Catalase (scavenger of H(2)O(2)), superoxide dismutase (SOD) (scavenger of O(2)(•-)), and SB203580 (inhibitor of p38) attenuated upregulation of p53 expression, suggesting that p53 might be partially regulated by ROS-p38 pathway. On the other hand, c-Jun N-terminal kinase (JNK) increased autophagic death in silibinin-treated cells, and JNK/p-JNK expression was upregulated by silibinin time-dependently. Inhibition of JNK by SP600125 did not influence generation of ROS. Scavengers of H(2)O(2) or O(2)(•-) showed no effect on expression of JNK/p-JNK, indicating that JNK might not correlate with ROS in this process. However, activation of p53 was suppressed by SP600125; therefore the function of p53 was possibly controlled by JNK as well. Western blotting analysis showed that PFT-α reduced activation of extracellular regulated kinase1/2 (ERK1/2) and expression of protein kinase B (PKB, or Akt)/p-Akt. PD98059 (inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK) and wortmannin (inhibitor of phosphoinositide 3-kinase (PI3K)/Akt) enhanced silibinin's cytotoxicity. Wortmannin augmented silibinin-induced autophagy, while PD98059 did not affect autophagic ratio. These results suggest that silibinin might induce p53-mediated autophagic cell death by activating ROS-p38 and JNK pathways, as well as inhibiting MEK/ERK and PI3K/Akt pathways.

  14. The c-Jun-N-terminal-Kinase inhibitor SP600125 enhances the butyrate derivative D1-induced apoptosis via caspase 8 activation in Kasumi 1 t(8;21) acute myeloid leukaemia cells.

    PubMed

    Rovida, Elisabetta; Gozzini, Antonella; Barbetti, Valentina; Giuntoli, Serena; Santini, Valeria; Dello Sbarba, Persio

    2006-12-01

    We recently showed that the histone deacetylase inhibitor D1 induced apoptosis in the t(8;21) Kasumi 1 acute myeloid leukaemia (AML) cell line and activated caspase 9. The present study characterised the effects of the combined administration of D1 with PD98059, SB203580 or SP600125, specific inhibitors of mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 or Jun N-terminal kinase (JNK), respectively. Among these inhibitors, SP600125 was the only one to markedly induce apoptosis and decrease cell proliferation. These experiments showed that SP600125 activated caspase 8 and confirmed that D1 activated the intrinsic pathway of apoptosis, as caspase 8 was not affected while Bcl-2 was down-regulated following D1 administration. The combination of the two drugs enhanced caspase-8 activation and induced apoptosis in an additive fashion. JNK was constitutively activated in the Kasumi 1, NB4, HL60 and THP-1 human AML cell lines, as well as in primary blasts from a t(8;21) AML patient. In all these cells, the pro-apoptotic effect of the two drugs alone was increased when they were combined. On this basis, the combined administration of D1 with SP600125 seems to be very promising as a potential anti-leukaemic tool in AML.

  15. MicroRNA-181a Regulates Apoptosis and Autophagy Process in Parkinson’s Disease by Inhibiting p38 Mitogen-Activated Protein Kinase (MAPK)/c-Jun N-Terminal Kinases (JNK) Signaling Pathways

    PubMed Central

    Liu, Ying; Song, Yanfeng; Zhu, Xiaotun

    2017-01-01

    Background microRNA (miR)-181a has been reported to be downregulated in Parkinson’s disease (PD), but the regulatory mechanism of miR-181a on neuron apoptosis and autophagy is still poorly understood. We aimed to investigate the neuroprotective effects of miR-181a on PD in vitro. Material/Methods Human SK-N-SH neuroblastoma cells were incubated with different concentrations of 1-methyl-4-phenylpyridinium ion (MPP+) to induce the PD model. The expression of miR-181a was then analyzed. After transfection with miR-181a mimic or scramble following MPP+ treatment, the expression of autophagy protein markers (LC3II, LC3I, and Beclin 1) and p38 mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinases (JNK) signaling proteins (p-p38, p38, p-JNK, and JNK) and cell apoptosis were detected. Furthermore, the cells were transfected with miR-181a inhibitor and cultured in the presence or absence of p38 inhibitor SB203582 or JNK inhibitor SP600125, and the cell apoptosis was tested again. Results The expression of miR-181a was gradually decreased with the increase of MPP+ concentration (P<0.05, P<0.01, or P<0.001). Overexpression of miR-181a significantly decreased the LC3II/LC3I ratio, Beclin 1 expression, cell apoptosis, and the expression of p-p38 and p-JNK compared to the MPP+ + miR-181a scramble group (all P<0.05). In addition, we observed that SB203582 or SP600125 showed no effects on cell apoptosis, but the effects of miR-181a inhibitor on cell apoptosis were reversed by administration of SB203582 or SP600125 compared to the scramble group (P<0.05). Conclusions Our results suggest that miR-181a regulates apoptosis and autophagy in PD by inhibiting the p38 MAPK/JNK pathway. PMID:28365714

  16. MicroRNA-181a Regulates Apoptosis and Autophagy Process in Parkinson's Disease by Inhibiting p38 Mitogen-Activated Protein Kinase (MAPK)/c-Jun N-Terminal Kinases (JNK) Signaling Pathways.

    PubMed

    Liu, Ying; Song, Yanfeng; Zhu, Xiaotun

    2017-04-02

    BACKGROUND microRNA (miR)-181a has been reported to be downregulated in Parkinson's disease (PD), but the regulatory mechanism of miR-181a on neuron apoptosis and autophagy is still poorly understood. We aimed to investigate the neuroprotective effects of miR-181a on PD in vitro. MATERIAL AND METHODS Human SK-N-SH neuroblastoma cells were incubated with different concentrations of 1-methyl-4-phenylpyridinium ion (MPP+) to induce the PD model. The expression of miR-181a was then analyzed. After transfection with miR-181a mimic or scramble following MPP+ treatment, the expression of autophagy protein markers (LC3II, LC3I, and Beclin 1) and p38 mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinases (JNK) signaling proteins (p-p38, p38, p-JNK, and JNK) and cell apoptosis were detected. Furthermore, the cells were transfected with miR-181a inhibitor and cultured in the presence or absence of p38 inhibitor SB203582 or JNK inhibitor SP600125, and the cell apoptosis was tested again. RESULTS The expression of miR-181a was gradually decreased with the increase of MPP+ concentration (P<0.05, P<0.01, or P<0.001). Overexpression of miR-181a significantly decreased the LC3II/LC3I ratio, Beclin 1 expression, cell apoptosis, and the expression of p-p38 and p-JNK compared to the MPP+ + miR-181a scramble group (all P<0.05). In addition, we observed that SB203582 or SP600125 showed no effects on cell apoptosis, but the effects of miR-181a inhibitor on cell apoptosis were reversed by administration of SB203582 or SP600125 compared to the scramble group (P<0.05). CONCLUSIONS Our results suggest that miR-181a regulates apoptosis and autophagy in PD by inhibiting the p38 MAPK/JNK pathway.

  17. Presynaptic c-Jun N-terminal Kinase 2 regulates NMDA receptor-dependent glutamate release

    PubMed Central

    Nisticò, Robert; Florenzano, Fulvio; Mango, Dalila; Ferraina, Caterina; Grilli, Massimo; Di Prisco, Silvia; Nobili, Annalisa; Saccucci, Stefania; D'Amelio, Marcello; Morbin, Michela; Marchi, Mario; Mercuri, Nicola B.; Davis, Roger J.; Pittaluga, Anna; Feligioni, Marco

    2015-01-01

    Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a critical step for neuronal death occurring in several neurological conditions. JNKs can be activated via receptor tyrosine kinases, cytokine receptors, G-protein coupled receptors and ligand-gated ion channels, including the NMDA glutamate receptors. While JNK has been generally associated with postsynaptic NMDA receptors, its presynaptic role remains largely unexplored. Here, by means of biochemical, morphological and functional approaches, we demonstrate that JNK and its scaffold protein JIP1 are also expressed at the presynaptic level and that the NMDA-evoked glutamate release is controlled by presynaptic JNK-JIP1 interaction. Moreover, using knockout mice for single JNK isoforms, we proved that JNK2 is the essential isoform in mediating this presynaptic event. Overall the present findings unveil a novel JNK2 localization and function, which is likely to play a role in different physiological and pathological conditions. PMID:25762148

  18. Cordycepin causes p21WAF1-mediated G2/M cell-cycle arrest by regulating c-Jun N-terminal kinase activation in human bladder cancer cells.

    PubMed

    Lee, Se-Jung; Kim, Si-Kwan; Choi, Won-Seok; Kim, Wun-Jae; Moon, Sung-Kwon

    2009-10-15

    Cordycepin (3'-deoxyadenosine), a bioactive compound of Cordyceps militaris, has many pharmacological activities. The present study reveals novel molecular mechanisms for the anti-tumor effects of cordycepin in two different bladder cancer cell lines, 5637 and T-24 cells. Cordycepin treatment, at a dose of 200 microM (IC(50)) during cell-cycle progression resulted in significant and dose-dependent growth inhibition, which was largely due to G2/M-phase arrest, and resulted in an up-regulation of p21WAF1 expression, independent of the p53 pathway. Moreover, treatment with cordycepin-induced phosphorylation of JNK (c-Jun N-terminal kinases). Blockade of JNK function using SP6001259 (JNK-specific inhibitor) and small interfering RNA (si-JNK1) rescued cordycepin-dependent p21WAF1 expression, inhibited cell growth, and decreased cell cycle proteins. These results suggest that cordycepin could be an effective treatment for bladder cancer.

  19. Crystal structure of the N-terminal SH3 domain of mouse {beta}PIX, p21-activated kinase-interacting exchange factor

    SciTech Connect

    Li Xiaofeng; Liu Xueqi; Sun Fei; Gao Jia; Zhou Hongwei; Gao, George F.; Bartlam, Mark; Rao Zihe . E-mail: raozh@xtal.tsinghua.edu.cn

    2006-01-06

    The mouse {beta}PIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3{sub 2}21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact {beta}-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse {beta}PIX-SH3 domain binding the way in which the {beta}PIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse {beta}PIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse {beta}PIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of {beta}PIX-SH3 domain resulting in its multiple biological functions.

  20. Mucin1 mediates autocrine transforming growth factor beta signaling through activating the c-Jun N-terminal kinase/activator protein 1 pathway in human hepatocellular carcinoma cells.

    PubMed

    Li, Qiongshu; Liu, Guomu; Shao, Dan; Wang, Juan; Yuan, Hongyan; Chen, Tanxiu; Zhai, Ruiping; Ni, Weihua; Tai, Guixiang

    2015-02-01

    In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-β) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-β signaling. To investigate the effect of MUC1 on TGF-β signaling, we determined TGF-β secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-β1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-β promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-β receptor (TβR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-β signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-β secretion and exogenous TGF-β1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TβR inhibitor or small interfering RNA silencing of TGF-β1 gene in HCC cells. The high correlation between MUC1 and TGF-β1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-β signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy.

  1. Arrestin-3 binds c-Jun N-terminal kinase 1 (JNK1) and JNK2 and facilitates the activation of these ubiquitous JNK isoforms in cells via scaffolding.

    PubMed

    Kook, Seunghyi; Zhan, Xuanzhi; Kaoud, Tamer S; Dalby, Kevin N; Gurevich, Vsevolod V; Gurevich, Eugenia V

    2013-12-27

    Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.

  2. c-Jun N-terminal kinase 1 promotes transforming growth factor-β1-induced epithelial-to-mesenchymal transition via control of linker phosphorylation and transcriptional activity of Smad3.

    PubMed

    Velden, Jos L J van der; Alcorn, John F; Guala, Amy S; Badura, Elsbeth C H L; Janssen-Heininger, Yvonne M W

    2011-04-01

    Transforming growth factor (TGF)-β1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-β1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-β1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-β1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-β1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-β1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-β1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-β1-induced EMT.

  3. Evolutionary Conserved Role of c-Jun-N-Terminal Kinase in CO2-Induced Epithelial Dysfunction

    PubMed Central

    Vadász, István; Dada, Laura A.; Briva, Arturo; Helenius, Iiro Taneli; Sharabi, Kfir; Welch, Lynn C.; Kelly, Aileen M.; Grzesik, Benno A.; Budinger, G. R. Scott; Liu, Jing; Seeger, Werner; Beitel, Greg J.; Gruenbaum, Yosef; Sznajder, Jacob I.

    2012-01-01

    Elevated CO2 levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO2 signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO2 levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO2 levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO2-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO2 signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO2 signaling in mammals, diptera and nematodes. PMID:23056407

  4. The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses

    SciTech Connect

    Muscarella, Donna E. Bloom, Stephen E.

    2008-04-01

    The c-Jun N-terminal kinase (JNK) pathway can play paradoxical roles as either a pro-survival or a pro-cell death pathway depending on type of stress and cell type. The goal of the present study was to determine the role of JNK pathway signaling for regulating B-cell apoptosis in two important but contrasting situations-global proteotoxic damage, induced by arsenite and hyperthermia, versus specific microtubule inhibition, induced by the anti-cancer drug vincristine, using the EW36 B-cell line. This cell line over-expresses the Bcl-2 protein and is a useful model to identify treatments that can overcome multi-drug resistance in lymphoid cells. Exposure of EW36 B-cells to arsenite or lethal hyperthermia resulted in activation of the JNK pathway and induction of apoptosis. However, pharmacological inhibition of the JNK pathway did not inhibit apoptosis, indicating that JNK pathway activation is not required for apoptosis induction by these treatments. In contrast, vincristine treatment of EW36 B-cells resulted in JNK activation and apoptosis that was suppressed by JNK inhibition. A critical difference between the two types of stress treatments was that only vincristine-induced JNK activation resulted in phosphorylation of Bcl-2 at threonine-56, a modification that can block its anti-apoptotic function. Importantly, Bcl-2 phosphorylation was attenuated by JNK inhibition implicating JNK as the upstream kinase. Furthermore, arsenite and hyperthermia treatments activated a p53/p21 pathway associated with apoptosis induction, whereas vincristine did not activate this pathway. These results reveal two stress-activated pathways, one JNK-dependent and another JNK-independent, either of which can bypass Bcl-2 mediated resistance, resulting in cell death.

  5. Phytosphingosine-1-phosphate represses the hydrogen peroxide-induced activation of c-Jun N-terminal kinase in human dermal fibroblasts through the phosphatidylinositol 3-kinase/Akt pathway.

    PubMed

    Lee, Jeong Pyo; Cha, Hwa Jun; Lee, Kwang Sik; Lee, Kun Kook; Son, Ju Hyun; Kim, Kwang Nyeon; Lee, Dong Kyu; An, Sungkwan

    2012-10-01

    Dermal fibroblasts are differentiated mesenchymal cells that regulate the extracellular matrix through the production of dermis components. Dermal fibroblasts can be damaged by reactive oxygen species induced by ultraviolet rays and chemicals. In addition to its effects on the dermis, oxidative stress poses a major threat to organisms and is believed to play an essential role in many disease processes. In this study, we show that human dermal fibroblasts (HDFs) express sphingosine-1-phosphate (S1P) receptors S1P(1), S1P(2), and S1P(3). In addition, cell viability of HDFs is increased by phytosphingosine-1-phosphate (PhS1P) via regulation of the Jun N-terminal kinase (JNK)/Akt pathway. Interestingly, regulation of the JNK/Akt pathway by PhS1P attenuated H(2)O(2)-induced cell growth arrest. Together, our data indicate that PhS1P attenuates H(2)O(2)-induced growth arrest through regulation of the signal molecules Akt and JNK, and suggest that PhS1P may have value as an anti-aging material in cosmetics and medicine.

  6. Independent repression of bile acid synthesis and activation of c-Jun N-terminal kinase (JNK) by activated hepatocyte fibroblast growth factor receptor 4 (FGFR4) and bile acids.

    PubMed

    Yu, Chundong; Wang, Fen; Jin, Chengliu; Huang, Xinqiang; McKeehan, Wallace L

    2005-05-06

    The fibroblast growth factor (FGF) receptor complex is a regulator of adult organ homeostasis in addition to its central role in embryonic development and wound healing. FGF receptor 4 (FGFR4) is the sole FGFR receptor kinase that is significantly expressed in mature hepatocytes. Previously, we showed that mice lacking mouse FGFR4 (mR4(-/-)) exhibited elevated fecal bile acids, bile acid pool size, and expression of liver cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for canonical neutral bile acid synthesis. To prove that hepatocyte FGFR4 was a negative regulator of cholesterol metabolism and bile acid synthesis independent of background, we generated transgenic mice overexpressing a constitutively active human FGFR4 (CahR4) in hepatocytes and crossed them with the FGFR4-deficient mice to generate CahR4/mR4(-/-) mice. In mice expressing active FGFR4 in liver, fecal bile acid excretion was 64%, bile acid pool size was 47%, and Cyp7a1 expression was 10-30% of wild-type mice. The repressed level of Cyp7a1 expression was resistant to induction by a high cholesterol diet relative to wild-type mice. Expression of CahR4 in mR4(-/-) mouse livers depressed bile acid synthesis below wild-type levels from the elevated levels observed in mR4(-/-). Levels of phosphorylated c-Jun N-terminal kinase (JNK), which is part of a pathway implicated in bile acid-mediated repression of synthesis, was 30% of wild-type levels in mR4(-/-) livers, whereas CahR4 livers exhibited an average 2-fold increase. However, cholate still strongly induced phospho-JNK in mR4(-/-) livers. These results confirm that hepatocyte FGFR4 regulates bile acid synthesis by repression of Cyp7a1 expression. Hepatocyte FGFR4 may contribute to the repression of bile acid synthesis through JNK signaling but is not required for activation of JNK signaling by bile acids.

  7. Constitutive ALK5-Independent c-Jun N-Terminal Kinase Activation Contributes to Endothelin-1 Overexpression in Pulmonary Fibrosis: Evidence of an Autocrine Endothelin Loop Operating through the Endothelin A and B Receptors

    PubMed Central

    Shi-Wen, Xu; Rodríguez-Pascual, Fernando; Lamas, Santiago; Holmes, Alan; Howat, Sarah; Pearson, Jeremy D.; Dashwood, Michael R.; du Bois, Roland M.; Denton, Christopher P.; Black, Carol M.; Abraham, David J.; Leask, Andrew

    2006-01-01

    The signal transduction mechanisms generating pathological fibrosis are almost wholly unknown. Endothelin-1 (ET-1), which is up-regulated during tissue repair and fibrosis, induces lung fibroblasts to produce and contract extracellular matrix. Lung fibroblasts isolated from scleroderma patients with chronic pulmonary fibrosis produce elevated levels of ET-1, which contribute to the persistent fibrotic phenotype of these cells. Transforming growth factor β (TGF-β) induces fibroblasts to produce and contract matrix. In this report, we show that TGF-β induces ET-1 in normal and fibrotic lung fibroblasts in a Smad-independent ALK5/c-Jun N-terminal kinase (JNK)/Ap-1-dependent fashion. ET-1 induces JNK through TAK1. Fibrotic lung fibroblasts display constitutive JNK activation, which was reduced by the dual ETA/ETB receptor inhibitor, bosentan, providing evidence of an autocrine endothelin loop. Thus, ET-1 and TGF-β are likely to cooperate in the pathogenesis of pulmonary fibrosis. As elevated JNK activation in fibrotic lung fibroblasts contributes to the persistence of the myofibroblast phenotype in pulmonary fibrosis by promoting an autocrine ET-1 loop, targeting the ETA and ETB receptors or constitutive JNK activation by fibrotic lung fibroblasts is likely to be of benefit in combating chronic pulmonary fibrosis. PMID:16809784

  8. N-Terminal Ubiquitination of Extracellular Signal-Regulated Kinase 3 and p21 Directs Their Degradation by the Proteasome

    PubMed Central

    Coulombe, Philippe; Rodier, Geneviève; Bonneil, Eric; Thibault, Pierre; Meloche, Sylvain

    2004-01-01

    Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH2 terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized. PMID:15226418

  9. The small GTPase RALA controls c-Jun N-terminal kinase-mediated FOXO activation by regulation of a JIP1 scaffold complex.

    PubMed

    van den Berg, Maaike C W; van Gogh, Inkie J A; Smits, Alida M M; van Triest, Miranda; Dansen, Tobias B; Visscher, Marieke; Polderman, Paulien E; Vliem, Marjolein J; Rehmann, Holger; Burgering, Boudewijn M T

    2013-07-26

    FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.

  10. The Small GTPase RALA Controls c-Jun N-terminal Kinase-mediated FOXO Activation by Regulation of a JIP1 Scaffold Complex*

    PubMed Central

    van den Berg, Maaike C. W.; van Gogh, Inkie J. A.; Smits, Alida M. M.; van Triest, Miranda; Dansen, Tobias B.; Visscher, Marieke; Polderman, Paulien E.; Vliem, Marjolein J.; Rehmann, Holger; Burgering, Boudewijn M. T.

    2013-01-01

    FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16. PMID:23770673

  11. Inhibition of c-Jun-N-terminal Kinase Increases Cardiac Peroxisome Proliferator-activated Receptor α Expression and Fatty Acid Oxidation and Prevents Lipopolysaccharide-induced Heart Dysfunction*

    PubMed Central

    Drosatos, Konstantinos; Drosatos-Tampakaki, Zoi; Khan, Raffay; Homma, Shunichi; Schulze, P. Christian; Zannis, Vassilis I.; Goldberg, Ira J.

    2011-01-01

    Septic shock results from bacterial infection and is associated with multi-organ failure, high mortality, and cardiac dysfunction. Sepsis causes both myocardial inflammation and energy depletion. We hypothesized that reduced cardiac energy production is a primary cause of ventricular dysfunction in sepsis. The JNK pathway is activated in sepsis and has also been implicated in impaired fatty acid oxidation in several tissues. Therefore, we tested whether JNK activation inhibits cardiac fatty acid oxidation and whether blocking JNK would restore fatty acid oxidation during LPS treatment. LPS treatment of C57BL/6 mice and adenovirus-mediated activation of the JNK pathway in cardiomyocytes inhibited peroxisome proliferator-activated receptor α expression and fatty acid oxidation. Surprisingly, none of the adaptive responses that have been described in other types of heart failure, such as increased glucose utilization, reduced αMHC:βMHC ratio or induction of certain microRNAs, occurred in LPS-treated mice. Treatment of C57BL/6 mice with a general JNK inhibitor (SP600125) increased fatty acid oxidation in mice and a cardiomyocyte-derived cell line. JNK inhibition also prevented LPS-mediated reduction in fatty acid oxidation and cardiac dysfunction. Inflammation was not alleviated in LPS-treated mice that received the JNK inhibitor. We conclude that activation of JNK signaling reduces fatty acid oxidation and prevents the peroxisome proliferator-activated receptor α down-regulation that occurs with LPS. PMID:21873422

  12. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner*

    PubMed Central

    Tirodkar, Tejas S.; Lu, Ping; Bai, Aiping; Scheffel, Matthew J.; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-01-01

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14–26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3–5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity. PMID:25839235

  13. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner.

    PubMed

    Tirodkar, Tejas S; Lu, Ping; Bai, Aiping; Scheffel, Matthew J; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-05-22

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity.

  14. Activation of Tax protein by c-Jun-N-terminal kinase is not dependent on the presence or absence of the early growth response-1 gene product.

    PubMed

    Parra, Eduardo; Gutierréz, Luís; Ferreira, Jorge

    2016-02-01

    The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells.

  15. Periostin promotes migration and invasion of renal cell carcinoma through the integrin/focal adhesion kinase/c-Jun N-terminal kinase pathway.

    PubMed

    Chuanyu, Sun; Yuqing, Zhu; Chong, Xu; Guowei, Xia; Xiaojun, Zhao

    2017-04-01

    Periostin (POSTN) is an extracellular matrix protein which is overexpressed in a variety of cancers and has been related to tumorigenesis of renal cell carcinoma. However, the involvement of POSTN in renal cell carcinoma migration, invasion, and their underlying mechanisms has not been established. In this study, renal cell carcinoma cell lines stably overexpressing POSTN were established using a lentiviral vector, and the effects of POSTN on renal cell carcinoma cell migration and invasion were investigated. POSTN overexpression increased the migration and invasion capabilities of renal cell carcinoma cell lines as well as activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. Integrin αvβ3 and αvβ5 antibodies inhibited POSTN overexpression or recombinant POSTN-induced focal adhesion kinase activation, cell migration, and invasion. Furthermore, lentivirus-mediated focal adhesion kinase knockdown and c-Jun N-terminal kinase inhibitor reduced POSTN-enhanced phosphorylation of c-Jun N-terminal kinase, matrix metalloproteinase-9 and matrix metalloproteinase-2 expressions, cell migration, and invasion. Our research thus indicates that POSTN promotes renal cell carcinoma cell migration and invasion through interaction with integrins αvβ3 and αvβ5 and subsequent activation of the focal adhesion kinase/c-Jun N-terminal kinase pathway. These results suggest that POSTN plays a critical role in renal cell carcinoma metastasis and may represent a potential target for novel therapeutic approaches against renal cell carcinoma.

  16. The c-Jun N-terminal kinase (JNK) pathway is activated in human interstitial cystitis (IC) and rat protamine sulfate induced cystitis

    PubMed Central

    Zhao, Jiang; Wang, Liang; Dong, Xingyou; Hu, Xiaoyan; Zhou, Long; Liu, Qina; Song, Bo; Wu, Qingjian; Li, Longkun

    2016-01-01

    The pathogenesis of bladder pain syndrome/interstitial cystitis (BPS/IC) is currently unclear. However, inflammation has been suggested to play an important role in BPS/IC. JNK downstream signaling plays an important role in numerous chronic inflammatory diseases. However, studies of the JNK pathway in BPS/IC are limited. In this study, we investigated the role of the JNK pathway in human BPS/IC and rat protamine sulfate (PS)-induced cystitis and examined the effect of the selective JNK inhibitor SP600125 on rat bladder cystitis. In our study, we demonstrated that the JNK signaling pathway was activated (the expression of JNK, c-Jun, p-JNK, p-c-Jun, IL-6 and TNF-α were significantly increasing in BPS/IC compared to the non-BPS/IC patients) and resulted in inflammation in human BPS/IC. Further animal models showed that the JNK pathway played an important role in the pathogenesis of cystitis. JNK inhibitors, SP600125, effectively inhibited the expression of p-JNK, p-c-Jun, IL-6 and TNF-α. The inhibition of these pathways had a protective effect on PS-induced rat cystitis by significantly decreasing histological score and mast cell count and improving bladder micturition function (micturition frequency significantly decreasing and bladder capacity significantly increasing). Therefore, JNK inhibition could be used as a potential treatment for BPS/IC. PMID:26883396

  17. The c-Jun N-terminal kinase (JNK) pathway is activated in human interstitial cystitis (IC) and rat protamine sulfate induced cystitis.

    PubMed

    Zhao, Jiang; Wang, Liang; Dong, Xingyou; Hu, Xiaoyan; Zhou, Long; Liu, Qina; Song, Bo; Wu, Qingjian; Li, Longkun

    2016-02-17

    The pathogenesis of bladder pain syndrome/interstitial cystitis (BPS/IC) is currently unclear. However, inflammation has been suggested to play an important role in BPS/IC. JNK downstream signaling plays an important role in numerous chronic inflammatory diseases. However, studies of the JNK pathway in BPS/IC are limited. In this study, we investigated the role of the JNK pathway in human BPS/IC and rat protamine sulfate (PS)-induced cystitis and examined the effect of the selective JNK inhibitor SP600125 on rat bladder cystitis. In our study, we demonstrated that the JNK signaling pathway was activated (the expression of JNK, c-Jun, p-JNK, p-c-Jun, IL-6 and TNF-α were significantly increasing in BPS/IC compared to the non-BPS/IC patients) and resulted in inflammation in human BPS/IC. Further animal models showed that the JNK pathway played an important role in the pathogenesis of cystitis. JNK inhibitors, SP600125, effectively inhibited the expression of p-JNK, p-c-Jun, IL-6 and TNF-α. The inhibition of these pathways had a protective effect on PS-induced rat cystitis by significantly decreasing histological score and mast cell count and improving bladder micturition function (micturition frequency significantly decreasing and bladder capacity significantly increasing). Therefore, JNK inhibition could be used as a potential treatment for BPS/IC.

  18. The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity

    PubMed Central

    Lee, Hyeon-Ju; Byun, Catherine Jeonghae; Park, Jung-Hyun; Park, Jae Hoon; Cho, Ho-Seong; Cho, Sung-Jin; Jo, Sangmee Ahn; Jo, Inho

    2015-01-01

    The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity. PMID:26375285

  19. The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

    PubMed

    Kim, Jee-Youn; Choi, Ji-Young; Lee, Hyeon-Ju; Byun, Catherine Jeonghae; Park, Jung-Hyun; Park, Jae Hoon; Cho, Ho-Seong; Cho, Sung-Jin; Jo, Sangmee Ahn; Jo, Inho

    2015-01-01

    The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.

  20. NADPH Oxidase 4 is required for interleukin-1β-mediated activation of protein kinase Cδ and downstream activation of c-Jun N-terminal kinase signaling in smooth muscle

    PubMed Central

    Ginnan, Roman; Jourd’heuil, Frances L.; Guikema, Benjamin; Simons, Malorie; Singer, Harold A.; Jourd’heuil, David

    2012-01-01

    Reactive oxygen species (ROS) are generated in the vascular wall upon stimulation by pro-inflammatory cytokines and are important mediators of diverse cellular responses that occur as a result of vascular injury. Member of the NADPH oxidase (NOX) family of proteins have been identified in vascular smooth muscle cells (VSM) as important sources of ROS. In this study, we tested the hypothesis that NOX4 is a proximal mediator of IL-1β-dependent activation of PKCδ and increases IL-1β stimulated c-Jun kinase (JNK) signaling in primary rat aortic VSM cells. We found that stimulation of VSM cells with IL-1β increased PKCδ activity and intracellular ROS generation. SiRNA silencing of NOX4 but not NOX1 ablated the IL-1β-dependent increase in ROS production. Pharmacological inhibition of PKCδ activity as well as siRNA depletion of PKCδ or NOX4 blocked the IL-1β-dependent activation of JNK. Further studies showed that the IL-1β-dependent upregulation of iNOS expression was inhibited through JNK inhibition and NOX4 silencing. Taken together, these results indicate that IL-1β-dependent activation of PKCδ is modulated by NOX4-derived ROS. Our study positions PKCδ as an important redox sensitive mediator of IL-1β-dependent signaling and downstream activation of inflammatory mediators in VSM cells. PMID:23022406

  1. Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death.

    PubMed

    Chang, Alice Y W

    2012-11-17

    Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague-Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73, rather than Elk-1 at

  2. Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death

    PubMed Central

    2012-01-01

    Background Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. Results An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague–Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73

  3. c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

    DTIC Science & Technology

    2014-10-01

    ABSTRACT Abstract: XXX aminopyrazoles, a new class of c-jun-N-terminal kinase (JNK) inhibitors, have been synthesized and the biochemical IC50 has been...have also been generated as backups. XXX compounds from the pyridopyrimidinone class have been synthesized and tested in biochemical and cell based...assays, and XXX compounds from the amino acid transporter analog class have been made and tested in biochemical assays. The goal of this work is to

  4. c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

    DTIC Science & Technology

    2013-10-01

    Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL INVESTIGATOR: Dr. Philip LoGrasso CONTRACTING ORGANIZATION: The Scripps Research... Lateral Sclerosis ” 5a. CONTRACT NUMBER W81XWH-12-1-0431 5b. GRANT NUMBER W81XWH-12-1-0431 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Philip...Annual 3. DATES COVERED 30September2012-29September2013 4. TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic

  5. c-Jun-N-Terminal Kinase Signaling Is Involved in Cyclosporine-Induced Epithelial Phenotypic Changes

    PubMed Central

    Pallet, Nicolas; Thervet, Eric; Anglicheau, Dany

    2012-01-01

    Tubular epithelial cells play a central role in the pathogenesis of chronic nephropathies. Previous toxicogenomic studies have demonstrated that cyclosporine- (CsA-) induced epithelial phenotypic changes (EPCs) are reminiscent of an incomplete epithelial to mesenchymal transition (EMT) in a TGF-β-independent manner. Furthermore, we identified endoplasmic reticulum (ER) stress as a potential mechanism that may participate in the modulation of tubular cell plasticity during CsA exposure. Because c-jun-N-terminal kinase (JNK), which is activated during ER stress, is implicated in kidney fibrogenesis, we undertook the current study to identify the role of JNK signaling in EPCs induced by CsA. In primary cultures of human renal epithelial cells, CsA activates JNK signaling, and the treatment with a JNK inhibitor reduces the occurrence of cell shape changes, E-cadherin downregulation, cell migration, and Snail-1 expression. Our results suggest that CsA activates JNK signaling, which, in turn, may participate in the morphological alterations through the regulation of Snail-1 expression. PMID:22028950

  6. c-Jun-N-Terminal Kinase Signaling Is Involved in Cyclosporine-Induced Epithelial Phenotypic Changes.

    PubMed

    Pallet, Nicolas; Thervet, Eric; Anglicheau, Dany

    2012-01-01

    Tubular epithelial cells play a central role in the pathogenesis of chronic nephropathies. Previous toxicogenomic studies have demonstrated that cyclosporine- (CsA-) induced epithelial phenotypic changes (EPCs) are reminiscent of an incomplete epithelial to mesenchymal transition (EMT) in a TGF-β-independent manner. Furthermore, we identified endoplasmic reticulum (ER) stress as a potential mechanism that may participate in the modulation of tubular cell plasticity during CsA exposure. Because c-jun-N-terminal kinase (JNK), which is activated during ER stress, is implicated in kidney fibrogenesis, we undertook the current study to identify the role of JNK signaling in EPCs induced by CsA. In primary cultures of human renal epithelial cells, CsA activates JNK signaling, and the treatment with a JNK inhibitor reduces the occurrence of cell shape changes, E-cadherin downregulation, cell migration, and Snail-1 expression. Our results suggest that CsA activates JNK signaling, which, in turn, may participate in the morphological alterations through the regulation of Snail-1 expression.

  7. Evidence of Presynaptic Localization and Function of the c-Jun N-Terminal Kinase

    PubMed Central

    Biggi, Silvia; Buccarello, Lucia; Sclip, Alessandra; Lippiello, Pellegrino; Rumio, Cristiano; Di Marino, Daniele

    2017-01-01

    The c-Jun N-terminal kinase (JNK) is part of a stress signalling pathway strongly activated by NMDA-stimulation and involved in synaptic plasticity. Many studies have been focused on the post-synaptic mechanism of JNK action, and less is known about JNK presynaptic localization and its physiological role at this site. Here we examined whether JNK is present at the presynaptic site and its activity after presynaptic NMDA receptors stimulation. By using N-SIM Structured Super Resolution Microscopy as well as biochemical approaches, we demonstrated that presynaptic fractions contained significant amount of JNK protein and its activated form. By means of modelling design, we found that JNK, via the JBD domain, acts as a physiological effector on T-SNARE proteins; then using biochemical approaches we demonstrated the interaction between Syntaxin-1-JNK, Syntaxin-2-JNK, and Snap25-JNK. In addition, taking advance of the specific JNK inhibitor peptide, D-JNKI1, we defined JNK action on the SNARE complex formation. Finally, electrophysiological recordings confirmed the role of JNK in the presynaptic modulation of vesicle release. These data suggest that JNK-dependent phosphorylation of T-SNARE proteins may have an important functional role in synaptic plasticity. PMID:28367336

  8. Saw palmetto extract suppresses insulin-like growth factor-I signaling and induces stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation in human prostate epithelial cells.

    PubMed

    Wadsworth, Teri L; Carroll, Julie M; Mallinson, Rebecca A; Roberts, Charles T; Roselli, Charles E

    2004-07-01

    A common alternative therapy for benign prostatic hyperplasia (BPH) is the extract from the fruit of saw palmetto (SPE). BPH is caused by nonmalignant growth of epithelial and stromal elements of the prostate. IGF action is important for prostate growth and development, and changes in the IGF system have been documented in BPH tissues. The main signaling pathways activated by the binding of IGF-I to the IGF-I receptor (IGF-IR) are the ERK arm of the MAPK cascade and the phosphoinositol-3-kinase (PI3K)/protein kinase B (PKB/Akt) cascade. We tested the hypothesis that SPE suppresses growth and induces apoptosis in the P69 prostate epithelial cell line by inhibiting IGF-I signaling. Treatment with 150 microg/ml SPE for 24 h decreased IGF-I-induced proliferation of P69 cells and induced cleavage of the enzyme poly(ADP-ribose)polymerase (PARP), an index of apoptosis. Treatment of serum-starved P69 cells with 150 microg/ml SPE for 6 h reduced IGF-I-induced phosphorylation of Akt (assessed by Western blot) and Akt activity (assessed by an Akt kinase assay). Western blot analysis showed that SPE reduced IGF-I-induced phosphorylation of the adapter protein insulin receptor substrate-1 and decreased downstream effects of Akt activation, including increased cyclin D1 levels and phosphorylation of glycogen synthase kinase-3 and p70(s6k). There was no effect on IGF-I-induced phosphorylation of MAPK, IGF-IR, or Shc. Treatment of starved cells with SPE alone induced phosphorylation the proapoptotic protein JNK. SPE treatment may relieve symptoms of BPH, in part, by inhibiting specific components of the IGF-I signaling pathway and inducing JNK activation, thus mediating antiproliferative and proapoptotic effects on prostate epithelia.

  9. PATHOPHYSIOLOGICAL SIGNIFICANCE OF C-JUN N-TERMINAL KINASE IN ACETAMINOPHEN HEPATOTOXICITY

    PubMed Central

    Du, Kuo; Xie, Yuchao; McGill, Mitchell R.; Jaeschke, Hartmut

    2016-01-01

    Background Acetaminophen (APAP) overdose is the leading cause of acute liver failure in the US. Although substantial progress regarding the mechanisms of APAP hepatotoxicity has been made in the past several decades, therapeutic options are still limited and novel treatments are clearly needed. c-jun N-terminal Kinase (JNK) has emerged as a promising therapeutic target in recent years. Areas covered Early studies established the critical role of JNK activation and mitochondrial translocation in APAP hepatotoxicity. However, this concept has also been challenged. Initial studies failed to reproduce the protection of JNK deficiency in APAP toxicity and concerns over off-target effects of JNK inhibitors and even in knock-out mice are increasing. Interestingly, recent studies have even shown that liver injury can be altered with or without effects on JNK activation. The current review addresses these discrepancies and tries to explain or reconcile some of the conflicting results. Expert opinion JNK is a potential therapeutic target for APAP poisoning. However, controversies still exist regarding its actual role in APAP hepatotoxicity. Future studies are warranted for more in-depth testing of specific inhibitors in well-defined preclinical models and human hepatocytes before JNK can be considered a relevant therapeutic target for APAP poisoning. PMID:26190663

  10. Alteration of Substrate Specificity: The Variable N-Terminal Domain of Tobacco Ca2+-Dependent Protein Kinase Is Important for Substrate Recognition[W

    PubMed Central

    Ito, Takeshi; Nakata, Masaru; Fukazawa, Jutarou; Ishida, Sarahmi; Takahashi, Yohsuke

    2010-01-01

    Protein kinases are major signaling molecules that are involved in a variety of cellular processes. However, the molecular mechanisms whereby protein kinases discriminate specific substrates are still largely unknown. Ca2+-dependent protein kinases (CDPKs) play central roles in Ca2+ signaling in plants. Previously, we found that a tobacco (Nicotiana tabacum) CDPK1 negatively regulated the transcription factor REPRESSION OF SHOOT GROWTH (RSG), which is involved in gibberellin feedback regulation. Here, we found that the variable N-terminal domain of CDPK1 is necessary for the recognition of RSG. A mutation (R10A) in the variable N-terminal domain of CDPK1 reduced both RSG binding and RSG phosphorylation while leaving kinase activity intact. Furthermore, the R10A mutation suppressed the in vivo function of CDPK1. The substitution of the variable N-terminal domain of an Arabidopsis thaliana CDPK, At CPK9, with that of Nt CDPK1 conferred RSG kinase activities. This chimeric CDPK behaved according to the identity of the variable N-terminal domain in transgenic plants. Our results open the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways. PMID:20442373

  11. The N-Terminal Domain of the Tomato Immune Protein Prf Contains Multiple Homotypic and Pto Kinase Interaction Sites*

    PubMed Central

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-01-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats). PMID:25792750

  12. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    PubMed

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Inhibitors of c-Jun N-terminal kinases: JuNK no more?

    PubMed

    Bogoyevitch, Marie A; Arthur, Peter G

    2008-01-01

    The c-Jun N-terminal kinases (JNKs) have been the subject of intense interest since their discovery in the early 1990s. Major research programs have been directed to the screening and/or design of JNK-selective inhibitors and testing their potential as drugs. We begin this review by considering the first commercially-available JNK ATP-competitive inhibitor, SP600125. We focus on recent studies that have evaluated the actions of SP600125 in lung, brain, kidney and liver following exposure to a range of stress insults including ischemia/reperfusion. In many but not all cases, SP600125 administration has proved beneficial. JNK activation can also follow infection, and we next consider recent examples that demonstrate the benefits of SP600125 administration in viral infection. Additional ATP-competitive JNK inhibitors have now been described following high throughput screening of small molecule libraries, but information on their use in biological systems remains limited and thus these inhibitors will require further evaluation. Peptide substrate-competitive ATP-non-competitive inhibitors of JNK have also now been described, and we discuss the recent advances in the use of JNK inhibitory peptides in the treatment of neuronal death, diabetes and viral infection. We conclude by raising a number of questions that should be considered in the quest for JNK-specific inhibitors.

  14. c-Jun N-terminal kinase - c-Jun pathway transactivates Bim to promote osteoarthritis.

    PubMed

    Ye, Zhiqiang; Chen, Yuxian; Zhang, Rongkai; Dai, Haitao; Zeng, Chun; Zeng, Hua; Feng, Hui; Du, Gengheng; Fang, Hang; Cai, Daozhang

    2014-02-01

    Osteoarthritis (OA) is a chronic degenerative joint disorder. Previous studies have shown abnormally increased apoptosis of chondrocytes in patients and animal models of OA. TNF-α and nitric oxide have been reported to induce chondrocyte ageing; however, the mechanism of chondrocyte apoptosis induced by IL-1β has remained unclear. The aim of this study is to identify the role of the c-Jun N-terminal kinase (JNK) - c-Jun pathway in regulating induction of Bim, and its implication in chondrocyte apoptosis. This study showed that Bim is upregulated in chondrocytes obtained from the articular cartilage of OA patients and in cultured mouse chondrocytes treated with IL-1β. Upregulation of Bim was found to be critical for chondrocyte apoptosis induced by IL-1β, as revealed by the genetic knockdown of Bim, wherein apoptosis was greatly reduced in the chondrocytes. Moreover, activation of the JNK-c-Jun pathway was observed under IL-1β treatment, as indicated by the increased expression levels of c-Jun protein. Suppression of the JNK-c-Jun pathway, using chemical inhibitors and RNA interference, inhibited the Bim upregulation induced by IL-1β. These findings suggest that the JNK-c-Jun pathway is involved in the upregulation of Bim during OA and that the JNK-c-Jun-Bim pathway is vital for chondrocyte apoptosis.

  15. Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality

    PubMed Central

    Pirianov, Grisha; MacIntyre, David A; Lee, Yun; Waddington, Simon N; Terzidou, Vasso; Mehmet, Huseyin; Bennett, Phillip R

    2015-01-01

    Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain. PMID:26183892

  16. Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality.

    PubMed

    Pirianov, Grisha; MacIntyre, David A; Lee, Yun; Waddington, Simon N; Terzidou, Vasso; Mehmet, Huseyin; Bennett, Phillip R

    2015-10-01

    Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain. © 2015 The authors.

