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Sample records for map kinase cascade

  1. The Potential for Signal Integration and Processing in Interacting Map Kinase Cascades

    PubMed Central

    Schwacke, John H.; Voit, Eberhard O.

    2009-01-01

    The cellular response to environmental stimuli requires biochemical information processing through which sensory inputs and cellular status are integrated and translated into appropriate responses by way of interacting networks of enzymes. One such network, the Mitogen Activated Protein (MAP) kinase cascade is a highly conserved signal transduction module that propagates signals from cell surface receptors to various cytosolic and nuclear targets by way of a phosphorylation cascade. We have investigated the potential for signal processing within a network of interacting feed-forward kinase cascades typified by the MAP kinase cascade. A genetic algorithm was used to search for sets of kinetic parameters demonstrating representative key input-output patterns of interest. We discuss two of the networks identified in our study, one implementing the exclusive-or function (XOR) and another implementing what we refer to as an in-band detector (IBD) or two-sided threshold. These examples confirm the potential for logic and amplitude-dependent signal processing in interacting MAP kinase cascades demonstrating limited cross-talk. Specifically, the XOR function allows the network to respond to either one, but not both signals simultaneously, while the IBD permits the network to respond exclusively to signals within a given range of strength, and to suppress signals below as well as above this range. The solution to the XOR problem is interesting in that it requires only two interacting pathways, crosstalk at only one layer, and no feedback or explicit inhibition. These types of responses are not only biologically relevant but constitute signal processing modules that can be combined to create other logical functions and that, in contrast to amplification, cannot be achieved with a single cascade or with two non-interacting cascades. Our computational results revealed surprising similarities between experimental data describing the JNK/MKK4/MKK7 pathway and the solution for

  2. Dual role for membrane localization in yeast MAP kinase cascade activation and its contribution to signaling fidelity.

    PubMed

    Lamson, Rachel E; Takahashi, Satoe; Winters, Matthew J; Pryciak, Peter M

    2006-03-21

    Distinct MAP kinase pathways in yeast share several signaling components , including the PAK Ste20 and the MAPKKK Ste11, yet signaling is specific. Mating pheromones trigger an initial step in which Ste20 activates Ste11 , and this requires plasma membrane recruitment of the MAP kinase cascade scaffold protein, Ste5 . Here, we demonstrate an additional role for Ste5 membrane localization. Once Ste11 is activated, signaling through the mating pathway remains minimal but is substantially amplified when Ste5 is recruited to the membrane either by the Gbetagamma dimer or by direct membrane targeting, even to internal membranes. Ste11 signaling is also amplified by Ste5 oligomerization and by a hyperactivating mutation in the Ste7 binding region of Ste5. We suggest a model in which membrane recruitment of Ste5 concentrates its binding partners and thereby amplifies signaling through the kinase cascade. We find similar behavior in the osmotically responsive HOG pathway. Remarkably, while both pheromone and hyperosmotic stimuli amplify signaling from constitutively active Ste11, the resulting signaling output remains pathway specific. These findings suggest a common mode of regulation in which pathway stimuli both initiate and amplify MAP kinase cascade signaling. The regulation of rate-limiting steps that lie after a branchpoint from shared components helps ensure signaling specificity.

  3. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade.

    PubMed Central

    Benn, J; Schneider, R J

    1994-01-01

    Hepatitis B virus produces a small (154-amino acid) transcriptional transactivating protein, HBx, which is required for viral infection and has been implicated in virus-mediated liver oncogenesis. However, the molecular mechanism for HBx activity and its possible influence on cell proliferation have remained obscure. A number of studies suggest that HBx may stimulate transcription by indirectly activating transcription factors, possibly by influencing cell signaling pathways. We now present biochemical evidence that HBx activates Ras and rapidly induces a cytoplasmic signaling cascade linking Ras, Raf, and mitogen-activated protein kinase (MAP kinase), leading to transcriptional transactivation. HBx strongly elevates levels of GTP-bound Ras, activated and phosphorylated Raf, and tyrosine-phosphorylated and activated MAP kinase. Transactivation of transcription factor AP-1 by HBx is blocked by inhibition of Ras or Raf activities but not by inhibition of Ca(2+)- and diacylglycerol-dependent protein kinase C. HBx was also found to stimulate DNA synthesis in serum-starved cells. The hepatitis B virus HBx protein therefore stimulates Ras-GTP complex formation and promotes downstream signaling through Raf and MAP kinases, and may influence cell proliferation. Images PMID:7937954

  4. EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity

    PubMed Central

    Zhao, Chunzhao; Nie, Haozhen; Shen, Qiujing; Zhang, Shuqun; Lukowitz, Wolfgang; Tang, Dingzhong

    2014-01-01

    Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity. PMID:24830651

  5. Turgor and net ion flux responses to activation of the osmotic MAP kinase cascade by fludioxonil in the filamentous fungus Neurospora crassa.

    PubMed

    Lew, Roger R

    2010-08-01

    The internal hydrostatic pressure (turgor) of the filamentous fungus Neurospora crassa is regulated at about 400-500 kiloPascals, primarily by an osmotic MAP kinase cascade which activates ion uptake from the extracellular medium and glycerol synthesis. In the absence of hyperosmotic stress, the phenylpyrrole fungicide fludioxonil activates the osmotic MAP kinase cascade, resulting in cell death. Turgor, the electrical potential and net ion fluxes were measured after treatment with fludioxonil. In wildtype, fludioxonil causes a hyperpolarization of the plasma membrane and net H(+) efflux from the cell, consistent with activation of the H(+)-ATPase. At the same time, net K(+) uptake occurs, and turgor increases (about 2-fold above normal levels). None of these changes are observed in the os-2 mutant (which lacks a functional MAP kinase, the last of the three kinases in the osmotic MAP kinase cascade). Tip growth ceases as hyperpolarization, net ion flux changes, and turgor increases begin. The inappropriate turgor increase is the probable cause of eventual lysis and death. The results corroborate a multi-pathway response to hyperosmotic stress that includes activation of plasma membrane transport. The relation to cell expansion (tip growth) is not direct. Increases in turgor due to ion transport might be expected to increase growth rate, but this does not occur. Instead, there must be a complex regulatory interplay between the growth and the turgor driving force, possibly mediated by regulation of cell wall extensibility.

  6. Activation of the MAP Kinase Cascade by Exogenous Calcium-Sensing Receptor

    SciTech Connect

    Hobson, Susan A.; Wright, Jay W.; Lee, Fred; Mcneil, Scott; Bilderback, Tim R.; Rodland, Karin D.

    2003-02-01

    In Rat-1 fibroblasts and ovarian surface epithelial cells, extracellular calcium induces a proliferative response which appears to be mediated by the G-protein coupled Calcium-sensing Receptor (CaR), as expression of the non-functional CaR-R795W mutant inhibits both thymidine incorporation and activation of the extracellular-regulated kinase (ERK) in response to calcium. In this report we utilized CaR-transfected HEK293 cells to demonstrate that functional CaR is necessary and sufficient for calcium-induced ERK activation. CaR-dependent ERK activation was blocked by co-expression of the Ras dominant-negative mutant, Ras N17, and by exposure to the phosphatidyl inositol 3' kinase inhibitors wortmannin and LY294002. In contrast to Rat-1 fibroblasts, CaR-mediated in vitro kinase activity of ERK2 was unaffected by tyrosine kinase inhibitor herbimycin in CaR-transfected HEK293 cells. These results suggest that usage of distinct pathways downstream of the CaR varies in a cell-type specific manner, suggesting a potential mechanism by which activation of the CaR could couple to distinct calcium-dependent responses.

  7. [Involvement of MAP-kinase cascades into the regulation of sodium butyrate induced premature cell senescence].

    PubMed

    Kochetkova, E Iu; Bykova, T V; Zubova, S G; Pospelova, T V

    2012-01-01

    We studied the role of p38 kinase and JNK1,2 in the activation of the complex mTORC1 and the program of senescence induced by histone deacetylase inhibitor, sodium butyrate (NaBut), in mouse embryonic fibroblasts transformed by E1A+cHa-Ras oncogenes. It was found that transformants from knockouts for the genes p38, were able to implement the program of NaBut-induced senescence, according to the data of the cell cycle arrest, inhibition of proliferation, hypertrophic changes associated with the activation of mTORC1 and SA-beta-galactosidase activity. According to the behavior of these markers, cell knockouts for the genes jnk1,2 were unable to implement NaBut-induced senescence. Induction of senescence closely correlates with the activation of the complex mTORC1, as it was shown by inhibiting mTORC1 with rapamycin. We believe that JNK 1,2 kinases are required for mTORC1 activation and acquiring the markers of premature senescence, induced by NaBut in the E1A+cHa-Ras transformants.

  8. A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival.

    PubMed

    Cherkasova, Vera A; McCully, Ryan; Wang, Yunmei; Hinnebusch, Alan; Elion, Elaine A

    2003-07-15

    In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival ([; see Figure S1 in the Supplemental Data available with this article online). Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Here, we report that Kss1 and Fus3 act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks. PMID:12867033

  9. A fungal cell wall integrity-associated MAP kinase cascade in Coniothyrium minitans is required for conidiation and mycoparasitism.

    PubMed

    Zeng, Fanyun; Gong, Xiaoyan; Hamid, Mahammad Imran; Fu, Yanping; Jiatao, Xie; Cheng, Jiasen; Li, Guoqing; Jiang, Daohong

    2012-05-01

    Coniothyrium minitans is an important biocontrol agent against Sclerotinia diseases. Previously, a conidiation-deficient mutant ZS-1T1000 was screened out from a T-DNA insertional library of C. minitans. CmBCK1, encoding MAP kinase kinase kinase and homologous to BCK1 of Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Targeted disruption of CmBCK1 led to the mutants undergoing autolysis and displaying hypersensitivity to the cell wall-degrading enzymes. The △CmBCK1 mutants lost the ability to produce pycnidia and conidia compared to the wild-type strain ZS-1. △CmBCK1 mutants could grow on the surface of sclerotia of Sclerotinia sclerotiorum but not form conidia, which resulted in much lower ability to reduce the viability of sclerotia of S. sclerotiorum. Furthermore, CmSlt2, a homolog of Slt2 encoding cell wall integrity-related MAP kinase and up-regulated by BCK1 in S. cerevisiae, was identified and targeted disrupted. The △CmSlt2 mutants had a similar phenotype to the △CmBCK1 mutants. The △CmSlt2 mutants also had autolytic aerial hyphae, hypersensitivity to cell wall-degrading enzymes, lack of conidiation and reduction of sclerotial mycoparasitism. Taken together, our results suggest that CmBCK1 and CmSlt2 are involved in conidiation and the hyperparasitic activities of C. minitans. PMID:22426009

  10. Kinase cascades regulating entry into apoptosis.

    PubMed Central

    Anderson, P

    1997-01-01

    All cells are constantly exposed to conflicting environment cues that signal cell survival or cell death. Survival signals are delivered by autocrine or paracrine factors that actively suppress a default death pathway. In addition to survival factor withdrawal, cell death can be triggered by environmental stresses such as heat, UV light, and hyperosmolarity or by dedicated death receptors (e.g., FAS/APO-1 and tumor necrosis factor [TNF] receptors) that are counterparts of growth factor or survival receptors at the cell surface. One of the ways that cells integrate conflicting exogenous stimuli is by phosphorylation (or dephosphorylation) of cellular constituents by interacting cascades of serine/threonine and tyrosine protein kinases (and phosphatases). Survival factors (e.g., growth factors and mitogens) activate receptor tyrosine kinases and selected mitogen-activated, cyclin-dependent, lipid-activated, nucleic acid-dependent, and cyclic AMP-dependent kinases to promote cell survival and proliferation, whereas environmental stress (or death factors such as FAS/APO-1 ligand and TNF-alpha) activates different members of these kinase families to inhibit cell growth and, under some circumstances, promote apoptotic cell death. Because individual kinase cascades can interact with one another, they are able to integrate conflicting exogenous stimuli and provide a link between cell surface receptors and the biochemical pathways leading to cell proliferation or cell death. PMID:9106363

  11. Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade.

    PubMed Central

    Enslen, H; Tokumitsu, H; Stork, P J; Davis, R J; Soderling, T R

    1996-01-01

    Membrane depolarization of NG108 cells gives rapid (< 5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK). To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively). In the absence of depolarization, CaM-KKc transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were co-transfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38). The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays. We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases. Mutation of the phosphorylation site (Thr196) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade. These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription. Images Fig. 1 Fig. 3 Fig. 4 PMID:8855261

  12. Map kinases in fungal pathogens.

    PubMed

    Xu, J R

    2000-12-01

    MAP kinases in eukaryotic cells are well known for transducing a variety of extracellular signals to regulate cell growth and differentiation. Recently, MAP kinases homologous to the yeast Fus3/Kss1 MAP kinases have been identified in several fungal pathogens and found to be important for appressorium formation, invasive hyphal growth, and fungal pathogenesis. This MAP kinase pathway also controls diverse growth or differentiation processes, including conidiation, conidial germination, and female fertility. MAP kinases homologous to yeast Slt2 and Hog1 have also been characterized in Candida albicans and Magnaporthe grisea. Mutants disrupted of the Slt2 homologues have weak cell walls, altered hyphal growth, and reduced virulence. The Hog1 homologues are dispensable for growth but are essential for regulating responses to hyperosmotic stress in C. albicans and M. grisea. Overall, recent studies have indicated that MAP kinase pathways may play important roles in regulating growth, differentiation, survival, and pathogenesis in fungal pathogens. PMID:11273677

  13. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  14. Regulation of carbamoyl phosphate synthetase by MAP kinase.

    PubMed

    Graves, L M; Guy, H I; Kozlowski, P; Huang, M; Lazarowski, E; Pope, R M; Collins, M A; Dahlstrand, E N; Earp, H S; Evans, D R

    2000-01-20

    The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by carbamoyl phosphate synthetase (CPS II), part of the multifunctional enzyme CAD. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo, CAD lost its feedback inhibition (which is dependent on uridine triphosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyrophosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triphosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides. PMID:10659854

  15. c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

    SciTech Connect

    Sprowles, Amy; Wu Yimi; Kung, H.-J.; Wisdom, Ron . E-mail: ronald.wisdom@ucdmc.ucdavis.edu

    2005-08-15

    The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis to define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli.

  16. Oregon Cascades Play Fairway Analysis: Faults and Heat Flow maps

    DOE Data Explorer

    Adam Brandt

    2015-11-15

    This submission includes a fault map of the Oregon Cascades and backarc, a probability map of heat flow, and a fault density probability layer. More extensive metadata can be found within each zip file.

  17. MAP4K Family Kinases in Immunity and Inflammation.

    PubMed

    Chuang, Huai-Chia; Wang, Xiaohong; Tan, Tse-Hua

    2016-01-01

    MAP kinase kinase kinase kinases (MAP4Ks) belong to the mammalian Ste20-like family of serine/threonine kinases. MAP4Ks including MAP4K1/HPK1, MAP4K2/GCK, MAP4K3/GLK, MAP4K4/HGK, MAP4K5/KHS, and MAP4K6/MINK have been reported to induce JNK activation through activating the MAP3K-MAP2K cascade. The physiological roles of MAP4Ks in immunity and inflammation are largely unknown until recent studies using biochemical approaches and knockout mice. Surprisingly, JNK is not the major target of MAP4Ks in immune cells; MAP4Ks regulate immune responses through novel targets. HPK1 inhibits T-cell receptor (TCR) signaling and B-cell receptor signaling via inducing phosphorylation/ubiquitination of SLP-76 and BLNK, respectively. GLK activates TCR signaling through phosphorylating/activating PKCθ. T-cell-mediated immune responses and Th17-mediated experimental autoimmune diseases are enhanced in HPK1 knockout mice but ameliorated in GLK knockout mice. Consistently, HPK1 levels are decreased in peripheral blood mononuclear cells and T cells from patients with psoriatic arthritis and systemic lupus erythematosus (SLE), respectively. Moreover, GLK levels are increased in T cells from patients with SLE, rheumatoid arthritis, or adult-onset Still's disease; the percentages of GLK-overexpression T cells are correlated with the disease activity. In addition, HGK phosphorylates and induces TRAF2 protein degradation, leading to negative regulation of IL-6 production in resting T cells. Loss of HGK in T cells results in spontaneous systemic inflammation and type 2 diabetes in mice. HGK is also involved in cancer cell migration. To date, the phenotypes of knockout mice for GCK, KHS, and MINK have not been reported; the roles of these three MAP4Ks in immune cell signaling are discussed in this review. Taken together, MAP4K family kinases play diverse roles in immune cell signaling, immune responses, and inflammation. PMID:26791862

  18. Identification of additional MAP kinases activated upon PAMP treatment

    PubMed Central

    Nitta, Yukino; Ding, Pingtao; Zhang, Yuelin

    2014-01-01

    Mitogen-activated protein (MAP) kinase cascades play important roles in plant immunity. Upon pathogen associated molecular pattern (PAMP) treatment, MPK3, MPK6 and MPK4 are quickly activated by upstream MKKs through phosphorylation. Western blot analysis using α-phospho-p44/42-ERK antibody suggests that additional MPKs with similar size as MPK4 are also activated upon PAMP perception. To identify these MAP kinases, 7 candidate MPKs with similar sizes as MPK4 were selected for further analysis. Transgenic plants expressing these MPKs with a ZZ-3xFLAG double tag of 17 kD were generated and analyzed by western blot. MPK1, MPK11 and MPK13 were found to be phosphorylated upon treatment with flg22. Our study revealed additional MAPKs being activated during PAMP-triggered immunity. PMID:25482788

  19. The MEKK1-MKK1/MKK2-MPK4 Kinase Cascade Negatively Regulates Immunity Mediated by a Mitogen-Activated Protein Kinase Kinase Kinase in Arabidopsis[C][W

    PubMed Central

    Kong, Qing; Qu, Na; Gao, Minghui; Zhang, Zhibin; Ding, Xiaojun; Yang, Fan; Li, Yingzhong; Dong, Oliver X.; Chen, She; Li, Xin; Zhang, Yuelin

    2012-01-01

    In Arabidopsis thaliana, the MEKK1-MKK1/MKK2-MPK4 mitogen-activated protein (MAP) kinase cascade represses cell death and immune responses. In mekk1, mkk1 mkk2, and mpk4 mutants, programmed cell death and defense responses are constitutively activated, but the mechanism by which MEKK1, MKK1/MKK2, and MPK4 negatively regulate cell death and immunity was unknown. From a screen for suppressors of mkk1 mkk2, we found that mutations in suppressor of mkk1 mkk2 1 (summ1) suppress the cell death and defense responses not only in mkk1 mkk2 but also in mekk1 and mpk4. SUMM1 encodes the MAP kinase kinase kinase MEKK2. It interacts with MPK4 and is phosphorylated by MPK4 in vitro. Overexpression of SUMM1 activates cell death and defense responses that are dependent on the nucleotide binding–leucine-rich repeat protein SUMM2. Taken together, our data suggest that the MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates MEKK2 and activation of MEKK2 triggers SUMM2-mediated immune responses. PMID:22643122

  20. CD28 signal transduction pathways. A comparison of B7-1 and B7-2 regulation of the map kinases: ERK2 and Jun kinases.

    PubMed

    Nunes, J A; Battifora, M; Woodgett, J R; Truneh, A; Olive, D; Cantrell, D A

    1996-01-01

    The present study compares the mitogen-activated protein (MAP) kinase responses in T cells activated with the CD28 ligands B7-1 (CD80) and B7-2/B70 (CD86). Ligands B7-1 and B7-2 do not activate the Raf-1/ERK2 cascade, but share the ability to activate related Jun kinases. These natural ligands for CD28 had no stimulatory effect alone on Jun kinase activation, but the data show that B7-1 and B7-2 could both co-operate with intracellular Ca2+ increase and protein kinase C (PKC) activation to stimulate Jun kinases. The present study shows that the interaction of CD28 with its ligands B7-1 and B7-2 can induce identical signal transduction through the MAP kinase cascades.

  1. Universal fractional map and cascade of bifurcations type attractors.

    PubMed

    Edelman, M

    2013-09-01

    We modified the way in which the Universal Map is obtained in the regular dynamics to derive the Universal α-Family of Maps depending on a single parameter α>0, which is the order of the fractional derivative in the nonlinear fractional differential equation describing a system experiencing periodic kicks. We consider two particular α-families corresponding to the Standard and Logistic Maps. For fractional α<2 in the area of parameter values of the transition through the period doubling cascade of bifurcations from regular to chaotic motion in regular dynamics corresponding fractional systems demonstrate a new type of attractors--cascade of bifurcations type trajectories.

  2. Geologic Map of the North Cascade Range, Washington

    USGS Publications Warehouse

    Haugerud, Ralph A.; Tabor, Rowland W.

    2009-01-01

    The North Cascade Range, commonly referred to as the North Cascades, is the northern part of the Cascade Range that stretches from northern California into British Columbia, where it merges with the Coast Mountains of British Columbia at the Fraser River. The North Cascades are generally characterized by exposure of plutonic and metamorphic rocks in contrast to the volcanic terrain to the south. The rocks of the North Cascades are more resistant to erosion, display greater relief, and show evidence of more pronounced uplift and recent glaciation. Although the total length of the North Cascade Range, extending north from Snoqualmie Pass in Washington, is about 200 mi (320 km), this compilation map at 1:200,000 scale covers only that part (~150 mi) in the United States. The compilation map is derived mostly from eight 1:100,000-scale quadrangle maps that include all of the North Cascade Range in Washington and a bit of the mostly volcanic part of the Cascade Range to the south (fig. 1, sheet 2). Overall, the area represented by this compilation is about 12,740 mi2 (33,000 km2). The superb alpine scenery of the North Cascade Range and its proximity to major population centers has led to designation of much of the area for recreational use or wilderness preservation. A major part of the map area is in North Cascade National Park. Other restricted use areas are the Alpine Lakes, Boulder River, Clearwater, Glacier Peak, Henry M. Jackson, Lake Chelan-Sawtooth, Mount Baker, Noisy-Diobsud, Norse Peak, and Pasayten Wildernesses and the Mount Baker, Lake Chelan, and Ross Lake National Recreation Areas. The valleys traversed by Washington State Highway 20 east of Ross Lake are preserved as North Cascades Scenic Highway. The map area is traversed by three major highways: U.S. Interstate 90, crossing Snoqualmie Pass; Washington State Highway 2, crossing Stevens Pass; and Washington State Highway 20, crossing Washington Pass. Major secondary roads, as well as a network of U

  3. A period-doubling cascade precedes chaos for planar maps.

    PubMed

    Sander, Evelyn; Yorke, James A

    2013-09-01

    A period-doubling cascade is often seen in numerical studies of those smooth (one-parameter families of) maps for which as the parameter is varied, the map transitions from one without chaos to one with chaos. Our emphasis in this paper is on establishing the existence of such a cascade for many maps with phase space dimension 2. We use continuation methods to show the following: under certain general assumptions, if at one parameter there are only finitely many periodic orbits, and at another parameter value there is chaos, then between those two parameter values there must be a cascade. We investigate only families that are generic in the sense that all periodic orbit bifurcations are generic. Our method of proof in showing there is one cascade is to show there must be infinitely many cascades. We discuss in detail two-dimensional families like those which arise as a time-2π maps for the Duffing equation and the forced damped pendulum equation. PMID:24089949

  4. A period-doubling cascade precedes chaos for planar maps

    NASA Astrophysics Data System (ADS)

    Sander, Evelyn; Yorke, James A.

    2013-09-01

    A period-doubling cascade is often seen in numerical studies of those smooth (one-parameter families of) maps for which as the parameter is varied, the map transitions from one without chaos to one with chaos. Our emphasis in this paper is on establishing the existence of such a cascade for many maps with phase space dimension 2. We use continuation methods to show the following: under certain general assumptions, if at one parameter there are only finitely many periodic orbits, and at another parameter value there is chaos, then between those two parameter values there must be a cascade. We investigate only families that are generic in the sense that all periodic orbit bifurcations are generic. Our method of proof in showing there is one cascade is to show there must be infinitely many cascades. We discuss in detail two-dimensional families like those which arise as a time-2π maps for the Duffing equation and the forced damped pendulum equation.

  5. MAP kinase pathways: The first twenty years

    PubMed Central

    Avruch, Joseph

    2007-01-01

    The MAP kinases, discovered approximately twenty years ago, together with their immediate upstream regulators, are among the most highly studied signal transduction molecules. This body of work has shaped many aspects of our present views of signal transduction by protein kinases. The effort expended in this area reflects the extensive participation of these regulatory modules in the control of cell fate decisions, i.e., proliferation, differentiation and death, across all eukaryotic phylla and in all tissues of metazoans. The discovery of these kinases is reviewed, followed by a discussion of some of the features of this signaling module that account for its broad impact on cell function and its enormous interest to many investigators. PMID:17229475

  6. Mitogen-Activated Protein Kinase Cascade MKK7-MPK6 Plays Important Roles in Plant Development and Regulates Shoot Branching by Phosphorylating PIN1 in Arabidopsis.

    PubMed

    Jia, Weiyan; Li, Baohua; Li, Shujia; Liang, Yan; Wu, Xiaowei; Ma, Mei; Wang, Jiyao; Gao, Jin; Cai, Yueyue; Zhang, Yuanya; Wang, Yingchun; Li, Jiayang; Wang, Yonghong

    2016-09-01

    Emerging evidences exhibit that mitogen-activated protein kinase (MAPK/MPK) signaling pathways are connected with many aspects of plant development. The complexity of MAPK cascades raises challenges not only to identify the MAPK module in planta but also to define the specific role of an individual module. So far, our knowledge of MAPK signaling has been largely restricted to a small subset of MAPK cascades. Our previous study has characterized an Arabidopsis bushy and dwarf1 (bud1) mutant, in which the MAP Kinase Kinase 7 (MKK7) was constitutively activated, resulting in multiple phenotypic alterations. In this study, we found that MPK3 and MPK6 are the substrates for phosphorylation by MKK7 in planta. Genetic analysis showed that MKK7-MPK6 cascade is specifically responsible for the regulation of shoot branching, hypocotyl gravitropism, filament elongation, and lateral root formation, while MKK7-MPK3 cascade is mainly involved in leaf morphology. We further demonstrated that the MKK7-MPK6 cascade controls shoot branching by phosphorylating Ser 337 on PIN1, which affects the basal localization of PIN1 in xylem parenchyma cells and polar auxin transport in the primary stem. Our results not only specify the functions of the MKK7-MPK6 cascade but also reveal a novel mechanism for PIN1 phosphorylation, establishing a molecular link between the MAPK cascade and auxin-regulated plant development. PMID:27618482

  7. Mitogen-Activated Protein Kinase Cascade MKK7-MPK6 Plays Important Roles in Plant Development and Regulates Shoot Branching by Phosphorylating PIN1 in Arabidopsis

    PubMed Central

    Liang, Yan; Wu, Xiaowei; Cai, Yueyue; Zhang, Yuanya; Wang, Yingchun; Li, Jiayang; Wang, Yonghong

    2016-01-01

    Emerging evidences exhibit that mitogen-activated protein kinase (MAPK/MPK) signaling pathways are connected with many aspects of plant development. The complexity of MAPK cascades raises challenges not only to identify the MAPK module in planta but also to define the specific role of an individual module. So far, our knowledge of MAPK signaling has been largely restricted to a small subset of MAPK cascades. Our previous study has characterized an Arabidopsis bushy and dwarf1 (bud1) mutant, in which the MAP Kinase Kinase 7 (MKK7) was constitutively activated, resulting in multiple phenotypic alterations. In this study, we found that MPK3 and MPK6 are the substrates for phosphorylation by MKK7 in planta. Genetic analysis showed that MKK7-MPK6 cascade is specifically responsible for the regulation of shoot branching, hypocotyl gravitropism, filament elongation, and lateral root formation, while MKK7-MPK3 cascade is mainly involved in leaf morphology. We further demonstrated that the MKK7-MPK6 cascade controls shoot branching by phosphorylating Ser 337 on PIN1, which affects the basal localization of PIN1 in xylem parenchyma cells and polar auxin transport in the primary stem. Our results not only specify the functions of the MKK7-MPK6 cascade but also reveal a novel mechanism for PIN1 phosphorylation, establishing a molecular link between the MAPK cascade and auxin-regulated plant development. PMID:27618482

  8. Mitogen-Activated Protein Kinase Cascade MKK7-MPK6 Plays Important Roles in Plant Development and Regulates Shoot Branching by Phosphorylating PIN1 in Arabidopsis.

    PubMed

    Jia, Weiyan; Li, Baohua; Li, Shujia; Liang, Yan; Wu, Xiaowei; Ma, Mei; Wang, Jiyao; Gao, Jin; Cai, Yueyue; Zhang, Yuanya; Wang, Yingchun; Li, Jiayang; Wang, Yonghong

    2016-09-01

    Emerging evidences exhibit that mitogen-activated protein kinase (MAPK/MPK) signaling pathways are connected with many aspects of plant development. The complexity of MAPK cascades raises challenges not only to identify the MAPK module in planta but also to define the specific role of an individual module. So far, our knowledge of MAPK signaling has been largely restricted to a small subset of MAPK cascades. Our previous study has characterized an Arabidopsis bushy and dwarf1 (bud1) mutant, in which the MAP Kinase Kinase 7 (MKK7) was constitutively activated, resulting in multiple phenotypic alterations. In this study, we found that MPK3 and MPK6 are the substrates for phosphorylation by MKK7 in planta. Genetic analysis showed that MKK7-MPK6 cascade is specifically responsible for the regulation of shoot branching, hypocotyl gravitropism, filament elongation, and lateral root formation, while MKK7-MPK3 cascade is mainly involved in leaf morphology. We further demonstrated that the MKK7-MPK6 cascade controls shoot branching by phosphorylating Ser 337 on PIN1, which affects the basal localization of PIN1 in xylem parenchyma cells and polar auxin transport in the primary stem. Our results not only specify the functions of the MKK7-MPK6 cascade but also reveal a novel mechanism for PIN1 phosphorylation, establishing a molecular link between the MAPK cascade and auxin-regulated plant development.

  9. VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells.

    PubMed

    Takahashi, T; Ueno, H; Shibuya, M

    1999-04-01

    KDR/FIk-1 tyrosine kinase, one of the two VEGF receptors induces mitogenesis and differentiation of vascular endothelial cells. We have previously reported that a major target molecule of KDR/Flk-1 kinase is PLC-gamma, and that VEGF induces activation of MAP kinase, mainly mediated by protein kinase C (PKC) in the NIH3T3 cells overexpressing KDR/FIk-1 (Takahashi and Shibuya, 1997). However, the signal transduction initiated from VEGF in endothelial cells remains to be elucidated. In primary sinusoidal endothelial cells which showed strictly VEGF-dependent growth, we found that VEGF stimulated the activation of Raf-1-MEK-MAP kinase cascade. To our surprise, an important regulator, Ras was not efficiently activated to a significant level in response to VEGF. Consistent with this, dominant-negative Ras did not block the VEGF-induced phosphorylation of MAP kinase. On the other hand, PKC-specific inhibitors severely reduced VEGF-dependent phosphorylation of MEK, activation of MAP kinase and subsequent DNA synthesis. A potent PI3 kinase inhibitor, Wortmannin, could not inhibit either of them. These results suggest that in primary endothelial cells, VEGF-induced activation of Raf-MEK-MAP kinase and DNA synthesis are mainly mediated by PKC-dependent pathway, much more than by Ras-dependent or PI3 kinase-dependent pathway.

  10. Mitogen-activated protein kinase cascades in signaling plant growth and development.

    PubMed

    Xu, Juan; Zhang, Shuqun

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling modules in eukaryotes. Early research of plant MAPKs has been focused on their functions in immunity and stress responses. Recent studies reveal that they also play essential roles in plant growth and development downstream of receptor-like protein kinases (RLKs). With only a limited number of MAPK components, multiple functional pathways initiated from different receptors often share the same MAPK components or even a complete MAPK cascade. In this review, we discuss how MAPK cascades function as molecular switches in response to spatiotemporal-specific ligand-receptor interactions and the availability of downstream substrates. In addition, we discuss other possible mechanisms governing the functional specificity of plant MAPK cascades, a question central to our understanding of MAPK functions.

  11. A Minimal Cascade Model for the Mitotic Oscillator Involving Cyclin and cdc2 Kinase

    NASA Astrophysics Data System (ADS)

    Goldbeter, Albert

    1991-10-01

    A minimal model for the mitotic oscillator is presented. The model, built on recent experimental advances, is based on the cascade of post-translational modification that modulates the activity of cdc2 kinase during the cell cycle. The model pertains to the situation encountered in early amphibian embryos, where the accumulation of cyclin suffices to trigger the onset of mitosis. In the first cycle of the bicyclic cascade model, cyclin promotes the activation of cdc2 kinase through reversible dephosphorylation, and in the second cycle, cdc2 kinase activates a cyclin protease by reversible phosphorylation. That cyclin activates cdc2 kinase while the kinase triggers the degradation of cyclin has suggested that oscillations may originate from such a negative feedback loop [Felix, M. A., Labbe, J. C., Doree, M., Hunt, T. & Karsenti, E. (1990) Nature (London) 346, 379-382]. This conjecture is corroborated by the model, which indicates that sustained oscillations of the limit cycle type can arise in the cascade, provided that a threshold exists in the activation of cdc2 kinase by cyclin and in the activation of cyclin proteolysis by cdc2 kinase. The analysis shows how mitotic oscillations may readily arise from time lags associated with these thresholds and from the delayed negative feedback provided by cdc2-induced cyclin degradation. A mechanism for the origin of the thresholds is proposed in terms of the phenomenon of zero-order ultrasensitivity previously described for biochemical systems regulated by covalent modification.

  12. Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase.