  17. Activation of the Stress Response Kinase JNK (c-Jun N-terminal Kinase) Attenuates Insulin Action in Retina through a p70S6K1-dependent Mechanism.

    PubMed

    Miller, William P; Ravi, Suhana; Martin, Tony D; Kimball, Scot R; Dennis, Michael D

    2017-02-03

    Despite recent advances in therapeutics, diabetic retinopathy remains a leading cause of vision impairment. Improvement in the treatment of diabetic retinopathy requires a better understanding of the molecular mechanisms that cause neurovascular complications, particularly in type 2 diabetes. Recent studies demonstrate that rodents fed a high fat diet exhibit retinal dysfunction concomitant with attenuated Akt phosphorylation. The purpose of the present study was to evaluate the impact of a high fat/high sucrose diet on retinal insulin signaling and evaluate the mechanism(s) responsible for the changes. Mice fed a high fat/sucrose diet exhibited attenuated Akt phosphorylation in the retina as compared with mice fed normal chow. Retinas of mice fed a high fat/sucrose diet also exhibited elevated levels of activated JNK as well as enhanced p70S6K1 autoinhibitory domain phosphorylation. In cells, JNK activation enhanced p70S6K1 phosphorylation and mTORC1-dependent activation of the kinase, as evidenced by enhanced phosphorylation of key substrates. Rictor phosphorylation by p70S6K1 was specifically enhanced by the addition of phosphomimetic mutations in the autoinhibitory domain and was more sensitive to inhibition of the kinase as compared with rpS6. Notably, rictor and IRS-1 phosphorylation by p70S6K1 attenuate insulin action through a negative feedback pathway. Indeed, p70S6K1 inhibition prevented the repressive effect of JNK activation on insulin action in retinas. Overall, the results identify the JNK/S6K1 axis as a key molecular mechanism whereby a high fat/sucrose diet impairs insulin action in retina.

  18. Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

    PubMed

    Vilitkevich, E L; Khapchaev, A Y; Kudryashov, D S; Nikashin, A V; Schavocky, J P; Lukas, T J; Watterson, D M; Shirinsky, V P

    2015-10-01

    High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

  19. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

    PubMed

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-02-26

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

  20. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis*

    PubMed Central

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy. PMID:26724804

  1. Involvement of c-Jun N-Terminal Kinase in TNF-α-Driven Remodeling.

    PubMed

    Eurlings, Irene M J; Reynaert, Niki L; van de Wetering, Cheryl; Aesif, Scott W; Mercken, Evi M; de Cabo, Rafael; van der Velden, Jos L; Janssen-Heininger, Yvonne M; Wouters, Emiel F M; Dentener, Mieke A

    2017-03-01

    Lung tissue remodeling in chronic obstructive pulmonary disease (COPD) is characterized by airway wall thickening and/or emphysema. Although the bronchial and alveolar compartments are functionally independent entities, we recently showed comparable alterations in matrix composition comprised of decreased elastin content and increased collagen and hyaluronan contents of alveolar and small airway walls. Out of several animal models tested, surfactant protein C (SPC)-TNF-α mice showed remodeling in alveolar and airway walls similar to what we observed in patients with COPD. Epithelial cells are able to undergo a phenotypic shift, gaining mesenchymal properties, a process in which c-Jun N-terminal kinase (JNK) signaling is involved. Therefore, we hypothesized that TNF-α induces JNK-dependent epithelial plasticity, which contributes to lung matrix remodeling. To this end, the ability of TNF-α to induce a phenotypic shift was assessed in A549, BEAS2B, and primary bronchial epithelial cells, and phenotypic markers were studied in SPC-TNF-α mice. Phenotypic markers of mesenchymal cells were elevated both in vitro and in vivo, as shown by the expression of vimentin, plasminogen activator inhibitor-1, collagen, and matrix metalloproteinases. Concurrently, the expression of the epithelial markers, E-cadherin and keratin 7 and 18, was attenuated. A pharmacological inhibitor of JNK attenuated this phenotypic shift in vitro, demonstrating involvement of JNK signaling in this process. Interestingly, activation of JNK signaling was also clearly present in lungs of SPC-TNF-α mice and patients with COPD. Together, these data show a role for TNF-α in the induction of a phenotypic shift in vitro, resulting in increased collagen production and the expression of elastin-degrading matrix metalloproteinases, and provide evidence for involvement of the TNF-α-JNK axis in extracellular matrix remodeling.

  2. Identification and characterization of a novel class of c-Jun N-terminal kinase inhibitors.

    PubMed

    Schepetkin, Igor A; Kirpotina, Liliya N; Khlebnikov, Andrei I; Hanks, Tracey S; Kochetkova, Irina; Pascual, David W; Jutila, Mark A; Quinn, Mark T

    2012-06-01

    In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC(50) values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4(+) T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs.

  3. Identification and Characterization of a Novel Class of c-Jun N-terminal Kinase Inhibitors

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Hanks, Tracey S.; Kochetkova, Irina; Pascual, David W.; Jutila, Mark A.

    2012-01-01

    In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC50 values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4+ T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs. PMID:22434859

  4. The Evi-1 oncoprotein inhibits c-Jun N-terminal kinase and prevents stress-induced cell death

    PubMed Central

    Kurokawa, Mineo; Mitani, Kinuko; Yamagata, Tetsuya; Takahashi, Tokiharu; Izutsu, Koji; Ogawa, Seishi; Moriguchi, Tetsuo; Nishida, Eisuke; Yazaki, Yoshio; Hirai, Hisamaru

    2000-01-01

    Evi-1 encodes a nuclear protein involved in leukemic transformation of hematopoietic cells. Evi-1 possesses two sets of zinc finger motifs separated into two domains, and its characteristics as a transcriptional regulator have been described. Here we show that Evi-1 acts as an inhibitor of c-Jun N-terminal kinase (JNK), a class of mitogen-activated protein kinases implicated in stress responses of cells. Evi-1 physically interacts with JNK, although it does not affect its phosphorylation. This interaction is required for inhibition of JNK. Evi-1 protects cells from stress-induced cell death with dependence on the ability to inhibit JNK. These results reveal a novel function of Evi-1, which provides evidence for inhibition of JNK by a nuclear oncogene product. Evi-1 blocks cell death by selectively inhibiting JNK, thereby contributing to oncogenic transformation of cells. PMID:10856240

  5. Evidence for N-Terminal Myristoylation of Tetrahymena Arginine Kinase Using Peptide Mass Fingerprinting Analysis.

    PubMed

    Motomura, Shou; Suzuki, Tomohiko

    2016-06-01

    In this study, we confirmed N-terminal myristoylation of Tetrahymena pyriformis arginine kinase (AK1) by identifying a myristoylation signal sequence at the N-terminus. A sufficient amount of modified enzyme was synthesized using an insect cell-free protein synthesis system that contains all of the elements necessary for post-transcriptional modification by fatty acids. Subsequent peptide mass fingerprinting (PMF) analyses were performed after digestion with trypsin. The PMF data covered 39 % (143 residues) of internal peptides. The target N-myristoylated peptide had a theoretical mass of 832.4477 and was clearly observed with an experimental mass (m/z-H(+)) of 832.4747. The difference between the two masses was 0.0271, supporting the accuracy of identification and indicating that the synthesized T. pyriformis AK1 is myristoylated. The fixed specimens of T. pyriformis were reacted with an anti-AK1 peptide antibody followed by a secondary antibody with a fluorescent chromophore and were observed using immunofluorescence microscope. In agreement with previous western blotting analyses, microscopic observations suggested that AK1 is localized in the cilia. The present PMF and microscopic analyses indicate that T. pyriformis AK1 may be localized and anchored to ciliary membranes via N-terminal myristoyl groups.

  6. ets-2 Is a Target for an Akt (Protein Kinase B)/Jun N-Terminal Kinase Signaling Pathway in Macrophages of motheaten-viable Mutant Mice

    PubMed Central

    Smith, James L.; Schaffner, Alicia E.; Hofmeister, Joseph K.; Hartman, Matthew; Wei, Guo; Forsthoefel, David; Hume, David A.; Ostrowski, Michael C.

    2000-01-01

    The transcription factor ets-2 was phosphorylated at residue threonine 72 in a colony-stimulating factor 1 (CSF-1)- and mitogen-activated protein kinase-independent manner in macrophages isolated from motheaten-viable (me-v) mice. The CSF-1 and ets-2 target genes coding for Bcl-x, urokinase plasminogen activator, and scavenger receptor were also expressed at high levels independent of CSF-1 addition to me-v cells. Akt (protein kinase B) was constitutively active in me-v macrophages, and an Akt immunoprecipitate catalyzed phosphorylation of ets-2 at threonine 72. The p54 isoform of c-jun N-terminal kinase–stress-activated kinase (JNK- SAPK) coimmunoprecipitated with Akt from me-v macrophages, and treatment of me-v cells with the specific phosphatidylinositol 3-kinase inhibitor LY294002 decreased cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bcl-x mRNA expression. Therefore, ets-2 is a target for phosphatidylinositol 3-kinase–Akt–JNK action, and the JNK p54 isoform is an ets-2 kinase in macrophages. Constitutive ets-2 activity may contribute to the pathology of me-v mice by increasing expression of genes like the Bcl-x gene that promote macrophage survival. PMID:11027273

  7. Protective role of c-Jun N-terminal kinase 2 in acetaminophen-induced liver injury

    SciTech Connect

    Bourdi, Mohammed Korrapati, Midhun C.; Chakraborty, Mala; Yee, Steven B.; Pohl, Lance R.

    2008-09-12

    Recent studies in mice suggest that stress-activated c-Jun N-terminal protein kinase 2 (JNK2) plays a pathologic role in acetaminophen (APAP)-induced liver injury (AILI), a major cause of acute liver failure (ALF). In contrast, we present evidence that JNK2 can have a protective role against AILI. When male C57BL/6J wild type (WT) and JNK2{sup -/-} mice were treated with 300 mg APAP/kg, 90% of JNK2{sup -/-} mice died of ALF compared to 20% of WT mice within 48 h. The high susceptibility of JNK2{sup -/-} mice to AILI appears to be due in part to deficiencies in hepatocyte proliferation and repair. Therefore, our findings are consistent with JNK2 signaling playing a protective role in AILI and further suggest that the use of JNK inhibitors as a potential treatment for AILI, as has been recommended by other investigators, should be reconsidered.

  8. Binding model for eriodictyol to Jun-N terminal kinase and its anti-inflammatory signaling pathway

    PubMed Central

    Lee, Eunjung; Jeong, Ki-Woong; Shin, Areum; Jin, Bonghwan; Jnawali, Hum Nath; Jun, Bong-Hyun; Lee, Jee-Young; Heo, Yong-Seok; Kim, Yangmee

    2013-01-01

    The anti-inflammatory activity of eriodictyol and its mode of action were investigated. Eriodictyol suppressed tumor necrosis factor (mTNF)-α, inducible nitric oxide synthase (miNOS), interleukin (mIL)-6, macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine release in LPS-stimulated macrophages. We found that the anti-inflammatory cascade of eriodictyol is mediated through the Toll-like Receptor (TLR)4/CD14, p38 mitogen-activated protein kinases (MAPK), extracellular-signalregulated kinase (ERK), Jun-N terminal kinase (JNK), and cyclooxygenase (COX)-2 pathway. Fluorescence quenching and saturation-transfer difference (STD) NMR experiments showed that eriodictyol exhibits good binding affinity to JNK, 8.79 × 105 M-1. Based on a docking study, we propose a model of eriodictyol and JNK binding, in which eriodictyol forms 3 hydrogen bonds with the side chains of Lys55, Met111, and Asp169 in JNK, and in which the hydroxyl groups of the B ring play key roles in binding interactions with JNK. Therefore, eriodictyol may be a potent anti-inflammatory inhibitor of JNK. [BMB Reports 2013; 46(12): 594-599] PMID:24195792

  9. c-Jun N-terminal kinase (JNK) is involved in immune defense against bacterial infection in Crassostrea hongkongensis.

    PubMed

    Qu, Fufa; Xiang, Zhiming; Xiao, Shu; Wang, Fuxuan; Li, Jun; Zhang, Yang; Zhang, Yuehuan; Qin, Yanping; Yu, Ziniu

    2017-02-01

    c-Jun N-terminal kinase (JNK) is a universal and essential subgroup of the mitogen-activated protein kinase (MAPK) superfamily, which is highly conserved from yeast to mammals and functions in a variety of physiological and pathological processes. In this study, we report the first oyster JNK gene homolog (ChJNK) and its biological functions in the Hong Kong oyster Crassostrea hongkongensis. The ChJNK protein consists of 383 amino acids and contains a conserved serine/threonine protein kinase (S_TKc) domain with a typical TPY motif. Phylogenetic analysis revealed that ChJNK shared a close evolutionary relationship with Crassostrea gigas JNK. Quantitative RT-PCR analyses revealed broad expression patterns of ChJNK mRNA in various adult tissues and different embryonic and larval stages of C. hongkongensis. When exposed to Vibrio alginolyticus or Staphylococcus haemolyticus, ChJNK mRNA expression levels were significantly up-regulated in the hemocytes and gills in a time-dependent manner. Additionally, subcellular localization studies that ChJNK is a cytoplasm-localized protein, and that its overexpression could significantly enhance the transcriptional activities of AP-1-Luc in HEK293T cells. In summary, this study provided the first experimental demonstration that oysters possess a functional JNK that participates in host defense against bacterial infection in C. hongkongensis.

  10. Antiepileptic action of c-Jun N-terminal kinase (JNK) inhibition in an animal model of temporal lobe epilepsy.

    PubMed

    Tai, Tina Y; Warner, Lindsay N; Jones, Terrance D; Jung, Sangwook; Concepcion, Francis A; Skyrud, David W; Fender, Jason; Liu, Yusha; Williams, Aaron D; Neumaier, John F; D'Ambrosio, Raimondo; Poolos, Nicholas P

    2017-02-22

    Several phosphorylation signaling pathways have been implicated in the pathogenesis of epilepsy arising from both genetic causes and acquired insults to the brain. Identification of dysfunctional signaling pathways in epilepsy may provide novel targets for antiepileptic therapies. We previously described a deficit in phosphorylation signaling mediated by p38 mitogen-activated protein kinase (p38 MAPK) that occurs in an animal model of temporal lobe epilepsy, and that produces neuronal hyperexcitability measured in vitro. We asked whether in vivo pharmacological manipulation of p38 MAPK activity would influence seizure frequency in chronically epileptic animals. Administration of a p38 MAPK inhibitor, SB203580, markedly worsened spontaneous seizure frequency, consistent with prior in vitro results. However, anisomycin, a non-specific p38 MAPK activator, significantly increased seizure frequency. We hypothesized that this unexpected result was due to activation of a related MAPK, c-Jun N-terminal kinase (JNK). Administration of JNK inhibitor SP600125 significantly decreased seizure frequency in a dose-dependent manner without causing overt behavioral abnormalities. Biochemical analysis showed increased JNK expression and activity in untreated epileptic animals. These results show for the first time that JNK is hyperactivated in an animal model of epilepsy, and that phosphorylation signaling mediated by JNK may represent a novel antiepileptic target.

  11. Deletion of the c-Jun N-terminal kinase 3 gene protects neonatal mice against cerebral hypoxic-ischaemic injury.

    PubMed

    Pirianov, Grisha; Brywe, Katarina G; Mallard, Carina; Edwards, A David; Flavell, Richard A; Hagberg, Henrik; Mehmet, Huseyin

    2007-05-01

    c-Jun N-terminal kinase 3 (JNK3) is a member of the stress-activated group of mitogen-activated protein kinases. c-Jun N-terminal kinase 3 is a potent mediator of apoptosis and the use of JNK inhibitors or jnk3 gene deletion each protect against brain injury in adults. However, little is known about the role of JNK3 or its mechanism of action in neonatal brain injury. The aim of the present study was to compare the vulnerability of neonatal JNK3 knockout (JNK3 KO) mice and wild-type (WT) mice to cerebral hypoxic-ischaemic injury (HII) using unilateral-carotid occlusion combined with transient hypoxia. The degree of neural tissue loss in JNK3 KO mice was substantially reduced compared with WT mice (JNK3 KO 27.8%+/-2.8% versus WT 48.3%+/-2.0%, Pactivity, with JNK3 as the major isoform. Significantly, in JNK3 KO animals there was no difference in the activation of the upstream kinases mitogen-activated protein kinase kinase (MKK4) or MKK7. Downstream of JNK3, HII lead to increased phosphorylation of the transcription factors c-Jun and adenovirus transcription factor-2 (ATF-2), which was attenuated in JNK3 KO mice. c-Jun N-terminal kinase 3 deletion also decrease caspase-3 cleavage and Bim/PUMA expression, coupled with a upregulation of AKT/FOXO3a levels, linking JNK3 to apoptosis. These findings implicate JNK3 involvement in neural cell loss resulting from cerebral HII in the developing brain.

  12. Expression and regulation of c-Jun N-terminal kinase (JNK) in endometrial cells in vivo and in vitro.

    PubMed

    Kizilay, Gulnur; Cakmak, Hakan; Yen, Chih-Feng; Atabekoglu, Cem; Arici, Aydin; Kayisli, Umit Ali

    2008-10-01

    JNK(c-Jun N-terminal kinase) is one of the main types of mitogen-activated protein kinases. JNK modulates inflammation and apoptosis in response to stress. Our hypothesis is that temporal and spatial changes in JNK activity regulate inflammation in human endometrium and that fluctuation in estrogen and progesterone levels may play a role in JNK activation. Therefore, we aimed to determine total-(t-) and active-(phosphorylated, p-) JNK expression in endometrial tissues in vivo by immunohistochemistry, and in vitro by immunocytochemistry and Western blot analysis. Immunohistochemistry revealed moderate cytoplasmic and nuclear t-JNK immunoreactivity, and mostly nuclear p-JNK immunoreactivity throughout the menstrual cycle and early pregnancy. The highest p- and t-JNK immunoreactivity was detected in late secretory phase (P < 0.05). We observed that endometrial stromal cell (ESC)s showed a significant increase in p-JNK expression following 48 h of estrogen combined with progesterone (E(2) + P(4)) withdrawal from the culture conditions, compared to control and non-withdrawal groups (P < 0.05). Upon treatment with JNK inhibitor SP600125, we observed a significantly decreased interleukin (IL)-8 level (P < 0.05) in the presence and absence of E(2). These results demonstrate that JNK expression increases during the late secretory phase when the inflammatory response is highest. Inhibition of IL-8 expression by SP600125 suggests that JNK is involved in regulation of proinflammatory mediators of endometrium.

  13. The c-Jun N-terminal kinase signaling pathway mediates chrysotile asbestos-induced alveolar epithelial cell apoptosis

    PubMed Central

    LI, PENG; LIU, TIE; KAMP, DAVID W.; LIN, ZIYING; WANG, YAHONG; LI, DONGHONG; YANG, LAWEI; HE, HUIJUAN; LIU, GANG

    2015-01-01

    Exposure to chrysotile asbestos exposure is associated with an increased risk of mortality in combination with pulmonary diseases including lung cancer, mesothelioma and asbestosis. Multiple mechanisms by which chrysotile asbestos fibers induce pulmonary disease have been identified, however the role of apoptosis in human lung alveolar epithelial cells (AEC) has not yet been fully explored. Accumulating evidence implicates AEC apoptosis as a crucial event in the development of both idiopathic pulmonary fibrosis and asbestosis. The aim of the present study was to determine whether chrysotile asbestos induces mitochondria-regulated (intrinsic) AEC apoptosis and, if so, whether this induction occurs via the activation of mitogen-activated protein kinases (MAPK). Human A549 bronchoalveolar carcinoma-derived cells with alveolar epithelial type II-like features were used. The present study showed that chrysotile asbestos induced a dose- and time-dependent decrease in A549 cell viability, which was accompanied by the activation of the MAPK c-Jun N-terminal kinases (JNK), but not the MAPKs extracellular signal-regulated kinase 1/2 and p38. Chrysotile asbestos was also shown to induce intrinsic AEC apoptosis, as evidenced by the upregulation of the pro-apoptotic genes Bax and Bak, alongside the activation of caspase-9, poly (ADP-ribose) polymerase (PARP), and the release of cytochrome c. Furthermore, the specific JNK inhibitor SP600125 blocked chrysotile asbestos-induced JNK activation and subsequent apoptosis, as assessed by both caspase-9 cleavage and PARP activation. The results of the present study demonstrated that chrysotile asbestos induces intrinsic AEC apoptosis by a JNK-dependent mechanism, and suggests a potential novel target for the modulation of chrysotile asbestos-associated lung diseases. PMID:25530474

  14. A Novel c-Jun N-terminal Kinase (JNK) Signaling Complex Involved in Neuronal Migration during Brain Development.

    PubMed

    Zhang, Feng; Yu, Jingwen; Yang, Tao; Xu, Dan; Chi, Zhixia; Xia, Yanheng; Xu, Zhiheng

    2016-05-27

    Disturbance of neuronal migration may cause various neurological disorders. Both the transforming growth factor-β (TGF-β) signaling and microcephaly-associated protein WDR62 are important for neuronal migration during brain development; however, the underlying molecular mechanisms involved remain unclear. We show here that knock-out or knockdown of Tak1 (TGFβ-activated kinase 1) and Jnk2 (c-Jun N-terminal kinase 2) perturbs neuronal migration during cortical development and that the migration defects incurred by knock-out and/or knockdown of Tβr2 (type II TGF-β receptor) or Tak1 can be partially rescued by expression of TAK1 and JNK2, respectively. Furthermore, TAK1 forms a protein complex with RAC1 and two scaffold proteins of the JNK pathway, the microcephaly-associated protein WDR62 and the RAC1-interacting protein POSH (plenty of Src homology). Components of the complex coordinate with each other in the regulation of TAK1 as well as JNK activities. We suggest that unique JNK protein complexes are involved in the diversified biological and pathological functions during brain development and pathogenesis of diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. The intermediate filament protein keratin 8 is a novel cytoplasmic substrate for c-Jun N-terminal kinase.

    PubMed

    He, Tao; Stepulak, Andrzej; Holmström, Tim H; Omary, M Bishr; Eriksson, John E

    2002-03-29

    Keratins 8 (K8) and 18 are the primary intermediate filaments of simple epithelia. Phosphorylation of keratins at specific sites affects their organization, assembly dynamics, and their interaction with signaling molecules. A number of keratin in vitro and in vivo phosphorylation sites have been identified. One example is K8 Ser-73, which has been implicated as an important phosphorylation site during mitosis, cell stress, and apoptosis. We show that K8 is strongly phosphorylated on Ser-73 upon stimulation of the pro-apoptotic cytokine receptor Fas/CD95/Apo-1 in HT-29 cells. Kinase assays showed that c-Jun N-terminal kinase (JNK) was also activated with activation kinetics corresponding to that of K8 phosphorylation. Furthermore, K8 was also phosphorylated on Ser-73 by JNK in vitro, yielding similar phosphopeptide maps as the in vivo phosphorylated material. In addition, co-immunoprecipitation studies revealed that part of JNK is associated with K8 in vivo, correlating with decreased ability of JNK to phosphorylate the endogenous c-Jun. Taken together, K8 is a new cytoplasmic target for JNK in Fas receptor-mediated signaling. The functional significance of this phosphorylation could relate to regulation of JNK signaling and/or regulation of keratin dynamics.

  16. N-terminal domain of complexin independently activates calcium-triggered fusion

    PubMed Central

    Lai, Ying; Choi, Ucheor B.; Zhang, Yunxiang; Zhao, Minglei; Pfuetzner, Richard A.; Wang, Austin L.; Brunger, Axel T.

    2016-01-01

    Complexin activates Ca2+-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca2+-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca2+-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca2+-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca2+-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca2+-triggered release. PMID:27444020

  17. The Ral/Exocyst Effector Complex Counters c-Jun N-Terminal Kinase-Dependent Apoptosis in Drosophila melanogaster▿ †

    PubMed Central

    Balakireva, Maria; Rossé, Carine; Langevin, Johanna; Chien, Yu-chen; Gho, Michel; Gonzy-Treboul, Geneviève; Voegeling-Lemaire, Stéphanie; Aresta, Sandra; Lepesant, Jean-Antoine; Bellaiche, Yohanns; White, Michael; Camonis, Jacques

    2006-01-01

    Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, we have generated null and hypomorph alleles of the Drosophila melanogaster Ral gene. Ral null animals were not viable. Reduced Ral expression in cells of the sensory organ lineage had no effect on cell division but led to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggested that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst is a Ral effector, and the epistasis between mutants of Ral and of msn, the fly ortholog of HGK, suggest the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. We conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. We propose that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK. PMID:17000765

  18. Novel role of c-jun N-terminal kinase in regulating the initiation of cap-dependent translation.

    PubMed

    Patel, Manish R; Sadiq, Ahad A; Jay-Dixon, Joe; Jirakulaporn, Tanawat; Jacobson, Blake A; Farassati, Faris; Bitterman, Peter B; Kratzke, Robert A

    2012-02-01

    Initiation of protein translation by the 5' mRNA cap is a tightly regulated step in cell growth and proliferation. Aberrant activation of cap-dependent translation is a hallmark of many cancers including non-small cell lung cancer. The canonical signaling mechanisms leading to translation initiation include activation of the Akt/mTOR pathway in response to the presence of nutrients and growth factors. We have previously observed that inhibition of c-jun N-terminal kinase (JNK) leads to inactivation of cap-dependent translation in mesothelioma cells. Since JNK is involved in the genesis of non-small cell lung cancer (NSCLC), we hypothesized that JNK could also be involved in activating cap-dependent translation in NSCLC cells and could represent an alternative pathway regulating translation. In a series of NSCLC cell lines, inhibition of JNK using SP600125 resulted in inhibition of 4E-BP1 phosphorylation and a decrease in formation of the cap-dependent translation complex, eIF4F. Furthermore, we show that JNK-mediated inhibition of translation is independent of mTOR. Our data provide evidence that JNK is involved in the regulation of translation and has potential as a therapeutic target in NSCLC.

  19. Pharmacological Inhibition of c-Jun N-terminal Kinase Reduces Food Intake and Sensitizes Leptin's Anorectic Signaling Actions.

    PubMed

    Gao, Su; Howard, Shannon; LoGrasso, Philip V

    2017-02-06

    The role for c-Jun N-terminal Kinase (JNK) in the control of feeding and energy balance is not well understood. Here, by use of novel and highly selective JNK inhibitors, we investigated the actions of JNK in the control of feeding and body weight homeostasis. In lean mice, intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of SR-3306, a brain-penetrant and selective pan-JNK (JNK1/2/3) inhibitor, reduced food intake and body weight. Moreover, i.p. and i.c.v. administrations of SR11935, a brain-penetrant and JNK2/3 isoform-selective inhibitor, exerted similar anorectic effects as SR3306, which suggests JNK2 or JNK3 mediates aspect of the anorectic effect by pan-JNK inhibition. Furthermore, daily i.p. injection of SR3306 (7 days) prevented the increases in food intake and weight gain in lean mice upon high-fat diet feeding, and this injection paradigm reduced high-fat intake and obesity in diet-induced obese (DIO) mice. In the DIO mice, JNK inhibition sensitized leptin's anorectic effect, and enhanced leptin-induced STAT3 activation in the hypothalamus. The underlying mechanisms likely involve the downregulation of SOCS3 by JNK inhibition. Collectively, our data suggest that JNK activity promotes positive energy balance, and the therapeutic intervention inhibiting JNK activities represents a promising approach to ameliorate diet-induced obesity and leptin resistance.

  20. c-Jun N-terminal kinase 3 (JNK3) Mediates Paraquat- and Rotenone-Induced Dopaminergic Neuron Death

    PubMed Central

    Choi, Won Seok; Abel, Glen; Klintworth, Heather; Flavell, Richard A.; Xia, Zhengui

    2011-01-01

    Mechanistic studies underlying dopaminergic neuron death may identify new drug targets for the treatment of Parkinson disease (PD). Epidemiological studies have linked pesticide exposure to increased risk for sporadic PD. Here, we investigated the role of c-Jun N-terminal kinase 3 (JNK3), a neural-specific JNK isoform, in dopaminergic neuron death induced by the pesticides rotenone and paraquat. The role of JNK3 was evaluated using RNA silencing and gene deletion to block JNK3 signaling. Using an antibody that recognizes all isoforms of activated JNKs, we found that paraquat and rotenone stimulate JNK phosphorylation in primary cultured dopaminergic neurons. In cultured neurons transfected with Jnk3-specific siRNA and in neurons from Jnk3−/− mice, JNK phosphorylation was nearly abolished, suggesting that JNK3 is the main JNK isoform activated in dopaminergic neurons by these pesticides. Paraquat- and rotenone-induced death of dopaminergic neurons was also significantly reduced by Jnk3 siRNA or Jnk3 gene deletion and deletion of the Jnk3 gene completely attenuated paraquat-induced dopaminergic neuron death and motor-deficits in vivo. Our data identify JNK3 as a common and critical mediator of dopaminergic neuron death induced by paraquat and rotenone, suggesting that it is a potential drug target for PD treatment. PMID:20418776

  1. Induction of experimental autoimmune encephalomyelitis in the absence of c-Jun N-terminal kinase 2.

    PubMed

    Nicolson, Kirsty; Freland, Sofia; Weir, Catherine; Delahunt, Brett; Flavell, Richard A; Bäckström, B Thomas

    2002-08-01

    Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-dependent, organ-specific autoimmune model commonly used to investigate mechanisms involved in the activation of autoreactive T(h)1 cells. Mitogen-activated protein kinases such as c-Jun N-terminal kinase (Jnk) 1 and 2 play an important role in the differentiation of naive precursors into T(h)1 or T(h)2 effector cells. To investigate the role of Jnk2 on autoimmunity, Jnk2(-/-) and wild-type mice were immunized with the myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide and the onset of EAE studied. Surprisingly, Jnk2(-/-) mice were as susceptible to EAE as wild-type mice, regardless of whether low or high antigen doses were used to induce disease. In vitro stimulation of lymph node cells from Jnk2(-/-) and wild-type mice resulted in comparable proliferation in response to MOG35-55, Mycobacterium tuberculosis and concanavalin A. MOG35-55-specific T cells lacking Jnk2 showed a T(h)1 cytokine profile with IFN-gamma, but no IL-4 or IL-5 production. No differences in the types of infiltrating cells or myelin destruction in the central nervous system were found between Jnk2(-/-) and wild-type mice, indicating that lack of Jnk2 does not alter the effector phase of EAE. Our results suggest that, despite involvement in T(h)1/T(h)2 differentiation in vitro, Jnk2 is necessary neither for the induction nor effector phase of MOG35-55-induced EAE and nor is it required for antigen-specific IFN-gamma production.

  2. Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death.

    PubMed

    Hashimoto, Yuichi; Tsuji, Osahiko; Niikura, Takako; Yamagishi, Yohichi; Ishizaka, Miho; Kawasumi, Masaoki; Chiba, Tomohiro; Kanekura, Kohsuke; Yamada, Marina; Tsukamoto, Emi; Kouyama, Keisuke; Terashita, Kenzo; Aiso, Sadakazu; Lin, Anning; Nishimoto, Ikuo

    2003-02-01

    Amyloid precursor protein (APP), the precursor of Abeta, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti-APP antibody. The c-Jun N-terminal kinase (JNK) can mediate various neurotoxic signals, including Abeta neurotoxicity. However, the relationship of APP-mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Abeta independent. Here we examined whether JNK is involved in APP-mediated neuronal cell death and found that: (i) neuronal cell death by antibody-bound APP was inhibited by dominant-negative JNK, JIP-1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK-mediated cell death closely coincided with that of APP-mediated cell death. Pertussis toxin (PTX) suppressed APP-mediated cell death but not caJNK-induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP-mediated cytotoxicity. In the presence of PTX, the PTX-resistant mutant of Galphao, but not that of Galphai, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP-mediated neuronal cell death as a downstream signal transducer of Go.

  3. Involvement of hippocampal jun-N terminal kinase pathway in the enhancement of learning and memory by nicotine.

    PubMed

    Kenney, Justin W; Florian, Cédrick; Portugal, George S; Abel, Ted; Gould, Thomas J

    2010-01-01

    Despite intense scrutiny over the past 20 years, the reasons for the high addictive liability of nicotine and extreme rates of relapse in smokers have remained elusive. One factor that contributes to the development and maintenance of nicotine addiction is the ability of nicotine to produce long-lasting modifications of behavior, yet little is known about the mechanisms by which nicotine alters the underlying synaptic plasticity responsible for behavioral changes. This study is the first to explore how nicotine interacts with learning to alter gene transcription, which is a process necessary for long-term memory consolidation. Transcriptional upregulation of hippocampal jun-N terminal kinase 1 (JNK1) mRNA was found in mice that learned contextual fear conditioning (FC) in the presence of nicotine, whereas neither learning alone nor nicotine administration alone exerted an effect. Furthermore, the upregulation of JNK1 was absent in beta2 nicotinic receptor subunit knockout mice, which are mice that do not show enhanced learning by nicotine. Finally, hippocampal JNK activation was increased in mice that were administered nicotine before conditioning, and the inhibition of JNK during consolidation prevented the nicotine-induced enhancement of contextual FC. These data suggest that nicotine and learning interact to alter hippocampal JNK1 gene expression and related signaling processes, thus resulting in strengthened contextual memories.

  4. Ion channel clustering by membrane-associated guanylate kinases. Differential regulation by N-terminal lipid and metal binding motifs.

    PubMed

    El-Husseini, A E; Topinka, J R; Lehrer-Graiwer, J E; Firestein, B L; Craven, S E; Aoki, C; Bredt, D S

    2000-08-04

    The postsynaptic density protein PSD-95 and related membrane-associated guanylate kinase (MAGUK) proteins assemble signal transduction complexes at sites of cell-cell contact including synapses. Whereas PSD-95 and PSD-93 occur only at postsynaptic sites in hippocampal neurons, SAP-102 also occurs in axons. In heterologous cells, PSD-95 and PSD-93 mediate cell surface ion channel clustering, but SAP-102 and SAP-97 do not. This selective ion channel clustering activity by MAGUKs is explained by differential palmitoylation, as PSD-93 and PSD-95 are palmitoylated though SAP-97, and SAP-102 are not. Rather than being palmitoylated, we find that N-terminal cysteines from SAP-102 tightly bind to zinc. And, appending the N terminus of SAP-102 to PSD-95 results in localization of the chimera to both axons and dendrites. These data suggest that lipid modifications and heavy metal associations with the N termini of MAGUKs mediate differential functions and subcellular localizations of these synaptic scaffolds.

  5. c-Jun N-terminal kinase has a key role in Alzheimer disease synaptic dysfunction in vivo

    PubMed Central

    Sclip, A; Tozzi, A; Abaza, A; Cardinetti, D; Colombo, I; Calabresi, P; Salmona, M; Welker, E; Borsello, T

    2014-01-01

    Altered synaptic function is considered one of the first features of Alzheimer disease (AD). Currently, no treatment is available to prevent the dysfunction of excitatory synapses in AD. Identification of the key modulators of synaptopathy is of particular significance in the treatment of AD. We here characterized the pathways leading to synaptopathy in TgCRND8 mice and showed that c-Jun N-terminal kinase (JNK) is activated at the spine prior to the onset of cognitive impairment. The specific inhibition of JNK, with its specific inhibiting peptide D-JNKI1, prevented synaptic dysfunction in TgCRND8 mice. D-JNKI1 avoided both the loss of postsynaptic proteins and glutamate receptors from the postsynaptic density and the reduction in size of excitatory synapses, reverting their dysfunction. This set of data reveals that JNK is a key signaling pathway in AD synaptic injury and that its specific inhibition offers an innovative therapeutic strategy to prevent spine degeneration in AD. PMID:24457963

  6. Thimerosal induces apoptosis in a neuroblastoma model via the cJun N-terminal kinase pathway.

    PubMed

    Herdman, Michelle L; Marcelo, Aileen; Huang, Ying; Niles, Richard M; Dhar, Sanjit; Kiningham, Kinsley Kelley

    2006-07-01

    The cJun N-terminal kinase (JNK)-signaling pathway is activated in response to a variety of stimuli, including environmental insults, and has been implicated in neuronal apoptosis. In this study, we investigated the role that the JNK pathway plays in neurotoxicity caused by thimerosal, an ethylmercury-containing preservative. SK-N-SH cells treated with thimerosal (0-10 microM) showed an increase in the phosphorylated (active) form of JNK and cJun with 5 and 10 microM thimerosal treatment at 2 and 4 h. To examine activator protein-1 (AP-1) transcription, cells were transfected with a pGL2 vector containing four AP-1 consensus sequences and then treated with thimerosal (0-2.5 microM) for 24 h. Luciferase studies showed an increase in AP-1 transcriptional activity upon thimerosal administration. To determine the components of the AP-1 complex, cells were transfected with a dominant negative to either cFos (A-Fos) or cJun (TAM67). Reporter analysis showed that TAM67, but not A-Fos, decreased AP-1 transcriptional activity, indicating a role for cJun in this pathway. To assess which components are essential to apoptosis, cells were treated with a cell-permeable JNK inhibitor II (SP600125) or transfected with TAM67, and the downstream effectors of apoptosis were analyzed. Cells pretreated with SP600125 showed decreases in activation of caspases 9 and 3, decreases in degradation of poly(ADP-ribose) polymerase (PARP), and decreased levels of proapoptotic Bim, in comparison to cells treated with thimerosal alone. However, cells transfected with TAM67 showed no changes in those same components. Taken together, these results indicate that thimerosal-induced neurotoxicity occurs through the JNK-signaling pathway, independent of cJun activation, leading ultimately to apoptotic cell death.

  7. c-Jun-N-terminal kinase 1 is necessary for nicotine-induced enhancement of contextual fear conditioning.

    PubMed

    Leach, Prescott T; Kenney, Justin W; Gould, Thomas J

    2016-08-03

    Acute nicotine enhances hippocampus-dependent learning. Identifying how acute nicotine improves learning will aid in understanding how nicotine facilitates the development of maladaptive memories that contribute to drug-seeking behaviors, help development of medications to treat disorders associated with cognitive decline, and advance understanding of the neurobiology of learning and memory. The effects of nicotine on learning may involve recruitment of signaling through the c-Jun N-terminal kinase family (JNK 1-3). Learning in the presence of acute nicotine increases the transcription of mitogen-activated protein kinase 8 (MAPK8, also known as JNK1), likely through a CREB-dependent mechanism. The functional significance of JNK1 in the effects of acute nicotine on learning, however, is unknown. The current studies undertook a backward genetic approach to determine the functional contribution JNK1 protein makes to nicotine-enhanced contextual fear conditioning. JNK1 wildtype (WT) and knockout (KO) mice were administered acute nicotine prior to contextual and cued fear conditioning. 24h later, mice were evaluated for hippocampus-dependent (contextual fear conditioning) and hippocampus-independent (cued fear conditioning) memory. Nicotine selectively enhanced contextual conditioning in WT mice, but not in KO mice. Nicotine had no effect on hippocampus-independent learning in either genotype. JNK1 KO and WT mice given saline showed similar levels of learning. These data suggest that JNK1 may be recruited by nicotine and is functionally necessary for the acute effects of nicotine on learning and memory. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways.

    PubMed

    Saberi, Behnam; Ybanez, Maria D; Johnson, Heather S; Gaarde, William A; Han, Derick; Kaplowitz, Neil

    2014-04-01

    This study examines the role of protein kinase C (PKC) and AMP-activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad-spectrum PKC inhibitors (Ro-31-8245, Go6983), protected against APAP cytotoxicity despite sustained c-jun-N-terminal kinase (JNK) activation. Broad-spectrum PKC inhibitor treatment enhanced p-AMPK levels and AMPK regulated survival-energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p-AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro-31-8245 treatment in mice up-regulated p-AMPK levels, increased autophagy (i.e., increased LC3-II formation, p62 degradation), and protected against APAP-induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC-α using antisense (ASO) in mice also protected against APAP-induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC-α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP-induced PKC-α translocation to mitochondria, suggesting PKC-α and JNK interplay in a feed-forward mechanism to mediate APAP-induced liver injury. PKC-α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP-induced liver injury. Copyright © 2014 by the American Association for the Study of Liver Diseases.

  9. Critical role of c-jun N-terminal protein kinase in promoting mitochondrial dysfunction and acute liver injury

    PubMed Central

    Jang, Sehwan; Yu, Li-Rong; Abdelmegeed, Mohamed A.; Gao, Yuan; Banerjee, Atrayee; Song, Byoung-Joon

    2015-01-01

    The mechanism by which c-Jun N-terminal protein kinase (JNK) promotes tissue injury is poorly understood. Thus we aimed at studying the roles of JNK and its phospho-target proteins in mouse models of acute liver injury. Young male mice were exposed to a single dose of CCl4 (50 mg/kg, IP) and euthanized at different time points. Liver histology, blood alanine aminotransferase, and other enzyme activities were measured in CCl4-exposed mice without or with the highly-specific JNK inhibitors. Phosphoproteins were purified from control or CCl4-exposed mice and analyzed by differential mass-spectrometry followed by further characterizations of immunoprecipitation and activity measurements. JNK was activated within 1 h while liver damage was maximal at 24 h post-CCl4 injection. Markedly increased phosphorylation of many mitochondrial proteins was observed between 1 and 8 h following CCl4 exposure. Pretreatment with the selective JNK inhibitor SU3327 or the mitochondria-targeted antioxidant mito-TEMPO markedly reduced the levels of p-JNK, mitochondrial phosphoproteins and liver damage in CCl4-exposed mice. Differential proteomic analysis identified many phosphorylated mitochondrial proteins involved in anti-oxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. Aldehyde dehydrogenase, NADH-ubiquinone oxidoreductase, and α-ketoglutarate dehydrogenase were phosphorylated in CCl4-exposed mice but dephosphorylated after SU3327 pretreatment. Consistently, the suppressed activities of these enzymes were restored by SU3327 pretreatment in CCl4-exposed mice. These data provide a novel mechanism by which JNK, rapidly activated by CCl4, promotes mitochondrial dysfunction and acute hepatotoxicity through robust phosphorylation of numerous mitochondrial proteins. PMID:26491845

  10. A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation.

    PubMed

    Lehnert, Mark; Relja, Borna; Sun-Young Lee, Veronika; Schwestka, Birgit; Henrich, Dirk; Czerny, Christoph; Froh, Matthias; Borsello, Tiziana; Marzi, Ingo

    2008-08-01

    Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

  11. Degradation of newly synthesized polypeptides by ribosome-associated RACK1/c-Jun N-terminal kinase/eukaryotic elongation factor 1A2 complex.

    PubMed

    Gandin, Valentina; Gutierrez, Gustavo J; Brill, Laurence M; Varsano, Tal; Feng, Yongmei; Aza-Blanc, Pedro; Au, Qingyan; McLaughlan, Shannon; Ferreira, Tiago A; Alain, Tommy; Sonenberg, Nahum; Topisirovic, Ivan; Ronai, Ze'ev A

    2013-07-01

    Folding of newly synthesized polypeptides (NSPs) into functional proteins is a highly regulated process. Rigorous quality control ensures that NSPs attain their native fold during or shortly after completion of translation. Nonetheless, signaling pathways that govern the degradation of NSPs in mammals remain elusive. We demonstrate that the stress-induced c-Jun N-terminal kinase (JNK) is recruited to ribosomes by the receptor for activated protein C kinase 1 (RACK1). RACK1 is an integral component of the 40S ribosome and an adaptor for protein kinases. Ribosome-associated JNK phosphorylates the eukaryotic translation elongation factor 1A isoform 2 (eEF1A2) on serines 205 and 358 to promote degradation of NSPs by the proteasome. These findings establish a role for a RACK1/JNK/eEF1A2 complex in the quality control of NSPs in response to stress.

  12. A new c-Jun N-terminal kinase (JNK)-interacting protein, Sab (SH3BP5), associates with mitochondria.

    PubMed

    Wiltshire, Carolyn; Matsushita, Masato; Tsukada, Satoshi; Gillespie, David A F; May, Gerhard H W

    2002-11-01

    We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Bruton's tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine-proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways.

  13. A new c-Jun N-terminal kinase (JNK)-interacting protein, Sab (SH3BP5), associates with mitochondria.

    PubMed Central

    Wiltshire, Carolyn; Matsushita, Masato; Tsukada, Satoshi; Gillespie, David A F; May, Gerhard H W

    2002-01-01

    We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Bruton's tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine-proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways. PMID:12167088

  14. Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation.

    PubMed

    Cripe, L D; Gelfanov, V M; Smith, E A; Spigel, D R; Phillips, C A; Gabig, T G; Jung, S-H; Fyffe, J; Hartman, A D; Kneebone, P; Mercola, D; Burgess, G S; Boswell, H S

    2002-05-01

    A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.

  15. Cyclooxygenase-2 utilizes Jun N-terminal kinases to induce invasion, but not tamoxifen resistance, in MCF-7 breast cancer cells.

    PubMed

    Gonzalez-Villasana, Vianey; Gutiérrez-Puente, Yolanda; Tari, Ana M

    2013-09-01

    Elevated cyclooxygenase-2 (COX-2) expression in breast tumors is associated with a lower survival rate in patients with estrogen receptor α (ERα)-positive tumors. We hypothesized that COX-2 reduces the survival rate of breast cancer patients with ERα-positive tumors since COX-2 increases the invasiveness of ERα-positive breast tumors and decreases tumor sensitivity to tamoxifen. Previously, we demonstrated that COX-2 stimulates the activity of protein kinase C (PKC) to increase the invasiveness of ERα-positive MCF-7 breast cancer cells and to decrease the sensitivity of MCF-7 cells to tamoxifen. High levels of COX-2 are associated with the activation of the mitogen-activated protein kinase (MAPK) family and the Akt kinase. However, it is not known whether these kinases mediate COX-2-induced invasive activity and tamoxifen resistance. In the present study, we report that COX-2 utilizes PKC to enhance the phosphorylation of Jun N-terminal kinases (JNKs), but not that of other MAPK family members or Akt. Inhibition aimed at JNKs reduced COX-2-induced invasion but not COX-2-induced tamoxifen resistance. We conclude that JNKs are essential for induced cell invasion by COX-2, but not tamoxifen resistance, in ERα-positive breast cancer cells.

  16. Hepatotoxicity mediated by pyrazole (cytochrome P450 2E1) plus tumor necrosis factor alpha treatment occurs in c-Jun N-terminal kinase 2-/- but not in c-Jun N-terminal kinase 1-/- mice.

    PubMed

    Wang, Xiaodong; Wu, Defeng; Yang, Lili; Cederbaum, Arthur I

    2011-11-01

    Cytochrome P450 2E1 (CYP2E1) induction and tumor necrosis factor alpha (TNF-α) production are key risk factors in alcoholic liver injury. Increased oxidative stress from CYP2E1 induction by pyrazole in vivo sensitizes the liver to TNF-α-induced hepatotoxicity by a mechanism involving the activation of c-jun N-terminal kinase (JNK) and mitochondrial damage. The aim of this study was to evaluate whether JNK1 or JNK2 plays a role in this potentiated hepatotoxicity. Wild-type (WT), jnk1(-/-) , and jnk2(-/-) mice were used to identify changes of hepatotoxicity, damage to mitochondria, and production of oxidative stress after pyrazole plus TNF-α treatment. Increased serum alanine aminotransferase, inflammatory infiltration, and central necrosis were observed in the jnk2(-/-) and WT mice treated with pyrazole plus TNF-α, but not in the jnk1(-/-) mice. Pyrazole elevated the activity and protein level of CYP2E1 in all mice. There was a significant increase of malondialdehyde, 4-hydroxynonenal adducts, 3-nitrotyrosine, and inducible nitric oxide synthase in the jnk2(-/-) and WT mice, compared to the jnk1(-/-) mice, upon pyrazole plus TNF-α treatment, or compared to mice treated with either pyrazole alone or TNF-α alone. The antioxidants, catalase, phospholipid hydroperoxide glutathione peroxidase, thioredoxin, and glutathione were lowered, and cytochrome c was released from the mitochondria in the jnk2(-/-) and WT mice. Mitochondrial production of superoxide was increased in the jnk2(-/-) and WT mice, compared to the jnk1(-/-) mice, upon pyrazole plus TNF-α treatment. Electron microscopy showed altered mitochondrial structure in the jnk2(-/-) and WT mice, but not the jnk1(-/-) mice. JNK1 plays a role in the hepatotoxicity, mitochondrial dysfunction, and oxidative stress mediated by pyrazole plus TNF-α treatment. These findings raise the question as to the potential mechanisms of JNK1 activation related to alcoholic liver injury. Copyright © 2011 American Association

  17. Signaling by the Engulfment Receptor Draper: A Screen in Drosophila melanogaster Implicates Cytoskeletal Regulators, Jun N-Terminal Kinase, and Yorkie

    PubMed Central

    Fullard, John F.; Baker, Nicholas E.

    2015-01-01

    Draper, the Drosophila melanogaster homolog of the Ced-1 protein of Caenorhabditis elegans, is a cell-surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, we screened chromosomal deficiencies to identify loci that dominantly modify the phenotype of overexpression of Draper isoform II (suppressed differentiation of the posterior crossvein in the wing). We found evidence for 43 genetic modifiers of Draper II. Twenty-four of the 37 suppressor loci and 3 of the 6 enhancer loci were identified. An additional 5 suppressors and 2 enhancers were identified among mutations in functionally related genes. These studies reveal positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott–Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in Draper signaling. Relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador/Warts/Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are also discussed. PMID:25395664

  18. Identification and Analysis of a Novel Dimerization Domain Shared by Various Members of c-Jun N-terminal Kinase (JNK) Scaffold Proteins*

    PubMed Central

    Cohen-Katsenelson, Ksenya; Wasserman, Tanya; Darlyuk-Saadon, Ilona; Rabner, Alona; Glaser, Fabian; Aronheim, Ami

    2013-01-01

    Mitogen-activated protein kinases (MAPKs) form a kinase tier module in which MAPK, MAP2K, and MAP3K are held by scaffold proteins. The scaffold proteins serve as a protein platform for selective and spatial kinase activation. The precise mechanism by which the scaffold proteins function has not yet been fully explained. WDR62 is a novel scaffold protein of the c-Jun N-terminal kinase (JNK) pathway. Recessive mutations within WDR62 result in severe cerebral cortical malformations. One of the WDR62 mutant proteins found in a patient with microcephaly encodes a C-terminal truncated protein that fails to associate efficiently with JNK and MKK7β1. The present article shows that the WDR62 C-terminal region harbors a novel dimerization domain composed of a putative loop-helix domain that is necessary and sufficient for WDR62 dimerization and is critical for its scaffolding function. The loop-helix domain is highly conserved between orthologues and is also shared by the JNK scaffold protein, JNKBP1/MAPKBP1. Based on the high sequence conservation of the loop-helix domain, our article shows that MAPKBP1 homodimerizes and heterodimerizes with WDR62. Endogenous WDR62 and MAPKBP1 co-localize to stress granules following arsenite treatment, but not during mitosis. This study proposes another layer of complexity, in which coordinated activation of signaling pathways is mediated by the association between the different JNK scaffold proteins depending on their biological function. PMID:23341463

  19. Mechanism of N-terminal modulation of activity at the melanocortin-4 receptor GPCR

    PubMed Central

    Ersoy, Baran A; Pardo, Leonardo; Zhang, Sumei; Thompson, Darren A; Millhauser, Glenn; Govaerts, Cedric; Vaisse, Christian

    2013-01-01

    Most of our understanding of G protein–coupled receptor (GPCR) activation has been focused on the direct interaction between diffusible ligands and their seven-transmembrane domains. However, a number of these receptors depend on their extracellular N-terminal domain for ligand recognition and activation. To dissect the molecular interactions underlying both modes of activation at a single receptor, we used the unique properties of the melanocortin-4 receptor (MC4R), a GPCR that shows constitutive activity maintained by its N-terminal domain and is physiologically activated by the peptide α-melanocyte stimulating hormone (αMSH). We find that activation by the N-terminal domain and αMSH relies on different key residues in the transmembrane region. We also demonstrate that agouti-related protein, a physiological antagonist of MC4R, acts as an inverse agonist by inhibiting N terminus–mediated activation, leading to the speculation that a number of constitutively active orphan GPCRs could have physiological inverse agonists as sole regulators. PMID:22729149

  20. Requirement of the N-terminal activation domain of herpes simplex virus ICP4 for viral gene expression.

    PubMed

    Wagner, Lauren M; Bayer, Avraham; Deluca, Neal A

    2013-01-01

    ICP4 is the major activator of herpes simplex virus (HSV) transcription. Previous studies have defined several regions of ICP4 that are important for viral gene expression, including a DNA binding domain and transactivation domains that are contained in the C-terminal and N-terminal 520 and 274 amino acids, respectively. Here we show that the N-terminal 210 amino acids of ICP4 are required for interactions with components of TFIID and mediator and, as a consequence, are necessary for the activation of viral genes. A mutant of ICP4 deleted for amino acids 30 to 210, d3-10, was unable to complement an ICP4 null virus at the level of viral replication. This was the result of a severe deficiency in viral gene and protein expression. The absence of viral gene expression coincided with a defect in the recruitment of RNA polymerase II to a representative early promoter (thymidine kinase [TK]). Affinity purification experiments demonstrated that d3-10 ICP4 was not found in complexes with components of TFIID and mediator, suggesting that the defect in RNA polymerase II (Pol II) recruitment was the result of ablated interactions between d3-10 and TFIID and mediator. Complementation assays suggested that the N-terminal and C-terminal regions of ICP4 cooperate to mediate gene expression. The complementation was the result of the formation of more functional heterodimers, which restored the ability of the d3-10-containing molecules to interact with TFIID. Together, these studies suggest that the N terminus contains a true activation domain, mediating interactions with TFIID, mediator, and perhaps other transcription factors, and that the C terminus of the molecule contains activities that augment the functions of the activation domain.

  1. Topology and Secondary Structure of the N-terminal Domain of Diacylglycerol Kinase

    SciTech Connect

    Oxenoid, Kirill; Soennichsen, Frank D.; Sanders, Charles R.

    2002-09-28

    Prokaryotic diacylglycerol kinase (DAGK) functions as a homotrimer of 13 kDa subunits, each of which has three transmembrane segments. This enzyme is conditionally essential to some bacteria and serves as a model system for studies of membrane protein biocatalysis, stability, folding, and misfolding. In this work, the detailed topology and secondary structure of DAGKs N-terminus up through the loop

  2. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain

    PubMed Central

    Stroehlein, Andreas J.; Young, Neil D.; Korhonen, Pasi K.; Chang, Bill C. H.; Sternberg, Paul W.; La Rosa, Giuseppe; Pozio, Edoardo; Gasser, Robin B.

    2016-01-01

    Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation. PMID:27412987

  3. The c-Jun N-terminal kinase prevents oxidative stress induced by UV and thermal stresses in corals and human cells.

    PubMed

    Courtial, Lucile; Picco, Vincent; Grover, Renaud; Cormerais, Yann; Rottier, Cécile; Labbe, Antoine; Pagès, Gilles; Ferrier-Pagès, Christine

    2017-04-04

    Coral reefs are of major ecological and socio-economic interest. They are threatened by global warming and natural pressures such as solar ultraviolet radiation. While great efforts have been made to understand the physiological response of corals to these stresses, the signalling pathways involved in the immediate cellular response exhibited by corals remain largely unknown. Here, we demonstrate that c-Jun N-terminal kinase (JNK) activation is involved in the early response of corals to thermal and UV stress. Furthermore, we found that JNK activity is required to repress stress-induced reactive oxygen species (ROS) accumulation in both the coral Stylophora pistillata and human skin cells. We also show that inhibiting JNK activation under stress conditions leads to ROS accumulation, subsequent coral bleaching and cell death. Taken together, our results suggest that an ancestral response, involving the JNK pathway, is remarkably conserved from corals to human, protecting cells from the adverse environmental effects.

  4. The c-Jun N-terminal kinase prevents oxidative stress induced by UV and thermal stresses in corals and human cells

    PubMed Central

    Courtial, Lucile; Picco, Vincent; Grover, Renaud; Cormerais, Yann; Rottier, Cécile; Labbe, Antoine; Pagès, Gilles; Ferrier-Pagès, Christine

    2017-01-01

    Coral reefs are of major ecological and socio-economic interest. They are threatened by global warming and natural pressures such as solar ultraviolet radiation. While great efforts have been made to understand the physiological response of corals to these stresses, the signalling pathways involved in the immediate cellular response exhibited by corals remain largely unknown. Here, we demonstrate that c-Jun N-terminal kinase (JNK) activation is involved in the early response of corals to thermal and UV stress. Furthermore, we found that JNK activity is required to repress stress-induced reactive oxygen species (ROS) accumulation in both the coral Stylophora pistillata and human skin cells. We also show that inhibiting JNK activation under stress conditions leads to ROS accumulation, subsequent coral bleaching and cell death. Taken together, our results suggest that an ancestral response, involving the JNK pathway, is remarkably conserved from corals to human, protecting cells from the adverse environmental effects. PMID:28374828

  5. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

    PubMed Central

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F.; Wahl, Markus C.

    2015-01-01

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  6. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    NASA Astrophysics Data System (ADS)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-06-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

  7. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    PubMed Central

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-01-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence. PMID:27345869

  8. N-Terminal Deletion of Peptide:N-Glycanase Results in Enhanced Deglycosylation Activity

    PubMed Central

    Wang, Shengjun; Xin, Fengxue; Liu, Xiaoyue; Wang, Yuxiao; An, Zhenyi; Qi, Qingsheng; Wang, Peng George

    2009-01-01

    Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the β-aspartylglucosaminyl bond. Peptide:N-glycanase in yeast binds to Rad23p through its N-terminus. In this study, the complex formed between Peptide:N-glycanase and Rad23p was found to exhibit enhanced deglycosylation activity, which suggests an important role for this enzyme in the misfolded glycoprotein degradation pathway in vivo. To investigate the role of this enzyme in this pathway, we made stepwise deletions of the N-terminal helices of peptide:N-glycanase. Enzymatic analysis of the deletion mutants showed that deletion of the N-terminal H1 helix (Png1p-ΔH1) enhanced the deglycosylation activity of N-glycanase towards denatured glycoproteins. In addition, this mutant exhibited high deglycosylation activity towards native glycoproteins. Dynamic simulations of the wild type and N-terminal H1 deletion mutant implied that Png1p-ΔH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-ΔH1 towards native and non-native glycoproteins offers a potential biotechnological application. PMID:20016784

  9. Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation.

    PubMed

    Höög, J O; Weis, M; Zeppezauer, M; Jörnvall, H; von Bahr-Lindström, H

    1987-12-01

    Human alcohol dehydrogenase (ADH, beta beta isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the beta-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3% of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic beta-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.

  10. Hsp72-Mediated Suppression of c-Jun N-Terminal Kinase Is Implicated in Development of Tolerance to Caspase-Independent Cell Death

    PubMed Central

    Gabai, Vladimir L.; Yaglom, Julia A.; Volloch, Vladimir; Meriin, Anatoli B.; Force, Thomas; Koutroumanis, Maria; Massie, Bernard; Mosser, Dick D.; Sherman, Michael Y.

    2000-01-01

    Pretreatment with mild heat shock is known to protect cells from severe stress (acquired thermotolerance). Here we addressed the mechanism of this phenomenon by using primary human fibroblasts. Severe heat shock (45°C, 75 min) of the fibroblasts caused cell death displaying morphological characteristics of apoptosis; however, it was caspase independent. This cell death process was accompanied by strong activation of Akt, extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38, and c-Jun N-terminal (JNK) kinases. Suppression of Akt or ERK1 and -2 kinases increased cell thermosensitivity. In contrast, suppression of stress kinase JNK rendered cells thermoresistant. Development of thermotolerance was not associated with Akt or ERK1 and -2 regulation, and inhibition of these kinases did not reduce acquired thermotolerance. On the other hand, acquired tolerance to severe heat shock was associated with downregulation of JNK. Using an antisense-RNA approach, we found that accumulation of the heat shock protein Hsp72 is necessary for JNK downregulation and is critical for thermotolerance. The capability of naive cells to withstand moderate heat treatment also appears to be dependent on the accumulation of Hsp72 induced by this stress. Indeed, exposure to 45°C for 45 min caused only transient JNK activation and was nonlethal, while prevention of Hsp72 accumulation prolonged JNK activation and led to massive cell death. We also found that JNK activation by UV irradiation, interleukin-1, or tumor necrosis factor was suppressed in thermotolerant cells and that Hsp72 accumulation was responsible for this effect. Hsp72-mediated suppression of JNK is therefore critical for acquired thermotolerance and may play a role in tolerance to other stresses. PMID:10958679

  11. Reactive oxygen species and c-Jun N-terminal kinases contribute to TEMPO-induced apoptosis in L5178Y cells.

    PubMed

    Guo, Xiaoqing; Chen, Si; Zhang, Zhuhong; Dobrovolsky, Vasily N; Dial, Stacey L; Guo, Lei; Mei, Nan

    2015-06-25

    The biological consequences of exposure to piperidine nitroxides is a concern, given their widespread use in manufacturing processes and their potential use in clinical applications. Our previous study reported that TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl), a low molecular weight free radical, possesses pro-oxidative activity in L5178Y cells. In this study, we investigated and characterized the role of reactive oxygen species (ROS) in TEMPO-induced toxicity in L5178Y cells. We found that TEMPO induced time- and concentration-dependent intracellular ROS production and glutathione depletion. TEMPO also induced apoptosis as demonstrated by increased caspase-3/7 activity, an increased proportion of annexin V stained cells, and decreased expression of anti-apoptotic proteins including Bcl-2, Bcl-xL and Mcl-1. N-acetylcysteine, a ROS scavenger, attenuated the ROS production and apoptosis induced by TEMPO. Moreover, Western blot analyses revealed that TEMPO activated γ-H2A.X, a hallmark of DNA damage, and c-Jun N-terminal kinases (JNK), a key member in the mitogen-activated protein kinase (MAPK) signaling pathway. Addition of SP600125, a JNK-specific inhibitor, blocked TEMPO-mediated JNK phosphorylation and also attenuated TEMPO-induced apoptosis. These findings indicate that both ROS production and JNK activation are involved in TEMPO-induced apoptosis, and may contribute to the toxicity of TEMPO in L5178Y cells.

  12. EGCG-targeted p57/KIP2 reduces tumorigenicity of oral carcinoma cells: Role of c-Jun N-terminal kinase

    SciTech Connect

    Yamamoto, Tetsuya; Digumarthi, Hari; Aranbayeva, Zina; Wataha, John; Lewis, Jill; Messer, Regina; Qin, Haiyan; Dickinson, Douglas; Osaki, Tokio; Schuster, George S.; Hsu, Stephen

    2007-11-01

    The green tea polyphenol epigallocatechin-3-gallate (EGCG) regulates gene expression differentially in tumor and normal cells. In normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. In humans, reduced expression of p57 often is associated with advanced tumors, and tumor cells with inactivated p57 undergo apoptosis when exposed to EGCG. The mechanism of p57 induction by EGCG is not well understood. Here, we show that in NHEK, EGCG-induces p57 via the p38 mitogen-activated protein kinase (MAPK) signaling pathway. In p57-negative tumor cells, JNK signaling mediates EGCG-induced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis via inhibition of c-Jun N-terminal kinase (JNK). We also found that restoration of p57 expression in tumor cells significantly reduced tumorigenicity in athymic mice. These results suggest that p57 expression may be an useful indicator for the clinical course of cancers, and could be potentially useful as a target for cancer therapies.

  13. The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus

    SciTech Connect

    Kutz, Helmut; Reisbach, Gilbert; Schultheiss, Ute; Kieser, Arnd

    2008-02-20

    The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) transforms cells activating signal transduction pathways such as NF-{kappa}B, PI3-kinase, or c-Jun N-terminal kinase (JNK). Here, we investigated the functional role of the LMP1-induced JNK pathway in cell transformation. Expression of a novel dominant-negative JNK1 allele caused a block of proliferation in LMP1-transformed Rat1 fibroblasts. The JNK-specific inhibitor SP600125 reproduced this effect in Rat1-LMP1 cells and efficiently interfered with proliferation of EBV-transformed lymphoblastoid cells (LCLs). Inhibition of the LMP1-induced JNK pathway in LCLs caused the downregulation of c-Jun and Cdc2, the essential G2/M cell cycle kinase, which was accompanied by a cell cycle arrest of LCLs at G2/M phase transition. Moreover, SP600125 retarded tumor growth of LCLs in a xenograft model in SCID mice. Our data support a critical role of the LMP1-induced JNK pathway for proliferation of LMP1-transformed cells and characterize JNK as a potential target for intervention against EBV-induced malignancies.

  14. Dexmedetomidine inhibits tumor necrosis factor-alpha and interleukin 6 in lipopolysaccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinases.

    PubMed

    Zhang, Xiaobao; Wang, Jun; Qian, Wenyi; Zhao, Jingjing; Sun, Li; Qian, Yanning; Xiao, Hang

    2014-06-01

    Astrocytes play an important role in immune regulation in the central nervous system (CNS). Dexmedetomidine (DEX) has been reported to exert anti-inflammatory effects on astrocytes stimulated by lipopolysaccharide (LPS) both in vitro and in vivo studies. However, the underlying molecular mechanisms remain poorly understood. This study was designed to evaluate the effects of DEX on tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) gene expressions in LPS-challenged astrocytes. Moreover, c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (MAPK) pathways in LPS-challenged astrocytes were also investigated. In the present study, astrocytes were stimulated with LPS in the absence and presence of various concentrations of DEX. With real-time PCR assay, we found that LPS significantly increased expressions of TNF-α and IL-6 in mRNA level; however, these effects could be attenuated by DEX. Furthermore, JNK pathway might be involved in LPS-induced astrocyte activation because JNK phosphorylation was significantly increased, and the inhibition of this pathway mediated by DEX as well as SP600125 (JNK inhibitor) decreased TNF-α and IL-6 expressions. Moreover, p38 MAPK was also activated by LPS; however, this pathway seemed to have not participated in DEX-mediated LPS-induced inflammation. These results, taken together, suggest that JNK rather than p38 MAPK signal pathway, provides the potential target for the therapeutic effects of DEX for neuronal inflammatory reactions.

  15. N-Terminal region is responsible for chemotaxis-inducing activity of flounder IL-8.

    PubMed

    Kurata, Osamu; Wada, Shinpei; Matsuyama, Tomomasa; Sakai, Takamitsu; Takano, Tomokazu

    2014-06-01

    The objective of this study was to locate the functional region responsible for the chemotaxis-inducing activity of flounder interleukin 8 (IL-8), which lacks the glutamic acid-leucine-arginine (ELR) motif essential for the induction of neutrophil migration by mammalian IL-8. Using a human cell line, we produced a secretory recombinant protein of flounder IL-8, and analyzed its chemotaxis-inducing activity on leukocytes collected from the flounder kidney. The recombinant IL-8 induced significant migration in neutrophils, which were morphologically and functionally characterized. Using the Edman degradation method, the N-terminal amino acid sequence of rIL-8 was identified as VSLRSLGV. To examine the significance of the N-terminal region for the bioactivity of flounder IL-8, we prepared several recombinant proteins that containing mutations at the N-terminus. Modification of three residues (residues 9-11: serine-leucine-histidine) corresponding in position to the ELR motif in mammalian IL-8 did not reduce its chemotaxis-inducing activity. However, deletion of the first six or more residues significantly reduced its chemotaxis-inducing activity. We propose that residue 6 (leucine) at the N-terminus is important for the chemotaxis-inducing activity of flounder IL-8.

  16. Novel Insights into Structure-Activity Relationships of N-Terminally Modified PACE4 Inhibitors.

    PubMed

    Kwiatkowska, Anna; Couture, Frédéric; Levesque, Christine; Ly, Kévin; Beauchemin, Sophie; Desjardins, Roxane; Neugebauer, Witold; Dory, Yves L; Day, Robert

    2016-02-04

    PACE4 plays important roles in prostate cancer cell proliferation. The inhibition of this enzyme has been shown to slow prostate cancer progression and is emerging as a promising therapeutic strategy. In previous work, we developed a highly potent and selective PACE4 inhibitor, the multi-Leu (ML) peptide, an octapeptide with the sequence Ac-LLLLRVKR-NH2 . Here, with the objective of developing a useful compound for in vivo administration, we investigate the effect of N-terminal modifications. The inhibitory activity, toxicity, stability, and cell penetration properties of the resulting analogues were studied and compared to the unmodified inhibitor. Our results show that the incorporation of a polyethylene glycol (PEG) moiety leads to a loss of antiproliferative activity, whereas the attachment of a lipid chain preserves or improves it. However, the lipidated peptides are significantly more toxic when compared with their unmodified counterparts. Therefore, the best results were achieved not by the N-terminal extension but by the protection of both ends with the d-Leu residue and 4-amidinobenzylamide, which yielded the most stable inhibitor, with an excellent activity and toxicity profile.

  17. Nobiletin, a dietary phytochemical, inhibits vascular smooth muscle cells proliferation via calcium-mediated c-Jun N-terminal kinases pathway.

    PubMed

    Zhou, Cheng-Hua; Wu, Xiao-Hong; Wu, Yu-Qing

    2009-08-01

    Dietary flavonoids have been shown to reduce risk of cardiovascular disease, but the underlying molecular mechanisms are not known. The objective of this study was to investigate the effect of nobiletin, a dietary phytochemical belonging to polymethoxy flavonoid from the peel of Citrus fruit, on vascular smooth muscle cells (VSMCs) proliferation and its mechanisms. VSMCs proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) and [(3)H]thymidine incorporation assay. The activity of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) were determined by western blotting. [Ca(2+)](i) was measured by laser scanning confocal microscopy. Our results showed that angiotensin II-induced VSMCs proliferation was inhibited by nobiletin. While no effect on ERK1/2 and p38 MAPK, nobiletin markedly inhibited angiotensin II-induced activation of JNK. Anthra[1-9-cd]pyrazol-6(2H)-one (SP600125), an inhibitor of JNK, decreased the [(3)H]thymidine incorporation induced by angiotensin II. Nobiletin also attenuated both the intracellular Ca(2+) mobilization and the extracellular Ca(2+) influx induced by angiotensin II. Furthermore, intracellular Ca(2+) chelation by BAPTA-AM, extracellular Ca(2+) chelation by EGTA or blockade of L-type Ca(2+) channel with verapamil inhibited angiotensin II-induced JNK activation. These findings suggest that the preventing effect of nobiletin on angiotensin II-induced VSMCs proliferation is attributed, in part, to its inhibitory effect on Ca(2+)-dependent JNK activation in VSMCs. Thus, inhibition of JNK by nobiletin may imply its usefulness for the treatment of cardiovascular diseases relevant to VSMCs growth.

  18. The N-terminal dimerization is required for TDP-43 splicing activity.

    PubMed

    Jiang, Lei-Lei; Xue, Wei; Hong, Jun-Ye; Zhang, Jun-Ting; Li, Min-Jun; Yu, Shao-Ning; He, Jian-Hua; Hu, Hong-Yu

    2017-07-21

    TDP-43 is a nuclear factor that functions in promoting pre-mRNA splicing. Deletion of the N-terminal domain (NTD) and nuclear localization signal (NLS) (i.e., TDP-35) results in mislocalization to cytoplasm and formation of inclusions. However, how the NTD functions in TDP-43 activity and proteinopathy remains largely unknown. Here, we studied the structure and function of the NTD in inclusion formation and pre-mRNA splicing of TDP-43 by using biochemical and biophysical approaches. We found that TDP-43 NTD forms a homodimer in solution in a concentration-dependent manner, and formation of intermolecular disulfide results in further tetramerization. Based on the NMR structure of TDP-43 NTD, the dimerization interface centered on Leu71 and Val72 around the β7-strand was defined by mutagenesis and size-exclusion chromatography. Cell experiments revealed that the N-terminal dimerization plays roles in protecting TDP-43 against formation of cytoplasmic inclusions and enhancing pre-mRNA splicing activity of TDP-43 in nucleus. This study may provide mechanistic insights into the physiological function of TDP-43 and its related proteinopathies.

  19. Design and characterization of a potent and selective dual ATP- and substrate-competitive subnanomolar bidentate c-Jun N-terminal kinase (JNK) inhibitor.

    PubMed

    Stebbins, John L; De, Surya K; Pavlickova, Petra; Chen, Vida; Machleidt, Thomas; Chen, Li-Hsing; Kuntzen, Christian; Kitada, Shinichi; Karin, Michael; Pellecchia, Maurizio

    2011-09-22

    c-Jun N-terminal kinases (JNKs) represent valuable targets in the development of new therapies. Present on the surface of JNK is a binding pocket for substrates and the scaffolding protein JIP1 in close proximity to the ATP binding pocket. We propose that bidentate compounds linking the binding energies of weakly interacting ATP and substrate mimetics could result in potent and selective JNK inhibitors. We describe here a bidentate molecule, 19, designed against JNK. 19 inhibits JNK kinase activity (IC(50) = 18 nM; K(i) = 1.5 nM) and JNK/substrate association in a displacement assay (IC(50) = 46 nM; K(i) = 2 nM). Our data demonstrate that 19 targets for the ATP and substrate-binding sites on JNK concurrently. Finally, compound 19 successfully inhibits JNK in a variety of cell-based experiments, as well as in vivo where it is shown to protect against Jo-2 induced liver damage and improve glucose tolerance in diabetic mice.

  20. Pinusolide isolated from Biota orientalis inhibits 5-lipoxygenase dependent leukotriene C4 generation by blocking c-Jun N-terminal kinase pathway in mast cells.

    PubMed

    Jin, Ye; Yang, Hyun Ok; Son, Jong Keun; Chang, Hyeun Wook

    2012-01-01

    Pinusolide, an herbal medicine isolated from Biota orientalis L. (B. orientalis), inhibited 5-lipoxygenase (5-LO)-dependent leukotriene C4 (LTC4) generation in immunoglobulin E (IgE)/Ag-induced bone marrow-derived mast cells (BMMCs) in a concentration-dependent manner. To clarify the action mechanism of pinusolide on the inhibition of LTC4 generation, we examined the effect of pinusolide on phosphorylation of cytosolic phospholipase A2 (cPLA2), as well as translocation phospho-cPLA2 and 5-LO to nucleus. Inhibition of LTC4 generation by pinusolide was accompanied by a decrease in cPLA2 phosphorylation which occurred via a decrease in intracellular Ca2+ influx and blocking the c-Jun N-terminal kinase (JNK) pathways. However, pinusolide had no effect on extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinas phosphorylation. Taken together, the present results suggest that potent inhibition of 5-LO dependent LTC4 generation by pinusolide requires both suppression of calcium influx and JNK phosphorylation.

  1. c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

    PubMed Central

    Suzuki, Akiko; Kakisaka, Keisuke; Suzuki, Yuji; Wang, Ting; Takikawa, Yasuhiro

    2016-01-01

    AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed “lipoapoptosis,” in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet (HFD) for 12 wk, after which the liver histology and expression of proteins such as p62 or LC3 were evaluated. Alpha mouse liver 12 (AML12) cells treated with palmitate (PA) were used as an in vitro model. RESULTS: LC3-II, p62, and Run domain Beclin-1 interacting and cysteine-rich containing (Rubicon) proteins increased in both the HFD mice and in AML12 cells in response to PA treatment. Rubicon expression was decreased upon c-Jun N-terminal kinase (JNK) inhibition at both the mRNA and the protein level in AML12 cells. Rubicon knockdown in AML12 cells with PA decreased the protein levels of both LC3-II and p62. Rubicon expression peaked at 4 h of PA treatment in AML12, and then decreased. Treatment with caspase-9 inhibitor ameliorated the decrease in Rubicon protein expression at 10 h of PA and resulted in enlarged AML12 cells under PA treatment. The enlargement of AML12 cells by PA with caspase-9 inhibition was canceled by Rubicon knockdown. CONCLUSION: The JNK-Rubicon axis enhanced lipoapoptosis, and caspase-9 inhibition and Rubicon had effects that were cytologically similar to hepatocyte ballooning. As ballooned hepatocytes secrete fibrogenic signals and thus might promote fibrosis in the liver, the inhibition of hepatocyte ballooning might provide anti-fibrosis in the NASH liver. PMID:27605885

  2. Recombinant N-Terminal Slit2 Inhibits TGF-β-Induced Fibroblast Activation and Renal Fibrosis.

    PubMed

    Yuen, Darren A; Huang, Yi-Wei; Liu, Guang-Ying; Patel, Sajedabanu; Fang, Fei; Zhou, Joyce; Thai, Kerri; Sidiqi, Ahmad; Szeto, Stephen G; Chan, Lauren; Lu, Mingliang; He, Xiaolin; John, Rohan; Gilbert, Richard E; Scholey, James W; Robinson, Lisa A

    2016-09-01

    Fibrosis and inflammation are closely intertwined injury pathways present in nearly all forms of CKD for which few safe and effective therapies exist. Slit glycoproteins signaling through Roundabout (Robo) receptors have been described to have anti-inflammatory effects through regulation of leukocyte cytoskeletal organization. Notably, cytoskeletal reorganization is also required for fibroblast responses to TGF-β Here, we examined whether Slit2 also controls TGF-β-induced renal fibrosis. In cultured renal fibroblasts, which we found to express Slit2 and Robo-1, the bioactive N-terminal fragment of Slit2 inhibited TGF-β-induced collagen synthesis, actin cytoskeletal reorganization, and Smad2/3 transcriptional activity, but the inactive C-terminal fragment of Slit2 did not. In mouse models of postischemic renal fibrosis and obstructive uropathy, treatment with N-terminal Slit2 before or after injury inhibited the development of renal fibrosis and preserved renal function, whereas the C-terminal Slit2 had no effect. Our data suggest that administration of recombinant Slit2 may be a new treatment strategy to arrest chronic injury progression after ischemic and obstructive renal insults by not only attenuating inflammation but also, directly inhibiting renal fibrosis. Copyright © 2016 by the American Society of Nephrology.

  3. Role of Wnt/β-catenin, Wnt/c-Jun N-terminal kinase and Wnt/Ca2+ pathways in cisplatin-induced chemoresistance in ovarian cancer

    PubMed Central

    Huang, Lu; Jin, Ye; Feng, Shujun; Zou, Yuqing; Xu, Sainan; Qiu, Shuang; Li, Ling; Zheng, Jianhua

    2016-01-01

    The aim of the present study was to explore the expression of Wnt signaling proteins β-catenin, c-Jun N-terminal kinase (JNK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in ovarian cancer cells, and assess the correlation between this expression and cisplatin-induced chemoresistance. SKOV3 ovarian carcinoma cells and SKOV3/DDP (cisplatin resistant) cells were treated with cisplatin in the absence or presence of a Wnt signaling activator (CHIR-99021, glycogen synthase kinase 3β inhibitor) or inhibitor (XAV-939, tankyrase inhibitor). Following incubation for 48 h, cell viability, proliferation and cytotoxicity were measured using a sensitive colorimetric cell counting kit. Expression levels of β-catenin, JNK and CaMKII were detected by western blot and immunofluorescence staining. The results of the current study identified that β-catenin and JNK expression levels were significantly higher (P<0.01 and P<0.05 respectively), while CaMKII expression was lower (P>0.05), in SKOV3/DDP cells compared with SKOV3 cells. Moreover, following treatment with 20 µM cisplatin, reduced expression of β-catenin and JNK (P<0.05 and P<0.01 respectively), and increased expression of CaMKII (P<0.01), was observed in SKOV3 and SKOV3/DPP cell lines. Furthermore, inhibition of β-catenin signaling by XAV-939 effectively reversed cisplatin chemoresistance in SKOV3/DDP cells. Similarly, XAV-939 downregulated JNK expression (P<0.001), but upregulated CaMKII expression (P<0.001), in SKOV3/DDP cells. In conclusion, abnormal activation of Wnt/β-catenin and Wnt/JNK signaling pathways in ovarian cancer cells promotes cisplatin resistance, while the Wnt/Ca2+ signaling pathway reduces cisplatin resistance. This indicates that β-catenin, JNK and CaMKII are potential therapeutic targets in chemoresistant ovarian cancers. PMID:28101169

  4. Upregulation of Connexin 43 Expression Via C-Jun N-Terminal Kinase Signaling in Prion Disease.

    PubMed

    Lee, Geon-Hwi; Jang, Byungki; Choi, Hong-Seok; Kim, Hee-Jun; Park, Jeong-Ho; Jeon, Yong-Chul; Carp, Richard I; Kim, Yong-Sun; Choi, Eun-Kyoung

    2016-01-01

    Prion infection leads to neuronal cell death, glial cell activation, and the accumulation of misfolded prion proteins. However, the altered cellular environments in animals with prion diseases are poorly understood. In the central nervous system, cells connect the cytoplasm of adjacent cells via connexin (Cx)-assembled gap junction channels to allow the direct exchange of small molecules, including ions, neurotransmitters, and signaling molecules, which regulate the activities of the connected cells. Here, we investigate the role of Cx43 in the pathogenesis of prion diseases. Upregulated Cx43 expression, which was dependent on c-Jun N-Terminal Kinase (JNK)/c-Jun signaling cascades, was found in prion-affected brain tissues and hippocampal neuronal cells. Scrapie infection-induced Cx43 formed aggregated plaques within the cytoplasmic compartments at the cell-cell interfaces. The ethidium bromide (EtBr) uptake assay and scrape-loading dye transfer assay demonstrated that increased Cx43 has functional consequences for the activity of Cx43 hemichannels. Interestingly, blockade of PrPSc accumulation reduced Cx43 expression through the inhibition of JNK signaling, indicating that PrPSc accumulation may be directly involved in JNK activation-mediated Cx43 upregulation. Overall, our findings describe a scrapie infection-mediated novel regulatory signaling pathway of Cx43 expression and may suggest a role for Cx43 in the pathogenesis of prion diseases.

  5. TGF-beta induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway.

    PubMed Central

    Hocevar, B A; Brown, T L; Howe, P H

    1999-01-01

    Transforming growth factor-beta (TGF-beta) exerts its effects on cell proliferation, differentiation and migration in part through its modulation of extracellular matrix components, such as fibronectin and plasminogen activator inhibitor-1 (PAI-1). Although the SMAD family of proteins recently has been shown to be a key participant in TGF-beta signaling, other signaling pathways have also been shown to be activated by TGF-beta. We report here that c-Jun N-terminal kinase (JNK), a member of the MAP kinase family, is activated in response to TGF-beta in the human fibrosarcoma HT1080-derived cell line BAHgpt. Stable expression of dominant-negative forms of JNK1 and MKK4, an upstream activator of JNK, results in loss of TGF-beta-stimulated fibronectin mRNA and protein induction, while having little effect on TGF-beta-induced levels of PAI-1. The human fibronectin promoter contains three CRE elements, one of which has been shown to bind a c-Jun-ATF-2 heterodimer. Utilizing a GAL4 fusion trans-reporting system, we demonstrate a decrease in transactivating potential of GAL4-c-Jun and GAL4-ATF-2 in dominant-negative JNK1- and MKK4-expressing cells. Finally, we show that TGF-beta-induced fibronectin synthesis is independent of Smad4. These results demonstrate that TGF-beta-mediated fibronectin induction requires activation of JNK which in turn modulates the activity of c-Jun and ATF-2 in a Smad4independent manner. PMID:10064600

  6. Arecoline-induced pro-fibrotic proteins in LLC-PK1 cells are dependent on c-Jun N-terminal kinase.

    PubMed

    Lin, Sheng-Hsuan; Chiou, Shean-Jaw; Ho, Wan-Ting; Chuang, Chao-Tang; Chuang, Lea-Yea; Guh, Jinn-Yuh

    2016-02-17

    Areca nut (AN) chewing is associated with chronic kidney disease (CKD). However, the molecular mechanisms of AN-induced CKD are not known. Thus, we studied the effects of arecoline, a major alkaloid of AN, on proximal tubule (LLC-PK1) cells in terms of cytotoxicity, fibrosis, transforming growth factor-β (TGF-β) and c-Jun N-terminal kinase (JNK). We found that arecoline dose (0.1-0.5mM) and time (24-72h)-dependently induced cytotoxicity without causing cell death. Arecoline (0.25 mM) also time-dependently (24-72h) increased fibronectin and plasminogen activator inhibitor-1 (PAI1) protein expressions. Arecoline (0.25 mM) time-dependently (24-72h) increased TGF-β gene transcriptional activity and supernatant levels of active TGF-β1. Moreover, arecoline (0.25 mM) activated JNK while SP600125 (a JNK inhibitor) attenuated arecoline-induced TGF-β gene transcriptional activity. SP600125, but not SB431542 (a TGF-β receptor type I kinase inhibitor), attenuated arecoline-induced fibronectin and PAI1 protein expressions. Finally, tubulointerstitial fibrosis occurred and renal cortical expressions of fibronectin and PAI1 proteins increased in arecoline-fed mice at 24 weeks. We concluded that arecoline induced tubulointerstitial fibrosis in mice while arecoline-induced TGF-β and pro-fibrotic proteins (fibronectin, PAI1) are dependent on JNK in LLC-PK1 cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Pharmacological Inhibition of c-Jun N-terminal Kinase Reduces Food Intake and Sensitizes Leptin’s Anorectic Signaling Actions

    PubMed Central

    Gao, Su; Howard, Shannon; LoGrasso, Philip V.

    2017-01-01

    The role for c-Jun N-terminal Kinase (JNK) in the control of feeding and energy balance is not well understood. Here, by use of novel and highly selective JNK inhibitors, we investigated the actions of JNK in the control of feeding and body weight homeostasis. In lean mice, intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of SR-3306, a brain-penetrant and selective pan-JNK (JNK1/2/3) inhibitor, reduced food intake and body weight. Moreover, i.p. and i.c.v. administrations of SR11935, a brain-penetrant and JNK2/3 isoform-selective inhibitor, exerted similar anorectic effects as SR3306, which suggests JNK2 or JNK3 mediates aspect of the anorectic effect by pan-JNK inhibition. Furthermore, daily i.p. injection of SR3306 (7 days) prevented the increases in food intake and weight gain in lean mice upon high-fat diet feeding, and this injection paradigm reduced high-fat intake and obesity in diet-induced obese (DIO) mice. In the DIO mice, JNK inhibition sensitized leptin’s anorectic effect, and enhanced leptin-induced STAT3 activation in the hypothalamus. The underlying mechanisms likely involve the downregulation of SOCS3 by JNK inhibition. Collectively, our data suggest that JNK activity promotes positive energy balance, and the therapeutic intervention inhibiting JNK activities represents a promising approach to ameliorate diet-induced obesity and leptin resistance. PMID:28165482

  8. Pim-2 Kinase Influences Regulatory T Cell Function and Stability by Mediating Foxp3 Protein N-terminal Phosphorylation*

    PubMed Central

    Deng, Guoping; Nagai, Yasuhiro; Xiao, Yan; Li, Zhiyuan; Dai, Shujia; Ohtani, Takuya; Banham, Alison; Li, Bin; Wu, Shiaw-Lin; Hancock, Wayne; Samanta, Arabinda; Zhang, Hongtao; Greene, Mark I.

    2015-01-01

    Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4+CD25+Foxp3+ regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses. PMID:25987564

  9. Pim-2 Kinase Influences Regulatory T Cell Function and Stability by Mediating Foxp3 Protein N-terminal Phosphorylation.