    PubMed

    Cappell, Steven D; Dohlman, Henrik G

    2011-04-29

    Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane. PMID:21388955

  13. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

  14. Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

    PubMed Central

    Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

    2015-01-01

    Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5–MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases. PMID:26136265

  15. Measuring MAP kinase activity in immune complex assays.

    PubMed

    Cherkasova, Vera A

    2006-11-01

    I present an overview of published methods for measuring mitogen activated protein (MAP) kinase activity on endogenous associated substrates, exogenously added substrates as well as determination of activation loop phosphorylation as a read-out of kinase activity in vivo. Detailed procedures for these assays are given for two MAP kinases (MAPKs) Fus3 and Kss1 and compared with other published protocols, including the protocols for Hog1 and Mpk1 MAPKs. Measuring kinase activity in immune complex assays can serve as an approach for identification of potential substrates of protein kinases as well as for detecting other kinase-associated proteins. PMID:16890454

  16. Comparative analysis of the activation of MAP/ERK kinases in the CNS of animals with different learning abilities.

    PubMed

    Grinkevich, L N; Lisachev, P D; Baranova, K A; Kharchenko, O A

    2007-09-01

    Western blot analysis was used to study the activation of MAP/ERK protein kinases responsible for controlling gene expression via phosphorylation of transcription factors CREB and ELK-1 in native common snails and animals with impaired abilities to form long-term types of conditioned aversive reflexes. Different periods of the formation of this reflex were found to be characterized by different levels of activation of MAP-ERK kinases. The extents of activation of MAP-ERK kinase cascade were different in ganglia (parietal-visceral, cerebral, and pedal) with different roles in the formation of this reflex. The dynamics of activation showed a wavelike nature, with peaks at 10 min and 4 h. Administration of the neurotoxin 5,7-DHT, which induces dysfunction of serotonin terminals and decreases the ability to acquire this type of learning, led to significant decreases in activation of the MAP-ERK kinase cascade at the early stages of learning, which is evidence for an important role for the serotoninergic system in inducing this cascade. Activation of the MAP/ERK kinase cascade 4 h after training was seen both in native and DHT-treated animals, which is probably evidence for activation of non-specific adaptive processes in response to the sensitizing unconditioned stimulus. Thus, the MAP/ERK kinase intracellular regulatory cascade, which plays an important role in the survival of neurons, the regeneration of neuron processes, and synaptic sprouting, also plays an important role in forming the serotonin-dependent food-aversive reflex in the common snail.

  17. Uncertainty propagation in a cascade modelling approach to flood mapping

    NASA Astrophysics Data System (ADS)

    Rodríguez-Rincón, J. P.; Pedrozo-Acuña, A.; Breña Naranjo, J. A.

    2014-07-01

    The purpose of this investigation is to study the propagation of meteorological uncertainty within a cascade modelling approach to flood mapping. The methodology is comprised of a Numerical Weather Prediction Model (NWP), a distributed rainfall-runoff model and a standard 2-D hydrodynamic model. The cascade of models is used to reproduce an extreme flood event that took place in the Southeast of Mexico, during November 2009. The event is selected as high quality field data (e.g. rain gauges; discharge) and satellite imagery are available. Uncertainty in the meteorological model (Weather Research and Forecasting model) is evaluated through the use of a multi-physics ensemble technique, which considers twelve parameterization schemes to determine a given precipitation. The resulting precipitation fields are used as input in a distributed hydrological model, enabling the determination of different hydrographs associated to this event. Lastly, by means of a standard 2-D hydrodynamic model, hydrographs are used as forcing conditions to study the propagation of the meteorological uncertainty to an estimated flooded area. Results show the utility of the selected modelling approach to investigate error propagation within a cascade of models. Moreover, the error associated to the determination of the runoff, is showed to be lower than that obtained in the precipitation estimation suggesting that uncertainty do not necessarily increase within a model cascade.

  18. HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    PubMed Central

    Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, David G.; Smith, Richard D.; Glass, N. Louise

    2014-01-01

    Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of

  19. MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation.

    PubMed

    Madhani, H D; Styles, C A; Fink, G R

    1997-11-28

    Filamentous invasive growth of S. cerevisiae requires multiple elements of the mitogen-activated protein kinase (MAPK) signaling cascade that are also components of the mating pheromone response pathway. Here we show that, despite sharing several constituents, the two pathways use different MAP kinases. The Fus3 MAPK regulates mating, whereas the Kss1 MAPK regulates filamentation and invasion. Remarkably, in addition to their kinase-dependent activation functions, Kss1 and Fus3 each have a distinct kinase-independent inhibitory function. Kss1 inhibits the filamentation pathway by interacting with its target transcription factor Ste12. Fus3 has a different inhibitory activity that prevents the inappropriate activation of invasion by the pheromone response pathway. In the absence of Fus3, there is erroneous crosstalk in which mating pheromone now activates filamentation-specific gene expression using the Kss1 MAPK. PMID:9393860

  20. Protein kinase cascades in the regulation of cardiac hypertrophy

    PubMed Central

    Dorn, Gerald W.; Force, Thomas

    2005-01-01

    In broad terms, there are 3 types of cardiac hypertrophy: normal growth, growth induced by physical conditioning (i.e., physiologic hypertrophy), and growth induced by pathologic stimuli. Recent evidence suggests that normal and exercise-induced cardiac growth are regulated in large part by the growth hormone/IGF axis via signaling through the PI3K/Akt pathway. In contrast, pathological or reactive cardiac growth is triggered by autocrine and paracrine neurohormonal factors released during biomechanical stress that signal through the Gq/phospholipase C pathway, leading to an increase in cytosolic calcium and activation of PKC. Here we review recent developments in the area of these cardiotrophic kinases, highlighting the utility of animal models that are helping to identify molecular targets in the human condition. PMID:15765134

  1. The role of mitogen-activated protein (MAP) kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea.

    PubMed

    Schamber, Astrid; Leroch, Michaela; Diwo, Janine; Mendgen, Kurt; Hahn, Matthias

    2010-01-01

    In all fungi studied so far, mitogen-activated protein (MAP) kinase cascades serve as central signalling complexes that are involved in various aspects of growth, stress response and infection. In this work, putative components of the yeast Fus3/Kss1-type MAP kinase cascade and the putative downstream transcription factor Ste12 were analysed in the grey mould fungus Botrytis cinerea. Deletion mutants of the MAP triple kinase Ste11, the MAP kinase kinase Ste7 and the MAP kinase adaptor protein Ste50 all resulted in phenotypes similar to that of the previously described BMP1 MAP kinase mutant, namely defects in germination, delayed vegetative growth, reduced size of conidia, lack of sclerotia formation and loss of pathogenicity. Mutants lacking Ste12 showed normal germination, but delayed infection as a result of low penetration efficiency. Two differently spliced ste12 transcripts were detected, and both were able to complement the ste12 mutant, except for a defect in sclerotium formation, which was only corrected by the full-sized transcript. Overexpression of the smaller ste12 transcript resulted in delayed germination and strongly reduced infection. Bc-Gas2, a homologue of Magnaporthe grisea Gas2 that is required for appressorial function, was found to be non-essential for growth and infection, but its expression was under the control of both Bmp1 and Ste12. In summary, the role and regulatory connections of the Fus3/Kss1-type MAP kinase cascade in B. cinerea revealed both common and unique properties compared with those of other plant pathogenic fungi, and provide evidence for a regulatory link between the BMP1 MAP kinase cascade and Ste12. PMID:20078780

  2. Crosstalk and Signaling Switches in Mitogen-Activated Protein Kinase Cascades

    PubMed Central

    Fey, Dirk; Croucher, David R.; Kolch, Walter; Kholodenko, Boris N.

    2012-01-01

    Mitogen-activated protein kinase (MAPK) cascades control cell fate decisions, such as proliferation, differentiation, and apoptosis by integrating and processing intra- and extracellular cues. However, similar MAPK kinetic profiles can be associated with opposing cellular decisions depending on cell type, signal strength, and dynamics. This implies that signaling by each individual MAPK cascade has to be considered in the context of the entire MAPK network. Here, we develop a dynamic model of feedback and crosstalk for the three major MAPK cascades; extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), c-Jun N-terminal kinase (JNK), and also include input from protein kinase B (AKT) signaling. Focusing on the bistable activation characteristics of the JNK pathway, this model explains how pathway crosstalk harmonizes different MAPK responses resulting in pivotal cell fate decisions. We show that JNK can switch from a transient to sustained activity due to multiple positive feedback loops. Once activated, positive feedback locks JNK in a highly active state and promotes cell death. The switch is modulated by the ERK, p38, and AKT pathways. ERK activation enhances the dual specificity phosphatase (DUSP) mediated dephosphorylation of JNK and shifts the threshold of the apoptotic switch to higher inputs. Activation of p38 restores the threshold by inhibiting ERK activity via the PP1 or PP2A phosphatases. Finally, AKT activation inhibits the JNK positive feedback, thus abrogating the apoptotic switch and allowing only proliferative signaling. Our model facilitates understanding of how cancerous deregulations disturb MAPK signal processing and provides explanations for certain drug resistances. We highlight a critical role of DUSP1 and DUSP2 expression patterns in facilitating the switching of JNK activity and show how oncogene induced ERK hyperactivity prevents the normal apoptotic switch explaining the failure of certain drugs to

  3. Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants

    PubMed Central

    Kovtun, Yelena; Chiu, Wan-Ling; Tena, Guillaume; Sheen, Jen

    2000-01-01

    Despite the recognition of H2O2 as a central signaling molecule in stress and wounding responses, pathogen defense, and regulation of cell cycle and cell death, little is known about how the H2O2 signal is perceived and transduced in plant cells. We report here that H2O2 is a potent activator of mitogen-activated protein kinases (MAPKs) in Arabidopsis leaf cells. Using epitope tagging and a protoplast transient expression assay, we show that H2O2 can activate a specific Arabidopsis mitogen-activated protein kinase kinase kinase, ANP1, which initiates a phosphorylation cascade involving two stress MAPKs, AtMPK3 and AtMPK6. Constitutively active ANP1 mimics the H2O2 effect and initiates the MAPK cascade that induces specific stress-responsive genes, but it blocks the action of auxin, a plant mitogen and growth hormone. The latter observation provides a molecular link between oxidative stress and auxin signal transduction. Finally, we show that transgenic tobacco plants that express a constitutively active tobacco ANP1 orthologue, NPK1, display enhanced tolerance to multiple environmental stress conditions without activating previously described drought, cold, and abscisic acid signaling pathways. Thus, manipulation of key regulators of an oxidative stress signaling pathway, such as ANP1/NPK1, provides a strategy for engineering multiple stress tolerance that may greatly benefit agriculture. PMID:10717008

  4. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

    DOE PAGES

    Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, II, David G.; Smith, Richard D.; Glass, N. Louise; Heitman, Joseph

    2014-11-20

    Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal

  5. Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation.

    PubMed

    Sabbagh, W; Flatauer, L J; Bardwell, A J; Bardwell, L

    2001-09-01

    Signals transmitted by common components often elicit distinct (yet appropriate) outcomes. In yeast, two developmental options-mating and invasive growth-are both regulated by the same MAP kinase cascade. Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway. Kss1 has been thought to participate in mating only when Fus3 is absent. Instead, we show that Kss1 is rapidly phosphorylated and potently activated by mating pheromone in wild-type cells, and that this is required for normal pheromone-induced gene expression. Signal identity is apparently maintained because active Fus3 limits the extent of Kss1 activation, thereby preventing inappropriate signal crossover. PMID:11583629

  6. Recent progress on MAP kinase pathway inhibitors.

    PubMed

    Uehling, David E; Harris, Philip A

    2015-10-01

    The RAS-RAF-MEK-ERK, or ERK signaling pathway propagates signals through an intracellular signal transduction cascade. Since approximately one third of human cancers are impacted by mutations in the ERK signaling pathway, intensive efforts to develop drugs targeting members of this cascade are ongoing. While efforts to develop drugs aimed at inhibiting RAS are still at an early stage, substantial progress in discovering clinical drugs targeting RAF, MEK, and ERK have been made. This review will highlight the recent progress in this area. PMID:26298497

  7. The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components.

    PubMed

    Lee, B N; Elion, E A

    1999-10-26

    In haploid Saccharomyces cerevisiae, the mating and invasive growth (IG) pathways use the same mitogen-activated protein kinase kinase kinase kinase (MAPKKKK, Ste20), MAPKKK (Ste11), MAPKK (Ste7), and transcription factor (Ste12) to promote either G(1) arrest and fusion or foraging in response to distinct stimuli. This exquisite specificity is the result of pathway-specific receptors, G proteins, scaffold protein, and MAPKs. It is currently not thought that the shared signaling components function under the basal conditions of vegetative growth. We tested this hypothesis by searching for mutations that cause lethality when the STE11 gene is deleted. Strikingly, we found that Ste11, together with Ste20, Ste7, Ste12, and the IG MAPK Kss1, functions in a third pathway that promotes vegetative growth and is essential in an och1 mutant that does not synthesize mannoproteins. We term this pathway the STE vegetative growth (SVG) pathway. The SVG pathway functions, in part, to promote cell wall integrity in parallel with the protein kinase C pathway. During vegetative growth, the SVG pathway is inhibited by the mating MAPK Fus3. By contrast, the SVG pathway is constitutively activated in an och1 mutant, suggesting that it senses intracellular changes arising from the loss of mannoproteins. We predict that general proliferative functions may also exist for other MAPK cascades thought only to perform specialized functions. PMID:10535982

  8. The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras.

    PubMed Central

    Jaiswal, R K; Moodie, S A; Wolfman, A; Landreth, G E

    1994-01-01

    Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway. Images PMID:7935411

  9. Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi.

    PubMed

    Lew, Roger R; Levina, Natalia N; Shabala, Lana; Anderca, Marinela I; Shabala, Sergey N

    2006-03-01

    Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca(2+) influx and the sustained hyperpolarization is due to H(+) efflux by activation of the plasma membrane H(+)-ATPase. Protein synthesis is not required for H(+)-ATPase activation. Net K(+) and Cl(-) uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl(-) uptake increases, but net K(+) flux barely changes and net H(+) efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H(+)-ATPase, and net K(+) and Cl(-) uptake during turgor regulation. Other pathways regulating turgor must also exist.

  10. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  11. Molecular basis of MAP kinase regulation

    PubMed Central

    Peti, Wolfgang; Page, Rebecca

    2013-01-01

    Mitogen-activated protein kinases (MAPKs; ERK1/2, p38, JNK, and ERK5) have evolved to transduce environmental and developmental signals (growth factors, stress) into adaptive and programmed responses (differentiation, inflammation, apoptosis). Almost 20 years ago, it was discovered that MAPKs contain a docking site in the C-terminal lobe that binds a conserved 13-16 amino acid sequence known as the D- or KIM-motif (kinase interaction motif). Recent crystal structures of MAPK:KIM-peptide complexes are leading to a precise understanding of how KIM sequences contribute to MAPK selectivity. In addition, new crystal and especially NMR studies are revealing how residues outside the canonical KIM motif interact with specific MAPKs and contribute further to MAPK selectivity and signaling pathway fidelity. In this review, we focus on these recent studies, with an emphasis on the use of NMR spectroscopy, isothermal titration calorimetry and small angle X-ray scattering to investigate these processes. PMID:24115095

  12. Redundancy in the World of MAP Kinases: All for One

    PubMed Central

    Saba-El-Leil, Marc K.; Frémin, Christophe; Meloche, Sylvain

    2016-01-01

    The protein kinases ERK1 and ERK2 are the effector components of the prototypical ERK1/2 mitogen-activated protein (MAP) kinase pathway. This signaling pathway regulates cell proliferation, differentiation and survival, and is essential for embryonic development and cellular homeostasis. ERK1 and ERK2 homologs share similar biochemical properties but whether they exert specific physiological functions or act redundantly has been a matter of controversy. However, recent studies now provide compelling evidence in support of functionally redundant roles of ERK1 and ERK2 in embryonic development and physiology. In this review, we present a critical assessment of the evidence for the functional specificity or redundancy of MAP kinase isoforms. We focus on the ERK1/ERK2 pathway but also discuss the case of JNK and p38 isoforms. PMID:27446918

  13. How MAP kinase modules function as robust, yet adaptable, circuits

    PubMed Central

    Tian, Tianhai; Harding, Angus

    2014-01-01

    Genetic and biochemical studies have revealed that the diversity of cell types and developmental patterns evident within the animal kingdom is generated by a handful of conserved, core modules. Core biological modules must be robust, able to maintain functionality despite perturbations, and yet sufficiently adaptable for random mutations to generate phenotypic variation during evolution. Understanding how robust, adaptable modules have influenced the evolution of eukaryotes will inform both evolutionary and synthetic biology. One such system is the MAP kinase module, which consists of a 3-tiered kinase circuit configuration that has been evolutionarily conserved from yeast to man. MAP kinase signal transduction pathways are used across eukaryotic phyla to drive biological functions that are crucial for life. Here we ask the fundamental question, why do MAPK modules follow a conserved 3-tiered topology rather than some other number? Using computational simulations, we identify a fundamental 2-tiered circuit topology that can be readily reconfigured by feedback loops and scaffolds to generate diverse signal outputs. When this 2-kinase circuit is connected to proximal input kinases, a 3-tiered modular configuration is created that is both robust and adaptable, providing a biological circuit that can regulate multiple phenotypes and maintain functionality in an uncertain world. We propose that the 3-tiered signal transduction module has been conserved through positive selection, because it facilitated the generation of phenotypic variation during eukaryotic evolution. PMID:25483189

  14. Geologic map of Three Sisters volcanic cluster, Cascade Range, Oregon

    USGS Publications Warehouse

    Hildreth, Wes; Fierstein, Judy; Calvert, Andrew T.