    PubMed

    Deng, Guoping; Nagai, Yasuhiro; Xiao, Yan; Li, Zhiyuan; Dai, Shujia; Ohtani, Takuya; Banham, Alison; Li, Bin; Wu, Shiaw-Lin; Hancock, Wayne; Samanta, Arabinda; Zhang, Hongtao; Greene, Mark I

    2015-08-14

    Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses.

  10. Effects of c-Jun N-terminal kinase on Activin A/Smads signaling in PC12 cell suffered from oxygen-glucose deprivation.

    PubMed

    Wang, J Q; Xu, Z H; Liang, W Z; He, J T; Cui, Y; Liu, H Y; Xue, L X; Shi, W; Shao, Y K; Mang, J; Xu, Z X

    2016-02-29

    Activin A (Act A), a member of transforming growth factor-β (TGF-β) superfamily, is an early gene in response to cerebral ischemia. Growing evidences confirm the neuroprotective effect of Act A in ischemic injury through Act A/Smads signal activation. In this process, regulation networks are involved in modulating the outcomes of Smads signaling. Among these regulators, crosstalk between c-Jun N-terminal kinase (JNK) and Smads signaling has been found in the TGF-β induced epithelial-mesenchymal transition. However, in neural ischemia, the speculative regulation between JNK and Act A/Smads signaling pathways has not been clarified. To explore this issue, an Oxygen Glucose Deprivation (OGD) model was introduced to nerve-like PC12 cells. We found that JNK signal activation occurred at the early time of OGD injury (1 h). Act A administration suppressed JNK phosphorylation. In addition, JNK inhibition could elevate the strength of Smads signaling and attenuate neural apoptosis after OGD injury. Our results indicated a negative regulation effect of JNK on Smads signaling in ischemic injury. Taken together, JNK, as a critical site for neural apoptosis and negative regulator for Act A/Smads signaling, was presumed to be a molecular therapeutic target for ischemia.

  11. Pulsed radiofrequency reduced complete Freund's adjuvant-induced mechanical hyperalgesia via the spinal c-Jun N-terminal kinase pathway.

    PubMed

    Chen, Kuan-Hung; Yang, Chien-Hui; Juang, Sin-Ei; Huang, Hui-Wen; Cheng, Jen-Kun; Sheen-Chen, Shyr-Ming; Cheng, Jiin-Tsuey; Lin, Chung-Ren

    2014-03-01

    Pulsed radiofrequency (PRF) treatment involves the pulsed application of a radiofrequency electric field to a nerve. The technology offers pain relief for patients suffering from chronic pain who do not respond well to conventional treatments. We tested whether PRF treatment attenuated complete Freund's adjuvant (CFA) induced inflammatory pain. The profile of spinal c-Jun N-terminal kinases (JNKs) phosphorylation was evaluated to elucidate the potential mechanism. Injection of CFA into the unilateral hind paw of rats induced mechanical hyperalgesia in both the ipsilateral and contralateral hind paws. We administered 500-kHz PRF treatment in 20-ms pulses, at a rate of 2 Hz (2 pulses per second) either to the sciatic nerve in the mid-thigh, or to the L4 anterior primary ramus just distal to the intervertebral foramen in both the CFA group and no-PRF group rats. Tissue samples were examined at 1, 3, 7, and 14 days following PRF treatments. Behavioral studies showed that PRF applied close to the dorsal root ganglion (DRG) significantly attenuated CFA-induced mechanical hyperalgesia compared to no-PRF group (P < .05). And western blotting revealed significant attenuation of the activation of JNK in the spinal dorsal horn compared to no-PRF group animals (P < .05). Application of PRF close to DRG provides an effective treatment for CFA-induced persistent mechanical hyperalgesia by attenuating JNK activation in the spinal dorsal horn.

  12. Small, N-Terminal Tags Activate Parkin E3 Ubiquitin Ligase Activity by Disrupting Its Autoinhibited Conformation

    PubMed Central

    Burchell, Lynn; Chaugule, Viduth K.; Walden, Helen

    2012-01-01

    Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species. PMID:22496854

  13. Rosiglitazone negatively regulates c-Jun N-terminal kinase and toll-like receptor 4 proinflammatory signalling during initiation of experimental aortic aneurysms.

    PubMed

    Pirianov, Grisha; Torsney, Evelyn; Howe, Franklyn; Cockerill, Gillian W

    2012-11-01

    Development and rupture of aortic aneurysms (AA) is a complex process involving inflammation, cell death, tissue and matrix remodelling. The thiazolidinediones (TZDs) including Rosiglitazone (RGZ) are a family of drugs which act as agonists of the nuclear peroxisome proliferator-activated receptors and have a broad spectrum of effects on a number of biological processes in the cardiovascular system. In our previous study we have demonstrated that RGZ has a marked effect on both aneurysm rupture and development, however, the precise mechanism of this is unknown. In the present study, we examined possible targets of RGZ action in the early stages of Angiotensin II-induced AA in apolipoprotein E-deficient mice. For this purpose we employed immunoblotting, ELISA and antibody array approaches. We found that RGZ significantly inhibited c-Jun N-terminal kinase (JNK) phosphorylation and down-regulated toll-like receptor 4 (TLR4) expression at the site of lesion formation in response to Angiotensin II infusion in the initiation stage (6-72 h) of experimental AA development. Importantly, this effect was also associated with a decrease of CD4 antigen and reduction in production of TLR4/JNK-dependant proinflammatory chemokines MCP-1 and MIP-1α. These data suggest that RGZ can modulate inflammatory processes by blocking TLR4/JNK signalling in initiation stages of AA development. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  14. The N-Terminal Non-Kinase-Domain-Mediated Binding of Haspin to Pds5B Protects Centromeric Cohesion in Mitosis.

    PubMed

    Zhou, Linli; Liang, Cai; Chen, Qinfu; Zhang, Zhenlei; Zhang, Bo; Yan, Haiyan; Qi, Feifei; Zhang, Miao; Yi, Qi; Guan, Youchen; Xiang, Xingfeng; Zhang, Xiaoqing; Ye, Sheng; Wang, Fangwei

    2017-04-03

    Sister-chromatid cohesion, mediated by the multi-subunit cohesin complex, must be precisely regulated to prevent chromosome mis-segregation. In prophase and prometaphase, whereas the bulk of cohesin on chromosome arms is removed by its antagonist Wapl, cohesin at centromeres is retained to ensure chromosome biorientation until anaphase onset. It remains incompletely understood how centromeric cohesin is protected against Wapl in mitosis. Here we show that the mitotic histone kinase Haspin binds to the cohesin regulatory subunit Pds5B through a conserved YGA/R motif in its non-catalytic N terminus, which is similar to the recently reported YSR-motif-dependent binding of Wapl to Pds5B. Knockout of Haspin or disruption of Haspin-Pds5B interaction causes weakened centromeric cohesion and premature chromatid separation, which can be reverted by centromeric targeting of a N-terminal short fragment of Haspin containing the Pds5B-binding motif or by prevention of Wapl-dependent cohesin removal. Conversely, excessive Haspin capable of binding Pds5B displaces Wapl from Pds5B and suppresses Wapl activity, and it largely bypasses the Wapl antagonist Sgo1 for cohesion protection. Taken together, these data indicate that the Haspin-Pds5B interaction is required to ensure proper sister-chromatid cohesion, most likely through antagonizing Wapl-mediated cohesin release from mitotic centromeres.

  15. DISCO interacting protein 2 determines direction of axon projection under the regulation of c-Jun N-terminal kinase in the Drosophila mushroom body.

    PubMed

    Nitta, Yohei; Sugie, Atsushi

    2017-04-08

    Precisely controlled axon guidance for complex neuronal wiring is essential for appropriate neuronal function. c-Jun N-terminal kinase (JNK) was found to play a role in axon guidance recently as well as in cell proliferation, protection and apoptosis. In spite of many genetic and molecular studies on these biological processes regulated by JNK, how JNK regulates axon guidance accurately has not been fully explained thus far. To address this question, we use the Drosophila mushroom body (MB) as a model since the α/β axons project in two distinct directions. Here we show that DISCO interacting protein 2 (DIP2) is required for the accurate direction of axonal guidance. DIP2 expression is under the regulation of Basket (Bsk), the Drosophila homologue of JNK. We additionally found that the Bsk/DIP2 pathway is independent from the AP-1 transcriptional factor complex pathway, which is directly activated by Bsk. In conclusion, our findings revealed DIP2 as a novel effector downstream of Bsk modulating the direction of axon projection.

  16. Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

    PubMed Central

    Hensmann, M.; Booker, G. W.; Panayotou, G.; Boyd, J.; Linacre, J.; Waterfield, M.; Campbell, I. D.

    1994-01-01

    The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study. PMID:7522724

  17. Protocatechuic aldehyde inhibits TNF-α-induced fibronectin expression in human umbilical vein endothelial cells via a c-Jun N-terminal kinase dependent pathway

    PubMed Central

    TONG, YUE-FENG; LIU, YONG; HU, ZHI-XING; LI, ZHE-CHENG; A, AGULA

    2016-01-01

    Fibronectin (FN) is one of the most important extracellular matrix proteins and plays an important role in the pathogenesis of atherosclerosis (AS). The aim of the present study was to evaluate the effect of a potent, water-soluble antioxidant, protocatechuic aldehyde (PA), which is derived from the Chinese herb Salvia miltiorrhiza, on the expression of FN in human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor-α (TNF-α). The pharmacological effects of PA on the production of FN were investigated using ELISA and western blot analysis. In addition, ELISA and western blot analysis were used to examine the activation and suppression of the mitogen-activated protein kinase (MAPK) pathways and nuclear factor (NF)-κB in TNF-α-stimulated HUVECs, in order to explore the underlying pharmacological mechanism of PA. The inhibitory effect of PA on the total generation of reactive oxygen species (ROS) in TNF-α-stimulated HUVECs was assessed using 2′,7′-dichlorofluorescein diacetate. Pretreatment of HUVECs with PA (0.15, 0.45 and 1.35 mM) for 18 h markedly attenuated the TNF-α-stimulated FN surface expression and secretion in a dose-dependent manner. Intracellular ROS generation and the expression of extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) were significantly induced by TNF-α (2 ng/ml) in HUVECs. TNF-α-induced ROS generation and JNK activation were inhibited by PA in a concentration-dependent manner. By contrast, ERK1/2 and p38 activation was not significantly affected by PA. Pretreatment of HUVECs with PA for 18 h markedly attenuated TNF-α-stimulated NF-κB activation. In conclusion, the present findings suggest that PA inhibits TNF-α-induced FN expression in HUVECs through a mechanism that involves ROS/JNK and NF-κB. PMID:26889254

  18. Protocatechuic aldehyde inhibits TNF-α-induced fibronectin expression in human umbilical vein endothelial cells via a c-Jun N-terminal kinase dependent pathway.

    PubMed

    Tong, Yue-Feng; Liu, Yong; Hu, Zhi-Xing; Li, Zhe-Cheng; A, Agula

    2016-01-01

    Fibronectin (FN) is one of the most important extracellular matrix proteins and plays an important role in the pathogenesis of atherosclerosis (AS). The aim of the present study was to evaluate the effect of a potent, water-soluble antioxidant, protocatechuic aldehyde (PA), which is derived from the Chinese herb Salvia miltiorrhiza, on the expression of FN in human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor-α (TNF-α). The pharmacological effects of PA on the production of FN were investigated using ELISA and western blot analysis. In addition, ELISA and western blot analysis were used to examine the activation and suppression of the mitogen-activated protein kinase (MAPK) pathways and nuclear factor (NF)-κB in TNF-α-stimulated HUVECs, in order to explore the underlying pharmacological mechanism of PA. The inhibitory effect of PA on the total generation of reactive oxygen species (ROS) in TNF-α-stimulated HUVECs was assessed using 2',7'-dichlorofluorescein diacetate. Pretreatment of HUVECs with PA (0.15, 0.45 and 1.35 mM) for 18 h markedly attenuated the TNF-α-stimulated FN surface expression and secretion in a dose-dependent manner. Intracellular ROS generation and the expression of extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) were significantly induced by TNF-α (2 ng/ml) in HUVECs. TNF-α-induced ROS generation and JNK activation were inhibited by PA in a concentration-dependent manner. By contrast, ERK1/2 and p38 activation was not significantly affected by PA. Pretreatment of HUVECs with PA for 18 h markedly attenuated TNF-α-stimulated NF-κB activation. In conclusion, the present findings suggest that PA inhibits TNF-α-induced FN expression in HUVECs through a mechanism that involves ROS/JNK and NF-κB.

  19. Modulation of nongenomic activation of PI3K signalling by tetramerization of N-terminally-cleaved RXRα

    PubMed Central

    Chen, Liqun; Aleshin, Alexander E.; Alitongbieke, Gulimiran; Zhou, Yuqi; Zhang, Xindao; Ye, Xiaohong; Hu, Mengjie; Ren, Gaoang; Chen, Ziwen; Ma, Yue; Zhang, Duo; Liu, Shuai; Gao, Weiwei; Cai, Lijun; Wu, Lingjuan; Zeng, Zhiping; Jiang, Fuquan; Liu, Jie; Zhou, Hu; Cadwell, Gregory; Liddington, Robert C.; Su, Ying; Zhang, Xiao-kun

    2017-01-01

    Retinoid X receptor-alpha (RXRα) binds to DNA either as homodimers or heterodimers, but it also forms homotetramers whose function is poorly defined. We previously discovered that an N-terminally-cleaved form of RXRα (tRXRα), produced in tumour cells, activates phosphoinositide 3-kinase (PI3K) signalling by binding to the p85α subunit of PI3K and that K-80003, an anti-cancer agent, inhibits this process. Here, we report through crystallographic and biochemical studies that K-80003 binds to and stabilizes tRXRα tetramers via a ‘three-pronged’ combination of canonical and non-canonical mechanisms. K-80003 binding has no effect on tetramerization of RXRα, owing to the head–tail interaction that is absent in tRXRα. We also identify an LxxLL motif in p85α, which binds to the coactivator-binding groove on tRXRα and dissociates from tRXRα upon tRXRα tetramerization. These results identify conformational selection as the mechanism for inhibiting the nongenomic action of tRXRα and provide molecular insights into the development of RXRα cancer therapeutics. PMID:28714476

  20. Modulation of nongenomic activation of PI3K signalling by tetramerization of N-terminally-cleaved RXRα.

    PubMed

    Chen, Liqun; Aleshin, Alexander E; Alitongbieke, Gulimiran; Zhou, Yuqi; Zhang, Xindao; Ye, Xiaohong; Hu, Mengjie; Ren, Gaoang; Chen, Ziwen; Ma, Yue; Zhang, Duo; Liu, Shuai; Gao, Weiwei; Cai, Lijun; Wu, Lingjuan; Zeng, Zhiping; Jiang, Fuquan; Liu, Jie; Zhou, Hu; Cadwell, Gregory; Liddington, Robert C; Su, Ying; Zhang, Xiao-Kun

    2017-07-17

    Retinoid X receptor-alpha (RXRα) binds to DNA either as homodimers or heterodimers, but it also forms homotetramers whose function is poorly defined. We previously discovered that an N-terminally-cleaved form of RXRα (tRXRα), produced in tumour cells, activates phosphoinositide 3-kinase (PI3K) signalling by binding to the p85α subunit of PI3K and that K-80003, an anti-cancer agent, inhibits this process. Here, we report through crystallographic and biochemical studies that K-80003 binds to and stabilizes tRXRα tetramers via a 'three-pronged' combination of canonical and non-canonical mechanisms. K-80003 binding has no effect on tetramerization of RXRα, owing to the head-tail interaction that is absent in tRXRα. We also identify an LxxLL motif in p85α, which binds to the coactivator-binding groove on tRXRα and dissociates from tRXRα upon tRXRα tetramerization. These results identify conformational selection as the mechanism for inhibiting the nongenomic action of tRXRα and provide molecular insights into the development of RXRα cancer therapeutics.

  1. The c-Jun N-terminal kinase signaling pathway mediates chrysotile asbestos-induced alveolar epithelial cell apoptosis.

    PubMed

    Li, Peng; Liu, Tie; Kamp, David W; Lin, Ziying; Wang, Yahong; Li, Donghong; Yang, Lawei; He, Huijuan; Liu, Gang

    2015-05-01

    Exposure to chrysotile asbestos exposure is associated with an increased risk of mortality in combination with pulmonary diseases including lung cancer, mesothelioma and asbestosis. Multiple mechanisms by which chrysotile asbestos fibers induce pulmonary disease have been identified, however the role of apoptosis in human lung alveolar epithelial cells (AEC) has not yet been fully explored. Accumulating evidence implicates AEC apoptosis as a crucial event in the development of both idiopathic pulmonary fibrosis and asbestosis. The aim of the present study was to determine whether chrysotile asbestos induces mitochondria‑regulated (intrinsic) AEC apoptosis and, if so, whether this induction occurs via the activation of mitogen‑activated protein kinases (MAPK). Human A549 bronchoalveolar carcinoma‑derived cells with alveolar epithelial type II‑like features were used. The present study showed that chrysotile asbestos induced a dose‑ and time‑dependent decrease in A549 cell viability, which was accompanied by the activation of the MAPK c‑Jun N‑terminal kinases (JNK), but not the MAPKs extracellular signal‑regulated kinase 1/2 and p38. Chrysotile asbestos was also shown to induce intrinsic AEC apoptosis, as evidenced by the upregulation of the pro‑apoptotic genes Bax and Bak, alongside the activation of caspase‑9, poly (ADP‑ribose) polymerase (PARP), and the release of cytochrome c. Furthermore, the specific JNK inhibitor SP600125 blocked chrysotile asbestos‑induced JNK activation and subsequent apoptosis, as assessed by both caspase‑9 cleavage and PARP activation. The results of the present study demonstrated that chrysotile asbestos induces intrinsic AEC apoptosis by a JNK‑dependent mechanism, and suggests a potential novel target for the modulation of chrysotile asbestos‑associated lung diseases.

  2. Trehalose induces functionally active conformation in the intrinsically disordered N-terminal domain of glucocorticoid receptor.

    PubMed

    Khan, Shagufta H; Jasuja, Ravi; Kumar, Raj

    2016-08-05

    Glucocorticoid receptor (GR) is a classic member of the nuclear receptor superfamily and plays pivotal roles in human physiology at the level of gene regulation. Various constellations of cellular cofactors are required to associate with GR to activate/repress genes. The effects of specific ligands on the AF2 structure and consequent preferential binding of co-activators or co-repressors have helped our understanding of the mechanisms involved. But the data so far fall short of fully explaining GR actions. We believe that this is because work so far has largely avoided detailed examination of the contributions of AF1 to overall GR actions. It has been shown that the GR containing only the N-terminal domain (NTD) and the DNA-binding domain (GR500) is constitutively quite active in stimulating transcription from simple promoters. However, we are only beginning to understand structure and functions of GR500 in spite of the fact that AF1 located within the NTD serves as major transactivation domain for GR. Lack of this information has hampered our complete understanding of how GR regulates its target gene(s). The major obstacle in determining GR500 structure has been due to its intrinsically disordered NTD conformation, frequently found in transcription factors. In this study, we tested whether a naturally occurring osmolyte, trehalose, can promote functionally ordered conformation in GR500. Our data show that in the presence of trehalose, GR500 is capable of formation of a native-like functionally folded conformation.

  3. Antcin H Protects Against Acute Liver Injury Through Disruption of the Interaction of c-Jun-N-Terminal Kinase with Mitochondria.

    PubMed

    Huo, Yazhen; Win, Sanda; Than, Tin Aung; Yin, Shutao; Ye, Min; Hu, Hongbo; Kaplowitz, Neil

    2017-02-10

    Antrodia Camphorate (AC) is a mushroom that is widely used in Asian countries to prevent and treat various diseases, including liver diseases. However, the active ingredients that contribute to the biological functions remain elusive. The purpose of the present study is to test the hepatoprotective effect of Antcin H, a major triterpenoid chemical isolated from AC, in murine models of acute liver injury. We found that Antcin H pretreatment protected against liver injury in both acetaminophen (APAP) and galactosamine/tumor necrosis factor (TNF)α models. More importantly, Antcin H also offered a significant protection against acetaminophen-induced liver injury when it was given 1 h after acetaminophen. The protection was verified in primary mouse hepatocytes. Antcin H prevented sustained c-Jun-N-terminal kinase (JNK) activation in both models. We excluded an effect of Antcin H on acetaminophen metabolism and TNF receptor signaling and excluded a direct effect as a free radical scavenger or JNK inhibitor. Since the sustained JNK activation through its interaction with mitochondrial Sab, leading to increased mitochondrial reactive oxygen species (ROS), is pivotal in both models, we examined the effect of Antcin H on p-JNK binding to mitochondria and impairment of mitochondrial respiration. Antcin H inhibited the direct effect of p-JNK on isolated mitochondrial function and binding to isolated mitochondria. Innovation and Conclusion: Our study has identified Antcin H as a novel active ingredient that contributes to the hepatoprotective effect of AC, and Antcin H protects against liver injury through disruption of the binding of p-JNK to Sab, which interferes with the ROS-dependent self-sustaining activation of MAPK cascade. Antioxid. Redox Signal. 26, 207-220.

  4. Proteolytic cleavage of ostrich and turkey pancreatic lipases: production of an active N-terminal domain.

    PubMed

    Ben Bacha, Abir; Fendri, Ahmed; Gargouri, Youssef; Mejdoub, Hafedh; Miled, Nabil

    2007-10-01

    The aim of this study was to check some biochemical and structural properties of ostrich and turkey pancreatic lipases (OPL and TPL, respectively). Limited proteolysis of OPL and TPL was performed in conditions similar to those reported for porcine pancreatic lipase. In the absence of bile salts and colipase, OPL failed to catalyze the hydrolysis of pure tributyrin or efficiently hydrolyze olive oil emulsion. When bile salts and colipase were preincubated with the substrate, the OPL kinetic behavior remained linear for more than 30 minutes. The enzyme presented a penetration power value into an egg phosphatidylcholine monomolecular film that was comparable to that of HPL and lower than that of TPL. Chymotrypsin, trypsin, and thermolysin were able to hydrolyze OPL and TPL in different ways. In both cases, only N-terminal fragments accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic cleavage of OPL and TPL completely degraded the enzymes. Nevertheless, chymotryptic attack generated 35-kd and 43-kd forms for TPL and OPL, respectively. Interestingly, the OPL 43-kd form was inactive, whereas the TPL 35-kd protein conserved its lipolytic activity. OPL, TPL, and mammal pancreatic lipases share a high amino acid sequence homology. Further investigations are, however, needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of lipases.

  5. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    SciTech Connect

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  6. c-Jun N-terminal kinase modulates oxidant stress and peroxynitrite formation independent of inducible nitric oxide synthase in acetaminophen hepatotoxicity

    SciTech Connect

    Saito, Chieko; Lemasters, John J.; Jaeschke, Hartmut

    2010-07-15

    Acetaminophen (APAP) overdose, which causes liver injury in animals and humans, activates c-jun N-terminal kinase (JNK). Although it was shown that the JNK inhibitor SP600125 effectively reduced APAP hepatotoxicity, the mechanisms of protection remain unclear. C57Bl/6 mice were treated with 10 mg/kg SP600125 or vehicle (8% dimethylsulfoxide) 1 h before 600 mg/kg APAP administration. APAP time-dependently induced JNK activation (detected by JNK phosphorylation). SP600125, but not the vehicle, reduced JNK activation, attenuated mitochondrial Bax translocation and prevented the mitochondrial release of apoptosis-inducing factor at 4-12 h. Nuclear DNA fragmentation, nitrotyrosine staining, tissue GSSG levels and liver injury (plasma ALT release and necrosis) were partially attenuated by the vehicle (- 65%) and completely eliminated by SP600125 (- 98%) at 6 and 12 h. Furthermore, SP600125 attenuated the increase of inducible nitric oxide synthase (iNOS) mRNA and protein. However, APAP did not enhance plasma nitrite + nitrate levels (NO formation); SP600125 had no effect on this parameter. The iNOS inhibitor L-NIL did not reduce NO formation or injury after APAP but prevented NO formation caused by endotoxin. Since SP600125 completely eliminated the increase in hepatic GSSG levels, an indicator of mitochondrial oxidant stress, it is concluded that the inhibition of peroxynitrite was mainly caused by reduced superoxide formation. Our data suggest that the JNK inhibitor SP600125 protects against APAP-induced liver injury in part by attenuation of mitochondrial Bax translocation but mainly by preventing mitochondrial oxidant stress and peroxynitrite formation and thereby preventing the mitochondrial permeability transition pore opening, a key event in APAP-induced cell necrosis.

  7. Momordica charantia polysaccharides could protect against cerebral ischemia/reperfusion injury through inhibiting oxidative stress mediated c-Jun N-terminal kinase 3 signaling pathway.

    PubMed

    Gong, Juanjuan; Sun, Fumou; Li, Yihang; Zhou, Xiaoling; Duan, Zhenzhen; Duan, Fugang; Zhao, Lei; Chen, Hansen; Qi, Suhua; Shen, Jiangang

    2015-04-01

    Momordica charantia (MC) is a medicinal plant for stroke treatment in Traditional Chinese Medicine, but its active compounds and molecular targets are unknown yet. M. charantia polysaccharide (MCP) is one of the important bioactive components in MC. In the present study, we tested the hypothesis that MCP has neuroprotective effects against cerebral ischemia/reperfusion injury through scavenging superoxide (O2(-)), nitric oxide (NO) and peroxynitrite (ONOO(-)) and inhibiting c-Jun N-terminal protein kinase (JNK3) signaling cascades. We conducted experiments with in vivo global and focal cerebral ischemia/reperfusion rat models and in vitro oxygen glucose deprivation (OGD) neural cells. The effects of MCP on apoptotic cell death and infarction volume, the bioactivities of scavenging O2(-), NO and ONOO(-), inhibiting lipid peroxidation and modulating JNK3 signaling pathway were investigated. Major results are summarized as below: (1) MCP dose-dependently attenuated apoptotic cell death in neural cells under OGD condition in vitro and reduced infarction volume in ischemic brains in vivo; (2) MCP had directing scavenging effects on NO, O2(-) and ONOO(-) and inhibited lipid peroxidation; (3) MCP inhibited the activations of JNK3/c-Jun/Fas-L and JNK3/cytochrome C/caspases-3 signaling cascades in ischemic brains in vivo. Taken together, we conclude that MCP could be a promising neuroprotective ingredient of M. charantia and its mechanisms could be at least in part attributed to its antioxidant activities and inhibiting JNK3 signaling cascades during cerebral ischemia/reperfusion injury. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Inhibition of c-Jun N-terminal kinase signaling suppresses skin flap apoptosis in a rat ischemia and/or reperfusion model.

    PubMed

    Bai, Ming; Liu, Yifang; Yin, Dechang; Zhang, Mingzi; Wang, Youbin; Ma, Xuemei; Liu, Yunqi; Zhao, Pengxiang

    2016-12-01

    The goals of this study were to validate the role of c-Jun N-terminal kinase (JNK) activation in skin flap apoptosis in a rat model of abdomen skin ischemia and/or reperfusion (IR) and to compare the protective effect of SP600125 and hydrogen-rich saline in skin IR injury. Male Sprague-Dawley rats were divided into five groups: one sham surgery group and four surgery groups. Before undergoing 3 h of IR management, the surgery groups were treated with normal saline (IR), dimethyl sulfoxide, SP600125 (SP), and hydrogen-rich saline (H). On the third postoperative day, blood perfusion of the flap was measured using Laser Doppler flowmeters. Hematoxylin and eosin staining was used to observe morphologic changes. Early apoptosis was observed using TdT-mediated dUTP-X nick end-labeling staining. pASK-1, pJNK, Bcl-2, and Bax were examined by immunodetection. Caspase-3 activity was also measured 24 h after reperfusion. Compared to the IR group and the dimethyl sulfoxide group, the SP group and the H group had larger skin flap survival area, more blood perfusion and lower levels of caspase-3 activity. The SP and the H groups had high expression levels of Bcl-2 and low expression levels of pASK-1 and pJNK. Bax was significantly decreased in the SP group. In addition, cell apoptosis was decreased in both the sham surgery and the H groups. IR-induced JNK phosphorylation was reduced by SP600125, indicating that JNK mediates the apoptosis pathways in rat skin. In the SP and the H groups, the apoptotic factors measured showed similar expression levels, indicating that JNK inhibition during IR may be associated with H-mediated protection against skin IR apoptosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. c-Jun N-terminal kinase-mediated anti-inflammatory effects of Garcinia subelliptica in macrophages.

    PubMed

    Cho, Young-Chang; Cho, Sayeon

    2016-03-01

    Garcinia plants have been traditionally used to treat inflammatory diseases, such as skin infections and pain, in many regions including South‑East Asia. Garcinia subelliptica, a plant of the Garcinia species widely distributed from Japan to Thailand, has been reported to contain components similar to other Garcinia plants that exhibit anti‑inflammatory effects. The present study aimed to explore the anti‑inflammatory effects of ethanol extracts of Garcinia subelliptica (EGS) in macrophages, as there are no previous systemic studies that have investigated the effects of Garcinia subelliptica on inflammation. Non‑cytotoxic concentrations of EGS (≤200 µg/ml) were observed to reduce nitric oxide production by modulating iNOS expression in lipopolysaccharide (LPS)‑stimulated RAW 264.7 macrophages. The expression of cyclooxygenase‑2, the enzyme responsible for the production of prostaglandin E2, was notably reduced by EGS. EGS treatment inhibited the production of pro‑inflammatory cytokines, including IL‑6 and IL‑1β, however, not TNF‑α. Reduced production of inflammatory mediators by EGS was followed by reduced phosphorylation of c‑Jun N‑terminal kinase (JNK) however, not of other mitogen‑activated protein kinases and nuclear factor‑κB. These results indicate that EGS selectively inhibits the excessive production of inflammatory mediators in LPS‑stimulated murine macrophages by reducing the activation of JNK, suggesting that EGS is a candidate for modulating severe inflammation.

  10. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    PubMed

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS. © 2014 S. Karger AG, Basel.

  11. The Roles of the RIIβ Linker and N-terminal Cyclic Nucleotide-binding Domain in Determining the Unique Structures of the Type IIβ Protein Kinase A

    PubMed Central

    Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; Zhang, Ping; Heller, William T.; Taylor, Susan S.

    2014-01-01

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. PMID:25112875

  12. Effect of Phenylephrine Pretreatment on the Expressions of Aquaporin 5 and c-Jun N-Terminal Kinase in Irradiated Submandibular Gland.

    PubMed

    Han, Lichi; Wang, Lei; Zhang, Fuyin; Liu, Ke Jian; Xiang, Bin

    2015-06-01

    Radiotherapy for malignant tumors of the head and neck commonly leads to radiation-induced sialadenitis as a result of radiation-induced salivary gland dysfunction. We demonstrated previously that phenylephrine could protect the irradiated submandibular gland against apoptosis, although the mechanism is unclear. In this study, we investigated the influence of phenylephrine pretreatment on the expressions of aquaporin 5 (AQP5) and c-Jun N-terminal kinase (JNK) that were presumed to have a role in radiation-induced salivary gland dysfunction. Rats pretreated with phenylephrine (5 mg/kg) were locally irradiated (20 Gy) in the head and neck region. The submandibular glands were removed on day 7 after irradiation. The expression of AQP5 and activation of JNK were measured by immunohistochemistry and Western blot. The localization of AQP5 at the apical and lateral plasma membrane of acinar cells was significantly reduced by irradiation, but markedly enhanced with phenylephrine pretreatment. The protein expression of AQP5 was decreased by 84.91% in irradiated glands, whereas it was fully recovered to the control level in phenylephrine-pretreated glands. Moreover, many acinar, ductal and granular convoluted tubular cells in the irradiated glands exhibited intense immunoreactivity for p-JNK, while in the phenylephrine-pretreated irradiated glands, only a few acinar cells exhibited very faint immunoreactivity for p-JNK. The protein expression level of p-JNK was increased by 41.65% in the irradiated alone glands, but was significantly decreased in the phenylephrine-pretreated irradiated glands. These results suggest that the protective mechanism of phenylephrine might be related to the improved expression of AQP5 and decreased activation of JNK. Pretreatment with phenylephrine in patients undergoing radiotherapy may provide a helpful strategy for suppression of radiation-induced sialadenitis.

  13. AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy.

    PubMed

    Lin, Ziying; Liu, Tie; Kamp, David W; Wang, Yahong; He, Huijuan; Zhou, Xu; Li, Donghong; Yang, Lawei; Zhao, Bin; Liu, Gang

    2014-07-01

    Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2(-/-) MEFs but not JNK1(-/-) MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting

  14. A non-catalytic N-terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα.

    PubMed

    Trosiuk, Tetiana V; Shalak, Vyacheslav F; Szczepanowski, Roman H; Negrutskii, Boris S; El'skaya, Anna V

    2016-02-01

    Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect.

  15. Phosphorylation of rat brain purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal kinase-3 modifies open-channel noise.

    PubMed

    Gupta, Rajeev

    2017-09-02

    The drift kinetic energy of ionic flow through single ion channels cause vibrations of the pore walls which are observed as open-state current fluctuations (open-channel noise) during single-channel recordings. Vibration of the pore wall leads to transitions among different conformational sub-states of the channel protein in the open-state. Open-channel noise analysis can provide important information about the different conformational sub-state transitions and how biochemical modifications of ion channels would affect their transport properties. It has been shown that c-Jun N-terminal kinase-3 (JNK3) becomes activated by phosphorylation in various neurodegenerative diseases and phosphorylates outer mitochondrion associated proteins leading to neuronal apoptosis. In our earlier work, JNK3 has been reported to phosphorylate purified rat brain mitochondrial voltage-dependent anion channel (VDAC) in vitro and modify its conductance and opening probability. In this article we have compared the open-state noise profile of the native and the JNK3 phosphorylated VDAC using Power Spectral Density vs frequency plots. Power spectral density analysis of open-state noise indicated power law with average slope value α ≈1 for native VDAC at both positive and negative voltage whereas average α value < 0.5 for JNK3 phosphorylated VDAC at both positive and negative voltage. It is proposed that 1/f(1) power law in native VDAC open-state noise arises due to coupling of ionic transport and conformational sub-states transitions in open-state and this coupling is perturbed as a result of channel phosphorylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Conformational changes in inositol 1,3,4,5,6-pentakisphosphate 2-kinase upon substrate binding: role of N-terminal lobe and enantiomeric substrate preference.

    PubMed

    Baños-Sanz, José Ignacio; Sanz-Aparicio, Julia; Whitfield, Hayley; Hamilton, Chris; Brearley, Charles A; González, Beatriz

    2012-08-24

    Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP(5) 2-K) catalyzes the synthesis of inositol 1,2,3,4,5,6-hexakisphosphate from ATP and IP(5). Inositol 1,2,3,4,5,6-hexakisphosphate is implicated in crucial processes such as mRNA export, DNA editing, and phosphorus storage in plants. We previously solved the first structure of an IP(5) 2-K, which shed light on aspects of substrate recognition. However, failure of IP(5) 2-K to crystallize in the absence of inositide prompted us to study putative conformational changes upon substrate binding. We have made mutations to residues on a region of the protein that produces a clasp over the active site. A W129A mutant allowed us to capture IP(5) 2-K in its different conformations by crystallography. Thus, the IP(5) 2-K apo-form structure displays an open conformation, whereas the nucleotide-bound form shows a half-closed conformation, in contrast to the inositide-bound form obtained previously in a closed conformation. Both nucleotide and inositide binding produce large conformational changes that can be understood as two rigid domain movements, although local changes were also observed. Changes in intrinsic fluorescence upon nucleotide and inositide binding are in agreement with the crystallographic findings. Our work suggests that the clasp might be involved in enzyme kinetics, with the N-terminal lobe being essential for inositide binding and subsequent conformational changes. We also show how IP(5) 2-K discriminates between inositol 1,3,4,5-tetrakisphosphate and 3,4,5,6-tetrakisphosphate enantiomers and that substrate preference can be manipulated by Arg(130) mutation. Altogether, these results provide a framework for rational design of specific inhibitors with potential applications as biological tools for in vivo studies, which could assist in the identification of novel roles for IP(5) 2-K in mammals.

  17. MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus

    PubMed Central

    Vacca, Barbara; Sanchez-Heras, Elena; Steed, Emily; Balda, Maria S.; Ohnuma, Shin-Ichi; Sasai, Noriaki; Mayor, Roberto

    2016-01-01

    ABSTRACT Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus. MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis. PMID:27870636

  18. Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity.

    PubMed

    Minagawa, Shun; Kondoh, Yasumitsu; Sueoka, Keigo; Osada, Hiroyuki; Nakamoto, Hitoshi

    2011-04-01

    Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.

  19. Arsenic trioxide inhibits Ewing's sarcoma cell invasiveness by targeting p38(MAPK) and c-Jun N-terminal kinase.

    PubMed

    Zhang, Shuai; Guo, Wei; Ren, Ting-Ting; Lu, Xin-Chang; Tang, Guo-Qing; Zhao, Fu-Long

    2012-01-01

    Ewing's sarcoma is the second most frequent primary malignant bone tumor, mainly affecting children and young adults. The notorious metastatic capability of this tumor aggravates patient mortality and remains a problem to be overcome. We investigated the effect of arsenic trioxide (As₂O₃) on the metastasis capability of Ewing's sarcoma cells. We performed 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide assays to choose appropriate concentrations of As₂O₃ for the experiments. Migration, invasion, and adhesion assays were performed to assess the effect of As₂O₃ on the metastasis of Ewing's sarcoma. Immunofluorescent staining was used to observe cytoskeleton reorganization in Ewing's sarcoma cells treated with As₂O₃. Changes in matrix metalloproteinase-9 expression and the mitogen-activated protein kinase (MAPK) pathway were investigated using western blot. Inhibitors of p38(MAPK) (sb202190) and c-Jun NH₂-terminal kinase (JNK, sp600125) were used in invasion assays to determine the effect of p38(MAPK) and JNK. We found that As₂O₃ may markedly inhibit the migration and invasion capacity of Ewing's sarcoma cells with structural rearrangements of the actin cytoskeleton. The expressions of matrix metalloproteinase-9, phosphor-p38(MAPK), and phosphor-JNK were suppressed by As₂O₃ treatment in a dose-dependent manner. The inhibitors of p38(MAPK) (sb202190) and JNK (sp600125) enhanced the inhibition induced by As₂O₃, which was counteracted by anisomycin, an activating agent of p38(MAPK) and JNK. Taken together, our results demonstrate that As₂O₃ can inhibit the metastasis capability of RD-ES and A-673 cells and may have new therapeutic value for Ewing's sarcoma.

  20. Classic phenotype of Coffin-Lowry syndrome in a female with stimulus-induced drop episodes and a genotype with preserved N-terminal kinase domain.

    PubMed

    Rojnueangnit, Kitiwan; Jones, Julie R; Basehore, Monica J; Robin, Nathaniel H

    2014-02-01

    An adolescent female presented with intellectual disability, stimulus-induced drop episodes (SIDEs), facial characteristics that include wide set eyes, short nose with wide columella, full and everted lips with wide mouth and progressive skeletal changes: scoliosis, spondylolisthesis and pectus excavatum. These findings were suggestive of Coffin-Lowry syndrome (CLS), and this was confirmed by the identification of a novel mutation in RPS6KA3, a heterozygous one basepair duplication at nucleotide 1570 (c.1570dupA). This mutation occurs within the C-terminal kinase domain of the protein, and, therefore contradicts the previous report that SIDEs is only associated with premature truncation of the protein in the N-terminal kinase domain or upstream of this domain. As CLS is X-linked, it is unusual for a female to have such a classic phenotype.

  1. Reactive Oxygen Species Control Senescence-Associated Matrix Metalloproteinase-1 through c-Jun-N-Terminal Kinase

    PubMed Central

    Dasgupta, Jaya; Kar, Supriya; Liu, Rong; Joseph, Joy; Kalayanaram, Balaraman; Remington, S. James; Chen, Cheshi; Melendez, J. Andres

    2010-01-01

    The lifetime exposure of organisms to oxidative stress influences many aging processes which involve the turnover of the extracellular matrix. In this study, we identify the redox-responsive molecular signals that drive senescence-associated (SA) matrix metalloproteinase-1 (MMP-1) expression. Precise biochemical monitoring revealed that senescent fibroblasts increase steady-state [H2O2] 3.5 fold (13.7→48.6 pM) relative to young cells. Restricting H2O2 production through low O2 exposure or by antioxidant treatments prevented SA increases in MMP-1 expression. The H2O2-dependent control of SA MMP-1 is attributed to sustained JNK activation and c-jun recruitment to the MMP-1 promoter. SA JNK activation corresponds to increases and decreases in the levels of its activating kinase (MKK-4) and inhibitory phosphatase (MKP-1), respectively. Enforced MKP-1 expression negates SA increases in JNK phosphorylation and MMP-1 production. Overall, these studies define redox-sensitive signaling networks regulating SA MMP-1 expression and link the free radical theory of aging to initiation of aberrant matrix turnover. PMID:20648623

  2. Lower susceptibility of female mice to acetaminophen hepatotoxicity: Role of mitochondrial glutathione, oxidant stress and c-jun N-terminal kinase

    SciTech Connect

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Jaeschke, Hartmut

    2014-11-15

    Acetaminophen (APAP) overdose causes severe hepatotoxicity in animals and humans. However, the mechanisms underlying the gender differences in susceptibility to APAP overdose in mice have not been clarified. In our study, APAP (300 mg/kg) caused severe liver injury in male mice but 69–77% lower injury in females. No gender difference in metabolic activation of APAP was found. Hepatic glutathione (GSH) was rapidly depleted in both genders, while GSH recovery in female mice was 2.6 fold higher in the mitochondria at 4 h, and 2.5 and 3.3 fold higher in the total liver at 4 h and 6 h, respectively. This faster recovery of GSH, which correlated with greater induction of glutamate-cysteine ligase, attenuated mitochondrial oxidative stress in female mice, as suggested by a lower GSSG/GSH ratio at 6 h (3.8% in males vs. 1.4% in females) and minimal centrilobular nitrotyrosine staining. While c-jun N-terminal kinase (JNK) activation was similar at 2 and 4 h post-APAP, it was 3.1 fold lower at 6 h in female mice. However, female mice were still protected by the JNK inhibitor SP600125. 17β-Estradiol pretreatment moderately decreased liver injury and oxidative stress in male mice without affecting GSH recovery. Conclusion: The lower susceptibility of female mice is achieved by the improved detoxification of reactive oxygen due to accelerated recovery of mitochondrial GSH levels, which attenuates late JNK activation and liver injury. However, even the reduced injury in female mice was still dependent on JNK. While 17β-estradiol partially protects male mice, it does not affect hepatic GSH recovery. - Highlights: • Female mice are less susceptible to acetaminophen overdose than males. • GSH depletion and protein adduct formation are similar in both genders. • Recovery of hepatic GSH levels is faster in females and correlates with Gclc. • Reduced oxidant stress in females leads to reduced JNK activation. • JNK activation and mitochondrial translocation are critical

  3. Regulation of Argonaute Slicer Activity by Guide RNA 3′ End Interactions with the N-terminal Lobe*

    PubMed Central

    Hur, Junho K.; Zinchenko, Michelle K.; Djuranovic, Sergej; Green, Rachel

    2013-01-01

    Structural studies indicate that binding of both the guide RNA (siRNA and miRNA) and the target mRNA trigger substantial conformational changes in the Argonaute proteins. Here we explore the role of the N-terminal lobe (and its PAZ domain) in these conformational changes using biochemical and cell culture-based approaches. In vitro, whereas deletion (or mutation) of the N-terminal lobe of DmAgo1 and DmAgo2 had no effect on binding affinity to guide RNAs, we observed a loss of protection of the 3′ end of the guide RNA and decreased target RNA binding; consistent with this, in cells, loss of function DmAgo1 PAZ variant proteins (PAZ6 and ΔN-PAZ) still bind RNA, although the RNAs are shorter than normal. We also find that deletion of the N-terminal lobe results in constitutive activation of endogenous PIWI domain-based cleavage activity in vitro, providing insights into how cleavage activity may be regulated in vivo in response to different types of pairing interactions with the target mRNAs. PMID:23329841

  4. The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

    PubMed Central

    Richardson, Brian C; Halaby, Steve L; Gustafson, Margaret A; Fromme, J Christopher

    2016-01-01

    The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth. DOI: http://dx.doi.org/10.7554/eLife.12411.001 PMID:26765562

  5. AKT/mTOR and C-Jun N-terminal Kinase (JNK) Signaling Pathways Are Required for Chrysotile Asbestos-Induced Autophagy

    PubMed Central

    Lin, Ziying; Liu, Tie; Kamp, David W; Wang, Yahong; He, Huijuan; Zhou, Xu; Li, Donghong; Yang, Lawei; Zhao, Bin; Liu, Gang

    2014-01-01

    Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In the present study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells, and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos-induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased A549 cell microtubule-associated protein 2 light chains 3 (LC3-II) expression, an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-P70s6k. Notably, AKT1/AKT2 double knockout (AKT DKO) murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2−/− MEFs but not JNK1−/− MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, NAC, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of p-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitors 3-methyladenine (3-MA) or ATG5 (autophagy-related gene 5) siRNA, indicating that chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical

  6. Hyperoside Downregulates the Receptor for Advanced Glycation End Products (RAGE) and Promotes Proliferation in ECV304 Cells via the c-Jun N-Terminal Kinases (JNK) Pathway Following Stimulation by Advanced Glycation End-Products In Vitro

    PubMed Central

    Zhang, Zhengyu; Sethiel, Mosha Silas; Shen, Weizhi; Liao, Sentai; Zou, Yuxiao

    2013-01-01

    Hyperoside is a major active constituent in many medicinal plants which are traditionally used in Chinese medicines for their neuroprotective, anti-inflammatory and antioxidative effects. The molecular mechanisms underlying these effects are unknown. In this study, quiescent ECV304 cells were treated in vitro with advanced glycation end products (AGEs) in the presence or absence of hyperoside. The results demonstrated that AGEs induced c-Jun N-terminal kinases (JNK) activation and apoptosis in ECV304 cells. Hyperoside inhibited these effects and promoted ECV304 cell proliferation. Furthermore, hyperoside significantly inhibited RAGE expression in AGE-stimulated ECV304 cells, whereas knockdown of RAGE inhibited AGE-induced JNK activation. These results suggested that AGEs may promote JNK activation, leading to viability inhibition of ECV304 cells via the RAGE signaling pathway. These effects could be inhibited by hyperoside. Our findings suggest a novel role for hyperoside in the treatment and prevention of diabetes. PMID:24252909

  7. Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis.

    PubMed

    Ayabe, T; Park, S K; Takenaka, H; Sumida, M; Uesugi, S; Takenaka, O; Hamada, M

    1996-11-01

    To elucidate lysine residues in the N-terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site-directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady-state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2-. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2-. In human AK, the epsilon-amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2- and AMP2- during transition state.

  8. c-Jun N-terminal Kinase (JNK) induces phosphorylation of amyloid precursor protein (APP) at Thr668, in okadaic acid-induced neurodegeneration

    PubMed Central

    Ahn, Ji-Hwan; So, Sang-Pil; Kim, Na-Young; Kim, Hyun-Ju; Yoon, Seung-Yong; Kim, Dong-Hou

    2016-01-01

    Several lines of evidence have revealed that phosphorylation of amyloid precursor protein (APP) at Thr668 is involved in the pathogenesis of Alzheimer’s disease (AD). Okadaic acid (OA), a protein phosphatase-2A inhibitor, has been used in AD research models to increase tau phosphorylation and induce neuronal death. We previously showed that OA increased levels of APP and induced accumulation of APP in axonal swellings. In this study, we found that in OA-treated neurons, phosphorylation of APP at Thr668 increased and accumulated in axonal swellings by c-jun N-terminal kinase (JNK), and not by Cdk5 or ERK/MAPK. These results suggest that JNK may be one of therapeutic targets for the treatment of AD. [BMB Reports 2016; 49(7): 376-381] PMID:26839154

  9. Impact of an N-terminal extension on the stability and activity of the GH11 xylanase from Thermobacillus xylanilyticus.

    PubMed

    Song, Letian; Dumon, Claire; Siguier, Béatrice; André, Isabelle; Eneyskaya, Elena; Kulminskaya, Anna; Bozonnet, Sophie; O'Donohue, Michael Joseph

    2014-03-20

    To understand structure-function relationships in the N-terminal region of GH11 xylanases, the 17 N-terminal amino acids of the GH11 xylanase from Neocallimastix patriciarum (Np-Xyn) have been grafted onto the N-terminal extremity of the untypically short GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn), creating a hybrid enzyme denoted NTfus. The hybrid xylanase displayed properties (pH and temperature optima) similar to those of the parental enzyme, although thermostability was lowered, with the Tm value, being reduced by 5°C. Kinetic assays using oNP-Xylo-oligosaccharides (DP2 and 3) indicated that the N-extension did not procure more extensive substrate binding, even when further mutagenesis was performed to promote this. However, these experiments confirmed weak subsite -3 for both NTfus and the parental enzyme. The catalytic efficiency of NTfus was shown to be 17% higher than that of the parental enzyme on low viscosity wheat arabinoxylan and trials using milled wheat straw as the substrate revealed that NTfus released more substituted oligosaccharide products (Xyl/Ara=8.97±0.13 compared to Xyl/Ara=9.70±0.21 for the parental enzyme), suggesting that the hybrid enzyme possesses wider substrate selectivity. Combining either the parental enzyme or NTfus with the cellulolytic cocktail Accellerase 1500 boosted the impact of the latter on wheat straw, procuring yields of solubilized xylose and glucose of 23 and 24% of theoretical yield, respectively, thus underlining the benefits of added xylanase activity when using this cellulase cocktail. Overall, in view of the results obtained for NTfus, we propose that the N-terminal extension leads to the modification of a putative secondary substrate binding site, a hypothesis that is highly consistent with previous data. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Molecular cloning and biologically active production of IpaD N-terminal region.

    PubMed

    Hesaraki, Mahdi; Saadati, Mojtaba; Honari, Hossein; Olad, Gholamreza; Heiat, Mohammad; Malaei, Fatemeh; Ranjbar, Reza

    2013-07-01

    Shigella is known as pathogenic intestinal bacteria in high dispersion and pathogenic bacteria due to invasive plasmid antigen (Ipa). So far, a number of Ipa proteins have been studied to introduce a new candidate vaccine. Here, for the first time, we examined whether the N-terminal region of IpaD(72-162) could be a proper candidate for Shigella vaccine. Initially, the DNA sequence coding N-terminal region was isolated by PCR from Shigella dysenteriae type I and cloned into pET-28a expression vector. Then, the heterologous protein was expressed, optimized and purified by affinity Ni-NTA column. Western blot analysis using, His-tag and IpaD(72-162) polyclonal antibodies, confirmed the purity and specificity of the recombinant protein, respectively. Subsequently, the high immunogenicity of the antigen was shown by ELISA. The results of the sereny test in Guinea pigs showed that IpaD(72-162) provides a protective system against Shigella flexneri 5a and S. dysenteriae type I. Copyright © 2013. Published by Elsevier Ltd.

  11. Acute inhibition of central c-Jun N-terminal kinase restores hypothalamic insulin signalling and alleviates glucose intolerance in diabetic mice.

    PubMed

    Benzler, J; Ganjam, G K; Legler, K; Stöhr, S; Krüger, M; Steger, J; Tups, A

    2013-05-01

    The hypothalamus has been identified as a main insulin target tissue for regulating normal body weight and glucose metabolism. Recent observations suggest that c-Jun-N-terminal kinase (JNK)-signalling plays a crucial role in the development of obesity and insulin resistance because neuronal JNK-1 ablation in the mouse prevented high-fat diet-induced obesity (DIO) and increased energy expenditure, as well as insulin sensitivity. In the present study, we investigated whether central JNK inhibition is associated with sensitisation of hypothalamic insulin signalling in mice fed a high-fat diet for 3 weeks and in leptin-deficient mice. We determined whether i.c.v. injection of a pharmacological JNK-inhibitor (SP600125) improved impaired glucose homeostasis. By immunohistochemistry, we first observed that JNK activity was increased in the arcuate nucleus (ARC) and the ventromedial hypothalamus (VMH) in both mouse models, relative to normoglycaemic controls. This suggests that up-regulation of JNK in these regions is associated with glucose intolerance and obesity, independent of leptin levels. Acute i.c.v. injection of SP600125 ameliorated glucose tolerance within 30 min in both leptin-deficient and DIO mice. Given the acute nature of i.c.v. injections, these effects cannot be attributed to changes in food intake or energy balance. In a hypothalamic cell line, and in the ARC and VMH of leptin-deficient mice, JNK inhibition by SP600125 consistently improved impaired insulin signalling. This was determined by a reduction of phospho-insulin receptor substrate-1 [IRS-1(Ser612)] protein in a hypothalamic cell line and a decline in the number of pIRS-1(Ser612) immunoreactive cells in the ARC and VMH. Serine 612 phosphorylation of IRS-1 is assumed to negatively regulate insulin signalling. In leptin-deficient mice, in both nuclei, central inhibition of JNK increased the number of cells immunoreactive for phospho-Akt (Ser473) and phospho-GSK-3β (Ser9), which are important

  12. N-terminal Serine Dephosphorylation Is Required for KCC3 Cotransporter Full Activation by Cell Swelling*

    PubMed Central

    Melo, Zesergio; de los Heros, Paola; Cruz-Rangel, Silvia; Vázquez, Norma; Bobadilla, Norma A.; Pasantes-Morales, Herminia; Alessi, Dario R.; Mercado, Adriana; Gamba, Gerardo

    2013-01-01

    The K+:Cl− cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity. PMID:24043619

  13. Modulation of thin filament activation of myosin ATP hydrolysis by N-terminal domains of cardiac myosin binding protein-C.

    PubMed

    Belknap, Betty; Harris, Samantha P; White, Howard D

    2014-10-28

    We have used enzyme kinetics to investigate the molecular mechanism by which the N-terminal domains of human and mouse cardiac MyBP-C (C0C1, C1C2, and C0C2) affect the activation of myosin ATP hydrolysis by F-actin and by native porcine thin filaments. N-Terminal domains of cMyBP-C inhibit the activation of myosin-S1 ATPase by F-actin. However, mouse and human C1C2 and C0C2 produce biphasic activating and inhibitory effects on the activation of myosin ATP hydrolysis by native cardiac thin filaments. Low ratios of MyBP-C N-terminal domains to thin filaments activate myosin-S1 ATP hydrolysis, but higher ratios inhibit ATP hydrolysis, as is observed with F-actin alone. These data suggest that low concentrations of C1C2 and C0C2 activate thin filaments by a mechanism similar to that of rigor myosin-S1, whereas higher concentrations inhibit the ATPase rate by competing with myosin-S1-ADP-Pi for binding to actin and thin filaments. In contrast to C0C2 and C1C2, the activating effects of the C0C1 domain are species-dependent: human C0C1 activates actomyosin-S1 ATPase rates, but mouse C0C1 does not produce significant activation or inhibition. Phosphorylation of serine residues in the m-linker between the C1 and C2 domains by protein kinase-A decreases the activation of thin filaments by huC0C2 at pCa > 8 but has little effect on the activation mechanism at pCa = 4. In sarcomeres, the low ratio of cMyBP-C to actin is expected to favor the activating effects of cMyBP-C while minimizing inhibition produced by competition with myosin heads.

  14. Effect of Tamoxifen and Brain-Penetrant Protein Kinase C and c-Jun N-Terminal Kinase Inhibitors on Tolerance to Opioid-Induced Respiratory Depression in Mice.

    PubMed

    Withey, Sarah L; Hill, Rob; Lyndon, Abigail; Dewey, William L; Kelly, Eamonn; Henderson, Graeme

    2017-04-01

    Respiratory depression is the major cause of death in opioid overdose. We have previously shown that prolonged treatment of mice with morphine induces profound tolerance to the respiratory-depressant effects of the drug (Hill et al., 2016). The aim of the present study was to investigate whether tolerance to opioid-induced respiratory depression is mediated by protein kinase C (PKC) and/or c-Jun N-terminal kinase (JNK). We found that although mice treated for up to 6 days with morphine developed tolerance, as measured by the reduced responsiveness to an acute challenge dose of morphine, administration of the brain-penetrant PKC inhibitors tamoxifen and calphostin C restored the ability of acute morphine to produce respiratory depression in morphine-treated mice. Importantly, reversal of opioid tolerance was dependent on the nature of the opioid ligand used to induce tolerance, as these PKC inhibitors did not reverse tolerance induced by prolonged treatment of mice with methadone nor did they reverse the protection to acute morphine-induced respiratory depression afforded by prolonged treatment with buprenorphine. We found no evidence for the involvement of JNK in morphine-induced tolerance to respiratory depression. These results indicate that PKC represents a major mechanism underlying morphine tolerance, that the mechanism of opioid tolerance to respiratory depression is ligand-dependent, and that coadministration of drugs with PKC-inhibitory activity and morphine (as well as heroin, largely metabolized to morphine in the body) may render individuals more susceptible to overdose death by reversing tolerance to the effects of morphine.

  15. Enhancing the antimicrobial activity of Sus scrofa lysozyme by N-terminal fusion of a sextuple unique homologous peptide.

    PubMed

    Zhu, Dewei; Cai, Guolin; Li, Xiaomin; Lu, Jian; Zhang, Liang

    2017-02-10

    Sus scrofa lysozyme (SSL), an important component of the pig immune system, is a potential candidate to replace antibiotics in feed. However, there is little antimicrobial activity of natural SSL against gram-negative bacteria, which limits its application. In this study, a unique peptide (A-W-V-A-W-K) with antimicrobial activity against gram-negative bacteria was discovered and purified from trypsin hydrolysate of natural SSL. This unique peptide was fused to natural SSL and the recombinant fused SSL exhibited improved activity against gram-negative bacteria. The N-terminal fusion likely increased the membrane penetrability and induced programmed bacterial cell death. The recombinant fused SSL also showed higher activity against some gram-positive bacteria with O-acetylation. By N-terminal fusion of the sextuple peptide, the anti-microbial activity, either to gram-positive or negative bacteria, of the recombinant SSL was higher than the fusion of only one copy of the peptide. This study provides a general, feasible, and highly useful strategy to enhance the antimicrobial activity of lysozyme.

  16. Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localisation of polycystin-2 in vivo and in vitro

    PubMed Central

    Streets, Andrew J; Moon, David J; Kane, Michelle E; Obara, Tomoko; Ong, Albert CM

    2008-01-01

    PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 (PC2), the PKD2 protein, is a nonselective Ca2+-permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser812) has been previously reported. Since we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser812 or all 5 predicted phosphorylation sites in the C-terminus, we hypothesised that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser76) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK-3). The consensus recognition sequence for GSK-3 (Ser76/Ser80) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK-3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without a change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK-3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology. PMID:16551655

  17. Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity

    PubMed Central

    D'Antuono, Alejandra; Loureiro, Maria Eugenia; Foscaldi, Sabrina; Marino-Buslje, Cristina

    2014-01-01

    ABSTRACT The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be

  18. Structural characterization and biological activity of recombinant human epidermal growth factor proteins with different N-terminal sequences.

    PubMed

    Svoboda, M; Bauhofer, A; Schwind, P; Bade, E; Rasched, I; Przybylski, M

    1994-05-18

    The primary structures and molecular homogeneity of recombinant human epidermal growth factors from different suppliers were characterized and their biological activities evaluated by a standard DNA synthesis assay. Molecular weight determinations using 252Cf-plasma-desorption and electrospray mass spectrometry in combination with N- and C-terminal sequence analysis and determination of intramolecular disulfide bridges revealed that one recombinant protein had the correct human-identical structure (54 aa residues; 6347 Da). In contrast, a second recombinant protein (7020 Da) was found to contain a pentapeptide (KKYPR) insert following its N-terminal methionine. This structural variant showed a significant reduction in its capacity to stimulate DNA synthesis.

  19. Effect of N-Terminal Myristoylation on the Active Conformation of Gαi1-GTP.

    PubMed

    van Keulen, Siri C; Rothlisberger, Ursula

    2017-01-10

    G proteins are part of the G-protein-coupled receptor (GPCR) signal transduction cascade in which they transfer a signal from the membrane-embedded GPCR to other proteins in the cell. In the case of the inhibitory G-protein heterotrimer, permanent N-terminal myristoylation can transiently localize the Gαi subunit at the membrane as well as crucially influence Gαi's function in the GTP-bound conformation. The attachment of lipids to proteins is known to be essential for membrane trafficking; however, our results suggest that lipidation is also important for protein-protein interactions during signal transduction. Here we investigate the effect of myristoylation on the structure and dynamics of soluble Gαi1 and its possible implication for signal transduction. A 2 μs classical molecular dynamics simulation of a myristoylated Gαi1-GTP complex suggests that the myristoyl-induced conformational changes of the switch II and alpha helical domains create new possibilities for protein-protein interactions and emphasize the importance of permanent lipid attachment for the conformation and functional tunability of signaling proteins.

  20. Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein

    PubMed Central

    Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C.; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

    2005-01-01

    We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561–565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an α-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38α or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa–egg interaction. PMID:15693750

  1. The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

    PubMed Central

    Landry, Aaron P.

    2014-01-01

    Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1. PMID:25147792

  2. A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic.

    PubMed

    Salazar, Héctor; Llorente, Itzel; Jara-Oseguera, Andrés; García-Villegas, Refugio; Munari, Mika; Gordon, Sharona E; Islas, León D; Rosenbaum, Tamara

    2008-03-01

    Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection.

  3. A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic

    PubMed Central

    Salazar, Héctor; Llorente, Itzel; Jara-Oseguera, Andrés; García-Villegas, Refugio; Munari, Mika; Gordon, Sharona E; Islas, León D; Rosenbaum, Tamara

    2015-01-01

    Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection. PMID:18297068

  4. The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

    PubMed

    Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

    2016-05-13

    Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species.

  5. Stronger learning recruits additional cell-signaling cascades: c-Jun-N-terminal kinase 1 (JNK1) is necessary for expression of stronger contextual fear conditioning.

    PubMed

    Leach, Prescott T; Kenney, Justin W; Gould, Thomas J

    2015-02-01

    Increased training often results in stronger memories but the neural changes responsible for these stronger memories are poorly understood. It is proposed here that higher levels of training that result in stronger memories recruit additional cell signaling cascades. This study specifically examined if c-Jun N-terminal kinase 1 (JNK1) is involved in the formation of stronger fear conditioning memories. Wildtype (WT), JNK1 heterozygous (Het), and JNK1 knockout (KO) mice were fear conditioned with 1 trial, 2 trials, or 4 trials. All mice learned both contextual (hippocampus-dependent) and cued (hippocampus-independent) fear conditioning but for contextual fear conditioning only, the JNK1 KO mice did not show higher levels of learning with increased trials. That is, WT mice showed a significant linear increase in contextual fear conditioning as training trials increased from 1 to 2 to 4 trials whereas KO mice showed the same level of contextual fear conditioning as WT mice for 1 trial training but did not have increased levels of contextual fear conditioning with additional trials. These data suggest that JNK1 may not be critical for learning but when higher levels of hippocampus-dependent learning occur, JNK1 signaling is recruited and is necessary for stronger hippocampus-dependent memory formation.

  6. Inhibition of development of experimental abdominal aortic aneurysm by c-jun N-terminal protein kinase inhibitor combined with lysyl oxidase gene modified smooth muscle progenitor cells.

    PubMed

    Chen, Feng; Zhang, ZhenDong; Zhu, XianHua

    2015-11-05

    Chronic inflammation, imbalance between the extracellular matrix synthesis and degradation, and loss of vascular smooth muscle cells (SMCs) contribute to the development of abdominal aortic aneurysm (AAA). The purpose of this study was to investigate the effect of the therapy with periaortic incubation of c-Jun N-terminal protein kinase inhibitor SP600125 infused from an osmotic pump and subadventitial injection of lysyl oxidase (LOX) gene modified autologous smooth muscle progenitor cells (SPCs) on treatment of AAA in a rabbit model. Obvious dilation of the abdominal aorta in the control group was caused by periaortic incubation of calcium chloride and elastase. But the progression of aortic dilation was significantly decreased after the treatment with SP600125 and LOX gene modified SPCs compared to the treatment with phosphate-buffered saline. This therapy could inhibit matrix metalloproteinases expression, enhance elastin synthesis, improve preservation of elastic laminar integrity, benefit SPCs survival and restore SMCs population. It seemed that this method might provide a novel therapeutic strategy to treat AAA.

  7. Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc.

    PubMed

    Mattila, Jaakko; Omelyanchuk, Leonid; Kyttälä, Satu; Turunen, Heikki; Nokkala, Seppo

    2005-01-01

    When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.

  8. cAMP-dependent protein kinase and c-Jun N-terminal kinase mediate stathmin phosphorylation for the maintenance of interphase microtubules during osmotic stress.

    PubMed

    Yip, Yan Y; Yeap, Yvonne Y C; Bogoyevitch, Marie A; Ng, Dominic C H

    2014-01-24

    Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.

  9. Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.

    PubMed

    Uchime, Onyinyechukwu; Dai, Zhou; Biris, Nikolaos; Lee, David; Sidhu, Sachdev S; Li, Sheng; Lai, Jonathan R; Gavathiotis, Evripidis

    2016-01-01

    The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site*

    PubMed Central

    Uchime, Onyinyechukwu; Dai, Zhou; Biris, Nikolaos; Lee, David; Sidhu, Sachdev S.; Li, Sheng; Lai, Jonathan R.; Gavathiotis, Evripidis

    2016-01-01

    The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation. PMID:26565029

  11. A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4.

    PubMed

    Sangawa, Takeshi; Nogi, Terukazu; Takagi, Junichi

    2008-10-01

    Abstract A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays an important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, suggesting its utility in the functional study of PAR4 signaling.

  12. A basic motif in the N-terminal region of RAG1 enhances V(D)J recombination activity.

    PubMed Central

    McMahan, C J; Difilippantonio, M J; Rao, N; Spanopoulou, E; Schatz, D G

    1997-01-01

    The variable portions of antigen receptor genes are assembled from component gene segments by a site-specific recombination reaction known as V(D)J recombination. The RAG1 and RAG2 proteins are the critical lymphoid cell-specific components of the recombination enzymatic machinery and are responsible for site-specific DNA recognition and cleavage. Previous studies had defined a minimal, recombinationally active core region of murine RAG1 consisting of amino acids 384 to 1008 of the 1,040-residue RAG1 protein. No recombination function has heretofore been ascribed to any portion of the 383-amino-acid N-terminal region that is missing from the core, but it seems likely to be of functional significance, based on its evolutionary conservation. Using extrachromosomal recombination substrates, we demonstrate here that the N-terminal region enhances the recombination activity of RAG1 by up to an order of magnitude in a variety of cell lines. Deletion analysis localized a region of the N terminus critical for this effect to amino acids 216 to 238, and further mutagenesis demonstrated that a small basic amino acid motif (BIIa) in this region is essential for enhancing the activity of RAG1. Despite the fact that BIIa is important for the interaction of RAG1 with the nuclear localization factor Srp-1, it does not appear to enhance recombination by facilitating nuclear transport of RAG1. A variety of models for how this region stimulates the recombination activity of RAG1 are considered. PMID:9234712

  13. Design and characterization of a potent and selective dual ATP- and substrate-competitive sub-nanomolar bi-dentate c-Jun N-terminal Kinase (JNK) inhibitor

    PubMed Central

    Stebbins, John L.; De, Surya K.; Pavlickova, Petra; Chen, Vida; Machleidt, Thomas; Chen, Li-Hsing; Kuntzen, Christian; Kitada, Shinichi; Karin, Michael; Pellecchia, Maurizio

    2011-01-01

    c-Jun N-terminal Kinases (JNKs) represent valuable targets in the development of new therapies. Present on the surface of JNK is a binding pocket for substrates and the scaffolding protein JIP1 in close proximity to the ATP binding pocket. We propose that bi-dentate compounds linking the binding energies of weakly interacting ATP and substrate mimetics could result in potent and selective JNK inhibitors. We describe here a bi-dentate molecule, 19, designed against JNK. 19 inhibits JNK kinase activity (IC50 = 18 nM; Ki = 1.5 nM) and JNK/substrate association in a displacement assay with a substrate peptide (compound 20; IC50 = 46 nM; Ki = 2 nM). Our data demonstrate that 19 targets for the ATP and substrate-binding sites on JNK concurrently. Finally, compound 19 not only inhibits JNK in a variety of cell-based experiments, but it elicits also in vivo activity where it is shown to improve glucose tolerance in diabetic mice. PMID:21815634

  14. Mouse hepatitis virus receptor activities of an MHVR/mph chimera and MHVR mutants lacking N-linked glycosylation of the N-terminal domain.

    PubMed Central

    Dveksler, G S; Basile, A A; Cardellichio, C B; Holmes, K V

    1995-01-01

    Mouse hepatitis virus binds to the N-terminal domain of its receptor, MHVR, a murine biliary glycoprotein with four immunoglobulin-like domains (G.S. Dveksler, M. N. Pensiero, C. W. Dieffenbach, C. B. Cardellichio, A.A. Basile, P.E. Elia, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 90:1716-1720, 1993). A recombinant protein with only the anchored N-terminal domain was not a functional receptor, but a recombinant protein with the N-terminal domain of MHVR linked to the second and third immunoglobulin-like domains and anchor from the mouse poliovirus receptor homolog, mph, was a functional receptor for mouse hepatitis virus. The native four-domain MHVR has 16 potential N-linked glycosylation sites, including three on the N-terminal domain. Recombinant proteins lacking each one of these three sites or all three of them were functional receptors. Thus, glycosylation of the N-terminal domain is not required, but a glycoprotein longer than the N-terminal domain is required for virus receptor activity. PMID:7983753

  15. c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis.

    PubMed Central

    Schroeter, Hagen; Boyd, Clinton S; Ahmed, Ruhi; Spencer, Jeremy P E; Duncan, Roger F; Rice-Evans, Catherine; Cadenas, Enrique

    2003-01-01

    The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis. PMID:12614194

  16. Anti-Inflammatory Effects and Joint Protection in Collagen-Induced Arthritis after Treatment with IQ-1S, a Selective c-Jun N-Terminal Kinase Inhibitor.

    PubMed

    Schepetkin, Igor A; Kirpotina, Liliya N; Hammaker, Deepa; Kochetkova, Irina; Khlebnikov, Andrei I; Lyakhov, Sergey A; Firestein, Gary S; Quinn, Mark T

    2015-06-01

    c-Jun N-terminal kinases (JNKs) participate in many physiologic and pathologic processes, including inflammatory diseases. We recently synthesized the sodium salt of IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime) and demonstrated that it is a high-affinity JNK inhibitor and inhibits murine delayed-type hypersensitivity. Here we show that IQ-1S is highly specific for JNK and that its neutral form is the most abundant species at physiologic pH. Molecular docking of the IQ-1S syn isomer into the JNK1 binding site gave the best pose, which corresponded to the position of cocrystallized JNK inhibitor SP600125 (1,9-pyrazoloanthrone). Evaluation of the therapeutic potential of IQ-1S showed that it inhibited matrix metalloproteinase 1 and 3 gene expression induced by interleukin-1β in human fibroblast-like synoviocytes and significantly attenuated development of murine collagen-induced arthritis (CIA). Treatment with IQ-1S either before or after induction of CIA resulted in decreased clinical scores, and joint sections from IQ-1S-treated CIA mice exhibited only mild signs of inflammation and minimal cartilage loss compared with those from control mice. Collagen II-specific antibody responses were also reduced by IQ-1S treatment. By contrast, the inactive ketone derivative 11H-indeno[1,2-b]quinoxalin-11-one had no effect on CIA clinical scores or collagen II-specific antibody titers. IQ-1S treatment also suppressed proinflammatory cytokine and chemokine levels in joints and lymph node cells. Finally, treatment with IQ-1S increased the number of Foxp3(+)CD4(+)CD25(+) regulatory T cells in lymph nodes. Thus, IQ-1S can reduce inflammation and cartilage loss associated with CIA and can serve as a small-molecule modulator for mechanistic studies of JNK function in rheumatoid arthritis.

  17. A Novel Dual NO-donating Oxime and c-Jun N-terminal Kinase Inhibitor Protects Against Cerebral Ischemia–Reperfusion Injury in Mice

    PubMed Central

    Atochin, Dmitriy N.; Schepetkin, Igor A.; Khlebnikov, Andrei I.; Seledtsov, Victor I.; Swanson, Helen; Quinn, Mark T.; Huang, Paul L.

    2017-01-01

    The c-Jun N-terminal kinase (JNK) has been shown to be an important regulator of neuronal cell death. Previously, we synthesized the sodium salt of 11H-indeno[1,2-b]quinoxalin-11-one (IQ-1S) and demonstrated that it was a high-affinity inhibitor of the JNK family. In the present work, we found that IQ-1S could release nitric oxide (NO) during its enzymatic metabolism by liver microsomes. Moreover, serum nitrite/nitrate concentration in mice increased after intraperitoneal injection of IQ-1S. Because of these dual actions as JNK inhibitor and NO-donor, the therapeutic potential of IQ-1S was evaluated in an animal stroke model. We subjected wild-type C57BL6 mice to focal ischemia (30 minutes) with subsequent reperfusion (48 hours). Mice were treated with IQ-1S (25 mg/kg) suspended in 10% solutol or with vehicle alone 30 minutes before and 24 hours after middle cerebral artery MCA) occlusion (MCAO). Using laser-Doppler flowmetry, we monitored cerebral blood flow (CBF) above the MCA during 30 minutes of MCAO provoked by a filament and during the first 30 minutes of subsequent reperfusion. In mice treated with IQ-1S, ischemic and reperfusion values of CBF were not different from vehicle-treated mice. However, IQ-1S treated mice demonstrated markedly reduced neurological deficit and infarct volumes as compared with vehicle-treated mice after 48 hours of reperfusion. Our results indicate that the novel JNK inhibitor releases NO during its oxidoreductive bioconversion and improves stroke outcome in a mouse model of cerebral reperfusion. We conclude that IQ-1S is a promising dual functional agent for the treatment of cerebral ischemia and reperfusion injury. PMID:26923672

  18. Anti-Inflammatory Effects and Joint Protection in Collagen-Induced Arthritis after Treatment with IQ-1S, a Selective c-Jun N-Terminal Kinase Inhibitor

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Hammaker, Deepa; Kochetkova, Irina; Khlebnikov, Andrei I.; Lyakhov, Sergey A.; Firestein, Gary S.

    2015-01-01

    c-Jun N-terminal kinases (JNKs) participate in many physiologic and pathologic processes, including inflammatory diseases. We recently synthesized the sodium salt of IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime) and demonstrated that it is a high-affinity JNK inhibitor and inhibits murine delayed-type hypersensitivity. Here we show that IQ-1S is highly specific for JNK and that its neutral form is the most abundant species at physiologic pH. Molecular docking of the IQ-1S syn isomer into the JNK1 binding site gave the best pose, which corresponded to the position of cocrystallized JNK inhibitor SP600125 (1,9-pyrazoloanthrone). Evaluation of the therapeutic potential of IQ-1S showed that it inhibited matrix metalloproteinase 1 and 3 gene expression induced by interleukin-1β in human fibroblast-like synoviocytes and significantly attenuated development of murine collagen-induced arthritis (CIA). Treatment with IQ-1S either before or after induction of CIA resulted in decreased clinical scores, and joint sections from IQ-1S–treated CIA mice exhibited only mild signs of inflammation and minimal cartilage loss compared with those from control mice. Collagen II–specific antibody responses were also reduced by IQ-1S treatment. By contrast, the inactive ketone derivative 11H-indeno[1,2-b]quinoxalin-11-one had no effect on CIA clinical scores or collagen II–specific antibody titers. IQ-1S treatment also suppressed proinflammatory cytokine and chemokine levels in joints and lymph node cells. Finally, treatment with IQ-1S increased the number of Foxp3+CD4+CD25+ regulatory T cells in lymph nodes. Thus, IQ-1S can reduce inflammation and cartilage loss associated with CIA and can serve as a small-molecule modulator for mechanistic studies of JNK function in rheumatoid arthritis. PMID:25784649

  19. Modulating the activity of short arginine-tryptophan containing antibacterial peptides with N-terminal metallocenoyl groups

    PubMed Central

    Albada, H Bauke; Chiriac, Alina-Iulia; Wenzel, Michaela; Penkova, Maya; Bandow, Julia E; Sahl, Hans-Georg

    2012-01-01

    Summary A series of small synthetic arginine and tryptophan containing peptides was prepared and analyzed for their antibacterial activity. The effect of N-terminal substitution with metallocenoyl groups such as ferrocene (FcCO) and ruthenocene (RcCO) was investigated. Antibacterial activity in different media, growth inhibition, and killing kinetics of the most active peptides were determined. The toxicity of selected derivatives was determined against erythrocytes and three human cancer cell lines. It was shown that the replacement of an N-terminal arginine residue with a metallocenoyl moiety modulates the activity of WRWRW-peptides against Gram-positive and Gram-negative bacteria. MIC values of 2–6 µM for RcCO-W(RW)2 and 1–11 µM for (RW)3 were determined. Interestingly, W(RW)2-peptides derivatized with ferrocene were significantly less active than those derivatized with ruthenocene which have similar structural but different electronic properties, suggesting a major influence of the latter. The high activities observed for the RcCO-W(RW)2- and (RW)3-peptides led to an investigation of the origin of activity of these peptides using several important activity-related parameters. Firstly, killing kinetics of the RcCO-W(RW)2-peptide versus killing kinetics of the (RW)3 derivative showed faster reduction of the colony forming units for the RcCO-W(RW)2-peptide, although MIC values indicated higher activity for the (RW)3-peptide. This was confirmed by growth inhibition studies. Secondly, hemolysis studies revealed that both peptides did not lead to significant destruction of erythrocytes, even up to 500 µg/mL for (RW)3 and 250 µg/mL for RcCO-W(RW)2. In addition, toxicity against three human cancer cell lines (HepG2, HT29, MCF7) showed that the (RW)3-peptide had an IC50 value of ~140 µM and the RcW(RW)2 one of ~90 µM, indicating a potentially interesting therapeutic window. Both the killing kinetics and growth inhibition studies presented in this work point to

  20. Trichodermin induces c-Jun N-terminal kinase-dependent apoptosis caused by mitotic arrest and DNA damage in human p53-mutated pancreatic cancer cells and xenografts.

    PubMed

    Chien, Ming-Hsien; Lee, Tzong-Huei; Lee, Wei-Jiunn; Yeh, Yen-Hsiu; Li, Tsai-Kun; Wang, Po-Chuan; Chen, Jih-Jung; Chow, Jyh-Ming; Lin, Yung-Wei; Hsiao, Michael; Wang, Shih-Wei; Hua, Kuo-Tai

    2017-03-01

    Pancreatic cancer is an aggressive malignancy, which generally responds poorly to chemotherapy. In this study, trichodermin, an endophytic fungal metabolite from Nalanthamala psidii, was identified as a potent and selective antitumor agent in human pancreatic cancer. Trichodermin exhibited antiproliferative effects against pancreatic cancer cells, especially p53-mutated cells (MIA PaCa-2 and BxPC-3) rather than normal pancreatic epithelial cells. We found that trichodermin induced caspase-dependent and mitochondrial intrinsic apoptosis. Trichodermin also increased apoptosis through mitotic arrest by activating Cdc2/cyclin B1 complex activity. Moreover, trichodermin promoted the activation of c-Jun N-terminal kinase (JNK), and inhibition of JNK by its inhibitor, shRNA, or siRNA significantly reversed trichodermin-mediated caspase-dependent apoptosis. Trichodermin triggered DNA damage stress to activate p53 function for executing apoptosis in p53-mutated cells. Importantly, we demonstrated that trichodermin with efficacy similar to gemcitabine, profoundly suppressed tumor growth through inducing intratumoral DNA damage and JNK activation in orthotopic pancreatic cancer model. Based on these findings, trichodermin is a potential therapeutic agent worthy of further development into a clinical trial candidate for treating cancer, especially the mutant p53 pancreatic cancer.

  1. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    PubMed Central

    Lübker, Carolin; Dove, Stefan; Tang, Wei-Jen; Urbauer, Ramona J. Bieber; Moskovitz, Jackob; Urbauer, Jeffrey L.; Seifert, Roland

    2015-01-01

    Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils. PMID:26184312

  2. Regulation of C-type Lectin Antimicrobial Activity by a Flexible N-terminal Prosegment*S⃞

    PubMed Central

    Mukherjee, Sohini; Partch, Carrie L.; Lehotzky, Rebecca E.; Whitham, Cecilia V.; Chu, Hiutung; Bevins, Charles L.; Gardner, Kevin H.; Hooper, Lora V.

    2009-01-01

    Members of the RegIII family of intestinal C-type lectins are directly antibacterial proteins that play a vital role in maintaining host-bacterial homeostasis in the mammalian gut, yet little is known about the mechanisms that regulate their biological activity. Here we show that the antibacterial activities of mouse RegIIIγ and its human ortholog, HIP/PAP, are tightly controlled by an inhibitory N-terminal prosegment that is removed by trypsin in vivo. NMR spectroscopy revealed a high degree of conformational flexibility in the HIP/PAP inhibitory prosegment, and mutation of either acidic prosegment residues or basic core protein residues disrupted prosegment inhibitory activity. NMR analyses of pro-HIP/PAP variants revealed distinctive colinear backbone amide chemical shift changes that correlated with antibacterial activity, suggesting that prosegment-HIP/PAP interactions are linked to a two-state conformational switch between biologically active and inactive protein states. These findings reveal a novel regulatory mechanism governing C-type lectin biological function and yield new insight into the control of intestinal innate immunity. PMID:19095652

  3. The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel*

    PubMed Central

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L.; He, Hong; Garcia, Guilherme J. M.; Kubelka, Jan; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2014-01-01

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation. PMID:24973914

  4. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    NASA Technical Reports Server (NTRS)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  5. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    NASA Technical Reports Server (NTRS)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  6. Molecular dynamics simulations of the active matrix metalloproteinase-2: positioning of the N-terminal fragment and binding of a small peptide substrate.

    PubMed

    Díaz, Natalia; Suárez, Dimas

    2008-07-01

    Herein we use different computational methods to study the structure and energetic stability of the catalytic domain of the active MMP-2 enzyme considering two different orientations of its N-terminal coil. The first orientation is largely solvent accessible and corresponds to that observed in the 1CK7 crystal structure of the proenzyme. In the second orientation, the N-terminal coil is packed against the Omega-loop and the alpha3-helix of the MMP-2 enzyme likewise in the so-called "superactivated" form of other MMPs. Binding to the MMP-2 catalytic domain of a short peptide substrate, which mimics the sequence of the alpha1 chain of collagen type I, is also examined considering again the two configurations of the N-terminal coil. All these MMP-2 models are subject to 20 ns molecular dynamics (MD) simulations followed by MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) calculations. The positioning of the N-terminal coil in the "superactivated" form is found to be energetically favored for the MMP-2 enzyme. Moreover, this configuration of the N-terminal moiety can facilitate the binding of peptide substrates. Globally, the results obtained in this study could be relevant for the structural-based design of specific MMP inhibitors.

  7. Cross monomer substrate contacts reposition the Hsp90 N-terminal domain and prime the chaperone activity

    PubMed Central

    Street, Timothy O.; Lavery, Laura A.; Verba, Kliment; Lee, Chung-Tien; Mayer, Matthias P.; Agard, David A.

    2012-01-01

    The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate catalyzed Hsp90 conformational changes are unknown. Here we find that substrate binding affects an intrinsically unfavorable rotation of the Hsp90 N-terminal domain (NTD) relative to the middle domain (MD) that is required for closure. We identify an MD substrate binding region on the interior cleft of the Hsp90 dimer and show that a secondary set of substrate contacts drive an NTD orientation change on the opposite monomer. These results suggest an Hsp90 activation mechanism in which cross-monomer contacts mediated by a partially structured substrate prime the chaperone for its functional activity. PMID:22063096

  8. Cross-monomer substrate contacts reposition the Hsp90 N-terminal domain and prime the chaperone activity.

    PubMed

    Street, Timothy O; Lavery, Laura A; Verba, Kliment A; Lee, Chung-Tien; Mayer, Matthias P; Agard, David A

    2012-01-06

    The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work, a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate-catalyzed Hsp90 conformational changes is unknown. Here, we find that substrate binding affects an intrinsically unfavorable rotation of the Hsp90 N-terminal domain (NTD) relative to the middle domain (MD) that is required for closure. We identify an MD substrate binding region on the interior cleft of the Hsp90 dimer and show that a secondary set of substrate contacts drives an NTD orientation change on the opposite monomer. These results suggest an Hsp90 activation mechanism in which cross-monomer contacts mediated by a partially structured substrate prime the chaperone for its functional activity.

  9. Role of c-Jun N-terminal kinase (JNK) in regulating tumor necrosis factor-alpha (TNF-alpha) mediated increase of acetaminophen (APAP) and chlorpromazine (CPZ) toxicity in murine hepatocytes.

    PubMed

    Gandhi, Adarsh; Guo, Tao; Ghose, Romi

    2010-04-01

    Drug induced liver injury (DILI) accounts for more than 50% of the cases of acute liver failure in this country, and is the major cause of drug withdrawal from the market. DILI has been associated with the induction of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha). Pro-inflammatory cytokines activate the mitogen activated protein kinase, c-Jun-N-terminal kinase (JNK) in the liver. Recent studies have shown that JNK can regulate the hepatotoxicity of the analgesic, acetaminophen (APAP). Several reports have shown that inflammation induced by the endotoxin, lipopolysaccharide (LPS) augments the toxic response to hepatotoxicants in vivo. However, the mechanism by which inflammation alters drug-induced hepatotoxicity is not known. This study investigated the role of inflammatory mediators in regulating the toxicity of the hepatotoxic drugs, APAP or chlorpromazine (CPZ) in primary mouse hepatocytes. We found that, pre-treatment with TNF-alpha resulted in approximately 50 to 60% increase in alanine aminotransferase (ALT) levels by APAP or CPZ, while interleukin-1beta (IL-1beta) or IL6 treatments showed only 15-20% increase in ALT release. The bacterial components, LPS or lipoteichoic acid (LTA) increased ALT release by approximately 35 to 38% upon drug treatment of the hepatocytes. The JNK inhibitor, SP600125 significantly diminished APAP and CPZ toxicity with or without TNF-alpha. Pre-treatment with TNF-alpha resulted in prolonged activation of JNK (upto 2 hr) in the presence of APAP or CPZ. These results show that TNF-alpha is the major cytokine involved in sensitizing hepatocytes to APAP- or CPZ-induced hepatotoxicity, likely by a mechanism involving sustained activation of JNK.

  10. [Effect of Acupuncture Intervention on c-jun N-terminal Kinase Signaling in the Hippocampus in Rats with Forced Swimming Stress].