    2012-01-01

    The cluster of glaciated stratovolcanoes called the Three Sisters—South Sister, Middle Sister, and North Sister—forms a spectacular 20-km-long reach along the crest of the Cascade Range in Oregon. The three eponymous stratocones, though contiguous and conventionally lumped sororally, could hardly display less family resemblance. North Sister (10,085 ft), a monotonously mafic edifice at least as old as 120 ka, is a glacially ravaged stratocone that consists of hundreds of thin rubbly lava flows and intercalated falls that dip radially and steeply; remnants of two thick lava flows cap its summit. Middle Sister (10,047 ft), an andesite-basalt-dacite cone built between 48 and 14 ka, is capped by a thick stack of radially dipping, dark-gray, thin mafic lava flows; asymmetrically glaciated, its nearly intact west flank contrasts sharply with its steep east face. Snow and ice-filled South Sister is a bimodal rhyolitic-intermediate edifice that was constructed between 50 ka and 2 ka; its crater (rim at 10,358 ft) was created between 30 and 22 ka, during the most recent of several explosive summit eruptions; the thin oxidized agglutinate that mantles its current crater rim protects a 150-m-thick pyroclastic sequence that helped fill a much larger crater. For each of the three, the eruptive volume is likely to have been in the range of 15 to 25 km³, but such estimates are fairly uncertain, owing to glacial erosion. The map area consists exclusively of Quaternary volcanic rocks and derivative surficial deposits. Although most of the area has been modified by glaciation, the volcanoes are young enough that the landforms remain largely constructional. Furthermore, twelve of the 145 eruptive units on the map are postglacial, younger than the deglaciation that was underway by about 17 ka. The most recent eruptions were of rhyolite near South Sister, about 2,000 years ago, and of mafic magma near McKenzie Pass, about 1,500 years ago. As observed by trailblazing volcanologist

  15. Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease.

    PubMed

    Shen, M R; Chou, C Y; Browning, J A; Wilkins, R J; Ellory, J C

    2001-12-01

    1. This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. 2. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl- channel. 4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl- channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. 5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. 6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells. PMID:11731569

  16. Gene expression related to synaptogenesis, neuritogenesis, and MAP kinase in behavioral sensitization to psychostimulants.

    PubMed

    Ujike, Hiroshi; Takaki, Manabu; Kodama, Masafumi; Kuroda, Shigetoshi

    2002-06-01

    The most important characteristic of behavioral sensitization to psychostimulants, such as amphetamine and cocaine, is the very long-lasting hypersensitivity to the drug after cessation of exposure. Rearrangement and structural modification of neural networks in CNS must be involved in behavioral sensitization. Previous microscopic studies have shown that the length of dendrites and density of dendritic spines increased in the nucleus accumbens and frontal cortex after repeated exposure to amphetamine and cocaine, but the molecular mechanisms responsible are not well understood. We investigated a set of genes related to synaptogenesis, neuritogenesis, and mitogen-activated protein (MAP) kinase after exposure to methamphetamine. Synaptophysin mRNA, but not VAMP2 (synaptobrevin 2) mRNA, which are considered as synaptogenesis markers, increased in the accumbens, striatum, hippocampus, and several cortices, including the medial frontal cortex, after a single dose of 4 mg/kg methamphetamine. Stathmin mRNA, but not neuritin or narp mRNA, which are markers for neuritic sprouting, increased in the striatum, hippocampus, and cortices after a single dose of methamphetamine. The mRNA of arc, an activity-regulated protein associated with cytoskeleton, but not of alpha-tubulin, as markers for neuritic elongation, showed robust increases in the striatum, hippocampus, and cortices after a single dose of methamphetamine. The mRNAs of MAP kinase phosphatase-1 (MKP-1), MKP-3, and rheb, a ras homologue abundant in brain, were investigated to assess the MAP kinase cascades. MKP-1 and MKP-3 mRNAs, but not rheb mRNA, increased in the striatum, thalamus, and cortices, and in the striatum, hippocampus, and cortices, respectively, after a single methamphetamine. Synaptophysin and stathmin mRNAs did not increase again after chronic methamphetamine administration, whereas the increases in arc, MKP-1, and MKP-3 mRNAs persisted in the brain regions after chronic methamphetamine administration

  17. Cascades/Aleutian Play Fairway Analysis: Data and Map Files

    SciTech Connect

    Lisa Shevenell

    2015-11-15

    Contains Excel data files used to quantifiably rank the geothermal potential of each of the young volcanic centers of the Cascade and Aleutian Arcs using world power production volcanic centers as benchmarks. Also contains shapefiles used in play fairway analysis with power plant, volcano, geochemistry and structural data.

  18. MAP kinase activity increases during mitosis in early sea urchin embryos.

    PubMed

    Philipova, R; Whitaker, M

    1998-09-01

    A MBP kinase activity increases at mitosis during the first two embryonic cell cycles of the sea urchin embryo. The activity profile of the MBP kinase is the same both in whole cell extracts and after immunoprecipitation with an anti-MAP kinase antibody (2199). An in-gel assay of MBP activity also shows the same activity profile. The activity is associated with the 44 kDa protein that cross-reacts with anti-MAP kinase antibodies. The 44 kDa protein shows cross-reactivity to anti-phosphotyrosine and MAP kinase-directed anti-phosphotyrosine/phosphothreonine antibodies at the times that MBP kinase activity is high. The 2199 antibody co-precipitates some histone H1 kinase activity, but the MBP kinase activity cannot be accounted for by histone H1 kinase-dependent phosphorylation of MBP. The MAP kinase 2199 antibody was used to purify the MBP kinase activity. Peptide sequencing after partial digestion shows the protein to be homologous to MAP kinases from other species. These data demonstrate that MAP kinase activation during nuclear division is not confined to meiosis, but also occurs during mitotic cell cycles. MAP kinase activity in immunoprecipitates also increases immediately after fertilization, which in the sea urchin egg occurs at interphase of the cell cycle. Treating unfertilized eggs with the calcium ionophore A23187 stimulates the increase in MAP kinase activity, demonstrating that a calcium signal can activate MAP kinase and suggesting that the activation of MAP kinase at fertilization is due to the fertilization-induced increase in cytoplasmic free calcium concentration. This signalling pathway must differ from the pathway responsible for calcium-induced inactivation of MAP kinase activity that is found in eggs that are fertilized in meiotic metaphase. PMID:9701549

  19. Arabidopsis MAP kinase phosphatase 1 and its target MAP kinases 3 and 6 antagonistically determine UV-B stress tolerance, independent of the UVR8 photoreceptor pathway.

    PubMed

    González Besteiro, Marina A; Bartels, Sebastian; Albert, Andreas; Ulm, Roman

    2011-11-01

    Plants perceive UV-B radiation as an informational signal by a pathway involving UVR8 as UV-B photoreceptor, activating photomorphogenic and acclimation responses. In contrast, the response to UV-B as an environmental stress involves mitogen-activated protein kinase (MAPK) signalling cascades. Whereas the perception pathway is plant specific, the UV-B stress pathway is more broadly conserved. Knowledge of the UV-B stress-activated MAPK signalling pathway in plants is limited, and its potential interplay with the UVR8-mediated pathway has not been defined. Here, we show that loss of MAP kinase phosphatase 1 in the mutant mkp1 results in hypersensitivity to acute UV-B stress, but without impairing UV-B acclimation. The MKP1-interacting proteins MPK3 and MPK6 are activated by UV-B stress and are hyperactivated in mkp1. Moreover, mutants mpk3 and mpk6 exhibit elevated UV-B tolerance and partially suppress the UV-B hypersensitivity of mkp1. We show further that the MKP1-regulated stress-response MAPK pathway is independent of the UVR8 photoreceptor, but that MKP1 also contributes to survival under simulated sunlight. We conclude that, whereas UVR8-mediated acclimation in plants promotes UV-B-induced defence measures, MKP1-regulated stress signalling results when UV-B protection and repair are insufficient and damage occurs. The combined activity of these two mechanisms is crucial to UV-B tolerance in plants.

  20. A MAPK cascade downstream of ERECTA receptor-like protein kinase regulates Arabidopsis inflorescence architecture by promoting localized cell proliferation.

    PubMed

    Meng, Xiangzong; Wang, Huachun; He, Yunxia; Liu, Yidong; Walker, John C; Torii, Keiko U; Zhang, Shuqun

    2012-12-01

    Spatiotemporal-specific cell proliferation and cell differentiation are critical to the formation of normal tissues, organs, and organisms. The highly coordinated cell differentiation and proliferation events illustrate the importance of cell-cell communication during growth and development. In Arabidopsis thaliana, ERECTA (ER), a receptor-like protein kinase, plays important roles in promoting localized cell proliferation, which determines inflorescence architecture, organ shape, and size. However, the downstream signaling components remain unidentified. Here, we report a mitogen-activated protein kinase (MAPK; or MPK) cascade that functions downstream of ER in regulating localized cell proliferation. Similar to an er mutant, loss of function of MPK3/MPK6 or their upstream MAPK kinases (MAPKKs; or MKKs), MKK4/MKK5, resulted in shortened pedicels and clustered inflorescences. Epistasis analysis demonstrated that the gain of function of MKK4 and MKK5 transgenes could rescue the loss-of-function er mutant phenotype at both morphological and cellular levels, suggesting that the MPK3/MPK6 cascade functions downstream of the ER receptor. Furthermore, YODA (YDA), a MAPKK kinase, was shown to be upstream of MKK4/MKK5 and downstream of ER in regulating inflorescence architecture based on both gain- and loss-of-function data. Taken together, these results suggest that the YDA-MKK4/MKK5-MPK3/MPK6 cascade functions downstream of the ER receptor in regulating localized cell proliferation, which further shapes the morphology of plant organs. PMID:23263767

  1. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways.

    PubMed

    Qiao, Xiaoyong; Yong Qiao, Xiao; Nie, Ying; Ma, Yaxian; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yin, Weiyao; Yao Yinrg, Wei; Hu, Ying; Xu, Wenming; Ming Xu, Wen; Xu, Liangzhi; Zhi Xu, Liang

    2016-01-07

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued.

  2. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways

    PubMed Central

    Yong Qiao, Xiao; Nie, Ying; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yao Yinrg, Wei; Hu, Ying; Ming Xu, Wen; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  3. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

    SciTech Connect

    Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, II, David G.; Smith, Richard D.; Glass, N. Louise; Heitman, Joseph

    2014-11-20

    Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal

  4. Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging.

    PubMed

    Slaughter, Brian D; Schwartz, Joel W; Li, Rong

    2007-12-18

    Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K(d) >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation. PMID:18077328

  5. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

    PubMed

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans.

  6. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

    PubMed

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. PMID:26907690

  7. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  8. The Croonian Lecture 1998. Identification of a protein kinase cascade of major importance in insulin signal transduction.

    PubMed Central

    Cohen, P

    1999-01-01

    Diabetes affects 3% of the European population and 140 million people worldwide, and is largely a disease of insulin resistance in which the tissues fail to respond to this hormone. This emphasizes the importance of understanding how insulin signals to the cell's interior. We have recently dissected a protein kinase cascade that is triggered by the formation of the insulin 'second messenger' phosphatidylinositide (3,4,5) trisphosphate (PtdIns (3,4,5)P3) and which appears to mediate many of the metabolic actions of this hormone. The first enzyme in the cascade is termed 3-phosphoinositide-dependent protein kinase-1 (PDK1), because it only activates protein kinase B (PKB), the next enzyme in the pathway, in the presence of PtdIns (3,4,5)P3. PKB then inactivates glycogen synthase kinase-3 (GSK3). PDK1, PKB and GSK3 regulate many physiological events by phosphorylating a variety of intracellular proteins. In addition, PKB plays an important role in mediating protection against apoptosis by survival factors, such as insulin-like growth factor-1. PMID:10212493

  9. Building Keypoint Mappings on Multispectral Images by a Cascade of Classifiers with a Resurrection Mechanism

    PubMed Central

    Li, Yong; Jing, Jing; Jin, Hongbin

    2015-01-01

    Inspired by the boosting technique for detecting objects, this paper proposes a cascade structure with a resurrection mechanism to establish keypoint mappings on multispectral images. The cascade structure is composed of four steps by utilizing best bin first (BBF), color and intensity distribution of segment (CIDS), global information and the RANSAC process to remove outlier keypoint matchings. Initial keypoint mappings are built with the descriptors associated with keypoints; then, at each step, only a small number of keypoint mappings of a high confidence are classified to be incorrect. The unclassified keypoint mappings will be passed on to subsequent steps for determining whether they are correct. Due to the drawback of a classification rule, some correct keypoint mappings may be misclassified as incorrect at a step. Observing this, we design a resurrection mechanism, so that they will be reconsidered and evaluated by the rules utilized in subsequent steps. Experimental results show that the proposed cascade structure combined with the resurrection mechanism can effectively build more reliable keypoint mappings on multispectral images than existing methods. PMID:26007729

  10. Identification of Phosphoinositide-Binding Protein PATELLIN2 as a Substrate of Arabidopsis MPK4 MAP Kinase during Septum Formation in Cytokinesis

    PubMed Central

    Suzuki, Takamasa; Matsushima, Chiyuki; Nishimura, Shingo; Higashiyama, Tetsuya; Sasabe, Michiko; Machida, Yasunori

    2016-01-01

    The phosphorylation of proteins by protein kinases controls many cellular and physiological processes, which include intracellular signal transduction. However, the underlying molecular mechanisms of such controls and numerous substrates of protein kinases remain to be characterized. The mitogen-activated protein kinase (MAPK) cascade is of particular importance in a variety of extracellular and intracellular signaling processes. In plant cells, the progression of cytokinesis is an excellent example of an intracellular phenomenon that requires the MAPK cascade. However, the way in which MAPKs control downstream processes during cytokinesis in plant cells remains to be fully determined. We show here that comparisons, by two-dimensional difference gel electrophoresis, of phosphorylated proteins from wild-type Arabidopsis thaliana and mutant plants defective in a MAPK cascade allow identification of substrates of a specific MAPK. Using this method, we identified the PATELLIN2 (PATL2) protein, which has a SEC14 domain, as a substrate of MPK4 MAP kinase. PATL2 was concentrated at the cell division plane, as is MPK4, and had binding affinity for phosphoinositides. This binding affinity was altered after phosphorylation of PATL2 by MPK4, suggesting a role for the MAPK cascade in the formation of cell plates via regeneration of membranes during cytokinesis. PMID:27335345

  11. ERK1 and ERK2 Map Kinases: Specific Roles or Functional Redundancy?

    PubMed Central

    Buscà, Roser; Pouysségur, Jacques; Lenormand, Philippe

    2016-01-01

    The MAP kinase signaling cascade Ras/Raf/MEK/ERK has been involved in a large variety of cellular and physiological processes that are crucial for life. Many pathological situations have been associated to this pathway. More than one isoform has been described at each level of the cascade. In this review we devoted our attention to ERK1 and ERK2, which are the effector kinases of the pathway. Whether ERK1 and ERK2 specify functional differences or are in contrast functionally redundant, constitutes an ongoing debate despite the huge amount of studies performed to date. In this review we compiled data on ERK1 vs. ERK2 gene structures, protein sequences, expression levels, structural and molecular mechanisms of activation and substrate recognition. We have also attempted to perform a rigorous analysis of studies regarding the individual roles of ERK1 and ERK2 by the means of morpholinos, siRNA, and shRNA silencing as well as gene disruption or gene replacement in mice. Finally, we comment on a recent study of gene and protein evolution of ERK isoforms as a distinct approach to address the same question. Our review permits the evaluation of the relevance of published studies in the field especially when measurements of global ERK activation are taken into account. Our analysis favors the hypothesis of ERK1 and ERK2 exhibiting functional redundancy and points to the concept of the global ERK quantity, and not isoform specificity, as being the essential determinant to achieve ERK function. PMID:27376062

  12. Geologic map of the Lassen region, Cascade Range, USA

    USGS Publications Warehouse

    Clynne, Michael; Muffler, L.J.

    1990-01-01

    A preliminary geologic map at 1:50,000 of the Lassen region encompasses 1400 km2. The map displays many small, monogenetic volcanoes of basalt to andesite as well as three major late Pliocene and Quaternary volcanic centers that have erupted products ranging from basaltic andesite to rhyolite. The youngest of these volcanic centers is the Lassen volcanic center, active from 600,000 years B.P. to the present. A major caldera formed at 400,000 years B.P. and has subsequently been filled with silicic lavas. The Lassen geothermal system, which consists of a central vapor-dominated reservoir at a temperature of 235??C underlain by a reservoir of hot water, is centered at Bumpass Hell within Lassen Volcanic National Park.