    PubMed

    Guo, Yu; Xu, Ke; Bao, Wu-ye; Wang, Yu; Zhang, Xu-hui; Xu, Ming-min; Yu, Miao; Zhang, Chun-tao; Zhao, Bing-cong; Wu, Ji-hong; Tu, Ya

    2016-02-01

    To observe the effect of acupuncture on c-jun N-terminal Kinase (JNK) signaling in the hippocampus in rats with forced-swimming stress, so as to reveal its underlying mechanism in relieving depression-like motor response. Forty-eight Sprague-Dawley rats were randomly divided into 8 groups as control, control + JNK inhibitor (SP 600125) , model, model + SP 600125, acupuncture, acupuncture + SP 600125, Fluoxetine (an anti-depressant) , and Fluoxetine + SP 600125 (n = 6 in each group). The depression-like behavior (immobility) model was established by forcing the rat to swim in a glass-cylinder and solitary raise. Acupuncture stimulation was applied to "Baihui" (GV-20) and "Yintang" (GV 29) for 20 min before forced swimming and once again 24 h later.. The rats of the Fluoxetine and Fluoxetine+ SP 600125 groups were treated by intragastric administration of fluoxetine 10 mL (1.8 mg)/kg before forced swimming and once again 24 h thereafter. The rats of the model + SP 600125 and acupuncture + SP 600125 groups were treated by intraperitoneal injection of SP 600125 (10 mg/kg) 90 min before forced swimming and 30 min before acupuncture intervention, respectively. The immobility duration of rats in the water glass-cylinder was used to assess their depression-like behavior response. The expression levels of protein kinase kinase 4 (MKK 4), MKK 7, JNK, and phosphorylated JNK (p-JNK) in the hippocampus were detected by Western blot. Compared to the control group, the duration of immobility, and the expression levels of hippocampal MKK 4, MKK 7, and p-JNK proteins were significantly increased in the model group (P < 0.01). While in comparison with the model group, the duration of immobility in the model + SP 600125, acupuncture, acupuncture + SP 600125, Fluoxetine and Fluoxetine + SP 600125 groups, the expression levels of hippocampal MKK 4 and MKK 7 proteins in the Fluoxetine + SP 600125 group, and those of p-JNK protein in the acupuncture, acupuncture + SP 600125, model + SP

  11. Biosynthesis, glycosylation, and partial N-terminal amino acid sequence of the T-cell-activating protein TAP

    SciTech Connect

    Reiser, H.; Coligan, J.; Benacerraf, B.; Rock, K.L.

    1987-05-01

    The authors have characterized the TAP molecule, an Ly-6 linked T-cell-activating glycoprotein. The three TAP bands that are precipitated from metabolically labeled cells display a common migration pattern in isoelectric focusing/NaDodSO/sub 4//PAGE gels and have common N-terminal sequences. This sequence is rich in cysteine and is homologous to that previously reported for the Ly-6.1E antigen. They therefore, compared TAP and Ly-6.1E biochemically and found them to be structurally distinct. Given the role of TAP in T-cell activation, they further studied whether the molecule was phosphorylated. We have not found evidence for phosphorylation of the TAP protein. The carbohydrates present on the TAP molecule are resistant to peptide N-glycosidase F in vitro and tunicamycin in vivo. The upper band of the TAP triplet is susceptible to treatment with trifluoromethanesulfonic acid and thus seems to be of the O-linked rather than of the N-linked variety. The biosynthetic processing of TAP was studied in pulse-chase experiments. The middle band of the TAP triplet appears to be the earliest detectable species. Its conversion to the O-linked high molecular weight species can be blocked by monensin.

  12. Biosynthesis, glycosylation, and partial N-terminal amino acid sequence of the T-cell-activating protein TAP.

    PubMed Central

    Reiser, H; Coligan, J; Benacerraf, B; Rock, K L

    1987-01-01

    We have characterized the TAP molecule, an Ly-6 linked T-cell-activating glycoprotein. The three TAP bands that are precipitated from metabolically labeled cells display a common migration pattern in isoelectric focusing/NaDodSO4/PAGE gels and have common N-terminal sequences. This sequence is rich in cysteine and is homologous to that previously reported for the Ly-6.1E antigen. We, therefore, compared TAP and Ly-6.1E biochemically and found them to be structurally distinct. Given the role of TAP in T-cell activation, we further studied whether the molecule was phosphorylated. We have not found evidence for phosphorylation of the TAP protein. The carbohydrates present on the TAP molecule are resistant to peptide N-glycosidase F in vitro and tunicamycin in vivo. The upper band of the TAP triplet is susceptible to treatment with trifluoromethanesulfonic acid and thus seems to be of the O-linked rather than of the N-linked variety. The biosynthetic processing of TAP was studied in pulse-chase experiments. The middle band of the TAP triplet appears to be the earliest detectable species. Its conversion to the O-linked high molecular weight species can be blocked by monensin. Images PMID:3033645

  13. The mitochondria of stallion spermatozoa are more sensitive than the plasmalemma to osmotic-induced stress: role of c-Jun N-terminal kinase (JNK) pathway.

    PubMed

    García, Beatriz Macías; Moran, Alvaro Miró; Fernández, Lauro González; Ferrusola, Cristina Ortega; Rodriguez, Antolin Morillo; Bolaños, Juan Maria Gallardo; da Silva, Carolina Maria Balao; Martínez, Heriberto Rodríguez; Tapia, Jose A; Peña, Fernando J

    2012-01-01

    Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa. Additionally, there is evidence that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa. The main sources of reactive oxygen species in mammalian sperm are the mitochondria. In view of this, the aim of our study was to test whether or not osmotic stress was able to induce mitochondrial damage and to explore the osmotic tolerance of the mitochondria of stallion spermatozoa. Ejaculates from 7 stallions were subjected to osmolalities ranging from 75 to 1500 mOsm/kg, and the effect on sperm membrane integrity and mitochondrial membrane potential was studied. Additionally, the effects of changes in osmolality from hyposmotic to isosmotic and from hyperosmotic to isosmotic solutions were studied (osmotic excursions). The cellular volume of stallion spermatozoa under isosmotic conditions was 20.4 ± 0.33 μm(3). When exposed to low osmolality, the stallion spermatozoa behaved like a linear osmometer, whereas exposure to high osmolalities up to 900 mOsm/kg resulted in decreased sperm volume. Although sperm membranes were relatively resistant to changes in osmolality, mitochondrial membrane potential decreased when osmolalities were low or very high (10.7 ± 1.74 and 16.5 ± 1.70 at 75 and 150 mOsm/kg, respectively, and 13.1 ± 1.83 at 1500 mOsm/kg), whereas in isosmolar controls the percentage of stallion sperm mitochondria with a high membrane potential was 41.1 ± 1.69 (P < .01). Osmotic excursions induced greater damage than exposure of spermatozoa to a given nonphysiologic osmolality, and again the mitochondria were more prone to damage induced by osmotic excursions than was the sperm plasma membrane. In search of intracellular components that could mediate these changes, we have detected for the first time the c-Jun N-terminal kinase 1/2 in stallion spermatozoa, which are apparently involved in the

  14. c-Jun N-terminal kinase 1 is required for cordycepin-mediated induction of G2/M cell-cycle arrest via p21WAF1 expression in human colon cancer cells.

    PubMed

    Lee, Se-Jung; Moon, Gi-Seong; Jung, Kyung-Hwan; Kim, Wun-Jae; Moon, Sung-Kwon

    2010-01-01

    Cordycepin (3'-deoxyadenosine) has many anti-cancer properties. However, neither its molecular mechanism nor its molecular targets are well understood. In the present study, we investigated novel molecular mechanisms for the anti-tumor effects of cordycepin in human colon cancer HCT116 cells. After treatment of cells with cordycepin, dose-dependent cell growth inhibition was observed at an IC(50) value of 200muM. Cordycepin treatment resulted in G2/M-phase cell-cycle arrest, which was associated with increased p21WAF1 levels and reduced amounts of cyclin B1, Cdc2, and Cdc25c in a p53-independent pathway. Moreover, cordycepin treatment induced activation of JNK (c-Jun N-terminal kinases). Pretreatment with SP600125, a JNK-specific inhibitor, abrogated cordycepin-mediated p21WAF1 expression, cell growth inhibition, and reduced cell-cycle proteins. Furthermore, JNK1 inhibition by small interfering RNA (siRNA) produced similar results: suppression of cordycepin-induced p21WAF1 expression, decreased cell growth, and reduced cell-cycle proteins. Together, these results suggest a critical role for JNK1 activation in cordycepin-induced inhibition of cell growth and G2/M-phase arrest in human colon cancer cells. Copyright 2009 Elsevier Ltd. All rights reserved.

  15. β-Amyloid Oligomers Induce Phosphorylation of Tau and Inactivation of Insulin Receptor Substrate via c-Jun N-Terminal Kinase Signaling: Suppression by Omega-3 Fatty Acids and Curcumin

    PubMed Central

    Ma, Qiu-Lan; Yang, Fusheng; Rosario, Emily R.; Ubeda, Oliver J.; Beech, Walter; Gant, Dana J.; Chen, Ping Ping; Hudspeth, Beverly; Chen, Cory; Zhao, Yongle; Vinters, Harry V.; Frautschy, Sally A.

    2009-01-01

    Both insulin resistance (type II diabetes) and β-amyloid (Aβ) oligomers are implicated in Alzheimer's disease (AD). Here, we investigate the role of Aβ oligomer-induced c-Jun N-terminal kinase (JNK) activation leading to phosphorylation and degradation of the adaptor protein insulin receptor substrate-1 (IRS-1). IRS-1 couples insulin and other trophic factor receptors to downstream kinases and neuroprotective signaling. Increased phospho-IRS-1 is found in AD brain and insulin-resistant tissues from diabetics. Here, we report Aβ oligomers significantly increased active JNK and phosphorylation of IRS-1 (Ser616) and tau (Ser422) in cultured hippocampal neurons, whereas JNK inhibition blocked these responses. The omega-3 fatty acid docosahexaenoic acid (DHA) similarly inhibited JNK and the phosphorylation of IRS-1 and tau in cultured hippocampal neurons. Feeding 3xTg-AD transgenic mice a diet high in saturated and omega-6 fat increased active JNK and phosphorylated IRS-1 and tau. Treatment of the 3xTg-AD mice on high-fat diet with fish oil or curcumin or a combination of both for 4 months reduced phosphorylated JNK, IRS-1, and tau and prevented the degradation of total IRS-1. This was accompanied by improvement in Y-maze performance. Mice fed with fish oil and curcumin for 1 month had more significant effects on Y-maze, and the combination showed more significant inhibition of JNK, IRS-1, and tau phosphorylation. These data indicate JNK mediates Aβ oligomer inactivation of IRS-1 and phospho-tau pathology and that dietary treatment with fish oil/DHA, curcumin, or a combination of both has the potential to improve insulin/trophic signaling and cognitive deficits in AD. PMID:19605645

  16. N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana.

    PubMed

    Otterhag, L; Sommarin, M; Pical, C

    2001-05-25

    Phosphoinositide-specific phospholipase C's (PI-PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI-PLCs have been identified, and the three-dimensional structure of one rat isozyme, PLC-delta1, determined. Plants appear to contain only one gene family encoding PI-PLCs. The catalytic properties of plant PI-PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI-PLCs. All plant PI-PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI-PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N-terminal domain may represent an EF-hand domain that is required for catalytic activity but not for lipid binding.

  17. Activation of c-Jun transcription factor by substitution of a charged residue in its N-terminal domain.

    PubMed Central

    Hoeffler, W K; Levinson, A D; Bauer, E A

    1994-01-01

    C-Jun is a cellular transcription factor that can control gene expression in response to treatment of cells with phorbol esters, growth factors, and expression of some oncogenes. The ability of c-Jun to catalyze the transcription of certain genes is controlled, in part, by changes in the phosphorylation state of specific amino acids in c-Jun. One of the major sites that is phosphorylated during signal response is Ser73. Here we show that substitution of a negatively charged aspartic acid residue at 73 constitutively increased transcriptional activity of c-Jun. The Asp73 substitution also enhanced its availability to bind to DNA in a whole cell extract without altering its intrinsic DNA binding activity since the intrinsic activity was unaltered for the c-Jun mutant proteins expressed in a bacterial system. The negatively charged Asp substitution may mimic the negative charge of a phosphorylated serine at 73. The substitution of an uncharged alanine at 73 resulted in lowered activities. The N-terminal end of c-Jun containing these substitutions was fused to the DNA-binding region of the bovine papilloma virus E2 protein, and was able to confer the same activation properties to the fusion protein at the heterologous E2 DNA-binding site. Ser73 lies in a region of c-Jun previously proposed to bind an uncharacterized inhibitor, perhaps related to a protein of approximately 17.5 kD that coprecipitates along with our c-Jun or the JunE2 fusion products. Images PMID:8165146

  18. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  19. The proximal N-terminal amino acid residues are required for the coupling activity of the bovine heart mitochondrial factor B.

    PubMed

    Belogrudov, Grigory I

    2008-05-01

    Treatment of the recombinant bovine factor B with trypsin yielded a fragment (amino acid residues 62-175) devoid of coupling activity. Removal of the N-terminal Trp2-Gly3-Trp4 peptide resulted in a significant loss of coupling activity in the FB(DeltaW)(2)(-W)(4) deletion mutant. Sucrose density gradient centrifugation demonstrated co-sedimentation of recombinant factor B with the ADP/ATP carrier, which is present in preparations of H(+)-translocating F(0)F(1)-ATPase, but not in preparations of complex V. The N-terminally truncated factor B mutant FB(DeltaW)(2)(-W)(4) did not co-sediment with the ADP/ATP carrier. Recombinant factor B co-sedimented with partially purified membrane sector F(0), extracted from F(1)-stripped bovine submitochondrial particles with n-dodecyl-beta-d-maltoside. Factor B inhibited the passive proton conductance catalyzed by F(0) reconstituted into asolectin liposomes. A factor B mutant, bearing a photoreactive unnatural amino acid pbenzoyl-l-phenylalanine (pBpa) substituted for Trp2, cross-linked with F(0) subunits e and g as well as the ADP/ATP carrier. These results suggest that the N-terminal domain and, in particular, the proximal N-terminal amino acids are important for the coupling activity and protein-protein interactions of bovine factor B.

  20. The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity

    PubMed Central

    Jiang, Xia-Yun; Hou, Fei; Shen, Xiao-Dan; Du, Xue-Di; Xu, Hai-Li; Zou, Shu-Ming

    2016-01-01

    Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%–25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition. PMID:27251101

  1. Plasmodium falciparum Werner homologue is a nuclear protein and its biochemical activities reside in the N-terminal region.

    PubMed

    Rahman, Farhana; Tarique, Mohammed; Ahmad, Moaz; Tuteja, Renu

    2016-01-01

    RecQ helicases, also addressed as a gatekeeper of genome, are an inevitable family of genome scrutiny proteins conserved from prokaryotes to eukaryotes and play a vital role in DNA metabolism. The deficiencies of three RecQ proteins out of five are involved in genetic abnormalities like Bloom syndrome (BS), Werner syndrome (WS), and Rothmund-Thomson syndrome (RTS). It is noteworthy that Plasmodium falciparum contains only two members of the RecQ family as opposed to five members present in the host Homo sapiens. In the present study, we report the biochemical characterization of the homologue of Werner (Wrn) helicase from P. falciparum 3D7 strain. Although there are significant sequence conservations between Wrn helicases of both H. sapiens and P. falciparum as well as among all the other Plasmodium species, they contain some peculiar differences also. In silico studies reveal that PfWrn is evolutionarily close to the bacterial RecQ protein. The N-terminal fragment (PfWrnN) contains all the helicase motifs along with all the functional domains and the predicted structure resembles with the human RecQ1 protein, whereas the C-terminal fragment (PfWrnC) contains no significant domain. Biochemical characterization further revealed that purified recombinant PfWrnN shows ATPase and DNA helicase activity in 3' to 5' direction, but PfWrnC lacks the ATPase and helicase activities. Immunofluorescence study shows that PfWrn is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain and localizes distinctly in the nucleus. This study can be used for further characterization of RecQ helicases that will aid in understanding the physiological significance of these helicases in the malaria parasite.

  2. Signal transduction cross talk mediated by Jun N-terminal kinase-interacting protein and insulin receptor substrate scaffold protein complexes.

    PubMed

    Standen, Claire L; Kennedy, Norman J; Flavell, Richard A; Davis, Roger J

    2009-09-01

    Scaffold proteins have been established as important mediators of signal transduction specificity. The insulin receptor substrate (IRS) proteins represent a critical group of scaffold proteins that are required for signal transduction by the insulin receptor, including the activation of phosphatidylinositol 3 kinase. The c-Jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) represent a different group of scaffold molecules that are implicated in the regulation of the JNK. These two signaling pathways are functionally linked because JNK can phosphorylate IRS1 on the negative regulatory site Ser-307. Here we demonstrate the physical association of these signaling pathways using a proteomic approach that identified insulin-regulated complexes of JIPs together with IRS scaffold proteins. Studies using mice with JIP scaffold protein defects confirm that the JIP1 and JIP2 proteins are required for normal glucose homeostasis. Together, these observations demonstrate that JIP proteins can influence insulin-stimulated signal transduction mediated by IRS proteins.

  3. Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

    PubMed

    Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

    2005-04-01

    After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

  4. Role of N-terminal methionine residues in the redox activity of copper bound to alpha-synuclein.

    PubMed

    Rodríguez, Esaú E; Arcos-López, Trinidad; Trujano-Ortiz, Lidia G; Fernández, Claudio O; González, Felipe J; Vela, Alberto; Quintanar, Liliana

    2016-09-01

    Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1-6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1-6) fragment, and in the redox activity of the Cu-AS(1-6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)-AS(1-6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu-AS(1-6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu-AS complex.

  5. Differential Inhibition of Macrophage Activation by Lymphocytic Choriomeningitis Virus and Pichinde Virus Is Mediated by the Z Protein N-Terminal Domain

    PubMed Central

    Xing, Junji; Chai, Zheng; Ly, Hinh

    2015-01-01

    Several arenavirus pathogens, such as Lassa and Junin viruses, inhibit macrophage activation, the molecular mechanism of which is unclear. We show that lymphocytic choriomeningitis virus (LCMV) can also inhibit macrophage activation, in contrast to Pichinde and Tacaribe viruses, which are not known to naturally cause human diseases. Using a recombinant Pichinde virus system, we show that the LCMV Z N-terminal domain (NTD) mediates the inhibition of macrophage activation and immune functions. PMID:26423945

  6. c-Jun N-terminal kinase (JNK)-dependent acute liver injury from acetaminophen or tumor necrosis factor (TNF) requires mitochondrial Sab protein expression in mice.

    PubMed

    Win, Sanda; Than, Tin Aung; Han, Derick; Petrovic, Lydia M; Kaplowitz, Neil

    2011-10-07

    Sustained JNK activation plays a critical role in hepatotoxicity by acetaminophen or GalN/TNF-α. To address the importance of JNK translocation to mitochondria that accompanies sustained activation in these models, we assessed the importance of the expression of a potential initial target of JNK in the outer membrane of mitochondria, namely Sab (SH3 domain-binding protein that preferentially associates with Btk), also known as Sh3bp5 (SH3 domain-binding protein 5). Silencing the expression of Sab in the liver using adenoviral shRNA inhibited sustained JNK activation and mitochondrial targeting of JNK and the upstream MKK4 (MAPK kinase 4), accompanied by striking protection against liver injury in vivo and in cultured hepatocytes in both toxicity models. We conclude that mitochondrial Sab may serve as a platform for the MAPK pathway enzymes and that the interaction of stress-activated JNK with Sab is required for sustained JNK activation and toxicity.

  7. c-Jun N-terminal Kinase (JNK)-dependent Acute Liver Injury from Acetaminophen or Tumor Necrosis Factor (TNF) Requires Mitochondrial Sab Protein Expression in Mice*

    PubMed Central

    Win, Sanda; Than, Tin Aung; Han, Derick; Petrovic, Lydia M.; Kaplowitz, Neil

    2011-01-01

    Sustained JNK activation plays a critical role in hepatotoxicity by acetaminophen or GalN/TNF-α. To address the importance of JNK translocation to mitochondria that accompanies sustained activation in these models, we assessed the importance of the expression of a potential initial target of JNK in the outer membrane of mitochondria, namely Sab (SH3 domain-binding protein that preferentially associates with Btk), also known as Sh3bp5 (SH3 domain-binding protein 5). Silencing the expression of Sab in the liver using adenoviral shRNA inhibited sustained JNK activation and mitochondrial targeting of JNK and the upstream MKK4 (MAPK kinase 4), accompanied by striking protection against liver injury in vivo and in cultured hepatocytes in both toxicity models. We conclude that mitochondrial Sab may serve as a platform for the MAPK pathway enzymes and that the interaction of stress-activated JNK with Sab is required for sustained JNK activation and toxicity. PMID:21844199

  8. c-Jun N-terminal kinase and Akt signalling pathways regulating tumour necrosis factor-α-induced interleukin-32 expression in human lung fibroblasts: implications in airway inflammation.

    PubMed

    Li, Dagen; Chen, Dapeng; Zhang, Xuemei; Wang, Hong; Song, Zixin; Xu, Wenchun; He, Yujuan; Yin, Yibing; Cao, Ju

    2015-02-01

    Airway inflammatory diseases such as chronic obstructive pulmonary disease (COPD) and asthma are associated with elevated expression of interleukin-32 (IL-32), a recently described cytokine that appears to play a critical role in inflammation. However, so far, the regulation of pulmonary IL-32 production has not been fully established. We examined the expression of IL-32 by tumour necrosis factor-α (TNF-α) in primary human lung fibroblasts. Human lung fibroblasts were cultured in the presence or absence of TNF-α and/or other cytokines/Toll-like receptor (TLR) ligands or various signalling molecule inhibitors to analyse the expression of IL-32 by quantitative RT-PCR and ELISA. Next, activation of Akt and c-Jun N-terminal kinase (JNK) signalling pathways was investigated by Western blot. Interleukin-32 mRNA of four spliced isoforms (α, β, γ and δ) was up-regulated upon TNF-α stimulation, which was associated with a significant IL-32 protein release from TNF-α-activated human lung fibroblasts. The combination of interferon-γ and TNF-α induced enhanced IL-32 release in human lung fibroblasts, whereas IL-4, IL-17A, IL-27 and TLR ligands did not alter IL-32 release in human lung fibroblasts either alone, or in combination with TNF-α. Furthermore, the activation of Akt and JNK pathways regulated TNF-α-induced IL-32 expression in human lung fibroblasts, and inhibition of the Akt and JNK pathways was able to suppress the increased release of IL-32 to nearly the basal level. These data suggest that TNF-α may be involved in airway inflammation via the induction of IL-32 by activating Akt and JNK signalling pathways. Therefore, the TNF-α/IL-32 axis may be a potential therapeutic target for airway inflammatory diseases. © 2014 John Wiley & Sons Ltd.

  9. Knockout of the c-Jun N-terminal Kinase 2 aggravates the development of mild chronic dextran sulfate sodium colitis independently of expression of intestinal cytokines TNFα, TGFB1, and IL-6

    PubMed Central

    Kersting, Sabine; Reinecke, Kirstin; Hilgert, Christoph; Janot, Monika S; Haarmann, Elisabeth; Albrecht, Martin; Müller, Annette M; Herdegen, Thomas; Mittelkötter, Ulrich; Uhl, Waldemar; Chromik, Ansgar M

    2013-01-01

    Introduction The c-Jun N-terminal kinases (JNKs) are involved in signal transduction of inflammatory bowel diseases. The aim of this study was to examine the function of JNKs by using a low-dose dextran sulfate sodium (DSS) model in JNK1 knockout mice (Mapk8−/−), JNK2 knockout mice (Mapk9−/−), and wild-type controls (WT1, WT2). Methods The animals were evaluated daily using a disease activity index. After 30 days, the intestine was evaluated histologically with a crypt damage score. CD4+ and CD8+ cells were quantified using immunofluorescence. Analysis of tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), and transforming growth factor β1 (TGFB1) expression was carried out using LightCycler® real-time polymerase chain reaction. Results Cyclic administration of low-dose DSS (1%) was not able to induce features of chronic colitis in Mapk8−/− WT2 mice. By contrast, DSS administration significantly increased the disease activity index in WT1 and Mapk9−/− mice. In Mapk9−/− mice, the crypt damage score and the number of CD4+ and CD8+ cells as features of chronic colitis/inflammation were also significantly elevated. Expression of TNFα, IL-6, and TGFB1 was not altered by the JNK knockout. Conclusion Administering DSS at a defined low concentration that is unable to induce colitis in WT animals leads to clinically and histologically detectable chronic colitis in Mapk9−/− mice. The reason for this disease-inducing effect resulting from the loss of JNK2 remains to be elucidated. Expression of TNFα, IL-6, and TGFB1 does not appear to be involved; proapoptotic JNK2 may prolong the activity of proinflammatory immune cells, leading to perpetuation of the inflammation. PMID:23426157

  10. N-terminal isoforms of the large-conductance Ca²⁺-activated K⁺ channel are differentially modulated by the auxiliary β1-subunit.

    PubMed

    Lorca, Ramón A; Stamnes, Susan J; Pillai, Meghan K; Hsiao, Jordy J; Wright, Michael E; England, Sarah K

    2014-04-04

    The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK(Ca) α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BK(Ca) α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μM and 100 μM Ca(2+). The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK(Ca) channel activity in cells, highlighting a novel mechanism of BK(Ca) channel regulation.

  11. The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium.

    PubMed

    Buchsbaum, R; Telliez, J B; Goonesekera, S; Feig, L A

    1996-09-01

    We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location. This requires the PH domain and the adjacent coiled-coil and IQ sequences. Remarkably, this form of Ras-GRF is constitutively activated. The PH and coiled-coil domains must also perform an additional function, since targeting to the particulate fraction of cells is not sufficient to allow Ras-GRF activation by calcium. A Ras-GRF mutant containing the PH domain from Ras-GTPase-activating protein in place of its own N-terminal PH domain localizes to the particulate fraction of cells but does not respond to calcium. Similar phenotypes are seen with mutant Ras-GRFs containing point mutations in either the PH or coiled-coil domain. These findings argue that the N-terminal PH, coiled-coil, and IQ domains of Ras-GRF function together to connect Ras-GRF to multiple components in the particulate fractions of cells that are required for responsiveness of the protein to calcium signaling.

  12. Structural and mechanistic insights into Mps1 kinase activation

    SciTech Connect

    Wang, Wei; Yang, Yuting; Gao, Yuefeng; Xu, Quanbin; Wang, Feng; Zhu, Songcheng; Old, William; Resing, Katheryn; Ahn, Natalie; Lei, Ming; Liu, Xuedong

    2010-11-05

    Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-{angstrom}-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the {alpha}C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices {alpha}EF and {alpha}F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.

  13. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  14. An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks

    PubMed Central

    Hegnauer, Anna Maria; Hustedt, Nicole; Shimada, Kenji; Pike, Brietta L; Vogel, Markus; Amsler, Philipp; Rubin, Seth M; van Leeuwen, Fred; Guénolé, Aude; van Attikum, Haico; Thomä, Nicolas H; Gasser, Susan M

    2012-01-01

    DNA replication fork stalling poses a major threat to genome stability. This is counteracted in part by the intra-S phase checkpoint, which stabilizes arrested replication machinery, prevents cell-cycle progression and promotes DNA repair. The checkpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLM contribute synergistically to fork maintenance on hydroxyurea (HU). Both enzymes interact with replication protein A (RPA). We identified and deleted the major interaction sites on Sgs1 for Rpa70, generating a mutant called sgs1-r1. In contrast to a helicase-dead mutant of Sgs1, sgs1-r1 did not significantly reduce recovery of DNA polymerase α at HU-arrested replication forks. However, the Sgs1 R1 domain is a target of Mec1 kinase, deletion of which compromises Rad53 activation on HU. Full activation of Rad53 is achieved through phosphorylation of the Sgs1 R1 domain by Mec1, which promotes Sgs1 binding to the FHA1 domain of Rad53 with high affinity. We propose that the recruitment of Rad53 by phosphorylated Sgs1 promotes the replication checkpoint response on HU. Loss of the R1 domain increases lethality selectively in cells lacking Mus81, Slx4, Slx5 or Slx8. PMID:22820947

  15. The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

    DOE PAGES

    Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; ...

    2014-08-11

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme ismore » much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.« less

  16. The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

    SciTech Connect

    Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; Zhang, Ping; Heller, William T.; Taylor, Susan S.

    2014-08-11

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.

  17. Retinol-Binding Protein 4 Inhibits Insulin Signaling in Adipocytes by Inducing Proinflammatory Cytokines in Macrophages through a c-Jun N-Terminal Kinase- and Toll-Like Receptor 4-Dependent and Retinol-Independent Mechanism

    PubMed Central

    Norseen, Julie; Hosooka, Tetsuya; Hammarstedt, Ann; Yore, Mark M.; Kant, Shashi; Aryal, Pratik; Kiernan, Urban A.; Phillips, David A.; Maruyama, Hiroshi; Kraus, Bettina J.; Usheva, Anny; Davis, Roger J.; Smith, Ulf

    2012-01-01

    Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1−/− JNK2−/− macrophages and TLR4−/− macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4. PMID:22431523

  18. The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

    PubMed

    Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

    2014-10-10

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.

  19. Advanced oxidation protein products induce intestine epithelial cell death through a redox-dependent, c-jun N-terminal kinase and poly (ADP-ribose) polymerase-1-mediated pathway.

    PubMed

    Xie, F; Sun, S; Xu, A; Zheng, S; Xue, M; Wu, P; Zeng, J H; Bai, L

    2014-01-16

    Advanced oxidation protein products (AOPPs), a novel protein marker of oxidative damage, have been confirmed to accumulate in patients with inflammatory bowel disease (IBD), as well as those with diabetes and chronic kidney disease. However, the role of AOPPs in the intestinal epithelium remains unclear. This study was designed to investigate whether AOPPs have an effect on intestinal epithelial cell (IEC) death and intestinal injury. Immortalized rat intestinal epithelial (IEC-6) cells and normal Sprague Dawley rats were treated with AOPP-albumin prepared by incubation of rat serum albumin (RSA) with hypochlorous acid. Epithelial cell death, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit activity, reactive oxygen species (ROS) generation, apoptosis-related protein expression, and c-jun N-terminal kinase (JNK) phosphorylation were detected both in vivo and in vitro. In addition, we measured AOPPs deposition and IEC death in 23 subjects with Crohn's disease (CD). Extracellular AOPP-RSA accumulation induced apoptosis in IEC-6 cultures. The triggering effect of AOPPs was mainly mediated by a redox-dependent pathway, including NADPH oxidase-derived ROS generation, JNK phosphorylation, and poly (ADP-ribose) polymerase-1 (PARP-1) activation. Chronic AOPP-RSA administration to normal rats resulted in AOPPs deposition in the villous epithelial cells and in inflammatory cells in the lamina propria. These changes were companied with IEC death, inflammatory cellular infiltration, and intestinal injury. Both cell death and intestinal injury were ameliorated by chronic treatment with apocynin. Furthermore, AOPPs deposition was also observed in IECs and inflammatory cells in the lamina propria of patients with CD. The high immunoreactive score of AOPPs showed increased apoptosis. Our results demonstrate that AOPPs trigger IEC death and intestinal tissue injury via a redox-mediated pathway. These data suggest that AOPPs may represent a novel pathogenic factor

  20. N-terminal domain of soluble epoxide hydrolase negatively regulates the VEGF-mediated activation of endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Hammock, Bruce D.; Su, Kou-Hui; Morisseau, Christophe; Kou, Yu Ru; Imaoka, Susumu; Oguro, Ami; Shyue, Song-Kun; Zhao, Jin-Feng; Lee, Tzong-Shyuan

    2012-01-01

    Aims The mammalian soluble epoxide hydrolase (sEH) has both an epoxide hydrolase and a phosphatase domain. The role of sEH hydrolase activity in the metabolism of epoxyeicosatrienoic acids (EETs) and the activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) has been well defined. However, far less is known about the role of sEH phosphatase activity in eNOS activation. In the present study, we investigated whether the phosphatase domain of sEH was involved in the eNOS activation in ECs. Methods and results The level of eNOS phosphorylation in aortas is higher in the sEH knockout (sEH−/−) mice than in wild-type mice. In ECs, pharmacological inhibition of sEH phosphatase or overexpressing sEH with an inactive phosphatase domain enhanced vascular endothelial growth factor (VEGF)-induced NO production and eNOS phosphorylation. In contrast, overexpressing the phosphatase domain of sEH prevented the VEGF-mediated NO production and eNOS phosphorylation at Ser617, Ser635, and Ser1179. Additionally, treatment with VEGF induced a c-Src kinase-dependent increase in transient tyrosine phosphorylation of sEH and the formation of a sEH–eNOS complex, which was abolished by treatment with a c-Src kinase inhibitor, PP1, or the c-Src dominant-negative mutant K298M. We also demonstrated that the phosphatase domain of sEH played a key role in VEGF-induced angiogenesis by detecting the tube formation in ECs and neovascularization in Matrigel plugs in mice. Conclusion In addition to epoxide hydrolase activity, phosphatase activity of sEH plays a pivotal role in the regulation of eNOS activity and NO-mediated EC functions. PMID:22072631

  1. Tyrosine kinase activity of a Ca{sup 2+}/calmodulin-dependent protein kinase II catalytic fragment

    SciTech Connect

    Sugiyama, Yasunori; Ishida, Atsuhiko; Sueyoshi, Noriyuki; Kameshita, Isamu

    2008-12-12

    A 30-kDa fragment of Ca{sup 2+}/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX{sub 40}-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca{sup 2+}/calmodulin-dependent protein kinase I (CX{sub 40}) were fused to the N-terminal end of 30K-CaMKII. Although CX{sub 40}-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX{sub 40} region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.

  2. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

  3. The Extended Transmembrane Orai1 N-terminal (ETON) Region Combines Binding Interface and Gate for Orai1 Activation by STIM1*♦

    PubMed Central

    Derler, Isabella; Plenk, Peter; Fahrner, Marc; Muik, Martin; Jardin, Isaac; Schindl, Rainer; Gruber, Hermann J.; Groschner, Klaus; Romanin, Christoph

    2013-01-01

    STIM1 and Orai1 represent the two molecular key components of the Ca2+ release-activated Ca2+ channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73–90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify “hot spot” residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca2+ selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca2+ selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76–90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate. PMID:23943619

  4. The effects of urotensin II on migration and invasion are mediated by NADPH oxidase-derived reactive oxygen species through the c-Jun N-terminal kinase pathway in human hepatoma cells.

    PubMed

    Li, Ying-Ying; Shi, Zheng-Ming; Yu, Xiao-Tong; Feng, Ping; Wang, Xue-Jiang

    2017-02-01

    Urotensin II (UII) is a vasoactive neuropeptide involved in migration and invasion in various cell types. However, the effects of UII on human hepatoma cells still remain unclear. The aim of this study was to investigate the role and mechanism of UII on migration and invasion in human hepatoma cells. Migration was measured by wound healing assays and a Transwell(®) methodology, and invasion was analyzed using Matrigel(®) invasion chambers. Reactive oxygen species (ROS) levels were detected using a 2', 7'-dichlorofluorescein diacetate probe, and flow cytometry, and protein expression levels were evaluated by western blotting. Cell proliferation and actin polymerization were examined using cell proliferation reagent WST-1 and F-actin immunohistochemistry staining. Exposure to UII promoted migration and invasion in hepatoma cells compared with that in cells without UII. UII also increased matrix metalloproteinase-2 (MMP2) expression in a time-independent manner. Furthermore, UII markedly enhanced ROS generation and NADPH oxidase subunit expression, and consequently facilitated the phosphorylation of c-Jun N-terminal kinase (JNK). The UT antagonist urantide or the antioxidant/NADPH oxidase inhibitor apocynin decreased UII-induced ROS production. JNK phosphorylation, migration, invasion, and MMP9/2 expression were also reversed by pretreatment with apocynin. Urantide and JNK inhibitor SP600125 abrogated migration, invasion, or MMP9/2 expression in response to UII. UII induced actin polymerization and fascin protein expression, and could be reversed by apocynin and SP600125. Exogenous UII induced migration and invasion in hepatoma cells that mainly involved NADPH oxidase-derived ROS through JNK activation. UT played an additional role in regulating hepatoma cells migration and invasion. Thus, our data suggested an important effect of UII in hepatocellular carcinoma metastasis. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Inhibiting c-Jun N-terminal kinase partially attenuates caffeine-dependent cell death without alleviating the caffeine-induced reduction in mitochondrial respiration in C2C12 skeletal myotubes.

    PubMed

    Downs, R M; Hughes, M A; Kinsey, S T; Johnson, M C; Baumgarner, B L

    2016-11-04

    Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release. Copyright © 2016 Elsevier Inc

  6. Inhibiting c-Jun N-terminal kinase partially attenuates caffeine-dependent cell death without alleviating the caffeine-induced reduction in mitochondrial respiration in C2C12 skeletal myotubes

    PubMed Central

    Downs, R.M.; Hughes, M.A.; Kinsey, S.T.; Johnson, M.C.; Baumgarner, B.L.

    2017-01-01

    Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release. PMID:27717822

  7. The N-terminal Capping Propensities of the D-helix Modulate the Allosteric Activation of the Escherichia coli cAMP Receptor Protein*

    PubMed Central

    Yu, Shaoning; Maillard, Rodrigo A.; Gribenko, Alexey V.; Lee, J. Ching

    2012-01-01

    Transduction of biological signals at the molecular level involves the activation and/or inhibition of allosteric proteins. In the transcription factor cAMP receptor protein (CRP) from Escherichia coli, the allosteric activation, or apo-holo transition, involves rigid body motions of domains and structural rearrangements within the hinge region connecting the cAMP- and DNA-binding domains. During this apo-holo transition, residue 138 is converted as part of the elongated D-helix to the position of the N-terminal capping residue of a shorter D-helix. The goal of the current study is to elucidate the role of residue 138 in modulating the allostery between cAMP and DNA binding. By systematically mutating residue 138, we found that mutants with higher N-terminal capping propensities lead to increased cooperativity of cAMP binding and a concomitant increase in affinity for lac-DNA. Furthermore, mutants with higher N-terminal capping propensity correlate with properties characteristic of holo-CRP, particularly, increase in protein structural dynamics. Overall, our results provide a quantitative characterization of the role of residue 138 in the isomerization equilibrium between the apo and holo forms of CRP, and in turn the thermodynamic underpin to the molecular model of allostery revealed by the high resolution structural studies. PMID:23035121

  8. Induction of a mesenchymal expression program in lung epithelial cells by wingless protein (Wnt)/β-catenin requires the presence of c-Jun N-terminal kinase-1 (JNK1).

    PubMed

    van der Velden, Jos L J; Guala, Amy S; Leggett, Susan E; Sluimer, Jasper; Badura, Elsbeth C H L; Janssen-Heininger, Yvonne M W

    2012-09-01

    Recent studies suggest the importance of the transition of airway epithelial cells (EMT) in pulmonary fibrosis, and also indicate a role for Wingless protein (Wnt)/β-catenin signaling in idiopathic pulmonary fibrosis. We investigated the possible role of the Wnt signaling pathway in inducing EMT in lung epithelial cells, and sought to unravel the role of c-Jun-N-terminal-kinase-1 (JNK1). The exposure of C10 lung epithelial cells or primary mouse tracheal epithelial cells (MTECs) to Wnt3a resulted in increases in JNK phosphorylation and nuclear β-catenin content. Because the role of β-catenin as a transcriptional coactivator is well established, we investigated T-cell factor/lymphocyte-enhancement factor (TCF/LEF) transcriptional activity in C10 lung epithelial cells after the activation of Wnt. TCF/LEF transcriptional activity was enhanced after the activation of Wnt, and this increase in TCF/LEF transcriptional activity was diminished after the small interfering (si)RNA-mediated ablation of JNK. The activation of the Wnt pathway by Wnt3a, or the expression of either wild-type or constitutively active β-catenin (S37A), led to the activation of an EMT transcriptome, manifested by the increased mRNA expression of CArG box-binding factor-A, fibroblast-specific protein (FSP)-1, α-smooth muscle actin (α-SMA), and vimentin, increases in the content of α-SMA and FSP1, and the concomitant loss of zona occludens-1. The siRNA-mediated ablation of β-catenin substantially decreased Wnt3a-induced EMT. The siRNA ablation of JNK1 largely abolished Wnt3a, β-catenin, and β-catenin S37a-induced EMT. In MTECs lacking Jnk1, Wnt3a-induced increases in nuclear β-catenin, EMT transcriptome, and the content of α-SMA or FSP1 were substantially diminished. These data show that the activation of the Wnt signaling pathway is capable of inducing an EMT program in lung epithelial cells through β-catenin, and that this process is controlled by JNK1.

  9. Identification of quercitrin as an inhibitor of the p90 S6 ribosomal kinase (RSK): structure of its complex with the N-terminal domain of RSK2 at 1.8 Å resolution

    SciTech Connect

    Derewenda, Urszula; Artamonov, Mykhaylo; Szukalska, Gabriela; Utepbergenov, Darkhan; Olekhnovich, Natalya; Parikh, Hardik I.; Kellogg, Glen E.; Somlyo, Avril V.; Derewenda, Zygmunt S.

    2013-02-01

    The crystal structure of quercitrin, a naturally occurring flavonol glycoside, has been determined in a complex with the N-terminal kinase domain of murine RSK2. The structure revealed that quercitrin inhibits the RSK2 kinase in the same fashion as another known inhibitor, SL0101. Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45–346) of mouse RSK2, or RSK2{sup NTKD}, has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3′′,4′′-di-O-acetyl-α-l-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-l-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2{sup NTKD} with a dissociation constant (K{sub d}) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K{sub d} for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca{sup 2+}.

  10. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

    PubMed

    Aleem, Saadat U; Craddock, Barbara P; Miller, W Todd

    2015-01-01

    The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

  11. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase

    PubMed Central

    Miller, W. Todd

    2015-01-01

    The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution. PMID:26090675

  12. Sepp1(UF) forms are N-terminal selenoprotein P truncations that have peroxidase activity when coupled with thioredoxin reductase-1.

    PubMed

    Kurokawa, Suguru; Eriksson, Sofi; Rose, Kristie L; Wu, Sen; Motley, Amy K; Hill, Salisha; Winfrey, Virginia P; McDonald, W Hayes; Capecchi, Mario R; Atkins, John F; Arnér, Elias S J; Hill, Kristina E; Burk, Raymond F

    2014-04-01

    Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1-239) that contains one selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain (residues 240-361) that contains nine selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up by renal proximal convoluted tubule (PCT) cells via megalin-mediated endocytosis. We purified Sepp1 forms from the urine of megalin(-/-) mice using a monoclonal antibody to the N-terminal domain. Mass spectrometry revealed that the purified urinary Sepp1 consisted of N-terminal fragments terminating at 11 sites between residues 183 and 208. They were therefore designated Sepp1(UF). Because the N-terminal domain of Sepp1 has a thioredoxin fold, Sepp1(UF) were compared with full-length Sepp1, Sepp1(Δ240-361), and Sepp1(U40S) as a substrate of thioredoxin reductase-1 (TrxR1). All forms of Sepp1 except Sepp1(U40S), which contains serine in place of the selenocysteine, were TrxR1 substrates, catalyzing NADPH oxidation when coupled with H2O2 or tert-butylhydroperoxide as the terminal electron acceptor. These results are compatible with proteolytic cleavage freeing Sepp1(UF) from full-length Sepp1, the form that has the role of selenium transport, allowing Sepp1(UF) to function by itself as a peroxidase. Ultimately, plasma Sepp1(UF) and small selenium-containing proteins are filtered by the glomerulus and taken up by PCT cells via megalin-mediated endocytosis, preventing loss of selenium in the urine and providing selenium for the synthesis of glutathione peroxidase-3. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Cooperation of the N-terminal Helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks.

    PubMed

    Komori, Kayoko; Hidaka, Masumi; Horiuchi, Takashi; Fujikane, Ryosuke; Shinagawa, Hideo; Ishino, Yoshizumi

    2004-12-17

    Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.