  13. Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade.

    PubMed

    Yamauchi, J; Tsujimoto, G; Kaziro, Y; Itoh, H

    2001-06-29

    Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.

  14. Salicylic acid activates a 48-kD MAP kinase in tobacco.

    PubMed

    Zhang, S; Klessig, D F

    1997-05-01

    The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors. Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate. This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase). Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not. Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation. The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells. The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity. Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene. Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses.

  15. Preliminary Geologic Map of the Mount Hood 30- by 60-minute Quadrangle, Northern Cascade Range, Oregon

    USGS Publications Warehouse

    Sherrod, David R.; Scott, William E.

    1995-01-01

    This map shows the geology of the central and eastern parts of the Cascade Range in northern Oregon. The Quaternary andesitic stratovolcano of Mount Hood dominates the northwest quarter of the quadrangle, but nearly the entire area is underlain by arc-related volcanic and volcaniclastic rocks of the Cascade Range. Most stratigraphic units were emplaced since middle Miocene time, and all are Oligocene or younger. Despite the proximity of the map area to the Portland metropolitan area, large parts remained virtually unstudied or known only from limited reconnaissance until the late 1970s. A notable exception is the area surrounding Mount Hood, where mapping and chemical analyses by Wise (1969) provided a framework for geologic interpretation. Mapping since 1975 was conducted first to understand the stratigraphy and structure of the Columbia River Basalt Group (Anderson, 1978; Vogt, 1981; J.L. Anderson, in Swanson and others, 1981; Vandiver-Powell, 1978; Burck, 1986) and later to examine the geothermal potential of Mount Hood (Priest and others, 1982). Additional mapping was completed in 1985 for a geologic map of the Cascade Range in Oregon (Sherrod and Smith, 1989). From 1987 to 1990, detailed mapping was conducted in three 15-minute quadrangles on a limited basis (D.R. Sherrod, unpublished mapping) (see fig. 1 for index to mapping). An ongoing volcanic hazards study of Mount Hood by the U.S. Geological Survey (Scott and others, 1994) has provided the catalyst for completing the geologic map of the Mount Hood 30-minute by 60-minute quadrangle. As of June 1994, only two broad areas still remain largely unmapped. One of these areas, labeled 'unmapped' on the geologic map, lies in the Salmon River valley south of Zigzag along the west margin of the quadrangle. Although strata of the Columbia River Basalt Group in the Salmon River valley were mapped in detail by Burck (1986), the overlying middle and upper(?) Miocene lava flows, volcaniclastic strata, and intrusions

  16. Signaling Network Map of Endothelial TEK Tyrosine Kinase.

    PubMed

    Khan, Aafaque Ahmad; Sandhya, Varot K; Singh, Priyata; Parthasarathy, Deepak; Kumar, Awinav; Advani, Jayshree; Gattu, Rudrappa; Ranjit, Dhanya V; Vaidyanathan, Rama; Mathur, Premendu Prakash; Prasad, T S Keshava; Mac Gabhann, F; Pandey, Akhilesh; Raju, Rajesh; Gowda, Harsha

    2014-01-01

    TEK tyrosine kinase is primarily expressed on endothelial cells and is most commonly referred to as TIE2. TIE2 is a receptor tyrosine kinase modulated by its ligands, angiopoietins, to regulate the development and remodeling of vascular system. It is also one of the critical pathways associated with tumor angiogenesis and familial venous malformations. Apart from the vascular system, TIE2 signaling is also associated with postnatal hematopoiesis. Despite the involvement of TIE2-angiopoietin system in several diseases, the downstream molecular events of TIE2-angiopoietin signaling are not reported in any pathway repository. Therefore, carrying out a detailed review of published literature, we have documented molecular signaling events mediated by TIE2 in response to angiopoietins and developed a network map of TIE2 signaling. The pathway information is freely available to the scientific community through NetPath, a manually curated resource of signaling pathways. We hope that this pathway resource will provide an in-depth view of TIE2-angiopoietin signaling and will lead to identification of potential therapeutic targets for TIE2-angiopoietin associated disorders.

  17. MAP KINASE ERK 1/2 INHIBITORS INDUCE DYSMORPHOLOGY IN MOUSE WHOLE EMBRYO CULTURE

    EPA Science Inventory

    ROSEN, M.B. and E. S. HUNTER. Reproductive Toxicology Division, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina. MAP kinase Erk1/2 inhibitors induce dysmorphology in mouse whole embryo culture.

    MAP Kinase signal transduction is associated with a variety ...

  18. Database for the Geologic Map of Upper Eocene to Holocene Volcanic and Related Rocks of the Cascade Range, Oregon

    USGS Publications Warehouse

    Nimz, Kathryn; Ramsey, David W.; Sherrod, David R.; Smith, James G.

    2008-01-01

    Since 1979, Earth scientists of the Geothermal Research Program of the U.S. Geological Survey have carried out multidisciplinary research in the Cascade Range. The goal of this research is to understand the geology, tectonics, and hydrology of the Cascades in order to characterize and quantify geothermal resource potential. A major goal of the program is compilation of a comprehensive geologic map of the entire Cascade Range that incorporates modern field studies and that has a unified and internally consistent explanation. This map is one of three in a series that shows Cascade Range geology by fitting published and unpublished mapping into a province-wide scheme of rock units distinguished by composition and age; map sheets of the Cascade Range in Washington (Smith, 1993) and California will complete the series. The complete series forms a guide to exploration and evaluation of the geothermal resources of the Cascade Range and will be useful for studies of volcano hazards, volcanology, and tectonics. This digital release contains all the information used to produce the geologic map published as U.S. Geological Survey Geologic Investigations Series I-2569 (Sherrod and Smith, 2000). The main component of this digital release is a geologic map database prepared using ArcInfo GIS. This release also contains files to view or print the geologic map and accompanying descriptive pamphlet from I-2569.

  19. Sub-nanometrically resolved chemical mappings of quantum-cascade laser active regions

    NASA Astrophysics Data System (ADS)

    Pantzas, Konstantinos; Beaudoin, Grégoire; Patriarche, Gilles; Largeau, Ludovic; Mauguin, Olivia; Pegolotti, Giulia; Vasanelli, Angela; Calvar, Ariane; Amanti, Maria; Sirtori, Carlo; Sagnes, Isabelle

    2016-05-01

    A procedure that produces sub-nanometrically resolved chemical mappings of MOCVD-grown InGaAs/InAlAs/InP quantum cascade lasers is presented. The chemical mappings reveal that, although the structure is lattice-matched to InP, the InAlAs barriers do not attain the nominal aluminum content—48%—and are, in fact, InGaAlAs quaternaries. This information is used to adjust the aluminum precursor flow and fine-tune the composition of the barriers, resulting in a significant improvement of the fabricated lasers.

  20. Novel screening cascade identifies MKK4 as key kinase regulating Tau phosphorylation at Ser422.

    PubMed

    Grueninger, Fiona; Bohrmann, Bernd; Christensen, Klaus; Graf, Martin; Roth, Doris; Czech, Christian

    2011-11-01

    Phosphorylation of Tau at serine 422 promotes Tau aggregation. The kinase that is responsible for this key phosphorylation event has so far not been identified but could be a potential drug target for Alzheimer's disease. We describe here an assay strategy to identify this kinase. Using a combination of screening a library of 65'000 kinase inhibitors and in vitro inhibitor target profiling of the screening hits using the Ambit kinase platform, MKK4 was identified as playing a key role in Tau-S422 phosphorylation in human neuroblastoma cells.

  1. The myxoma virus thymidine kinase gene: sequence and transcriptional mapping.

    PubMed

    Jackson, R J; Bults, H G

    1992-02-01

    The myxoma virus thymidine kinase (TK) gene is encoded on a 1.6 kb SacI-SalI restriction fragment located between 57.7 and 59.3 kb on the 163 kb genomic map. The nucleotide sequence of this fragment as well as 228 bp from the adjacent SalI-AA2 fragment was determined and found to encode four major open reading frames (ORFs). Three of these ORFs are similar in nucleotide sequence to ORFs L5R and J1R, and the TK gene of vaccinia virus (VV). The fourth ORF, MF8a, shows similarity to the ORFs found in the same position relative to the TK genes of Shope fibroma virus, Kenya sheep-1 virus and swine-pox virus. A search of the complete VV nucleotide sequence for regions of similarity to MF8a identified the host specificity gene C7L. Northern blot analysis of early viral RNA identified transcripts of approximately 700 nucleotides for both the TK gene and ORF MF8a. The 5' ends of the TK gene and ORF MF8a early mRNAs were mapped by primer extension to initiation sites 13 nucleotides downstream of sequences with similarity to the VV early promoter consensus. The sizes of the TK and MF8a mRNAs are consistent with transcription termination and polyadenylation occurring downstream of the sequence TTTTTNT, which is identical to the consensus sequence for the VV transcription termination signal.

  2. Translational control of myelin basic protein expression by ERK2 MAP kinase regulates timely remyelination in the adult brain.

    PubMed

    Michel, Kelly; Zhao, Tianna; Karl, Molly; Lewis, Katherine; Fyffe-Maricich, Sharyl L

    2015-05-20

    Successful myelin repair in the adult CNS requires the robust and timely production of myelin proteins to generate new myelin sheaths. The underlying regulatory mechanisms and complex molecular basis of myelin regeneration, however, remain poorly understood. Here, we investigate the role of ERK MAP kinase signaling in this process. Conditional deletion of Erk2 from cells of the oligodendrocyte lineage resulted in delayed remyelination following demyelinating injury to the adult mouse corpus callosum. The delayed repair occurred as a result of a specific deficit in the translation of the major myelin protein, MBP. In the absence of ERK2, activation of the ribosomal protein S6 kinase (p70S6K) and its downstream target, ribosomal protein S6 (S6RP), was impaired at a critical time when premyelinating oligodendrocytes were transitioning to mature cells capable of generating new myelin sheaths. Thus, we have described an important link between the ERK MAP kinase signaling cascade and the translational machinery specifically in remyelinating oligodendrocytes in vivo. These results suggest an important role for ERK2 in the translational control of MBP, a myelin protein that appears critical for ensuring the timely generation of new myelin sheaths following demyelinating injury in the adult CNS.

  3. ALTERED PHOSPHORYLATION OF MAP KINASE AFTER ACUTE EXPOSURE TO PCB153.

    EPA Science Inventory

    Long-term potentiation (LTP) is a model of synaptic plasticity believed to encompass the physiological substrate of memory. The mitogen-activated protein kinase (ERK1/2) signalling cascade contributes to synaptic plasticity and to long-term memory formation. Learning and LTP st...

  4. TGF-β activates Erk MAP kinase signalling through direct phosphorylation of ShcA

    PubMed Central

    Lee, Matt K; Pardoux, Cécile; Hall, Marie C; Lee, Pierre S; Warburton, David; Qing, Jing; Smith, Susan M; Derynck, Rik

    2007-01-01

    Erk1/Erk2 MAP kinases are key regulators of cell behaviour and their activation is generally associated with tyrosine kinase signalling. However, TGF-β stimulation also activates Erk MAP kinases through an undefined mechanism, albeit to a much lower level than receptor tyrosine kinase stimulation. We report that upon TGF-β stimulation, the activated TGF-β type I receptor (TβRI) recruits and directly phosphorylates ShcA proteins on tyrosine and serine. This dual phosphorylation results from an intrinsic TβRI tyrosine kinase activity that complements its well-defined serine-threonine kinase function. TGF-β-induced ShcA phosphorylation induces ShcA association with Grb2 and Sos, thereby initiating the well-characterised pathway linking receptor tyrosine kinases with Erk MAP kinases. We also found that TβRI is tyrosine phosphorylated in response to TGF-β. Thus, TβRI, like the TGF-β type II receptor, is a dual-specificity kinase. Recruitment of tyrosine kinase signalling pathways may account for aspects of TGF-β biology that are independent of Smad signalling. PMID:17673906

  5. Computational insights for the discovery of non-ATP competitive inhibitors of MAP kinases.

    PubMed

    Schnieders, Michael J; Kaoud, Tamer S; Yan, Chunli; Dalby, Kevin N; Ren, Pengyu

    2012-01-01

    Due to their role in cellular signaling mitogen activated protein (MAP) kinases represent targets of pharmaceutical interest. However, the majority of known MAP kinase inhibitors compete with cellular ATP and target an ATP binding pocket that is highly conserved in the 500 plus representatives of the human protein kinase family. Here we review progress toward the development of non-ATP competitive MAP kinase inhibitors for the extracellular signal regulated kinases (ERK1/2), the c-jun N-terminal kinases (JNK1/2/3) and the p38 MAPKs (α, β, γ, and δ). Special emphasis is placed on the role of computational methods in the drug discovery process for MAP kinases. Topics include recent advances in X-ray crystallography theory that improve the MAP kinase structures essential to structurebased drug discovery, the use of molecular dynamics to understand the conformational heterogeneity of the activation loop and inhibitors discovered by virtual screening. The impact of an advanced polarizable force field such as AMOEBA used in conjunction with sophisticated kinetic and thermodynamic simulation methods is also discussed.

  6. Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

    PubMed Central

    Schnieders, Michael J.; Kaoud, Tamer S.; Yan, Chunli; Dalby, Kevin N.; Ren, Pengyu

    2014-01-01

    Due to their role in cellular signaling mitogen activated protein (MAP) kinases represent targets of pharmaceutical interest. However, the majority of known MAP kinase inhibitors compete with cellular ATP and target an ATP binding pocket that is highly conserved in the 500 plus representatives of the human protein kinase family. Here we review progress toward the development of non-ATP competitive MAP kinase inhibitors for the extracellular signal regulated kinases (ERK1/2), the c-jun N-terminal kinases (JNK1/2/3) and the p38 MAPKs (α, β, γ, and δ). Special emphasis is placed on the role of computational methods in the drug discovery process for MAP kinases. Topics include recent advances in X-ray crystallography theory that improve the MAP kinase structures essential to structure-based drug discovery, the use of molecular dynamics to understand the conformational heterogeneity of the activation loop and inhibitors discovered by virtual screening. The impact of an advanced polarizable force field such as AMOEBA used in conjunction with sophisticated kinetic and thermodynamic simulation methods is also discussed. PMID:22316156

  7. p38 Mitogen-activated protein kinase inhibitors: a review on pharmacophore mapping and QSAR studies.

    PubMed

    Gangwal, Rahul P; Bhadauriya, Anuseema; Damre, Mangesh V; Dhoke, Gaurao V; Sangamwar, Abhay T

    2013-01-01

    p38 mitogen-activated protein (MAP) kinases are the serine/threonine protein kinases, which play a vital role in cellular responses to external stress signals. p38 MAP kinase inhibitors have shown anti-inflammatory effects in the preclinical disease models, primarily through inhibition of the expression of inflammatory mediators. A number of structurally diverse p38 MAP kinase inhibitors have been developed as potential anti-inflammatory agents. Most of the inhibitors have failed in the clinical trials either due to poor pharmacokinetic profile or selectivity issue, which makes p38 MAP kinase a promising target for molecular modelling studies. Several quantitative structure activity relationships (QSAR) and pharmacophore models have been developed to identify the structural requirements essential for p38 MAP kinase inhibitory activity. In this review, we provide an overview of the presently known p38 MAP kinase inhibitors and how QSAR analyses among series of compounds have led to the development of molecular models and pharmacophores, allowing the design of novel inhibitors.

  8. FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

    PubMed Central

    Worthen, G S; Avdi, N; Buhl, A M; Suzuki, N; Johnson, G L

    1994-01-01

    Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants. Images PMID:8040337

  9. Preparation of Residual Gravity Maps for the Southern Cascade Mountains, Washington Using Fourier Analysis

    SciTech Connect

    Dishberger, Debra McLean

    1983-04-01

    This report represents a continuation of gravity work in the Cascade Mountains of Washington supported by the Division of Geology and Earth Resources since 1974. The purpose of this research has been collection of baseline gravity data for use in geothermal resource evaluation. Results of the Division's gravity studies to date are given in Danes and Phillips (1983a, 1983b). One of the problems encountered when analyzing gravity data is distinguishing between those parts of the data that represent geologic structures of interest, and those that do not. In many cases, the features of interest are relatively small, near-surface features, such as those sought in mineral, petroleum, or geothermal exploration. Gravity anomalies caused by such structures may be distorted or masked by anomalies caused by larger, deeper geologic structures. Gravity anomalies caused by relatively shallow, small geologic structures are termed residual anomalies. Those due to broad, deep-seated features can be described as regional anomalies. The purpose of this report is to describe a Fourier analysis method for separating residual and regional gravity anomalies from a complete Bouguer gravity anomaly field. The technique has been applied to gravity data from the Southern Cascade Mountains, Washington. Residual gravity anomaly maps at a scale of 1:250,000 are presented for various regional wavelength filters, and a power spectrum of the frequency components in the South Cascade gravity data is displayed. No attempt is made to interpret the results of this study in terms of geologic structures.

  10. Hydrological-hydraulic model cascading for pan-European flood hazard mapping

    NASA Astrophysics Data System (ADS)

    Alfieri, Lorenzo; Salamon, Peter; Bianchi, Alessandra; Pappenberger, Florian; Wetterhall, Fredrik

    2013-04-01

    Flood hazard maps at trans-national and continental scale have potential for a large number of applications ranging from climate change studies, aid to emergency planning for major flood crisis, early damage assessment and urban development, among others. However, such maps are usually available at rather coarse resolution, which limits their applications to rough assessments. At finer resolution, maps are often limited to country boundaries, due to limited data sharing and specific cooperation programs at trans-national level. The European Floods Directive 2007/60/EC requires EU Member States to map the potential flood extent for all water courses by the end of 2013. In this work we derive a pan-European flood hazard map at 100 m resolution, covering most of the European territory. The proposed approach is based on expanding the cascade model presented by Barredo et al. (2007). First, a pan-European distributed rainfall-runoff model with a resolution of 5x5km is set up and calibrated using discharge observations at 481 gauging sites. Then, by using 21-year meteorological climatology we derived a long term discharge simulation. A generalized extreme value fitting is applied to estimate flood peaks with 100-year return period for each river pixel in the model. This data is downscaled to the river network at 100 m resolution and design flood hydrographs are derived for 100-year return period event along the entire pan-European river network. Design flood hydrographs are then used to perform small-scale floodplain hydraulic simulations every 5 km along the river network using a two-dimensional hydraulic model. Finally, output maps of more than 35000 hydraulic simulations are merged into a pan-European flood hazard map. The quality of this map is evaluated for selected areas against the flood hazard maps provided by national/regional authorities. Finally, limitations of the approach and future directions of research are discussed.

  11. Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel Is Regulated by the Phototransduction Cascade and Involves Several Protein Kinases and Phosphatases

    PubMed Central

    Voolstra, Olaf; Bartels, Jonas-Peter; Oberegelsbacher, Claudia; Pfannstiel, Jens; Huber, Armin

    2013-01-01

    Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation. PMID:24040070

  12. Highly sensitive detection of T4 polynucleotide kinase activity by coupling split DNAzyme and ligation-triggered DNAzyme cascade amplification.

    PubMed

    Liu, Shufeng; Ming, Jingjing; Lin, Ying; Wang, Chunfeng; Cheng, Chuanbin; Liu, Tao; Wang, Li

    2014-05-15

    In current study, a dual strategy for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity was proposed, which combined split DNAzyme-based background reduction with ligation-triggered DNAzyme cascade for signal amplification. The 8-17 DNAzyme is split into two separate oligonucleotide fragments, which can be separately hybridized to the template DNA to form a ligatable nick after one of the fragments is phosphorylated at the 5at the yl by T4 PNK. With the further addition of Escherichia coli DNA ligase, the two oligonucleotides can be ligated to produce the activated 8-17 DNAzyme, the amount of which is positively related to the activity of T4 PNK. The signal amplification can be achieved through the cyclic cleavage of 8-17 DNAzyme toward the molecular beacon substrate, resulting in an evident fluorescence signal enhancement. The current dual strategy can significantly improve the detection sensitivity of the sensing systems, resulting in a detection limit of 0.001 U mL(-1) for T4 PNK activity, which is superior or comparable to the reported methods. Furthermore, the inhibition effects of adenosine diphosphate and sodium hydrogen phosphate on T4 PNK activity have also been demonstrated with satisfactory results. The current method may be further developed as a universal protocol for monitoring activity and inhibition of nucleotide kinase, and may show the huge potentials in biological process researches, drug discovery, and clinic diagnostics.

  13. Reciprocal Regulation of AKT and MAP Kinase Dictates Virus-Host Cell Fusion ▿

    PubMed Central

    Sharma, Nishi R.; Mani, Prashant; Nandwani, Neha; Mishra, Rajakishore; Rana, Ajay; Sarkar, Debi P.

    2010-01-01

    Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy. PMID:20164223

  14. Synaptic generation of an intracellular retrograde signal requires activation of the tyrosine kinase and mitogen-activated protein kinase signaling cascades in Aplysia.

    PubMed

    Stough, Shara; Kopec, Ashley M; Carew, Thomas J

    2015-11-01

    Cellular changes underlying memory formation can be generated in an activity-dependent manner at specific synapses. Thus an important question concerns the mechanisms by which synaptic signals communicate with the cell body to mediate these cellular changes. A monosynaptic circuit that is enhanced by sensitization in Aplysia is well-suited to study this question because three different subcellular compartments: (i) the sensorimotor SN-MN synapses, (ii) the SN projections to MNs via axonal connections, (iii) the SN cell bodies, can all be manipulated and studied independently. Here, we report that activity-dependent (AD) training in either the entire SN-MN circuit or in only the synaptic compartment, activates MAPK in a temporally and spatially specific pattern. Specifically, we find (i) MAPK activation is first transiently generated at SN-MN synapses during training, (ii) immediately after training MAPK is transiently activated in SN-MN axonal connections and persistently activated in SN cell bodies, and finally, (iii) MAPK is activated in SN cell bodies and SN-MN synapses 1h after training. These data suggest that there is an intracellularly transported retrograde signal generated at the synapse which is later responsible for delayed MAPK activation at SN somata. Finally, we find that this retrograde signal requires activation of tyrosine kinase (TK) and MEK signaling cascades at the synapses.

  15. Targeting the Raf kinase cascade in cancer therapy--novel molecular targets and therapeutic strategies.

    PubMed

    Lee, John T; McCubrey, James A

    2002-12-01

    The mitogen-activated protein kinases (MAPKs) are a group of signal transducers with oncogenic potential in an assortment of cell types. Dysregulated signalling from any of the members of this family has been shown to result in development of human malignancies. Consequently, the collective goal of the scientific community is to inhibit aberrant signalling initiated from these molecules whilst minimising toxicity associated with such inhibition. This review covers events responsible for MAPK activation in detail, with an emphasis placed upon possible points of pharmacological intervention. A discussion addressing numerous chemotherapeutic approaches that have been developed over the previous decade for MAPK inhibition is also included. In addition, emphasis is placed upon the various arrays of kinase inhibitors, small molecule inhibitors, competitive inhibitors, nucleic acid aptamers and other molecules which have been proven effective in prevention of MAPK signalling. Finally, the potential therapeutic promise of many of these compounds is addressed in a manner that encompasses the complexities of MAPK signal transduction, in addition to concerns surrounding the development of drug resistance.

  16. Inhibition of the PI3 kinase cascade in corticolimbic circuit: temporal and differential effects on contextual fear and extinction.

    PubMed

    Kritman, Milly; Maroun, Mouna

    2013-05-01

    We studied the role of PI3K cascade in the basolateral amygdala (BLA) and the infralimbic region of the medial prefrontal cortex (IL-mPFC), in contextual fear learning and extinction in the rat. To that end, we micro-infused the phosphoinositide-3-kinase (PIK3) inhibitor LY294002 into either the mPFC or the BLA. Infusion of LY294002 into the BLA following fear conditioning was associated with enhanced freezing levels and impaired extinction in the subsequent sessions. Similarly, inhibition of PI3K in the BLA before the retrieval of fear memory was associated with impaired retrieval of the fear memory, which was expressed as reduced freezing levels that persisted over 2 d. In the IL-mPFC, only consolidation of fear extinction was impaired: micro-infusion of PI3K inhibitor following the retrieval of fear was associated with impaired extinction on the following days. These results indicate differences in the temporal parameters of the effects of PI3K inhibition in the IL-mPFC and in the BLA, which suggest differential involvement of these structures in long-term fear and in extinction of fear memory. Our findings provide additional evidence for the critical roles played by PI3K in intact formation of fear memory and in its extinction and add new evidence for a role of PI3K in consolidation of memory of extinction. Better understanding of the differential involvement of the PI3K cascade during acquisition and extinction of fear conditioning in the mPFC-amygdala circuit could potentially contribute to the understanding and treatment of anxiety disorders.

  17. Integration of Apoptosis Signal-Regulating Kinase 1-Mediated Stress Signaling with the Akt/Protein Kinase B-IκB Kinase Cascade

    PubMed Central

    Puckett, Mary C.; Goldman, Erinn H.; Cockrell, Lisa M.; Huang, Bei; Kasinski, Andrea L.; Du, Yuhong; Wang, Cun-Yu; Lin, Anning; Ichijo, Hidenori; Khuri, Fadlo

    2013-01-01

    Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of κB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-κB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development. PMID:23530055

  18. MAP4K family kinases act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway

    PubMed Central

    Meng, Zhipeng; Moroishi, Toshiro; Mottier-Pavie, Violaine; Plouffe, Steven W.; Hansen, Carsten G.; Hong, Audrey W.; Park, Hyun Woo; Mo, Jung-Soon; Lu, Wenqi; Lu, Shicong; Flores, Fabian; Yu, Fa-Xing; Halder, Georg; Guan, Kun-Liang

    2015-01-01

    The Hippo pathway plays a central role in tissue homoeostasis, and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include a kinase cascade of MST1/2 and LATS1/2 and the transcription co-activators YAP/TAZ. In response to stimulation, LATS1/2 phosphorylate and inhibit YAP/TAZ, the main effectors of the Hippo pathway. Accumulating evidence suggests that MST1/2 are not required for the regulation of YAP/TAZ. Here we show that deletion of LATS1/2 but not MST1/2 abolishes YAP/TAZ phosphorylation. We have identified MAP4K family members—Drosophila Happyhour homologues MAP4K1/2/3 and Misshapen homologues MAP4K4/6/7—as direct LATS1/2-activating kinases. Combined deletion of MAP4Ks and MST1/2, but neither alone, suppresses phosphorylation of LATS1/2 and YAP/TAZ in response to a wide range of signals. Our results demonstrate that MAP4Ks act in parallel to and are partially redundant with MST1/2 in the regulation of LATS1/2 and YAP/TAZ, and establish MAP4Ks as components of the expanded Hippo pathway. PMID:26437443

  19. Phosphodiesterase MoPdeH targets MoMck1 of the conserved mitogen-activated protein (MAP) kinase signalling pathway to regulate cell wall integrity in rice blast fungus Magnaporthe oryzae.

    PubMed

    Yin, Ziyi; Tang, Wei; Wang, Jingzhen; Liu, Xinyu; Yang, Lina; Gao, Chuyun; Zhang, Jinlong; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2016-06-01

    In the rice blast fungus Magnaporthe oryzae, the high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance through an unknown mechanism. By utilizing affinity purification, we found that MoPdeH interacts with MoMck1, one of the components of the mitogen-activated protein (MAP) kinase cascade that regulates CWI. Overexpression of MoMCK1 suppressed defects in autolysis and pathogenicity of the ΔMopdeH mutant, although partially, suggesting that MoPdeH plays a critical role in CWI maintenance mediated by the MAP kinase pathway. We found that MoMck1 and two other MAP kinase cascade components, MoMkk1 and MoMps1, modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop. In addition, disruption of MoMKK1 resulted in less aerial hyphal formation, defective asexual development and attenuated pathogenicity. Moreover, MoMkk1 plays a role in the response to osmotic stress via regulation of MoOsm1 phosphorylation levels, whereas endoplasmic reticulum (ER) stress enhances MoMps1 phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response (UPR) pathway. Taken together, our findings demonstrate that MoPdeH functions upstream of the MoMck1-MoMkk1-MoMps1 MAP kinase pathway to regulate CWI, and that MoPdeH also mediates crosstalk between the cAMP signalling pathway, the osmotic sensing high osmolarity glycerol (HOG) pathway and the dithiothreitol (DTT)-induced UPR pathway in M. oryzae.

  20. Phosphodiesterase MoPdeH targets MoMck1 of the conserved mitogen-activated protein (MAP) kinase signalling pathway to regulate cell wall integrity in rice blast fungus Magnaporthe oryzae.

    PubMed

    Yin, Ziyi; Tang, Wei; Wang, Jingzhen; Liu, Xinyu; Yang, Lina; Gao, Chuyun; Zhang, Jinlong; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2016-06-01

    In the rice blast fungus Magnaporthe oryzae, the high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance through an unknown mechanism. By utilizing affinity purification, we found that MoPdeH interacts with MoMck1, one of the components of the mitogen-activated protein (MAP) kinase cascade that regulates CWI. Overexpression of MoMCK1 suppressed defects in autolysis and pathogenicity of the ΔMopdeH mutant, although partially, suggesting that MoPdeH plays a critical role in CWI maintenance mediated by the MAP kinase pathway. We found that MoMck1 and two other MAP kinase cascade components, MoMkk1 and MoMps1, modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop. In addition, disruption of MoMKK1 resulted in less aerial hyphal formation, defective asexual development and attenuated pathogenicity. Moreover, MoMkk1 plays a role in the response to osmotic stress via regulation of MoOsm1 phosphorylation levels, whereas endoplasmic reticulum (ER) stress enhances MoMps1 phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response (UPR) pathway. Taken together, our findings demonstrate that MoPdeH functions upstream of the MoMck1-MoMkk1-MoMps1 MAP kinase pathway to regulate CWI, and that MoPdeH also mediates crosstalk between the cAMP signalling pathway, the osmotic sensing high osmolarity glycerol (HOG) pathway and the dithiothreitol (DTT)-induced UPR pathway in M. oryzae. PMID:27193947

  1. Fungal Communication Requires the MAK-2 Pathway Elements STE-20 and RAS-2, the NRC-1 Adapter STE-50 and the MAP Kinase Scaffold HAM-5

    PubMed Central

    Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

    2014-01-01

    Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes. PMID:25411845

  2. Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

    PubMed Central

    Roth Flach, Rachel J.; Skoura, Athanasia; Matevossian, Anouch; Danai, Laura V.; Zheng, Wei; Cortes, Christian; Bhattacharya, Samit K.; Aouadi, Myriam; Hagan, Nana; Yawe, Joseph C.; Vangala, Pranitha; Menendez, Lorena Garcia; Cooper, Marcus P.; Fitzgibbons, Timothy P.; Buckbinder, Leonard; Czech, Michael P.

    2015-01-01

    Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe−/− mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe−/− and Ldlr−/− mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis. PMID:26688060

  3. A Cell-Autonomous Molecular Cascade Initiated by AMP-Activated Protein Kinase Represses Steroidogenesis

    PubMed Central

    Abdou, Houssein S.; Bergeron, Francis

    2014-01-01

    Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production. PMID:25225331

  4. Brain tissue energy dependence of CaM kinase IV cascade activation during hypoxia in the cerebral cortex of newborn piglets.

    PubMed

    Delivoria-Papadopoulos, Maria; Ashraf, Qazi M; Mishra, Om Prakash

    2011-03-17

    The present study aims to investigate the dependence of CaM kinase IV cascade activation during hypoxia and tests the hypothesis that hypoxia-induced tyrosine phosphorylation of CaM and CaM kinase IV, activation of CaM kinase IV and phosphorylation of CREB protein during hypoxia increases as a function of increase in cerebral tissue hypoxia as measured by decrease in tissue ATP and phosphocreatine (PCr). 3-5 days old newborn piglets were divided into normoxic (Nx, FiO₂ of 0.21 for 1h) and hypoxic (Hx, FiO₂ of 0.07 for 1h) groups. Cerebral tissue hypoxia was documented by determining the levels of high energy phosphates ATP and phosphocreatine (PCr). Cerebral cortical neuronal nuclei were isolated and purified, and tyrosine phosphorylation of calmodulin (Tyr⁹⁹), the activator of CaM kinase IV, and CaM kinase IV determined by Western blot using anti-phospho-(pTyr⁹⁹)-calmodulin, anti-pTyrosine and anti-CaM kinase IV antibodies. The activity of CaM kinase IV and its consequence the phosphorylation of CREB protein at Ser¹³³ were determined. The levels of ATP (μmole/g brain) ranged from 3.48 to 5.28 in Nx, and 0.41 to 2.26 in Hx. The levels of PCr (μmole/g brain) ranged from 2.46 to 3.91 in Nx and 0.72 to 1.20 in Hx. The pTyr⁹⁹ calmodulin (OD x mm²) ranged from 20.35 to 54.47.60 in Nx, and 84.52 to 181.42 in Hx (r²=0.5309 vs ATP and r²=0.6899 vs PCr). Expression of tyrosine phosphorylated CaM kinase IV ranged from 32.86 to 82.46 in Nx and 96.70 to 131.62 in Hx (r²=0.5132 vs ATP and r²=0.4335 vs PCr). The activity of CaM kinase IV (pmole/mg protein/min) ranged from 1263 to 3448 in Nx and 3767 to 6633 in Hx (r²=0.7113 vs ATP and r²=0.6182 vs PCr). The expression of p-CREB at Ser¹³³ ranged from 44.26 to 70.28 in Nx and 82.70 to 182.86 in Hx (r²=0.6621 vs ATP and r²=0.5485 vs PCr). The data show that hypoxia results in increased tyrosine phosphorylation of calmodulin (Tyr⁹⁹), increased tyrosine phosphorylation of CaM kinase IV, increased

  5. Structural Bioinformatics-Based Prediction of Exceptional Selectivity of p38 MAP Kinase Inhibitor PH-797804

    SciTech Connect

    Xing, Li; Shieh, Huey S.; Selness, Shaun R.; Devraj, Rajesh V.; Walker, John K.; Devadas, Balekudru; Hope, Heidi R.; Compton, Robert P.; Schindler, John F.; Hirsch, Jeffrey L.; Benson, Alan G.; Kurumbail, Ravi G.; Stegeman, Roderick A.; Williams, Jennifer M.; Broadus, Richard M.; Walden, Zara; Monahan, Joseph B.; Pfizer

    2009-07-24

    PH-797804 is a diarylpyridinone inhibitor of p38{alpha} mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38{alpha} inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38{alpha} kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180{sup o} rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38{alpha} kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38{alpha} kinase inhibitors.