  14. Auto-phosphorylation Represses Protein Kinase R Activity.

    PubMed

    Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J

    2017-03-10

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

  15. Auto-phosphorylation Represses Protein Kinase R Activity

    PubMed Central

    Wang, Die; de Weerd, Nicole A.; Willard, Belinda; Polekhina, Galina; Williams, Bryan R. G.; Sadler, Anthony J.

    2017-01-01

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity. PMID:28281686

  16. Osmotic Stress Regulates Mammalian Target of Rapamycin (mTOR) Complex 1 via c-Jun N-terminal Kinase (JNK)-mediated Raptor Protein Phosphorylation*

    PubMed Central

    Kwak, Dongoh; Choi, Sunkyu; Jeong, Heeyoon; Jang, Jin-Hyeok; Lee, Youngmi; Jeon, Hyeona; Lee, Mi Nam; Noh, Jungeun; Cho, Kun; Yoo, Jong Shin; Hwang, Daehee; Suh, Pann-Ghill; Ryu, Sung Ho

    2012-01-01

    mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway. PMID:22493283

  17. Dichotomal effect of space flight-associated microgravity on stress-activated protein kinases in innate immunity.

    PubMed

    Verhaar, Auke P; Hoekstra, Elmer; Tjon, Angela S W; Utomo, Wesley K; Deuring, J Jasper; Bakker, Elvira R M; Muncan, Vanesa; Peppelenbosch, Maikel P

    2014-06-27

    Space flight strongly moderates human immunity but is in general well tolerated. Elucidation of the mechanisms by which zero gravity interacts with human immunity may provide clues for developing rational avenues to deal with exaggerated immune responses, e.g. as in autoimmune disease. Using two sounding rockets and one manned Soyuz launch, the influence of space flight on immunological signal transduction provoked by lipopolysaccharide (LPS) stimulation was investigated in freshly isolated peripheral blood monocytes and was compared to samples obtained from on-board centrifuge-loaded 1 g controls. The effect of microgravity on immunological signal transduction is highly specific, since LPS dependent Jun-N-terminal kinase activation is impaired in the 0 g condition, while the corresponding LPS dependent activation of p38 MAP kinase remains unaffected. Thus our results identify Jun-N-terminal kinase as a relevant target in immunity for microgravity and support using Jun-N-terminal kinase specific inhibitors for combating autoimmune disease.

  18. Structural Insight into the Critical Role of the N-Terminal Region in the Catalytic Activity of Dual-Specificity Phosphatase 26

    PubMed Central

    Won, Eun-Young; Lee, Sang-Ok; Lee, Dong-Hwa; Lee, Daeyoup; Bae, Kwang-Hee; Lee, Sang Chul; Kim, Seung Jun; Chi, Seung-Wook

    2016-01-01

    Human dual-specificity phosphatase 26 (DUSP26) is a novel target for anticancer therapy because its dephosphorylation of the p53 tumor suppressor regulates the apoptosis of cancer cells. DUSP26 inhibition results in neuroblastoma cell cytotoxicity through p53-mediated apoptosis. Despite the previous structural studies of DUSP26 catalytic domain (residues 61–211, DUSP26-C), the high-resolution structure of its catalytically active form has not been resolved. In this study, we determined the crystal structure of a catalytically active form of DUSP26 (residues 39–211, DUSP26-N) with an additional N-terminal region at 2.0 Å resolution. Unlike the C-terminal domain-swapped dimeric structure of DUSP26-C, the DUSP26-N (C152S) monomer adopts a fold-back conformation of the C-terminal α8-helix and has an additional α1-helix in the N-terminal region. Consistent with the canonically active conformation of its protein tyrosine phosphate-binding loop (PTP loop) observed in the structure, the phosphatase assay results demonstrated that DUSP26-N has significantly higher catalytic activity than DUSP26-C. Furthermore, size exclusion chromatography-multiangle laser scattering (SEC-MALS) measurements showed that DUSP26-N (C152S) exists as a monomer in solution. Notably, the crystal structure of DUSP26-N (C152S) revealed that the N-terminal region of DUSP26-N (C152S) serves a scaffolding role by positioning the surrounding α7-α8 loop for interaction with the PTP-loop through formation of an extensive hydrogen bond network, which seems to be critical in making the PTP-loop conformation competent for phosphatase activity. Our study provides the first high-resolution structure of a catalytically active form of DUSP26, which will contribute to the structure-based rational design of novel DUSP26-targeting anticancer therapeutics. PMID:27583453

  19. p21-Activated Kinase 1 Plays a Critical Role in Cellular Activation by Nef

    PubMed Central

    Fackler, Oliver T.; Lu, Xiaobin; Frost, Jeffrey A.; Geyer, Matthias; Jiang, Bing; Luo, Wen; Abo, Arie; Alberts, Arthur S.; Peterlin, B. Matija

    2000-01-01

    The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef. PMID:10713183

  20. Inhibition of c-Jun N-terminal kinase attenuates low shear stress-induced atherogenesis in apolipoprotein E-deficient mice.

    PubMed

    Wang, Juan; An, Feng Shuang; Zhang, Wei; Gong, Lei; Wei, Shu Jian; Qin, Wei Dong; Wang, Xu Ping; Zhao, Yu Xia; Zhang, Yun; Zhang, Cheng; Zhang, Ming-Xiang

    2011-01-01

    Atherosclerosis begins as local inflammation of arterial walls at sites of disturbed flow, such as vessel curvatures and bifurcations with low shear stress. c-Jun NH₂-terminal kinase (JNK) is a major regulator of flow-dependent gene expression in endothelial cells in atherosclerosis. However, little is known about the in vivo role of JNK in low shear stress in atherosclerosis. We aimed to observe the effect of JNK on low shear stress-induced atherogenesis in apolipoprotein E-deficient (ApoE(-/-)) mice and investigate the potential mechanism in human umbilical vein endothelial cells (HUVECs). We divided 84 male ApoE(-/-) mice into two groups for treatment with normal saline (NS) (n = 42) and JNK inhibitor SP600125 (JNK-I) (n = 42). Perivascular shear stress modifiers were placed around the right carotid arteries, and plaque formation was studied at low shear stress regions. The left carotid arteries without modifiers represented undisturbed shear stress as a control. The NS group showed atherosclerotic lesions in arterial regions with low shear stress, whereas the JNK-I group showed almost no atherosclerotic lesions. Corresponding to the expression of proatherogenic vascular cell adhesion molecule 1 (VCAM-1), phospho-JNK (p-JNK) level was higher in low shear stress regions with NS than with JNK-I inhibitor. In HUVECs under low shear stress, siRNA knockdown and SP600125 inhibition of JNK attenuated nuclear factor (NF)-κB activity and VCAM-1 expression. Furthermore, siRNA knockdown of platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31) reduced p-JNK and VCAM-1 levels after low shear stress stimulation. JNK may play a critical role in low shear stress-induced atherogenesis by a PECAM-1-dependent mechanosensory pathway and modulating NF-κB activity and VCAM-1 expression.

  1. Inhibition of c-Jun N-terminal kinase sensitizes tumor cells to flavonoid-induced apoptosis through down-regulation of JunD

    SciTech Connect

    Kook, Sung-Ho; Son, Young-Ok; Jang, Yong-Suk; Lee, Kyung-Yeol; Lee, Seung-Ah; Kim, Beom-Soo; Lee, Hyun-Jeong; Lee, Jeong-Chae

    2008-03-15

    Reduction of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun NH{sub 2}-terminal kinase (JNK) has been implicated in stress-induced apoptosis. However, many studies also emphasize the role of JNK on cell survival, although its mechanisms are not completely understood. Previously, we found that inhibition of JNK activity promotes flavonoid-mediated apoptosis of human osteosarcoma cells. We thus determined whether inhibition of JNK sensitizes tumor cells to a bioflavonoid-induced apoptosis, and whether this effect of JNK is a general effect. As the results, quercetin and genistein as well as a flavonoid fraction induced apoptosis of tumor cells, which was further accelerated by specific JNK inhibitor, SP600125 or by small interfering RNA specific to JNK1/2. This effect was specific to types of cells because it was further apparent in tumorigenic cell lines. Inhibition of JNK by SP600125 also reduced flavonoid-stimulated nuclear induction of JunD which was known to have protective role in apoptosis, whereas JNK inhibition alone had little effect on apoptosis. The flavonoid-induced apoptosis of tumor cells was significantly enhanced by transfecting them with antisense JunD oligonucleotides. These results suggest that inhibition of JNK facilitates flavonoid-induced apoptosis through down-regulation of JunD, which is further sensitive to tumor cells. Therefore, combination with a specific JNK inhibitor further enhances the anti-cancer and chemopreventive potential of bio-flavonoids.

  2. Effect of N-terminal truncation on antibacterial activity, cytotoxicity and membrane perturbation activity of Cc-CATH3.

    PubMed

    Jittikoon, Jiraphun; Ngamsaithong, Narumon; Pimthon, Jutarat; Vajragupta, Opa

    2015-10-01

    A series of amino-terminal truncated analogues of quail antimicrobial peptide Cc-CATH3(1-29) were created and examined antibacterial activity against Gram-positive bacteria, cytotoxicity against mouse fibroblast cell line, and membrane perturbation activity against various membrane models. Parent peptide Cc-CATH3(1-29) and the first four-residue truncated peptide Cc-CATH3(5-29) were active in all tested experiments. In contrast, the eight- and twelve-residue truncated variants Cc-CATH3(9-29) and Cc-CATH3(13-29) appeared to have lost activities. Cc-CATH3(1-29) and Cc-CATH3(5-29) possessed antibacterial activity with minimum inhibitory concentrations of 2-4 and 1-2 µM, respectively. For cytotoxicity, Cc-CATH3(1-29) and Cc-CATH3(5-29) displayed cytotoxicity with the IC50 values of 9.33 and 4.93 μM, respectively. Cc-CATH3(5-29) induced greater liposome membranes disruption than Cc-CATH3(1-29) regardless of lipid type and composition. The leakage results of Cc-CATH3(1-29) share a similar trend with that in Cc-CATH3(5-29); they exhibit no preferential binding to anionic phospholipids. In conclusion, the results suggested that the first four residues at the N-terminus "RVRR" is not essential for presenting all test activities. In contrast, residues five to eight of "FWPL" are necessary as the exclusion of this short motif in Cc-CATH3(9-29) and Cc-CATH3(13-29) leads to a loss of activities. This study will be beneficial for further design and development of Cc-CATH3 to be novel antibiotic.

  3. A novel N-terminal motif is responsible for the evolution of neural crest-specific gene-regulatory activity in vertebrate FoxD3.

    PubMed

    Ono, Hiroki; Kozmik, Zbynek; Yu, Jr-Kai; Wada, Hiroshi

    2014-01-15

    The neural crest is unique to vertebrates and has allowed the evolution of their complicated craniofacial structures. During vertebrate evolution, the acquisition of the neural crest must have been accompanied by the emergence of a new gene regulatory network (GRN). Here, to investigate the role of protein evolution in the emergence of the neural crest GRN, we examined the neural crest cell (NCC) differentiation-inducing activity of chordate FoxD genes. Amphioxus and vertebrate (Xenopus) FoxD proteins both exhibited transcriptional repressor activity in Gal4 transactivation assays and bound to similar DNA sequences in vitro. However, whereas vertebrate FoxD3 genes induced the differentiation of ectopic NCCs when overexpressed in chick neural tube, neither amphioxus FoxD nor any other vertebrate FoxD paralogs exhibited this activity. Experiments using chimeric proteins showed that the N-terminal portion of the vertebrate FoxD3 protein is critical to its NCC differentiation-inducing activity. Furthermore, replacement of the N-terminus of amphioxus FoxD with a 39-amino-acid segment from zebrafish FoxD3 conferred neural crest-inducing activity on amphioxus FoxD or zebrafish FoxD1. Therefore, fixation of this N-terminal amino acid sequence may have been crucial in the evolutionary recruitment of FoxD3 to the vertebrate neural crest GRN. © 2013 Published by Elsevier Inc.

  4. Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure.

    PubMed

    Sakaguchi, Masayoshi; Matsushima, Yudai; Nagamine, Yusuke; Matsuhashi, Tomoki; Honda, Shotaro; Okuda, Shoi; Ohno, Misa; Sugahara, Yasusato; Shin, Yongchol; Oyama, Fumitaka; Kawakita, Masao

    2017-03-01

    Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.

  5. Synthesis and optimization of thiadiazole derivatives as a novel class of substrate competitive c-Jun N-terminal kinase inhibitors

    PubMed Central

    De, Surya K.; Chen, Vida; Stebbins, John L.; Chen, Li-Hsing; Cellitti, Jason F.; Machleidt, Thomas; Barile, Elisa; Riel-Mehan, Megan; Dahl, Russell; Yang, Li; Emdadi, Aras; Murphy, Ria; Pellecchia, Maurizio

    2009-01-01

    A series of thiadiazole derivatives has been designed as potential allosteric, substrate competitive inhibitors of the protein kinase JNK. We report on the synthesis, characterization and evaluation of a series of compounds that resulted in the identification of potent and selective JNK inhibitors targeting its JIP-1 docking site. PMID:20045647

  6. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  7. N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile.

    PubMed

    Weltrowska, Grazyna; Nguyen, Thi M-D; Chung, Nga N; Wilkes, Brian C; Schiller, Peter W

    2013-09-15

    Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist activity. Guanidinylation of the mixed μ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) and H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]) converted them to mixed μ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH2 and Guan-DIPP-NH2 in the δ receptor binding site. Lys(3)-analogues of DIPP-NH2 and DIPP-NH2[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed μ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction.

  8. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    SciTech Connect

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F.

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  9. Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

    SciTech Connect

    Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya; Szukalska, Gabriela; Banerjee, Budhaditya; Hilinski, Michael K.; Lannigan, Deborah A.; Stukenberg, P. Todd; Derewenda, Zygmunt S.

    2012-09-11

    The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-a-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 {angstrom} resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the aD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.

  10. The drosophila T-box transcription factor midline functions within Insulin/Akt and c-Jun-N terminal kinase stress-reactive signaling pathways to regulate interommatial bristle formation and cell survival

    PubMed Central

    Chen, Q. Brent; Das, Sudeshna; Visic, Petra; Buford, Kendrick D.; Zong, Yan; Buti, Wisam; Odom, Kelly R.; Lee, Hannah; Leal, Sandra M.

    2015-01-01

    We recently reported that the T-box transcription factor midline (mid) functions within the Notch-Delta signaling pathway to specify sensory organ precursor (SOP) cell fates in early-staged pupal eye imaginal discs and to suppress apoptosis (Das et al.). From genetic and allelic modifier screens, we now report that mid interacts with genes downstream of the insulin receptor(InR)/Akt, c-Jun-N-terminal kinase (JNK) and Notch signaling pathways to regulate interommatidial bristle (IOB) formation and cell survival. One of the most significant mid-interacting genes identified from the modifier screen is dFOXO, a transcription factor exhibiting a nucleocytoplasmic subcellular distribution pattern. In common with dFOXO, we show that Mid exhibits a nucleocytoplasmic distribution pattern within WT third-instar larval (3°L) tissue homogenates. Because dFOXO is a stress-responsive factor, we assayed the effects of either oxidative or metabolic stress responses on modifying the mid mutant phenotype which is characterized by a 50% loss of IOBs within the adult compound eye. While metabolic starvation stress does not affect the mid mutant phenotype, either 1 mM paraquat or 20% coconut oil, oxidative stress inducers, partially suppresses the mid mutant phenotype resulting in a significant recovery of IOBs. Another significant mid-interacting gene we identified is groucho (gro). Mid and Gro are predicted to act as corepressors of the enhancer-of-split gene complex downstream of Notch. Immunolabeling WT and dFOXO null 3°L eye-antennal imaginal discs with anti-Mid and anti-Engrailed (En) antibodies indicate that dFOXO is required to activate Mid and En expression within photoreceptor neurons of the eye disc. Taken together, these studies show that Mid and dFOXO serve as critical effectors of cell fate specification and survival within integrated Notch, InR/dAkt, and JNK signaling pathways during 3°L and pupal eye imaginal disc development. PMID:25748605

  11. The drosophila T-box transcription factor midline functions within Insulin/Akt and c-Jun-N terminal kinase stress-reactive signaling pathways to regulate interommatial bristle formation and cell survival.

    PubMed

    Chen, Q Brent; Das, Sudeshna; Visic, Petra; Buford, Kendrick D; Zong, Yan; Buti, Wisam; Odom, Kelly R; Lee, Hannah; Leal, Sandra M

    2015-05-01

    We recently reported that the T-box transcription factor midline (mid) functions within the Notch-Delta signaling pathway to specify sensory organ precursor (SOP) cell fates in early-staged pupal eye imaginal discs and to suppress apoptosis (Das et al.). From genetic and allelic modifier screens, we now report that mid interacts with genes downstream of the insulin receptor(InR)/Akt, c-Jun-N-terminal kinase (JNK) and Notch signaling pathways to regulate interommatidial bristle (IOB) formation and cell survival. One of the most significant mid-interacting genes identified from the modifier screen is dFOXO, a transcription factor exhibiting a nucleocytoplasmic subcellular distribution pattern. In common with dFOXO, we show that Mid exhibits a nucleocytoplasmic distribution pattern within WT third-instar larval (3(o)L) tissue homogenates. Because dFOXO is a stress-responsive factor, we assayed the effects of either oxidative or metabolic stress responses on modifying the mid mutant phenotype which is characterized by a 50% loss of IOBs within the adult compound eye. While metabolic starvation stress does not affect the mid mutant phenotype, either 1 mM paraquat or 20% coconut oil, oxidative stress inducers, partially suppresses the mid mutant phenotype resulting in a significant recovery of IOBs. Another significant mid-interacting gene we identified is groucho (gro). Mid and Gro are predicted to act as corepressors of the enhancer-of-split gene complex downstream of Notch. Immunolabeling WT and dFOXO null 3(o)L eye-antennal imaginal discs with anti-Mid and anti-Engrailed (En) antibodies indicate that dFOXO is required to activate Mid and En expression within photoreceptor neurons of the eye disc. Taken together, these studies show that Mid and dFOXO serve as critical effectors of cell fate specification and survival within integrated Notch, InR/dAkt, and JNK signaling pathways during 3(o)L and pupal eye imaginal disc development.

  12. Regulation of multidrug resistance protein 1 by tumor necrosis factor alpha in cultured glial cells: involvement of nuclear factor-kappaB and c-Jun N-terminal kinase signaling pathways.

    PubMed

    Ronaldson, Patrick T; Ashraf, Tamima; Bendayan, Reina

    2010-04-01

    Pharmacotherapy of brain HIV-1 infection may be limited by ABC transporters [i.e., P-glycoprotein (P-gp), multidrug resistance protein 1 (Mrp1)] that export antiretroviral drugs from HIV-1 brain cellular targets (i.e., astrocytes, microglia). Using an in vitro astrocyte model of an HIV-1 associated inflammatory response, our laboratory has shown that cytokines [i.e., tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta, IL-6], which are secreted in response to HIV-1 envelope glycoprotein gp120 exposure, can decrease P-gp functional expression; however, it is unknown whether these same cytokines can alter expression and/or activity of other ABC transporters (i.e., Mrp1). In primary cultures of rat astrocytes, Mrp1 expression was increased by TNF-alpha (2.7-fold) but was not altered by IL-1 beta or IL-6. Cellular retention of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, an Mrp substrate, was reduced in TNF-alpha-treated astrocytes, suggesting increased Mrp-mediated transport. Pharmacologic inhibition of nuclear factor-kappaB (NF-kappaB) signaling with SN50 prevented both TNF-alpha release and Mrp1 expression changes in astrocytes triggered with gp120; however, SN50 did not attenuate Mrp1 expression in cells triggered with TNF-alpha. In contrast, Mrp1 functional expression was not altered in the presence of gp120 or TNF-alpha when astrocyte cultures were pretreated with 1,9-pyrazoloanthrone (SP600125), an established c-Jun N-terminal kinase (JNK) inhibitor. SP600125 did not affect TNF-alpha release from cultured astrocytes triggered with gp120. Mrp1 mRNA expression was increased after treatment with gp120 (1.6-fold) or TNF-alpha (1.7-fold), suggesting altered Mrp1 gene transcription. These data suggest that gp120 and TNF-alpha can up-regulate Mrp1 expression in cultured astrocytes. Furthermore, our results imply that both NF-kappaB and JNK signaling are involved in Mrp1 regulation during an HIV-1 associated inflammatory response.

  13. P21 activated kinases

    PubMed Central

    Rane, Chetan K; Minden, Audrey

    2014-01-01

    The p21 activated kinases (Paks) are well known effector proteins for the Rho GTPases Cdc42 and Rac. The Paks contain 6 members, which fall into 2 families of proteins. The first family consists of Paks 1, 2, and 3, and the second consists of Paks 4, 5, and 6. While some of the Paks are ubiquitously expressed, others have more restrictive tissue specificity. All of them are found in the nervous system. Studies using cell culture, transgenic mice, and knockout mice, have revealed important roles for the Paks in cytoskeletal organization and in many aspects of cell growth and development. This review discusses the basic structures of the Paks, and their roles in cell growth, development, and in cancer. PMID:24658305

  14. 4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage.

    PubMed

    Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

    2017-07-14

    γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

  15. 4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

    PubMed Central

    Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

    2017-01-01

    γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C–C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria. PMID:28706296

  16. 4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

    NASA Astrophysics Data System (ADS)

    Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

    2017-07-01

    γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

  17. Raman optical activity demonstrates poly(L-proline) II helix in the N-terminal region of the ovine prion protein: implications for function and misfunction.

    PubMed

    Blanch, Ewan W; Gill, Andrew C; Rhie, Alexandre G O; Hope, James; Hecht, Lutz; Nielsen, Kurt; Barron, Laurence D

    2004-10-15

    The aqueous solution structure of the full-length recombinant ovine prion protein PrP(25-233), together with that of the N-terminal truncated version PrP(94-233), have been studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UVCD). A sharp positive band at approximately 1315 cm(-1) characteristic of poly(L-proline) II (PPII) helix that is present in the ROA spectrum of the full-length protein is absent from that of the truncated protein, together with bands characteristic of beta-turns. Although it is not possible similarly to identify PPII helix in the full-length protein directly from its UVCD spectrum, subtraction of the UVCD spectrum of PrP(94-233) from that of PrP(25-233) yields a difference UVCD spectrum also characteristic of PPII structure and very similar to the UVCD spectrum of murine PrP(25-113). These results provide confirmation that a major conformational element in the N-terminal region is PPII helix, but in addition show that the PPII structure is interspersed with beta-turns and that little PPII structure is present in PrP(94-233). A principal component analysis of the ROA data indicates that the alpha-helix and beta-sheet content, located in the structured C-terminal domain, of the full-length and truncated proteins are similar. The flexibility imparted by the high PPII content of the N-terminal domain region may be an essential factor in the function and possibly also the misfunction of prion proteins.

  18. Evidence of mineralization activity and supramolecular assembly by the N-terminal sequence of ACCBP, a biomineralization protein that is homologous to the acetylcholine binding protein family.

    PubMed

    Amos, Fairland F; Ndao, Moise; Evans, John Spencer

    2009-12-14

    Several biomineralization proteins that exhibit intrinsic disorder also possess sequence regions that are homologous to nonmineral associated folded proteins. One such protein is the amorphous calcium carbonate binding protein (ACCBP), one of several proteins that regulate the formation of the oyster shell and exhibit 30% conserved sequence identity to the acetylcholine binding protein sequences. To gain a better understanding of the ACCBP protein, we utilized bioinformatic approaches to identify the location of disordered and folded regions within this protein. In addition, we synthesized a 50 AA polypeptide, ACCN, representing the N-terminal domain of the mature processed ACCBP protein. We then utilized this polypeptide to determine the mineralization activity and qualitative structure of the N-terminal region of ACCBP. Our bioinformatic studies indicate that ACCBP consists of a ten-stranded beta-sandwich structure that includes short disordered sequence blocks, two of which reside within the primarily helical and surface-accessible ACCN sequence. Circular dichroism studies reveal that ACCN is partially disordered in solution; however, ACCN can be induced to fold into an alpha helix in the presence of TFE. Furthermore, we confirm that the ACCN sequence is multifunctional; this sequence promotes radial calcite polycrystal growth on Kevlar threads and forms supramolecular assemblies in solution that contain amorphous-appearing deposits. We conclude that the partially disordered ACCN sequence is a putative site for mineralization activity within the ACCBP protein and that the presence of short disordered sequence regions within the ACCBP fold are essential for function.

  19. Characterization of an invertase with pH tolerance and truncation of its N-terminal to shift optimum activity toward neutral pH.

    PubMed

    Du, Liqin; Pang, Hao; Wang, Zilong; Lu, Jian; Wei, Yutuo; Huang, Ribo

    2013-01-01

    Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.

  20. Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins.

    PubMed

    Vickaryous, Nicola K; Teh, Evelyn M; Stewart, Bruce; Dolphin, Peter J; Too, Catherine K L; McLeod, Roger S

    2003-03-21

    Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the

  1. Autophosphorylation Activity of a Soluble Hexameric Histidine Kinase Correlates with the Shift in Protein Conformational Equilibrium

    PubMed Central

    Wojnowska, Marta; Yan, Jun; Sivalingam, Ganesh N.; Cryar, Adam; Gor, Jayesh; Thalassinos, Konstantinos; Djordjevic, Snezana

    2013-01-01

    Summary In a commonly accepted model, in response to stimuli, bacterial histidine kinases undergo a conformational transition between an active and inactive form. Structural information on histidine kinases is limited. By using ion mobility-mass spectrometry (IM-MS), we demonstrate an exchange between two conformational populations of histidine kinase ExsG that are linked to different levels of kinase activity. ExsG is an atypical signaling protein that incorporates an uncommon histidine kinase catalytic core at the C terminus preceded by an N-terminal “receiver domain” that is normally associated with the response regulator proteins in two-component signal transduction systems. IM-MS analysis and enzymatic assays indicate that phosphorylation of the ExsG receiver domain stabilizes the “compact” form of the protein and inhibits kinase core activity; in contrast, nucleotide binding required for kinase activity is associated with the more open conformation of ExsG. PMID:24210218

  2. MED14 tethers mediator to the N-terminal domain of peroxisome proliferator-activated receptor gamma and is required for full transcriptional activity and adipogenesis.

    PubMed

    Grøntved, Lars; Madsen, Maria S; Boergesen, Michael; Roeder, Robert G; Mandrup, Susanne

    2010-05-01

    The Mediator subunit MED1/TRAP220/DRIP205/PBP interacts directly with many nuclear receptors and was long thought to be responsible for tethering Mediator to peroxisome proliferator-activated receptor (PPAR)-responsive promoters. However, it was demonstrated recently that PPARgamma can recruit Mediator by MED1-independent mechanisms. Here, we show that target gene activation by ectopically expressed PPARgamma and PPARalpha is independent of MED1. Consistent with this finding, recruitment of PPARgamma, MED6, MED8, TATA box-binding protein (TBP), and RNA polymerase II (RNAPII) to the enhancer and proximal promoter of the PPARgamma target gene Fabp4 is also independent of MED1. Using a small interfering RNA (siRNA)-based approach, we identify MED14 as a novel critical Mediator component for PPARgamma-dependent transactivation, and we demonstrate that MED14 interacts directly with the N terminus of PPARgamma in a ligand-independent manner. Interestingly, MED14 knockdown does not affect the recruitment of PPARgamma, MED6, and MED8 to the Fabp4 enhancer but does reduce their occupancy of the Fabp4 proximal promoter. In agreement with the necessity of MED14 for PPARgamma transcriptional activity, we show that knockdown of MED14 impairs adipogenesis of 3T3-L1 cells. Thus, MED14 constitutes a novel anchoring point between Mediator and the N-terminal domain of PPARgamma that is necessary for functional PPARgamma-mediated recruitment of Mediator and transactivation of PPARgamma subtype-specific target genes.

  3. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: The N-terminal region is more important than enzymatic activity

    PubMed Central

    Rouault, Morgane; Rash, Lachlan D.; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H.; Lambeau, Gérard

    2009-01-01

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, an homologous but non toxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1–22) of OS2, but not the central one (residues 58–89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102–119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. PMID:16669624

  4. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.

    PubMed

    Rouault, Morgane; Rash, Lachlan D; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H; Lambeau, Gérard

    2006-05-09

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity.

  5. Role of N-Terminal Hydrophobic Region in Modulating the Subcellular Localization and Enzyme Activity of the Bisphosphate Nucleotidase from Debaryomyces hansenii

    PubMed Central

    Aggarwal, Monika; Mondal, Alok K.

    2006-01-01

    3′, 5′-Bisphosphate nucleotidase is a ubiquitous enzyme that converts 3′-phosphoadenosine-5′-phosphate to adenosine-5′-phosphate and inorganic phosphate. These enzymes are highly sensitive to sodium and lithium and, thus, perform a crucial rate-limiting metabolic step during salt stress in yeast. Recently, we have identified a bisphosphate nucleotidase gene (DHAL2) from the halotolerant yeast Debaryomyces hansenii. One of the unique features of Dhal2p is that it contains an N-terminal 54-amino-acid-residue hydrophobic extension. In this study, we have shown that Dhal2p exists as a cytosolic as well as a membrane-bound form and that salt stress markedly influences the accumulation of the latter form in the cell. We have demonstrated that the N-terminal hydrophobic region was necessary for the synthesis of the membrane-bound isoform. It appeared that an alternative translation initiation was the major mechanism for the synthesis of these two forms. Moreover, the two forms exhibit significant differences in their substrate specificity. Unlike the cytosolic form, the membrane-bound form showed very high activity against inositol-1,4-bisphosphate. Thus, the present study for the first time reports the existence of multiple forms of a bisphosphate nucleotidase in any organism. PMID:16467467

  6. Human/bovine chimeric MxA-like GTPases reveal a contribution of N-terminal domains to the magnitude of anti-influenza A activity.

    PubMed

    Garigliany, Mutien-Marie; Cornet, Anne; Desmecht, Daniel

    2012-07-01

    Type I interferons (IFN-α/β) provide powerful and universal innate intracellular defense mechanisms against viruses. Among the antiviral effectors induced by IFN-α/β, Mx proteins of some species appear as key components of defense against influenza A viruses. The body of work published to date suggests that to exert anti-influenza activity, an Mx protein should possess a GTP-binding site, structural bases allowing multimerisation, and a specific C-terminal GTPase effector domain (GED). Both the human MxA and bovine Mx1 proteins meet these minimal requirements, but the bovine protein is more active against influenza viruses. Here, we measured the anti-influenza activity exerted by 2 human/bovine chimeric Mx proteins. We show that substituting the bovine GED for the human one in human MxA does not affect the magnitude of anti-influenza activity. Strikingly, however, substituting the human GED for the bovine one in bovine Mx1 yields a chimeric protein with a much higher anti-influenza activity than the human protein. We conclude, in contradiction to the hypothesis currently in vogue in the literature, that the GED is not the sole determinant controlling the magnitude of the anti-influenza activity exercised by an Mx protein that can bind GTP and multimerise. Our results suggest that 1 or several motifs that remain to be discovered, located N-terminally with regard to the GED, may interact with a viral component or a cellular factor so as to alter the viral cycle. Identifying, in the N-terminal portion of bovine Mx1, the motif(s) responsible for its higher anti-influenza activity could contribute to the development of new anti-influenza molecules.

  7. The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.

    PubMed

    Jongruja, Nujarin; You, Dong-Ju; Kanaya, Eiko; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2010-11-01

    Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) contains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-terminal domain containing the HBD (Tma-ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg(2+) to Mn(2+) for activity, and specifically loses most of the Mg(2+)-dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage-site specificities of Tma-RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma-RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg(2+)-dependent activity and substrate binding affinity. Tma-CD was as stable as Tma-RNase HI, indicating that the HBD is not important for stability. The HBD of Tma-RNase HI is important not only for substrate binding, but also for Mg(2+)-dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg(2+) ion are arranged ideally.

  8. The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity.

    PubMed

    Yu, Rongjie; Liu, Hongyu; Peng, Xinhe; Cui, Yue; Song, Suqin; Wang, Like; Zhang, Huahua; Hong, An; Zhou, Tianhong

    2017-06-27

    VPAC1 is class B G protein-coupled receptors (GPCR) shared by pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). The first cysteine (Cys37) in the N-terminal extracellular domain of mature VPAC1 is a free Cys not involved in the formation of conserved intramolecular disulfide bonds. In order to investigate the biological role of this Cys37 in VPAC1, the wild-type VPAC1 and Cys37/Ala mutant (VPAC1-C37/A) were expressed stably as fusion proteins with enhanced yellow fluorescent protein (EYFP) respectively in Chinese hamster ovary (CHO) cells. Both VPAC1-EYFP and VPAC1-C37/A-EYFP trafficked to the plasma membrane normally, and CHO cells expressing VPAC1-EYFP displayed higher anti-apoptotic activity against camptothecin (CPT) induced apoptosis than the cells expressing VPAC1-C37/A-EYFP, while VPAC1-C37/A-CHO cells showed higher proliferative activity than VPAC1-CHO cells. Confocal microscopic analysis, western blotting and fluorescence quantification assay showed VPAC1-EYFP displayed significant nuclear translocation while VPAC1-C37/A-EYFP did not transfer into nucleus under the stimulation of VIP (0.1 nM). Acyl-biotin exchange assay and click chemistry-based palmitoylation assay confirmed for the first time the palmitoylation of Cys37, which has been predicted by bioinformatics analysis. And the palmitoylation inhibitor 2-bromopalmitate significantly inhibited the nuclear translocation of VPAC1-EYFP and its anti-apoptotic activity synchronously. These results indicated the palmitoylation of the Cys37 in the N-terminal extracellular domain of VPAC1 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity. These findings reveal for the first time the lipidation-mediating nuclear translocation of VPAC1 produces a novel anti-apoptotic signal pathway, which may help to promote new drug development strategy targeting VPAC1.

  9. An Ankyrin-G N-terminal Gate and Protein Kinase CK2 Dually Regulate Binding of Voltage-gated Sodium and KCNQ2/3 Potassium Channels.

    PubMed

    Xu, Mingxuan; Cooper, Edward C

    2015-07-03

    In many mammalian neurons, fidelity and robustness of action potential generation and conduction depends on the co-localization of voltage-gated sodium (Nav) and KCNQ2/3 potassium channel conductance at the distal axon initial segment (AIS) and nodes of Ranvier in a ratio of ∼40 to 1. Analogous "anchor" peptides within intracellular domains of vertebrate KCNQ2, KCNQ3, and Nav channel α-subunits bind Ankyrin-G (AnkG), thereby mediating concentration of those channels at AISs and nodes of Ranvier. Here, we show that the channel anchors bind at overlapping but distinct sites near the AnkG N terminus. In pulldown assays, the rank order of AnkG binding strength is Nav1.2 ≫ KCNQ3 > KCNQ2. Phosphorylation of KCNQ2 and KCNQ3 anchor domains by protein kinase CK2 (CK2) augments binding, as previously shown for Nav1.2. An AnkG fragment comprising ankyrin repeats 1 through 7 (R1-7) binds phosphorylated Nav or KCNQ anchors robustly. However, mutational analysis of R1-7 reveals differences in binding mechanisms. A smaller fragment, R1-6, exhibits much-diminished KCNQ3 binding but binds Nav1.2 well. Two lysine residues at the tip of repeat 2-3 β-hairpin (residues 105-106) are critical for Nav1.2 but not KCNQ3 channel binding. Another dibasic motif (residues Arg-47, Arg-50) in the repeat 1 front α-helix is crucial for KCNQ2/3 but not Nav1.2 binding. AnkG's alternatively spliced N terminus selectively gates access to those sites, blocking KCNQ but not Nav channel binding. These findings suggest that the 40:1 Nav:KCNQ channel conductance ratio at the distal AIS and nodes arises from the relative strength of binding to AnkG.

  10. The Anti-Candida Vaccine Based on the Recombinant N-Terminal Domain of Als1p Is Broadly Active against Disseminated Candidiasis

    PubMed Central

    Ibrahim, Ashraf S.; Spellberg, Brad J.; Avanesian, Valentina; Fu, Yue; Edwards, John E.

    2006-01-01

    We have previously shown that vaccination with a vaccine based on the recombinant N-terminal domain of Als1p (rAls1p-N) protected BALB/c mice against disseminated infection caused by a single strain of Candida albicans (A. S. Ibrahim, B. J. Spellberg, V. Avenissian, Y. Fu, S. G. Filler, and J. E. Edwards, Jr., Infect. Immun. 73:999-1005, 2005, and B. J. Spellberg, A. S. Ibrahim, V. Avenissian, S. G. Filler, C. Myers, Y. Fu, and J. E. Edwards, Jr., Infect. Immun. 73:6191-6193, 2005). Here we show that the rAls1p-N vaccine also improves survival of outbred mice from disseminated candidiasis and that it is active against multiple virulent strains of C. albicans and non-C. albicans spp. PMID:16622247

  11. Unveiling the Membrane-Binding Properties of N-Terminal and C-Terminal Regions of G Protein-Coupled Receptor Kinase 5 by Combined Optical Spectroscopies

    PubMed Central

    2015-01-01

    G protein-coupled receptor kinase 5 (GRK5) is thought to associate with membranes in part via N- and C-terminal segments that are typically disordered in available high-resolution crystal structures. Herein we investigate the interactions of these regions with model cell membrane using combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy. It was found that both regions associate with POPC lipid bilayers but adopt different structures when doing so: GRK5 residues 2–31 (GRK52–31) was in random coil whereas GRK5546–565 was partially helical. When the subphase for the GRK52–31 peptide was changed to 40% TFE/60% 10 mM phosphate pH 7.4 buffer, a large change in the SFG amide I signal indicated that GRK52–31 became partially helical. By inspecting the membrane behavior of two different segments of GRK52–31, namely, GRK52–24 and GRK525–31, we found that residues 25–31 are responsible for membrane binding, whereas the helical character is imparted by residues 2–24. With SFG, we deduced that the orientation angle of the helical segment of GRK52–31 is 46 ± 1° relative to the surface normal in 40% TFE/60% 10 mM phosphate pH = 7.4 buffer but increases to 78 ± 11° with higher ionic strength. We also investigated the effect of PIP2 in the model membrane and concluded that the POPC:PIP2 (9:1) lipid bilayer did not change the behavior of either peptide compared to a pure POPC lipid bilayer. With ATR-FTIR, we also found that Ca2+·calmodulin is able to extract both peptides from the POPC lipid bilayer, consistent with the role of this protein in disrupting GRK5 interactions with the plasma membrane in cells. PMID:24401145

  12. Role of the N-terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin.

    PubMed Central

    Findlay, W. A.; Gradwell, M. J.; Bayley, P. M.

    1995-01-01

    The binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk-MLCK) target sequence has been studied using 1H-NMR. The 18-residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C-domain and the Phe 17 side chain with the N-domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H-NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C-domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C-domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H-NMR spectra of CaM bound to WFF, a Trp 4-->Phe analogue (FFF), or a Trp 4-->Phe/Phe 17-->Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C-domain and the side chain in position 17 interacting with the N-domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34:2087-2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N-domain of CaM. PMID:8563635

  13. Identifying the activation motif in the N-terminal of rainbow trout and zebrafish melanocortin-2 receptor accessory protein 1 (MRAP1) orthologs.

    PubMed

    Dores, Robert M; Liang, Liang; Hollmann, Rebecca E; Sandhu, Navdeep; Vijayan, Mathilakath M

    2016-08-01

    The activation of mammalian melanocortin-2 receptor (MC2R) orthologs is dependent on a four-amino acid activation motif (LDYL/I) located in the N-terminal of mammalian MRAP1 (melanocortin-2 receptor accessory protein). Previous alanine substitution analysis had shown that the Y residue in this motif appears to be the most important for mediating the activation of mammalian MC2R orthologs. Similar, but not identical amino acid motifs were detected in rainbow trout MRAP1 (YDYL) and zebrafish MRAP1 (YDYV). To determine the importance of these residues in the putative activation motifs, rainbow trout and zebrafish MRAP1 orthologs were individually co-expressed in CHO cells with rainbow trout MC2R, and the activation of this receptor with either the wild-type MRAP1 ortholog or alanine-substituted analogs of the two teleost MRAP1s was analyzed. Alanine substitutions at all four amino acid positions in rainbow trout MRAP1 blocked activation of the rainbow trout MC2R. Single alanine substitutions of the D and Y residues in rainbow trout and zebrafish MRAP1 indicate that these two residues play a significant role in the activation of rainbow trout MC2R. These observations indicate that there are subtle differences in the way that teleost and mammalian MRAPs are involved in the activation of their corresponding MC2R orthologs. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Increase of RhoB in gamma-radiation-induced apoptosis is regulated by c-Jun N-terminal kinase in Jurkat T cells.

    PubMed

    Kim, Chun-Ho; Won, Misun; Choi, Chung-Hae; Ahn, Jiwon; Kim, Bo-Kyung; Song, Kyung-Bin; Kang, Chang-Mo; Chung, Kyung-Sook

    2010-01-08

    The Ras-related small GTP-binding protein RhoB is known to be a pro-apoptotic protein and immediate-early inducible by genotoxic stresses. In addition, JNK activation is known to function in gamma-radiation-induced apoptosis. However, it is unclear how JNK activation and gamma-radiation-dependent RhoB induction are related. Here we verified the relationship between JNK activation and RhoB induction. RhoB induction by gamma-radiation occurred at the transcriptional level and transcriptional activation of RhoB was concomitant with an increase in RhoB protein. gamma-Radiation-induced RhoB expression was markedly attenuated by pretreatment with a JNK-specific inhibitor, SP600125, but not by a p38 MAPK inhibitor, SB203580. Inhibition of JNK caused a decrease in early apoptotic cell death that correlated with RhoB expression. However, PI3K inhibition had no significant effects, indicating that the AKT survival pathway was not involved. The siRNA knockdown of JNK resulted in a decrease in RhoB expression and the siRNA knockdown of RhoB restored cell growth even in the gamma-irradiated cells. These results suggest that RhoB regulation involves the JNK pathway and contributes to the early apoptotic response of Jurkat T cells to gamma-radiation. Copyright 2009 Elsevier Inc. All rights reserved.

  15. Oncoprotein protein kinase

    DOEpatents

    Karin, M.; Hibi, M.; Lin, A.

    1997-02-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

  16. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  18. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  19. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  20. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  1. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  2. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  3. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  4. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression

    PubMed Central

    Pfefferle, Adam D.; Perou, Charles M.; Van Den Berg, Carla Lynn

    2015-01-01

    Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis. PMID:25970777

  6. TBP Is Differentially Regulated by c-Jun N-Terminal Kinase 1 (JNK1) and JNK2 through Elk-1, Controlling c-Jun Expression and Cell Proliferation▿

    PubMed Central

    Zhong, Shuping; Fromm, Jody; Johnson, Deborah L.

    2007-01-01

    Emerging evidence supports the idea that the c-Jun N-terminal kinases (JNKs) possess overlapping but distinct functions. The potential roles of the ubiquitously expressed JNK1 and JNK2 in regulating expression of the central transcription initiation factor, TATA-binding protein (TBP), were examined. Relative to wild-type fibroblasts, TBP was decreased in Jnk1−/− cells and increased in Jnk2−/− cells. Similarly, reduction of JNK1 in human hepatoma cells decreased TBP expression, whereas reduction of JNK2 enhanced it. JNK-mediated regulation of TBP expression occurs at the transcriptional level through their ability to target Elk-1, which directly regulates the TBP promoter in response to epidermal growth factor stimulation. JNK1 increases, whereas JNK2 decreases, the phosphorylation state of Elk-1, which differentially affects Elk-1 occupancy at a defined site within the TBP promoter. These JNK-mediated alterations in TBP expression, alone, serve to regulate c-Jun expression and fibroblast proliferation rates. These studies uncovered several new molecular events that distinguish the functions of JNK1 and JNK2 that are critical for their regulation of cellular proliferation. PMID:17074809

  7. c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression.