  6. ERK5 MAP Kinase Regulates Neurogenin1 during Cortical Neurogenesis

    PubMed Central

    Cundiff, Paige; Liu, Lidong; Wang, Yupeng; Zou, Junhui; Pan, Yung-Wei; Abel, Glen; Duan, Xin; Ming, Guo-li; Englund, Chris; Hevner, Robert; Xia, Zhengui

    2009-01-01

    The commitment of multi-potent cortical progenitors to a neuronal fate depends on the transient induction of the basic-helix-loop-helix (bHLH) family of transcription factors including Neurogenin 1 (Neurog1). Previous studies have focused on mechanisms that control the expression of these proteins while little is known about whether their pro-neural activities can be regulated by kinase signaling pathways. Using primary cultures and ex vivo slice cultures, here we report that both the transcriptional and pro-neural activities of Neurog1 are regulated by extracellular signal-regulated kinase (ERK) 5 signaling in cortical progenitors. Activation of ERK5 potentiated, while blocking ERK5 inhibited Neurog1-induced neurogenesis. Furthermore, endogenous ERK5 activity was required for Neurog1-initiated transcription. Interestingly, ERK5 activation was sufficient to induce Neurog1 phosphorylation and ERK5 directly phosphorylated Neurog1 in vitro. We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1. Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1. Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors. PMID:19365559

  7. Macrophage signalling upon mycobacterial infection: the MAP kinases lead the way.

    PubMed

    Schorey, Jeffrey S; Cooper, Andrea M

    2003-03-01

    Mycobacteria activate a series of macrophage signalling pathways upon engaging host cell receptors and during the invasion process. These signals initiate a cascade of events leading to the production of immune effector molecules including cytokines, chemokines and reactive nitrogen intermediates. This response by the macrophage is critical for the control of the mycobacterial infection and, not surprisingly, pathogenic mycobacteria have evolved mechanisms to limit this macrophage activation. Recent data has suggested that macrophages infected with pathogenic compared to non-pathogenic mycobacteria are restricted in their activation of the mitogen activated protein kinase (MAPK) pathways. Mitogen activated protein kinase activation in macrophages appears to play an important role in promoting antimycobacterial activity and in the production of various effector molecules following a mycobacterial infection. Therefore, the ability of pathogenic mycobacteria to limit MAPK activity is likely an important virulence mechanism and may be a potential therapeutic target.

  8. Database for the geologic map of upper Eocene to Holocene volcanic and related rocks in the Cascade Range, Washington

    USGS Publications Warehouse

    Barron, Andrew D.; Ramsey, David W.; Smith, James G.

    2014-01-01

    This digital database contains information used to produce the geologic map published as Sheet 1 in U.S. Geological Survey Miscellaneous Investigations Series Map I-2005. (Sheet 2 of Map I-2005 shows sources of geologic data used in the compilation and is available separately). Sheet 1 of Map I-2005 shows the distribution and relations of volcanic and related rock units in the Cascade Range of Washington at a scale of 1:500,000. This digital release is produced from stable materials originally compiled at 1:250,000 scale that were used to publish Sheet 1. The database therefore contains more detailed geologic information than is portrayed on Sheet 1. This is most noticeable in the database as expanded polygons of surficial units and the presence of additional strands of concealed faults. No stable compilation materials exist for Sheet 1 at 1:500,000 scale. The main component of this digital release is a spatial database prepared using geographic information systems (GIS) applications. This release also contains links to files to view or print the map sheet, main report text, and accompanying mapping reference sheet from Map I-2005. For more information on volcanoes in the Cascade Range in Washington, Oregon, or California, please refer to the U.S. Geological Survey Volcano Hazards Program website.

  9. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site. PMID:17062013

  10. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site.

  11. Differential regulation of Jun N-terminal kinase and p38MAP kinase by Galpha12.

    PubMed

    Dermott, Jonathan M; Ha, Ji Hee; Lee, Chang Ho; Dhanasekaran, N

    2004-01-01

    Based on the findings that the overexpression of the wild-type Galpha(12) (Galpha(12)WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Galpha(12) can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Galpha(12)WT or the expression of an activated mutant of Galpha(12) (Galpha(12)QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Galpha(12)WT or the expression of Galpha(12)QL results in a potent inhibition of p38MAPK, and that the mechanism by which Galpha(12) inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Galpha(12) attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Galpha(12) activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Galpha(12) differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Galpha(12) stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK- and JNK-mediated signaling pathways. PMID:14712227

  12. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

    PubMed

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus; Nielsen, Henrik Bjørn; Botanga, Christopher J; Thorgrimsen, Stephan; Palma, Kristoffer; Suarez-Rodriguez, Maria Cristina; Sandbech-Clausen, Signe; Lichota, Jacek; Brodersen, Peter; Grasser, Klaus D; Mattsson, Ole; Glazebrook, Jane; Mundy, John; Petersen, Morten

    2008-08-20

    Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

  13. Feedback phosphorylation of an RGS protein by MAP kinase in yeast.

    PubMed

    Garrison, T R; Zhang, Y; Pausch, M; Apanovitch, D; Aebersold, R; Dohlman, H G

    1999-12-17

    Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity in vitro and to promote G protein desensitization in vivo. Less is known about how RGS proteins are themselves regulated. To address this question we purified the RGS in yeast, Sst2, and used electrospray ionization mass spectrometry to identify post-translational modifications. This analysis revealed that Sst2 is phosphorylated at Ser-539 and that phosphorylation occurs in response to pheromone stimulation. Ser-539 lies within a consensus mitogen-activated protein (MAP) kinase phosphorylation site, Pro-X-Ser-Pro. Phosphorylation is blocked by mutations in the MAP kinase genes (FUS3, KSS1), as well as by mutations in components needed for MAP kinase activation (STE11, STE7, STE4, STE18). Phosphorylation is also blocked by replacing Ser-539 with Ala, Asp, or Glu (but not Thr). These point mutations do not alter pheromone sensitivity, as determined by growth arrest and reporter transcription assays. However, phosphorylation appears to slow the rate of Sst2 degradation. These findings indicate that the G protein-regulated MAP kinase in yeast can act as a feedback regulator of Sst2, itself a regulator of G protein signaling. PMID:10593933

  14. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation.

    PubMed

    Eichel, K; Jullié, D; von Zastrow, M

    2016-03-01

    β-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-β-arrestin complexes. We show here, using β1-adrenergic receptors, that β-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and β-arrestin-dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a β-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery.

  15. Resveratrol amplifies BMP-4-stimulated osteoprotegerin synthesis via p38 MAP kinase in osteoblasts.

    PubMed

    Kuroyanagi, Gen; Tokuda, Haruhiko; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Mizutani, Jun; Kozawa, Osamu; Otsuka, Takanobu

    2015-09-01

    Resveratrol is a naturally occurring polyphenol that possesses health‑related properties, and is predominantly found in grapes and berries. Bone morphogenetic protein‑4 (BMP‑4) stimulates osteocalcin synthesis via p38 mitogen‑activated protein (MAP) kinase in osteoblast‑like MC3T3‑E1 cells. The present study aimed to investigate the effects of resveratrol on BMP‑4‑induced osteoprotegerin (OPG) synthesis in MC3T3‑E1 cells. Resveratrol alone had no effect on OPG expression levels, but significantly enhanced BMP‑4‑induced OPG release. In addition, resveratrol markedly amplified the mRNA expression levels of BMP‑4‑induced OPG. SB203580 is an inhibitor of p38 MAP kinase, which was shown to suppress BMP‑4‑stimulated OPG release. BMP‑4‑induced phosphorylation of p38 MAP kinase was also enhanced by resveratrol. Furthermore, SB203580 significantly reduced the resveratrol‑induced amplification of BMP‑4‑stimulated OPG release. These results suggested that resveratrol was able to upregulate BMP‑4‑stimulated OPG synthesis via the amplification of p38 MAP kinase activity in osteoblasts.

  16. β-arrestin drives MAP kinase signaling from clathrin-coated structures after GPCR dissociation

    PubMed Central

    Eichel, K.; Jullié, D.

    2016-01-01

    β-arrestins critically regulate G protein-coupled receptor (GPCR) signaling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G protein-independent signal via MAP kinase1–3. Despite enormous recent progress toward understanding biophysical aspects of arrestin function4,5, its cell biology remains relatively poorly understood. Two key tenets underlie the present dogma: (1) β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and (2) MAP kinase activation requires endocytosis of formed GPCR - β-arrestin complexes6–9. We show here, using β1-adrenergic receptors, that β-arrestin-2 (Arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin and β-arrestin -dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signaling from CCSs. We propose a β-arrestin signaling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  17. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation.

    PubMed

    Eichel, K; Jullié, D; von Zastrow, M

    2016-03-01

    β-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-β-arrestin complexes. We show here, using β1-adrenergic receptors, that β-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and β-arrestin-dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a β-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  18. Smooth-muscle mitogen-activated protein (MAP) kinase: purification and characterization, and the phosphorylation of caldesmon.

    PubMed Central

    Childs, T J; Mak, A S

    1993-01-01

    A single 42 kDa isoform of mitogen-activated protein (MAP) kinase is expressed in both embryonic and adult chicken gizzard. The gizzard MAP kinase, which cross-reacts with anti-p44mpk antibody, has been purified from adult chicken gizzard and partially characterized. The purification protocol employs phenyl-Sepharose, polylysine-agarose, hydroxyapatite, Mono-Q and phenyl-Superose column chromatography. The purified enzyme phosphorylates myelin basic protein and gizzard high-molecular-mass (h-)caldesmon. Sea-star p44mpk and gizzard MAP kinase phosphorylate h-caldesmon at identical sites at the C-terminal domain, as revealed by tryptic-peptide mapping of the phosphorylated protein. Phosphorylation of h-caldesmon by gizzard MAP kinase abolishes its interaction with polymerized tubulin. The specific activity of the purified gizzard kinase toward myelin basic protein is similar to that of brain tau kinase, but is only a fraction of that of activated sea-star p44mpk. This suggests that, although a large amount of MAP kinase is present in the gizzard, only a small percentage of the enzyme is activated normally. Autophosphorylation of the gizzard kinase, at least in part on tyrosine residues, activates its kinase activity. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 PMID:8280072

  19. Parallelizing Backpropagation Neural Network Using MapReduce and Cascading Model

    PubMed Central

    Liu, Yang; Jing, Weizhe; Xu, Lixiong

    2016-01-01

    Artificial Neural Network (ANN) is a widely used algorithm in pattern recognition, classification, and prediction fields. Among a number of neural networks, backpropagation neural network (BPNN) has become the most famous one due to its remarkable function approximation ability. However, a standard BPNN frequently employs a large number of sum and sigmoid calculations, which may result in low efficiency in dealing with large volume of data. Therefore to parallelize BPNN using distributed computing technologies is an effective way to improve the algorithm performance in terms of efficiency. However, traditional parallelization may lead to accuracy loss. Although several complements have been done, it is still difficult to find out a compromise between efficiency and precision. This paper presents a parallelized BPNN based on MapReduce computing model which supplies advanced features including fault tolerance, data replication, and load balancing. And also to improve the algorithm performance in terms of precision, this paper creates a cascading model based classification approach, which helps to refine the classification results. The experimental results indicate that the presented parallelized BPNN is able to offer high efficiency whilst maintaining excellent precision in enabling large-scale machine learning. PMID:27217823

  20. Parallelizing Backpropagation Neural Network Using MapReduce and Cascading Model.

    PubMed

    Liu, Yang; Jing, Weizhe; Xu, Lixiong

    2016-01-01

    Artificial Neural Network (ANN) is a widely used algorithm in pattern recognition, classification, and prediction fields. Among a number of neural networks, backpropagation neural network (BPNN) has become the most famous one due to its remarkable function approximation ability. However, a standard BPNN frequently employs a large number of sum and sigmoid calculations, which may result in low efficiency in dealing with large volume of data. Therefore to parallelize BPNN using distributed computing technologies is an effective way to improve the algorithm performance in terms of efficiency. However, traditional parallelization may lead to accuracy loss. Although several complements have been done, it is still difficult to find out a compromise between efficiency and precision. This paper presents a parallelized BPNN based on MapReduce computing model which supplies advanced features including fault tolerance, data replication, and load balancing. And also to improve the algorithm performance in terms of precision, this paper creates a cascading model based classification approach, which helps to refine the classification results. The experimental results indicate that the presented parallelized BPNN is able to offer high efficiency whilst maintaining excellent precision in enabling large-scale machine learning. PMID:27217823

  1. Biological outcome and mapping of total factor cascades in response to HIF induction during regenerative angiogenesis

    PubMed Central

    Khatib, Abdel-Majid; Lahlil, Rachid; Hagedorn, Martin; Delomenie, Claudine; Christophe, Olivier; Denis, Cecile; Siegfried, Geraldine

    2016-01-01

    Hypoxia Inducible Factor (HIF) is the main transcription factor that mediates cell response to hypoxia. Howeverthe complex factor cascades induced by HIF during regenerative angiogenesis are currently incompletely mapped and the biological outcome mediated by chronic HIF induction during vessel regeneration are not well known. Here, we investigated the biological impact of HIF induction on vascular regeneration and identified the differentially regulated genes during regeneration, HIF induction and hypoxic regeneration. The use of the fin zebrafish regeneration model revealed that exposure to HIF inducer (cobalt chloride) prevents vessel differentiation by maintaining their vascular plexuses in an immature state. The regenerated fins are easily breakable, lacking completely endochondral ossification. Gene expression arrays combined to gene functional enrichment analysis revealed that regenerative process and HIF induction shared the regulation of common genes mainly involved in DNA replication and proteasome complex. HIF induction during regeneration affected the expression of exclusive genes involved in cell differentiation and communication, consistent with the observed immature vascular plexuses of the regenerated fins during HIF induction. The use of morpholino (MO) knockdown strategy revealed that the expression of some of these genes such as tubulin and col10a1 are required for fin regeneration. Taken together, this study revealed the impact of HIF induction on regenerative angiogenesis and provided a framework to develop a gene network leading to regenerative process during HIF expression. PMID:26933814

  2. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel; Busenlehner, Laura; Marcus, Stevan

    2010-08-20

    Research highlights: {yields} Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. {yields} The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. {yields} Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. {yields} PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  3. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

    PubMed Central

    Migliaccio, A; Di Domenico, M; Castoria, G; de Falco, A; Bontempo, P; Nola, E; Auricchio, F

    1996-01-01

    The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor. Images PMID:8635462

  4. Short-term cascaded hydroelectric system scheduling based on chaotic particle swarm optimization using improved logistic map

    NASA Astrophysics Data System (ADS)

    He, Yaoyao; Yang, Shanlin; Xu, Qifa

    2013-07-01

    In order to solve the model of short-term cascaded hydroelectric system scheduling, a novel chaotic particle swarm optimization (CPSO) algorithm using improved logistic map is introduced, which uses the water discharge as the decision variables combined with the death penalty function. According to the principle of maximum power generation, the proposed approach makes use of the ergodicity, symmetry and stochastic property of improved logistic chaotic map for enhancing the performance of particle swarm optimization (PSO) algorithm. The new hybrid method has been examined and tested on two test functions and a practical cascaded hydroelectric system. The experimental results show that the effectiveness and robustness of the proposed CPSO algorithm in comparison with other traditional algorithms.

  5. Prostaglandin F2alpha stimulates the Raf/MEK1/mitogen-activated protein kinase signaling cascade in bovine luteal cells.

    PubMed

    Chen, D B; Westfall, S D; Fong, H W; Roberson, M S; Davis, J S

    1998-09-01

    Upon binding to its G protein-coupled transmembrane receptors, the actions of PGF2alpha on the corpus luteum are initiated by the phospholipase C/diacylglycerol-inositol 1,4,5-trisphosphate (InsP3)/Ca2+-protein kinase C (PKC) pathway. However, little is known about the downstream intracellular signaling events that can lead to transcriptional activation in response to PGF2alpha. The present study was conducted to examine the involvement of the mitogen-activated protein kinase (MAPK) signaling cascade in the corpus luteum. Three isoforms of the Raf family of oncoprotein kinases (A-Raf, B-Raf, and Raf-1 or c-Raf) were detected in bovine luteal cells. Raf-1 and B-Raf, but not A-Raf, were activated by PGF2alpha (1 microM) and the pharmacological PKC activator phorbol myristate acetate (PMA, 20 nM). Kinetic analysis revealed that PGF2alpha rapidly and transiently activated Raf-1. In vitro protein kinase assays demonstrated that activation of Raf-1 and B-Raf resulted in the phosphorylation and activation of MAPK kinase (MEK1), which subsequently phosphorylated p42mapk. As determined by hyperphosphorylation, tyrosine phosphorylation, and enzymatic activity, p42mapk and p44mapk were rapidly and transiently activated by both PGF2alpha (1 microM) and PMA (20 nM). Additionally, both PGF2alpha (1 microM) and PMA (20 nM) stimulated phosphorylation of Raf-1, MEK1, and p42mapk in 32P-labeled cells. Our data demonstrate that PGF2alpha activates the Raf/MEK1/p42/44mapk signaling cascade in bovine luteal cells and that the actions of PGF2alpha are mimicked by the PKC activator PMA. Activation of the Raf/MEK1/MAPK signaling cascade by PGF2alpha in luteal cells provides a mechanism to transduce signals initiated by PGF2alpha receptors on the cell surface into the nucleus. Activation of the Raf/MEK1/MAPK signaling cascade may be associated with transcriptional activation of luteal genes possessing activator protein-1-binding sites.