    PubMed

    Cantrell, Michael A; Ebelt, Nancy D; Pfefferle, Adam D; Perou, Charles M; Van Den Berg, Carla Lynn

    2015-05-20

    Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis.

  8. A major transactivator of varicella-zoster virus, the immediate-early protein IE62, contains a potent N-terminal activation domain.

    PubMed Central

    Perera, L P; Mosca, J D; Ruyechan, W T; Hayward, G S; Straus, S E; Hay, J

    1993-01-01

    Accumulating evidence indicates that the product of the putative immediate-early gene ORF62 (IE62) activates varicella-zoster virus (VZV) genes thought to represent all three kinetic classes, namely, immediate-early (alpha), early (beta), and late (gamma) classes, of VZV genes as well as a variety heterologous gene promoters. However, the mechanism(s) by which IE62 protein mediates transactivation of these diverse VZV and heterologous gene promoters remains to be elucidated. In this study, by using yeast GAL4 protein chimeras, the coding regions of VZV ORF62 possessing activation domains have been assessed. We demonstrate that the VZV IE62 protein contains a potent activation domain in the N-terminal portion of the molecule, encoded within the first 86 codons of ORF62. The predicted secondary structure profile and the acid-base composition of this IE62 domain resemble those of other transregulatory proteins whose activation is mediated through acidic, hydrophobic elements. In addition, we show that deletion of this activation domain from the 1,310-residue native IE62 protein results in ablation of the transactivator function of IE62. We also present evidence that the mutant IE62 protein lacking the activation domain, though devoid of transactivation ability, was still capable of interfering with the activation of target promoters by the native, full-length IE62. Images PMID:8392592

  9. Pharmacokinetic and tissue distribution studies of 1,9-pyrazoloanthrone, a c-Jun-N-terminal kinase inhibitor in Wistar rats by a simple and sensitive HPLC method.

    PubMed

    Ambhore, Nilesh Sudhakar; Yamjala, Karthik; Mohire, Shubhashri; Raju, Kalidhindi Rama Satyanarayana; Mulukutla, Shashank; Murthy, Vishakantha; Tondhawada, Mahesh; Elango, Kannan

    2016-02-20

    JNK pathway activates c-Jun(s) which are responsible for cell apoptosis; as a result, inhibitors of JNK pathway have the potential to prevent dopaminergic neurons from death and decrease the loss of dopamine in substantia nigra pars compacta (SNpc). Recent in-vitro studies show that 1,9-pyrazoloanthrone (1,9-P) a potent JNK-3 inhibitor prevents the apoptosis of dopaminergic cells of brain. In the present study we formulated liposomes to increase the bioavailability of 1,9-P in the brain and developed a simple, sensitive and selective high performance liquid chromatographic method and validated for the estimation of 1,9-P in Wistar rat plasma and tissue samples. Plasma and tissue samples were extracted by protein precipitation technique using acetonitrile (ACN) and rasagiline as the internal standards. Chromatography was performed on Hibar C18 column with mobile phase of ammonium acetate (10mM, pH 8.0 adjusted with ammonia) and ACN at a flow rate of 1mL/min. The lower limit of quantification of the developed method was found to be 2.0ng/mL and 4.0ng/g in plasma and tissue samples respectively. The liposomes of 1,9-P administered to animals at the dose equivalent to 15mg/kg orally demonstrated remarkable absorption into the systemic circulation with maximum concentration (∼7500ng/mL) within 2.0h. The order of the area under curve was found to be kidney>liver>brain>lungs>spleen>heart. The liposomes of 1,9-P were rapidly taken up into brain and showed a good brain concentration after 2.0h; sustenance up to 4.0h was achieved which is better than 1,9-P solution.

  10. A multilayered regulatory mechanism for the autoinhibition and activation of a plant CC-NB-LRR resistance protein with an extra N-terminal domain.

    PubMed

    Chen, Xiaojiao; Zhu, Min; Jiang, Lei; Zhao, Wenyang; Li, Jia; Wu, Jianyan; Li, Chun; Bai, Baohui; Lu, Gang; Chen, Hongyu; Moffett, Peter; Tao, Xiaorong

    2016-10-01

    The tomato resistance protein Sw-5b differs from the classical coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) resistance proteins by having an extra N-terminal domain (NTD). To understand how NTD, CC and NB-LRR regulate autoinhibition and activation of Sw-5b, we dissected the function(s) of each domain. When viral elicitor was absent, Sw-5b LRR suppressed the central NB-ARC to maintain autoinhibition of the NB-LRR segment. The CC and NTD domains independently and additively enhanced the autoinhibition of NB-LRR. When viral elicitor was present, the NB-LRR segment of Sw-5b was specifically activated to trigger a hypersensitive response. Surprisingly, Sw-5b CC suppressed the activation of NB-LRR, whereas the extra NTD of Sw-5b became a positive regulator and fully activated the resistance protein, probably by relieving the inhibitory effects of the CC. In infection assays of transgenic plants, the NB-LRR segment alone was insufficient to confer resistance against Tomato spotted wilt tospovirus; the layers of NTD and CC regulation on NB-LRR were required for Sw-5b to confer resistance. Based on these findings, we propose that, to counter the negative regulation of the CC on NB-LRR, Sw-5b evolved an extra NTD to coordinate with the CC, thus developing a multilayered regulatory mechanism to control autoinhibition and activation.

  11. Lipid sulfates and sulfonates are allosteric competitive inhibitors of the N-terminal phosphatase activity of the mammalian soluble epoxide hydrolase.

    PubMed

    Tran, Katherine L; Aronov, Pavel A; Tanaka, Hiromasa; Newman, John W; Hammock, Bruce D; Morisseau, Christophe

    2005-09-13

    The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with K(I) in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH.

  12. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703.

    PubMed

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-03-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase's properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca(2+) were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center.

  13. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703

    PubMed Central

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-01-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase’s properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca2+ were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center. PMID:26926401

  14. Lipid Sulfates and Sulfonates Are Allosteric Competitive Inhibitors of the N-Terminal Phosphatase Activity of the Mammalian Soluble Epoxide Hydrolase†

    PubMed Central

    Tran, Katherine L.; Aronov, Pavel A.; Tanaka, Hiromasa; Newman, John W.; Hammock, Bruce D.; Morisseau, Christophe

    2006-01-01

    The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with KI in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH. PMID:16142916

  15. Structure of the active N-terminal domain of Ezrin. Conformational and mobility changes identify keystone interactions.

    PubMed

    Smith, William James; Nassar, Nicolas; Bretscher, Anthony; Cerione, Richard A; Karplus, P Andrew

    2003-02-14

    Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins that cross-link the actin cytoskeleton to the plasma membrane and also may function in signaling cascades that regulate the assembly of actin stress fibers. Here, we report a crystal structure for the free (activated) FERM domain (residues 2-297) of recombinant human ezrin at 2.3 A resolution. Structural comparison among the dormant moesin FERM domain structure and the three known active FERM domain structures (radixin, moesin, and now ezrin) allows the clear definition of regions that undergo structural changes during activation. The key regions affected are residues 135-150 and 155-180 in lobe F2 and residues 210-214 and 235-267 in lobe F3. Furthermore, we show that a large increase in the mobilities of lobes F2 and F3 accompanies activation, suggesting that their integrity is compromised. This leads us to propose a new concept that we refer to as keystone interactions. Keystone interactions occur when one protein (or protein part) contributes residues that allow another protein to complete folding, meaning that it becomes an integral part of the structure and would rarely dissociate. Such interactions are well suited for long-lived cytoskeletal protein interactions. The keystone interactions concept leads us to predict two specific docking sites within lobes F2 and F3 that are likely to bind target proteins.

  16. The peroxisome proliferator-activated receptor N-terminal domain controls isotype-selective gene expression and adipogenesis.

    PubMed

    Hummasti, Sarah; Tontonoz, Peter

    2006-06-01

    Peroxisome proliferator-activated receptors (PPARgamma, PPARalpha, and PPARdelta) are important regulators of lipid metabolism. Although they share significant structural similarity, the biological effects associated with each PPAR isotype are distinct. For example, PPARalpha and PPARdelta regulate fatty acid catabolism, whereas PPARgamma controls lipid storage and adipogenesis. The different functions of PPARs in vivo can be explained at least in part by the different tissue distributions of the three receptors. The question of whether the receptors have different intrinsic activities and regulate distinct target genes, however, has not been adequately explored. We have engineered cell lines that express comparable amounts of each receptor. Transcriptional profiling of these cells in the presence of selective agonists reveals partially overlapping but distinct patterns of gene regulation by the three PPARs. Moreover, analysis of chimeric receptors points to the N terminus of each receptor as the key determinant of isotype-selective gene expression. For example, the N terminus of PPARgamma confers the ability to promote adipocyte differentiation when fused to the PPARdelta DNA binding domain and ligand binding domain, whereas the N terminus of PPARdelta leads to the inappropriate expression of fatty acid oxidation genes in differentiated adipocytes when fused to PPARgamma. Finally, we demonstrate that the N terminus of each receptor functions in part to limit receptor activity because deletion of the N terminus leads to nonselective activation of target genes. A more detailed understanding of the mechanisms by which the individual PPARs differentially regulate gene expression should aid in the design of more effective drugs, including tissue- and target gene-selective PPAR modulators.

  17. Site-specific phosphorylation induces functionally active conformation in the intrinsically disordered N-terminal activation function (AF1) domain of the glucocorticoid receptor.

    PubMed

    Garza, Anna M S; Khan, Shagufta H; Kumar, Raj

    2010-01-01

    Intrinsically disordered (ID) regions are disproportionately higher in cell signaling proteins and are predicted to have much larger frequency of phosphorylation sites than ordered regions, suggesting an important role in their regulatory capacity. In this study, we show that AF1, an ID activation domain of the glucocorticoid receptor (GR), adopts a functionally folded conformation due to its site-specific phosphorylation by p38 mitogen-activated protein kinase, which is involved in apoptotic and gene-inductive events initiated by the GR. Further, we show that site-specific phosphorylation-induced secondary and tertiary structure formation specifically facilitates AF1's interaction with critical coregulatory proteins and subsequently its transcriptional activity. These data demonstrate a mechanism through which ID activation domain of the steroid receptors and other similar transcription factors may adopt a functionally active conformation under physiological conditions.

  18. An N-terminal partial sequence of the 13 kDa Pycnopodia helianthoides sperm chemoattractant 'startrak' possesses sperm-attracting activity.

    PubMed

    Miller, R L; Vogt, R

    1996-02-01

    Freshwater extracts of starfish ovaries were used to purify the sperm-attracting peptide 'startrak' from Pycnopodia helianthoides using hydrophobic interaction chromatography and DEAE-high-pressure liquid chromatography. Partially purified attractant had a molecular mass of 13 kDa, estimated from gel filtration and polyacrylamide gel electrophoresis results. The purified attractant was subjected to amino acid analysis and direct sequencing, and was found to consist largely of a single peptide composed of an estimated 127 residues based on a molecular mass of 13kDa. An N-terminal sequence of amino acids from positions 3 to 34 was obtained and synthesized as: NH2-Ala-Glu-Leu-Gly-Leu-Cys-Ile-Ala-Arg-Val-Arg-Gln-Gln-Asn-Gln-Gly-Gln- Asp-Asp-Val-Ser-Ile-Tyr-Gln-Ala-Ile-Met-Ser-Gln-Cys-Gln-Ser-COOH. The synthetic peptide possessed sperm-attracting activity 130 times greater than the activity of partially purified startrak and showed a pattern of species-specificity of sperm chemotaxis similar to that of startrak. Antibody prepared against synthetic peptide removed the sperm-attracting activity from crude and partially purified preparations of startrak. The partial sequence of startrak was not homologous with that of any of the known echinoid sperm motility-activating peptides.

  19. Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity

    SciTech Connect

    Gay, Sean C.; Segel, Irwin H.; Fisher, Andrew J.

    2009-10-01

    APS kinase from Thiobacillus denitrificans contains an inactive N-terminal ATP sulfurylase domain. The structure presented unveils the first hexameric assembly for an APS kinase, and reveals that structural changes in the N-terminal domain disrupt the ATP sulfurylase active site thus prohibiting activity. The Tbd-0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 Å resolution X-ray crystal structure reported here revealed a hexameric assembly with D{sub 3} symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.

  20. Structural insights into the N-terminal GIY–YIG endonuclease activity of Arabidopsis glutaredoxin AtGRXS16 in chloroplasts

    PubMed Central

    Liu, Xi; Liu, Shian; Feng, Yingang; Liu, Jian-Zhong; Pham, Khanh; Deng, Haiteng; Hirschi, Kendal D.; Wang, Xinquan; Cheng, Ninghui

    2013-01-01

    Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions. A chloroplastic monothiol glutaredoxin, AtGRXS16 from Arabidopsis thaliana, comprises two distinct functional domains, an N-terminal domain (NTD) with GlyIleTyr-TyrIleGly (GIY-YIG) endonuclease motif and a C-terminal Grx module, to coordinate redox regulation and DNA cleavage in chloroplasts. Structural determination of AtGRXS16-NTD showed that it possesses a GIY–YIG endonuclease fold, but the critical residues for the nuclease activity are different from typical GIY–YIG endonucleases. AtGRXS16-NTD was able to cleave λDNA and chloroplast genomic DNA, and the nuclease activity was significantly reduced in AtGRXS16. Functional analysis indicated that AtGRXS16-NTD could inhibit the ability of AtGRXS16 to suppress the sensitivity of yeast grx5 cells to oxidative stress; however, the C-terminal Grx domain itself and AtGRXS16 with a Cys123Ser mutation were active in these cells and able to functionally complement a Grx5 deficiency in yeast. Furthermore, the two functional domains were shown to be negatively regulated through the formation of an intramolecular disulfide bond. These findings unravel a manner of regulation for Grxs and provide insights into the mechanistic link between redox regulation and DNA metabolism in chloroplasts. PMID:23690600

  1. Defining the roles of the N-terminal region and the helicase activity of RECQ4A in DNA repair and homologous recombination in Arabidopsis.

    PubMed

    Schröpfer, Susan; Kobbe, Daniela; Hartung, Frank; Knoll, Alexander; Puchta, Holger

    2014-02-01

    RecQ helicases are critical for the maintenance of genomic stability. The Arabidopsis RecQ helicase RECQ4A is the functional counterpart of human BLM, which is mutated in the genetic disorder Bloom's syndrome. RECQ4A performs critical roles in regulation of homologous recombination (HR) and DNA repair. Loss of RECQ4A leads to elevated HR frequencies and hypersensitivity to genotoxic agents. Through complementation studies, we were now able to demonstrate that the N-terminal region and the helicase activity of RECQ4A are both essential for the cellular response to replicative stress induced by methyl methanesulfonate and cisplatin. In contrast, loss of helicase activity or deletion of the N-terminus only partially complemented the mutant hyper-recombination phenotype. Furthermore, the helicase-deficient protein lacking its N-terminus did not complement the hyper-recombination phenotype at all. Therefore, RECQ4A seems to possess at least two different and independent sub-functions involved in the suppression of HR. By in vitro analysis, we showed that the helicase core was able to regress an artificial replication fork. Swapping of the terminal regions of RECQ4A with the closely related but functionally distinct helicase RECQ4B indicated that in contrast to the C-terminus, the N-terminus of RECQ4A was required for its specific functions in DNA repair and recombination.

  2. Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

    PubMed

    Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

    1998-08-15

    Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

  3. Proline-directed phosphorylation of the dopamine transporter N-terminal domain

    PubMed Central

    Gorentla, Balachandra K.; Moritz, Amy E.; Foster, James D.; Vaughan, Roxanne A.

    2009-01-01

    Phosphorylation of the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with PKC- and substrate-induced alterations in transporter activity, subcellular distribution, and dopamine efflux, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCα, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCα-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses. PMID:19146407

  4. Implications of mitogen-activated protein kinase signaling in glioma.

    PubMed

    Pandey, Vimal; Bhaskara, Vasantha Kumar; Babu, Phanithi Prakash

    2016-02-01

    Gliomas are the most common primary central nervous system tumors. Gliomas originate from astrocytes, oligodendrocytes, and neural stem cells or their precursors. According to WHO classification, gliomas are classified into four different malignant grades ranging from grade I to grade IV based on histopathological features and related molecular aberrations. The induction and maintenance of these tumors can be attributed largely to aberrant signaling networks. In this regard, the mitogen-activated protein kinase (MAPK) network has been widely studied and is reported to be severely altered in glial tumors. Mutations in MAPK pathways most frequently affect RAS and B-RAF in the ERK, c-Jun N-terminal kinase (JNK), and p38 pathways leading to malignant transformation. Also, it is linked to both inherited and sequential accumulations of mutations that control receptor tyrosine kinase (RTK)-activated signal transduction pathways, cell cycle growth arrest pathways, and nonresponsive cell death pathways. Genetic alterations that modulate RTK signaling can also alter several downstream pathways, including RAS-mediated MAP kinases along with JNK pathways, which ultimately regulate cell proliferation and cell death. The present review focuses on recent literature regarding important deregulations in the RTK-activated MAPK pathway during gliomagenesis and progression.

  5. Glycogen synthase kinase 3β suppresses polyglutamine aggregation by inhibiting Vaccinia-related kinase 2 activity

    PubMed Central

    Lee, Eunju; Ryu, Hye Guk; Kim, Sangjune; Lee, Dohyun; Jeong, Young-Hun; Kim, Kyong-Tai

    2016-01-01

    Huntington’s disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the N-terminal of huntingtin. The amount of aggregate-prone protein is controlled by various mechanisms, including molecular chaperones. Vaccinia-related kinase 2 (VRK2) is known to negatively regulate chaperonin TRiC, and VRK2-facilitated degradation of TRiC increases polyQ protein aggregation, which is involved in HD. We found that VRK2 activity was negatively controlled by glycogen synthase kinase 3β (GSK3β). GSK3β directly bound to VRK2 and inhibited the catalytic activity of VRK2 in a kinase activity-independent manner. Furthermore, GSK3β increased the stability of TRiC and decreased the formation of HttQ103-GFP aggregates by inhibiting VRK2. These results indicate that GSK3β signaling may be a regulatory mechanism of HD progression and suggest targets for further therapeutic trials for HD. PMID:27377031

  6. Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain.

    PubMed

    Yousefi, Reza; Shchutskaya, Yulia Y; Zimny, Jaroslaw; Gaudin, Jean-Charles; Moosavi-Movahedi, Ali A; Muronetz, Vladimir I; Zuev, Yuriy F; Chobert, Jean-Marc; Haertlé, Thomas

    2009-08-01

    As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

  7. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit*

    PubMed Central

    Cuevas-Velazquez, Cesar L.; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A.

    2016-01-01

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. PMID:27006402

  8. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    PubMed

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. UV activation of receptor tyrosine kinase activity.

    PubMed

    Coffer, P J; Burgering, B M; Peppelenbosch, M P; Bos, J L; Kruijer, W

    1995-08-03

    The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumor promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as AP-1 and NF-kappa B, as well as activation of the Src tyrosine kinase. This 'UV-response' has been well studied in mammalian cells and furthermore is conserved in yeast, however the most upstream components of this signal transduction pathway have remained elusive. Here we show that UV rapidly activates both the EGF receptor and insulin receptor, as shown by tyrosine phosphorylation of these receptors. We demonstrate that this activation is due to autophosphorylation as it only occurs in cells containing receptors with a functional kinase domain. We have further analysed the propagation of the UV-induced signal to downstream events such as, IRS-1 and Shc tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, leukotriene synthesis, MAP kinase activation and gene induction all of which are activated by UV irradiation. Importantly, we demonstrate that in cells expressing a 'kinase-dead' receptor mutant the UV-response is inhibited, blocking leukotriene synthesis, MAP kinase activation and transcriptional induction. Furthermore, prior-stimulation of cells with UV appears to reduce further responsiveness to addition of growth factor suggesting a common signaling pathway. These data demonstrate a critical role for receptor-mediated events in regulating the response mammalian cells to UV exposure.

  10. Sphingosine Kinase Protects Lipopolysaccharide-Activated Macrophages from Apoptosis

    PubMed Central

    Wu, Weicheng; Mosteller, Raymond D.; Broek, Daniel

    2004-01-01

    Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-κB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria. PMID:15314148

  11. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation

    PubMed Central

    Harris, Samantha P.; Belknap, Betty; Van Sciver, Robert E.; White, Howard D.; Galkin, Vitold E.

    2016-01-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure–function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the “open” structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca2+ have been achieved. We suggest that Ca2+ modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  12. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    PubMed

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-09

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

  13. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    SciTech Connect

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam

    2010-07-13

    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  14. Crystal structure of full-length Mycobacterium tuberculosis H37Rv glycogen branching enzyme: insights of N-terminal beta-sandwich in substrate specificity and enzymatic activity.

    PubMed

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam

    2010-07-02

    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an alpha-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1-->4 bond and making a new 1-->6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-A resolution. MtbGlgBWT contains four domains: N1 beta-sandwich, N2 beta-sandwich, a central (beta/alpha)(8) domain that houses the catalytic site, and a C-terminal beta-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) MtbDelta108GlgB protein. The N1 beta-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 beta-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and MtbDelta108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1-->4 bond breakage) and isomerization (1-->6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and MtbDelta108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (ECDelta112GlgB).

  15. Histone recognition and nuclear receptor co-activator functions of Drosophila Cara Mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3

    PubMed Central

    Chauhan, Chhavi; Zraly, Claudia B.; Parilla, Megan; Diaz, Manuel O.; Dingwall, Andrew K.

    2012-01-01

    MLL2 and MLL3 histone lysine methyltransferases are conserved components of COMPASS-like co-activator complexes. In vertebrates, the paralogous MLL2 and MLL3 contain multiple domains required for epigenetic reading and writing of the histone code involved in hormone-stimulated gene programming, including receptor-binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and MLL3 arose from a common ancestor. Phylogenetic analyses reveal that the ancestral gene underwent a fission event in some Brachycera dipterans, including Drosophila species, creating two independent genes corresponding to the N- and C-terminal portions. In Drosophila, the C-terminal SET domain is encoded by trithorax-related (trr), which is required for hormone-dependent gene activation. We identified the cara mitad (cmi) gene, which encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor-binding motifs. The cmi gene is essential and its functions are dosage sensitive. CMI associates with TRR, as well as the EcR-USP receptor, and is required for hormone-dependent transcription. Unexpectedly, although the CMI and MLL2 PHDf3 domains could bind histone H3, neither showed preference for trimethylated lysine 4. Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone-stimulated transcription requires chromatin binding by CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of MLL2 into two distinct genes in Drosophila provides important insight into distinct epigenetic functions of conserved readers and writers of the histone code. PMID:22569554

  16. Structurally Conserved Nop56/58 N-terminal Domain Facilitates Archaeal Box C/D Ribonucleoprotein-guided Methyltransferase Activity*

    PubMed Central

    Gagnon, Keith T.; Biswas, Shyamasri; Zhang, Xinxin; Brown, Bernard A.; Wollenzien, Paul; Mattos, Carla; Maxwell, E. Stuart

    2012-01-01

    Box C/D RNA-protein complexes (RNPs) guide the 2′-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C′/D′ RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity. PMID:22496443

  17. The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity

    PubMed Central

    Takaku, Hiroaki; Minagawa, Asako; Takagi, Masamichi; Nashimoto, Masayuki

    2004-01-01

    Transfer RNA (tRNA) 3′ processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3′ trailer from pre-tRNA. There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300–400 amino acids, and the other is a long form (tRNase ZL) that contains 800–900 amino acids. Here we investigated whether the short and long forms have different preferences for various RNA substrates. We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNAArg and the RNase 65 activity. All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA. We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA. These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL. Furthermore, the optimal concentrations of NaCl, MgCl2 and MnCl2 differed between tRNase ZSs and tRNase ZLs, and the Km values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs. PMID:15317868

  18. Histone recognition and nuclear receptor co-activator functions of Drosophila cara mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3.

    PubMed

    Chauhan, Chhavi; Zraly, Claudia B; Parilla, Megan; Diaz, Manuel O; Dingwall, Andrew K

    2012-06-01

    MLL2 and MLL3 histone lysine methyltransferases are conserved components of COMPASS-like co-activator complexes. In vertebrates, the paralogous MLL2 and MLL3 contain multiple domains required for epigenetic reading and writing of the histone code involved in hormone-stimulated gene programming, including receptor-binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and MLL3 arose from a common ancestor. Phylogenetic analyses reveal that the ancestral gene underwent a fission event in some Brachycera dipterans, including Drosophila species, creating two independent genes corresponding to the N- and C-terminal portions. In Drosophila, the C-terminal SET domain is encoded by trithorax-related (trr), which is required for hormone-dependent gene activation. We identified the cara mitad (cmi) gene, which encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor-binding motifs. The cmi gene is essential and its functions are dosage sensitive. CMI associates with TRR, as well as the EcR-USP receptor, and is required for hormone-dependent transcription. Unexpectedly, although the CMI and MLL2 PHDf3 domains could bind histone H3, neither showed preference for trimethylated lysine 4. Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone-stimulated transcription requires chromatin binding by CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of MLL2 into two distinct genes in Drosophila provides important insight into distinct epigenetic functions of conserved readers and writers of the histone code.

  19. Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester.

    PubMed

    Muto, Akihiro; Ruland, Jürgen; McAllister-Lucas, Linda M; Lucas, Peter C; Yamaoka, Shoji; Chen, Felicia F; Lin, Amy; Mak, Tak W; Núñez, Gabriel; Inohara, Naohiro

    2002-08-30

    Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.

  20. Structural insights into the N-terminal GIY-YIG endonuclease activity of "Arabidopsis" glutaredoxin AtGRXS16 in chloroplasts

    USDA-ARS?s Scientific Manuscript database

    Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions. A chloroplastic monothiol glutaredoxin, AtGRXS16 from "Arabidopsis thaliana", comprises two distinct functional domains, an N-terminal domain (NTD) with GlyIleTyr-TyrIleGly (GIY-YIG) endo...

  1. Prevention of neuronal apoptosis by phorbol ester-induced activation of protein kinase C: blockade of p38 mitogen-activated protein kinase.

    PubMed

    Behrens, M M; Strasser, U; Koh, J Y; Gwag, B J; Choi, D W

    1999-01-01

    Consistent with previous studies on cell lines and non-neuronal cells, specific inhibitors of protein kinase C induced mouse primary cultured neocortical neurons to undergo apoptosis. To examine the complementary hypothesis that activating protein kinase C would attenuate neuronal apoptosis, the cultures were exposed for 1 h to phorbol-12-myristate-13-acetate, which activated protein kinase C as evidenced by downstream enhancement of the mitogen-activated protein kinase pathway. Exposure to phorbol-12-myristate-13-acetate, or another active phorbol ester, phorbol-12,13-didecanoate, but not to the inactive ester, 4alpha-phorbol-12,13-didecanoate, markedly attenuated neuronal apoptosis induced by serum deprivation. Phorbol-12-myristate-13-acetate also attenuated neuronal apoptosis induced by exposure to beta-amyloid peptide 1-42, or oxygen-glucose deprivation in the presence of glutamate receptor antagonists. The neuroprotective effects of phorbol-12-myristate-13-acetate were blocked by brief (non-toxic) concurrent exposure to the specific protein kinase C inhibitors, but not by a specific mitogen-activated protein kinase 1 inhibitor. Phorbol-12-myristate-13-acetate blocked the induction of p38 mitogen-activated protein kinase activity and specific inhibition of this kinase by SB 203580 attenuated serum deprivation-induced apoptosis. c-Jun N-terminal kinase 1 activity was high at rest and not modified by phorbol-12-myristate-13-acetate treatment. These data strengthen the idea that protein kinase C is a key modulator of several forms of central neuronal apoptosis, in part acting through inhibition of p38 mitogen-activated protein kinase regulated pathways.

  2. Oncoprotein protein kinase antibody kit

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  3. A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells.

    PubMed

    Dias, Juliana Vieira; Benslimane-Ahmim, Zahia; Egot, Marion; Lokajczyk, Anna; Grelac, Françoise; Galy-Fauroux, Isabelle; Juliano, Luiz; Le-Bonniec, Bernard; Takiya, Cristina Maeda; Fischer, Anne-Marie; Blanc-Brude, Olivier; Morandi, Verônica; Boisson-Vidal, Catherine

    2012-10-15

    Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 μg/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  5. A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity.

    PubMed

    McKell, Allison O; LaConte, Leslie E W; McDonald, Sarah M

    2017-04-01

    Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase.IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP

  6. A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity

    PubMed Central

    McKell, Allison O.; LaConte, Leslie E. W.

    2017-01-01

    ABSTRACT Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase. IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid

  7. Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2α Kinases PKR and GCN2

    PubMed Central

    Romano, Patrick R.; Garcia-Barrio, Minerva T.; Zhang, Xiaolong; Wang, Qizhi; Taylor, Deborah R.; Zhang, Fan; Herring, Christopher; Mathews, Michael B.; Qin, Jun; Hinnebusch, Alan G.

    1998-01-01

    The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit α (eIF2α) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2α kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop. PMID:9528799

  8. NAK is an IkappaB kinase-activating kinase.

    PubMed

    Tojima, Y; Fujimoto, A; Delhase, M; Chen, Y; Hatakeyama, S; Nakayama, K; Kaneko, Y; Nimura, Y; Motoyama, N; Ikeda, K; Karin, M; Nakanishi, M

    2000-04-13

    Phosphorylation of IkappaB by the IkappaB kinase (IKK) complex is a critical step leading to IkappaB degradation and activation of transcription factor NF-kappaB. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, the latter being indispensable for NF-kappaB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKbeta activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-kappaB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IkappaB degradation and NF-kappaB activity through IKKbeta. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-kappaB by protein kinase C-epsilon (PKCepsilon). Thus, NAK is an IKK kinase that may mediate IKK and NF-kappaB activation in response to growth factors that stimulate PKCepsilon activity.

  9. Treponema denticola activates mitogen-activated protein kinase signal pathways through Toll-like receptor 2.

    PubMed

    Ruby, John; Rehani, Kunal; Martin, Michael

    2007-12-01

    Treponema denticola, a spirochete indigenous to the oral cavity, is associated with host inflammatory responses to anaerobic polymicrobial infections of the root canal, periodontium, and alveolar bone. However, the cellular mechanisms responsible for the recognition of T. denticola by the innate immune system and the underlying cell signaling pathways that regulate the inflammatory response to T. denticola are currently unresolved. In this study, we demonstrate that T. denticola induces innate immune responses via the utilization of Toll-like receptor 2 (TLR2) but not TLR4. Assessment of TLR2/1 and TLR2/6 heterodimers revealed that T. denticola predominantly utilizes TLR2/6 for the induction of cellular responses. Analysis of the mitogen-activated protein kinase (MAPK) signaling pathway in T. denticola-stimulated monocytes identified a prolonged up-regulation of the MAPK extracellular signal-related kinase 1/2 (ERK1/2) and p38, while no discernible increase in phospho-c-Jun N-terminal kinase 1/2 (JNK1/2) levels was observed. With the aid of pharmacological inhibitors selectively targeting ERK1/2 via the mitogen-activated protein kinase/extracellular signal-related kinase 1/2 kinase and p38, we further demonstrate that ERK1/2 and p38 play a major role in T. denticola-mediated pro- and anti-inflammatory cytokine production.

  10. N-Terminal Methionine Processing.

    PubMed

    Wingfield, Paul T

    2017-04-03

    Protein synthesis is initiated by methionine in eukaryotes and by formylmethionine in prokaryotes. N-terminal methionine can be co-translationally cleaved by the enzyme methionine aminopeptidase (MAP). When recombinant proteins are expressed in bacterial and mammalian expression systems, there is a simple universal rule that predicts whether the initiating methionine will be processed by MAP based on the size of the residue adjacent (penultimate) to the N-methionine. In general, if the side chains of the penultimate residues have a radius of gyration of 1.29 Å or less, methionine is cleaved. © 2017 by John Wiley & Sons, Inc.

  11. Induction of mitogen-activated protein kinases is proportional to the amount of pressure overload.

    PubMed

    Esposito, Giovanni; Perrino, Cinzia; Schiattarella, Gabriele Giacomo; Belardo, Lorena; di Pietro, Elisa; Franzone, Anna; Capretti, Giuliana; Gargiulo, Giuseppe; Pironti, Gianluigi; Cannavo, Alessandro; Sannino, Anna; Izzo, Raffaele; Chiariello, Massimo

    2010-01-01

    Pressure overload has been shown to induce mitogen activated protein kinases (MAPKs) and reactivate the atrial natriuretic factor in the heart. To test the sensitivity of these signals to pressure overload, we assayed the activity of MAPKs extracellular signal-regulated kinase, c-Jun N-terminal kinase 1, and p38 in protein lysates from the left ventricle (LV) or white blood cells (WBC) isolated from aortic banded mice with varying levels of pressure overload. In separated mice we measured atrial natriuretic factor mRNA levels by Northern blotting. As expected, a significant induction of atrial natriuretic factor mRNA levels was observed after aortic banding, and it significantly correlated with the trans-stenotic systolic pressure gradient but not with the LV weight:body weight ratio. In contrast, a significant correlation with systolic pressure gradient or LV weight:body weight ratio was observed for all of the MAPK activity detected in LV samples or WBCs. Importantly, LV activation of MAPKs significantly correlated with their activation in WBCs from the same animal. To test whether MAPK activation in WBCs might reflect uncontrolled blood pressure levels in humans, we assayed extracellular signal-regulated kinase, c-Jun N-terminal kinase 1, and p38 activation in WBCs isolated from normotensive volunteers, hypertensive patients with controlled blood pressure values, or hypertensive patients with uncontrolled blood pressure values. Interestingly, in hypertensive patients with controlled blood pressure values, LV mass and extracellular signal-regulated kinase phosphorylation were significantly reduced compared with those in hypertensive patients with uncontrolled blood pressure values. These results suggest that MAPKs are sensors of pressure overload and that extracellular signal-regulated kinase activation in WBCs might be used as a novel surrogate biomarker of uncontrolled human hypertension.

  12. Physical Activity in Older Men: Longitudinal Associations with Inflammatory and Hemostatic Biomarkers, N-Terminal Pro-Brain Natriuretic Peptide, and Onset of Coronary Heart Disease and Mortality

    PubMed Central

    Jefferis, Barbara J; Whincup, Peter H; Lennon, Lucy T; Papacosta, Olia; Goya Wannamethee, S

    2014-01-01

    Objectives To examine associations between habitual physical activity (PA) and changes in PA and onset of coronary heart disease (CHD) and the pathways linking PA to CHD. Design British Regional Heart Study population-based cohort; men completed questionnaires in 1996 and 1998 to 2000, attended rescreen in 1998 to 2000, and were followed up to June 2010. Setting Community. Participants Of 4,252 men recruited from primary care centers (77% of those invited and eligible) who were rescreened in 1998 to 2000, 3,320 were ambulatory and free from CHD, stroke, and heart failure and participated in the current study. Measurements Usual PA (regular walking and cycling, recreational activity and sport). Outcome was first fatal or nonfatal myocardial infarction. Results In 3,320 ambulatory men, 303 first and 184 fatal CHD events occurred during a median of 11 years of follow-up; 9% reported no usual PA, 23% occasional PA, and 68% light or more-intense PA. PA was inversely associated with novel risk markers C-reactive protein, D-dimer, von Willebrand Factor and N-terminal pro-brain natriuretic peptide (NT-proBNP). Compared with no usual PA, hazard ratios (HRs) for CHD events, adjusted for age and region, were 0.52 (95% confidence interval (CI) = 0.34–0.79) for occasional PA, 0.47 (95% CI = 0.30–0.74) for light PA, 0.51 (95% CI = 0.32–0.82) for moderate PA, and 0.44 (95% CI = 0.29–0.65) for moderately vigorous or vigorous PA (P for linear trend = .004). Adjustment for established and novel risk markers somewhat attenuated HRs and abolished linear trends. Compared with men who remained inactive, men who maintained at least light PA had an HR for CHD events of 0.73 (95% CI = 0.53–1.02) and men whose PA level increased had an HR of 0.86 (95% CI = 0.55–1.35). Conclusion Even light PA was associated with significantly lower risk of CHD events in healthy older men, partly through inflammatory and hemostatic mechanisms and cardiac function (NT-proBNP). PMID

  13. Physical activity in older men: longitudinal associations with inflammatory and hemostatic biomarkers, N-terminal pro-brain natriuretic peptide, and onset of coronary heart disease and mortality.

    PubMed

    Jefferis, Barbara J; Whincup, Peter H; Lennon, Lucy T; Papacosta, Olia; Goya Wannamethee, S

    2014-04-01

    To examine associations between habitual physical activity (PA) and changes in PA and onset of coronary heart disease (CHD) and the pathways linking PA to CHD. British Regional Heart Study population-based cohort; men completed questionnaires in 1996 and 1998 to 2000, attended rescreen in 1998 to 2000, and were followed up to June 2010. Community. Of 4,252 men recruited from primary care centers (77% of those invited and eligible) who were rescreened in 1998 to 2000, 3,320 were ambulatory and free from CHD, stroke, and heart failure and participated in the current study. Usual PA (regular walking and cycling, recreational activity and sport). Outcome was first fatal or nonfatal myocardial infarction. In 3,320 ambulatory men, 303 first and 184 fatal CHD events occurred during a median of 11 years of follow-up; 9% reported no usual PA, 23% occasional PA, and 68% light or more-intense PA. PA was inversely associated with novel risk markers C-reactive protein, D-dimer, von Willebrand Factor and N-terminal pro-brain natriuretic peptide (NT-proBNP). Compared with no usual PA, hazard ratios (HRs) for CHD events, adjusted for age and region, were 0.52 (95% confidence interval (CI) = 0.34-0.79) for occasional PA, 0.47 (95% CI = 0.30-0.74) for light PA, 0.51 (95% CI = 0.32-0.82) for moderate PA, and 0.44 (95% CI = 0.29-0.65) for moderately vigorous or vigorous PA (P for linear trend = .004). Adjustment for established and novel risk markers somewhat attenuated HRs and abolished linear trends. Compared with men who remained inactive, men who maintained at least light PA had an HR for CHD events of 0.73 (95% CI = 0.53-1.02) and men whose PA level increased had an HR of 0.86 (95% CI = 0.55-1.35). Even light PA was associated with significantly lower risk of CHD events in healthy older men, partly through inflammatory and hemostatic mechanisms and cardiac function (NT-proBNP). © 2014, Copyright the Authors. Journal compilation © 2014, The American Geriatrics Society.

  14. The Role of Specific Mitogen-Activated Protein Kinase Signaling Cascades in the Regulation of Steroidogenesis

    PubMed Central

    Manna, Pulak R.; Stocco, Douglas M.

    2011-01-01

    Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface to the nucleus. The MAPK family includes four distinct MAPK cascades, that is, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase or stress-activated protein kinase, and ERK5. These MAPKs are essentially operated through three-tiered consecutive phosphorylation events catalyzed by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs lie in protein kinase cascades. The MAPK signaling pathways have been demonstrated to be associated with events regulating the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenesis in steroidogenic tissues. However, it has become clear that the regulation of MAPK-dependent StAR expression and steroid synthesis is a complex process and is context dependent. This paper summarizes the current level of understanding concerning the roles of the MAPK signaling cascades in the regulation of StAR expression and steroidogenesis in different steroidogenic cell models. PMID:21637381

  15. In vivo association of ATFa with JNK/SAP kinase activities.

    PubMed

    Bocco, J L; Bahr, A; Goetz, J; Hauss, C; Kallunki, T; Kedinger, C; Chatton, B

    1996-05-02

    The human ATFa proteins belong to the CREB/ATF family of transcription factors. We have previously shown that the ATFa proteins may contribute to the modulation of the transcriptional activity of the Jun/Fos complexes (Chatton et al. (1994). Oncogene, 9, 375-385). We now show that a protein kinase activity is strongly associated with ATFa in vivo, as revealed by coimmunoprecipitation of ATFa/kinase complexes from whole cell extracts, with antibodies against ATFa. Two independent regions were found to be implicated in kinase binding: a major interaction site is located within the N-terminal 82 residues comprising an important metal-chelating element; a weaker binding site corresponds to the basic sequence element preceding the C-terminal leucine-zipper of ATFa. Induction experiments suggest that each of these ATFa domains may interact with different kinases. The major activity is associated with the ATFa N-terminal domain. Based on its response to various inducers, on both in vitro and in vivo binding assays, and on its immunological properties, this activity most likely corresponds to the 54/55 kDa JNK2 protein. Taken together, these observations suggest that the ATFa proteins, among other CREB/ATF proteins, may be important effectors of cell signalling pathways.

  16. Rassf Proteins as Modulators of Mst1 Kinase Activity

    PubMed Central

    Bitra, Aruna; Sistla, Srinivas; Mariam, Jessy; Malvi, Harshada; Anand, Ruchi

    2017-01-01

    Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway. PMID:28327630

  17. Rassf Proteins as Modulators of Mst1 Kinase Activity.

    PubMed

    Bitra, Aruna; Sistla, Srinivas; Mariam, Jessy; Malvi, Harshada; Anand, Ruchi

    2017-03-22

    Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway.

  18. Phosphorylation of the mitochondrial protein Sab by stress-activated protein kinase 3.

    PubMed

    Court, Naomi W; Kuo, Ivana; Quigley, Oonagh; Bogoyevitch, Marie A

    2004-06-18

    Mitogen-activated protein kinases (MAPKs) transduce extracellular signals into responses such as growth, differentiation, and death through their phosphorylation of specific substrate proteins. Early studies showed the consensus sequence (Pro/X)-X-(Ser/Thr)-Pro to be phosphorylated by MAPKs. Docking domains such as the "kinase interaction motif" (KIM) also appear to be crucial for efficient substrate phosphorylation. Here, we show that stress-activated protein kinase-3 (SAPK3), a p38 MAPK subfamily member, localizes to the mitochondria. Activated SAPK3 phosphorylates the mitochondrial protein Sab, an in vitro substrate of c-Jun N-terminal kinase (JNK). Sab phosphorylation by SAPK3 was dependent on the most N-terminal KIM (KIM1) of Sab and occurred primarily on Ser321. This appeared to be dependent on the position of Ser321 within Sab and the sequence immediately surrounding it. Our results suggest that SAPK3 and JNK may share a common target at the mitochondria and provide new insights into the substrate recognition by SAPK3.

  19. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

  20. Dichotomal effect of space flight-associated microgravity on stress-activated protein kinases in innate immunity

    PubMed Central

    Verhaar, Auke P.; Hoekstra, Elmer; Tjon, Angela S. W.; Utomo, Wesley K.; Deuring, J. Jasper; Bakker, Elvira R. M.; Muncan, Vanesa; Peppelenbosch, Maikel P.

    2014-01-01

    Space flight strongly moderates human immunity but is in general well tolerated. Elucidation of the mechanisms by which zero gravity interacts with human immunity may provide clues for developing rational avenues to deal with exaggerated immune responses, e.g. as in autoimmune disease. Using two sounding rockets and one manned Soyuz launch, the influence of space flight on immunological signal transduction provoked by lipopolysaccharide (LPS) stimulation was investigated in freshly isolated peripheral blood monocytes and was compared to samples obtained from on-board centrifuge-loaded 1 g controls. The effect of microgravity on immunological signal transduction is highly specific, since LPS dependent Jun-N-terminal kinase activation is impaired in the 0 g condition, while the corresponding LPS dependent activation of p38 MAP kinase remains unaffected. Thus our results identify Jun-N-terminal kinase as a relevant target in immunity for microgravity and support using Jun-N-terminal kinase specific inhibitors for combating autoimmune disease. PMID:24968806