  6. Cross-interactions of two p38 mitogen-activated protein (MAP) kinase inhibitors and two cholecystokinin (CCK) receptor antagonists with the CCK1 receptor and p38 MAP kinase.

    PubMed

    Morel, Caroline; Ibarz, Géraldine; Oiry, Catherine; Carnazzi, Eric; Bergé, Gilbert; Gagne, Didier; Galleyrand, Jean-Claude; Martinez, Jean

    2005-06-01

    Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.

  7. How can yeast cells decide between three activated MAP kinase pathways? A model approach.

    PubMed

    Rensing, Ludger; Ruoff, Peter

    2009-04-21

    In yeast (Saccharomyces cerevisiae), the regulation of three MAP kinase pathways responding to pheromones (Fus3 pathway), carbon/nitrogen starvation (Kss1 pathway), and high osmolarity/osmotic stress (Hog1 pathway) is the subject of intensive research. We were interested in the question how yeast cells would respond when more than one of the MAP kinase pathways are activated simultaneously. Here, we give a brief overview over the regulatory mechanisms of the yeast MAP kinase pathways and investigate a kinetic model based on presently known molecular interactions and feedbacks within and between the three mitogen-activated protein kinases (MAPK) pathways. When two pathways are activated simultaneously with the osmotic stress response as one of them, the model predicts that the osmotic stress response (Hog1 pathway) is turned on first. The same is true when all three pathways are activated at the same time. When testing simultaneous stimulations by low nitrogen and pheromones through the Kss1 and Fus3 pathways, respectively, the low nitrogen response dominates over the pheromone response. Due to its autocatalytic activation mechanism, the pheromone response (Fus3 pathway) shows typical sigmoid response kinetics and excitability. In the presence of a small but sufficient amount of activated Fus3, a stimulation by pheromones will lead to a rapid self-amplification of the pheromone response. This 'excitability' appears to be a feature of the pheromone pathway that has specific biological significance. PMID:19322936

  8. BAFF activation of the ERK5 MAP kinase pathway regulates B cell survival.

    PubMed

    Jacque, Emilie; Schweighoffer, Edina; Tybulewicz, Victor L J; Ley, Steven C

    2015-06-01

    B cell activating factor (BAFF) stimulation of the BAFF receptor (BAFF-R) is essential for the homeostatic survival of mature B cells. Earlier in vitro experiments with inhibitors that block MEK 1 and 2 suggested that activation of ERK 1 and 2 MAP kinases is required for BAFF-R to promote B cell survival. However, these inhibitors are now known to also inhibit MEK5, which activates the related MAP kinase ERK5. In the present study, we demonstrated that BAFF-induced B cell survival was actually independent of ERK1/2 activation but required ERK5 activation. Consistent with this, we showed that conditional deletion of ERK5 in B cells led to a pronounced global reduction in mature B2 B cell numbers, which correlated with impaired survival of ERK5-deficient B cells after BAFF stimulation. ERK5 was required for optimal BAFF up-regulation of Mcl1 and Bcl2a1, which are prosurvival members of the Bcl-2 family. However, ERK5 deficiency did not alter BAFF activation of the PI3-kinase-Akt or NF-κB signaling pathways, which are also important for BAFF to promote mature B cell survival. Our study reveals a critical role for the MEK5-ERK5 MAP kinase signaling pathway in BAFF-induced mature B cell survival and homeostatic maintenance of B2 cell numbers.

  9. Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast.

    PubMed

    Millar, J B; Buck, V; Wilkinson, M G

    1995-09-01

    Simultaneous inactivation of pyp1 and pyp2 PTPases in fission yeast leads to aberrant cell morphology and growth arrest. Spontaneous recessive mutations that bypass the requirement for pyp1 and pyp2 and reside in two complementation groups were isolated, sty1 and sty2. sty1- and sty2- mutant cells are substantially delayed in the timing of mitotic initiation. We have isolated the sty1 gene, which encodes a MAP kinase that is closely related to a subfamily of MAP kinases regulated by osmotic stress including Saccharomyces cervisiae HOG1 and human CSBP1. We find that sty2 is allelic to the wis1 MAP kinase kinase and that delta sty1 and delta wis1 cells are unable to grow in high osmolarity medium. Osmotic stress induces both tyrosine phosphorylation of Sty1 and a reduction in cell size at division. Pyp2 associates with and tyrosine dephosphorylates Sty1 in vitro. We find that wis1-dependent induction of pyp2 mRNA is responsible for tyrosine dephosphorylation of Sty1 in vivo on prolonged exposure to osmotic stress. We conclude that Pyp1 and Pyp2 are tyrosine-specific MAP kinase phosphatases that inactivate an osmoregulated MAP kinase, Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast. PMID:7657164

  10. The apoptotic volume decrease is an upstream event of MAP kinase activation during Staurosporine-induced apoptosis in HeLa cells.

    PubMed

    Hasegawa, Yuichi; Shimizu, Takahiro; Takahashi, Nobuyuki; Okada, Yasunobu

    2012-01-01

    Persistent cell shrinkage, called apoptotic volume decrease (AVD), is a pivotal event of apoptosis. Activation of the volume-sensitive outwardly rectifying Cl(-) channel (VSOR) is involved in the AVD induction. On the other hand, activation of the MAP kinase (MAPK) cascade is also known to play a critical role in apoptosis. In the present study, we investigated the relationship between the AVD induction and the stress-responsive MAPK cascade activation during the apoptosis process induced by staurosporine (STS) in HeLa cells. STS was found to induce AVD within 2-5 min and phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK after over 20-30 min. VSOR blockers suppressed not only STS-induced AVD but also phosphorylation of JNK and p38 as well as activation of caspase-3/7. Moreover, a p38 inhibitor, SB203580, and a JNK inhibitor, SP600125, failed to affect STS-induced AVD, whereas these compounds reduced STS-induced activation of caspase-3/7. Also, treatment with ASK1-specific siRNA suppressed STS-induced caspase-3/7 activation without affecting the AVD induction. Furthermore, sustained osmotic cell shrinkage per se was found to trigger phosphorylation of JNK and p38, caspase activation, and cell death. Thus, it is suggested that activation of p38 and JNK is a downstream event of AVD for the STS-induced apoptosis of HeLa cells.

  11. NMR Characterization of Information Flow and Allosteric Communities in the MAP Kinase p38γ.

    PubMed

    Aoto, Phillip C; Martin, Bryan T; Wright, Peter E

    2016-01-01

    The intramolecular network structure of a protein provides valuable insights into allosteric sites and communication pathways. However, a straightforward method to comprehensively map and characterize these pathways is not currently available. Here we present an approach to characterize intramolecular network structure using NMR chemical shift perturbations. We apply the method to the mitogen activated protein kinase (MAPK) p38γ. p38γ contains allosteric sites that are conserved among eukaryotic kinases as well as unique to the MAPK family. How these regulatory sites communicate with catalytic residues is not well understood. Using our method, we observe and characterize for the first time information flux between regulatory sites through a conserved kinase infrastructure. This network is accessed, reinforced, and broken in various states of p38γ, reflecting the functional state of the protein. We demonstrate that the approach detects critical junctions in the network corresponding to biologically significant allosteric sites and pathways. PMID:27353957

  12. NMR Characterization of Information Flow and Allosteric Communities in the MAP Kinase p38γ

    PubMed Central

    Aoto, Phillip C.; Martin, Bryan T.; Wright, Peter E.

    2016-01-01

    The intramolecular network structure of a protein provides valuable insights into allosteric sites and communication pathways. However, a straightforward method to comprehensively map and characterize these pathways is not currently available. Here we present an approach to characterize intramolecular network structure using NMR chemical shift perturbations. We apply the method to the mitogen activated protein kinase (MAPK) p38γ. p38γ contains allosteric sites that are conserved among eukaryotic kinases as well as unique to the MAPK family. How these regulatory sites communicate with catalytic residues is not well understood. Using our method, we observe and characterize for the first time information flux between regulatory sites through a conserved kinase infrastructure. This network is accessed, reinforced, and broken in various states of p38γ, reflecting the functional state of the protein. We demonstrate that the approach detects critical junctions in the network corresponding to biologically significant allosteric sites and pathways. PMID:27353957

  13. RKIP regulates MAP kinase signaling in cells with defective B-Raf activity.

    PubMed

    Zeng, Lingchun; Ehrenreiter, Karin; Menon, Jyotsana; Menard, Ray; Kern, Florian; Nakazawa, Yoko; Bevilacqua, Elena; Imamoto, Akira; Baccarini, Manuela; Rosner, Marsha Rich

    2013-05-01

    MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.

  14. Structural Requirements for Yersinia YopJ Inhibition of MAP Kinase Pathways

    PubMed Central

    Burdette, Dara; Mukherjee, Sohini; Keitany, Gladys; Goldsmith, Elizabeth; Orth, Kim

    2008-01-01

    MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G α-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition. PMID:18167536

  15. Principal facts and a discussion of terrain correction methods for the complete Bouguer gravity anomaly map of the Cascade Mountains, Washington

    SciTech Connect

    Danes, Z.F.; Phillips, W.M.

    1983-02-01

    Since 1974, the Division of Geology and Earth Resources, in conjunction with the US Department of Energy, has supported gravity studies in the Cascade Mountains of Washington State. Results of the Cascade gravity project are summarized graphically as a complete Bouguer gravity anomaly map of the Cascade Mountains, Washington (Danes and Phillips, 1983). This report provides supplementary data and documentation for the complete Bouguer gravity anomaly map. Presented are principal gravity facts, simple Bouguer and Free-air gravity anomalies, computational methods, error analysis and a discussion of terrain corrections.

  16. The regulation of runt-related transcription factor 2 by fibroblast growth factor-2 and connexin43 requires the inositol polyphosphate/protein kinasecascade.

    PubMed

    Niger, Corinne; Luciotti, Maria A; Buo, Atum M; Hebert, Carla; Ma, Vy; Stains, Joseph P

    2013-06-01

    Connexin43 (Cx43) plays a critical role in osteoblast function and bone mass accrual, yet the identity of the second messengers communicated by Cx43 gap junctions, the targets of these second messengers and how they regulate osteoblast function remain largely unknown. We have shown that alterations of Cx43 expression in osteoblasts can impact the responsiveness to fibroblast growth factor-2 (FGF2), by modulating the transcriptional activity of runt-related transcription factor 2 (Runx2). In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/protein kinase C delta (PKCδ) cascade to the Cx43-dependent transcriptional response of MC3T3 osteoblasts to FGF2. Knockdown of expression and/or inhibition of function of phospholipase Cγ1, inositol polyphosphate multikinase, which generates inositol 1,3,4,5-tetrakisphosphate (InsP₄) and InsP₅, and inositol hexakisphosphate kinase 1/2, which generates inositol pyrophosphates, prevented the ability of Cx43 to potentiate FGF2-induced signaling through Runx2. Conversely, overexpression of phospholipase Cγ1 and inositol hexakisphosphate kinase 1/2 enhanced FGF2 activation of Runx2 and the effect of Cx43 overexpression on this response. Disruption of these pathways blocked the nuclear accumulation of PKCδ and the FGF2-dependent interaction of PKCδ and Runx2, reducing Runx2 transcriptional activity. These data reveal that FGF2-signaling involves the inositol polyphosphate cascade, including inositol hexakisphosphate kinase (IP6K), and demonstrate that IP6K regulates Runx2 and osteoblast gene expression. Additionally, these data implicate the water-soluble inositol polyphosphates as mediators of the Cx43-dependent amplification of the osteoblast response to FGF2, and suggest that these low molecular weight second messengers may be biologically relevant mediators of osteoblast function that are communicated by Cx43-gap junctions.

  17. Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis.

    PubMed

    Kachaner, David; Pinson, Xavier; El Kadhi, Khaled Ben; Normandin, Karine; Talje, Lama; Lavoie, Hugo; Lépine, Guillaume; Carréno, Sébastien; Kwok, Benjamin H; Hickson, Gilles R; Archambault, Vincent

    2014-10-27

    Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.

  18. hKSR-2 inhibits MEKK3-activated MAP kinase and NF-kappaB pathways in inflammation.

    PubMed

    Channavajhala, Padma L; Rao, Vikram R; Spaulding, Vikki; Lin, Lih-Ling; Zhang, Y George

    2005-09-01

    Kinase suppressor of ras (KSR) and MEKK3 (MAP kinase kinase kinase) are integral members of the MAP kinase pathway. We have recently identified a new isoform of the KSR family named human kinase suppressor of ras-2 (hKSR-2), and demonstrated that hKSR-2 negatively regulates Cot, a MAP3K family member which is important in inflammation and oncogenesis [P.L. Channavajhala, L. Wu, J.W. Cuozzo, J.P. Hall, W. Liu, L.L. Lin, Y. Zhang, J. Biol. Chem. 278 (2003) 47089-47097]. In this report, we provide evidence that hKSR-2 also regulates the activity of MEKK3 (another MAP3K family member) in HEK-293T cells. We demonstrate that hKSR-2 is a negative regulator of MEKK3-mediated activation of MAP kinase (specifically ERK and JNK) and NF-kappaB pathways, and concurrently inhibits MEKK3-mediated interleukin-8 production. We find that while hKSR-2 blocks MEKK3 activation, it has little to no effect on other members of the MAP3K family, including MEKK4, TAK1, and Ras-Raf, suggesting that its effects are selective. PMID:16039990

  19. Sevoflurane Stimulates MAP Kinase Signal transduction through the Activation of PKC α and βII in Fetal Rat Cerebral Cortex Cultured Neuron

    PubMed Central

    Hasegawa, Jun; Takekoshi, Susumu; Nagata, Hidetaka; Osamura, R. Yoshiyuki; Suzuki, Toshiyasu

    2006-01-01

    Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral ­cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (α, βI, βII, γ, δ, ɛ and ζ) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC α and PKC βII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC α and βII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5–10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10–15 min after sevoflurane treatments. That of ERK proteins was induced at 15–60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the ­localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0

  20. The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis.

    PubMed

    Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B; Ferrandiz, Nuria; Crawley, Oliver; Montoya, Alex; Faull, Peter; Snijders, Ambrosius P; Cutillas, Pedro R; Jambhekar, Ashwini; Blower, Michael D; Martinez-Perez, Enrique; Harper, J Wade; Colaiacovo, Monica P

    2016-02-27

    Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation.

  1. The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

    PubMed Central

    Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B; Ferrandiz, Nuria; Crawley, Oliver; Montoya, Alex; Faull, Peter; Snijders, Ambrosius P; Cutillas, Pedro R; Jambhekar, Ashwini; Blower, Michael D; Martinez-Perez, Enrique; Harper, J Wade; Colaiacovo, Monica P

    2016-01-01

    Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.12039.001 PMID:26920220

  2. Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation.

    PubMed

    Coyle, Scott M; Flores, Jonathan; Lim, Wendell A

    2013-08-15

    Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets coevolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and to the evolution of distinct signaling behaviors. PMID:23953117

  3. Exploitation of latent protein allostery enables the evolution of novel and divergent MAP kinase regulation

    PubMed Central

    Coyle, Scott M.; Flores, Jonathan; Lim, Wendell A.

    2013-01-01

    SUMMARY Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets co-evolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and the evolution of distinct signaling behaviors. PMID:23953117

  4. Identification of p38α MAP kinase inhibitors by pharmacophore based virtual screening.

    PubMed

    Gangwal, Rahul P; Das, Nihar R; Thanki, Kaushik; Damre, Mangesh V; Dhoke, Gaurao V; Sharma, Shyam S; Jain, Sanyog; Sangamwar, Abhay T

    2014-04-01

    The p38α mitogen-activated protein (MAP) kinase plays a vital role in treating many inflammatory diseases. In the present study, a combined ligand and structure based pharmacophore model was developed to identify potential DFG-in selective p38 MAP kinase inhibitors. Conformations of co-crystallised inhibitors were used in the development and validation of ligand and structure based pharmacophore modeling approached. The validated pharmacophore was utilized in database screening to identify potential hits. After Lipinski's rule of five filter and molecular docking analysis, nineteen hits were purchased and selected for in vitro analysis. The virtual hits exhibited promising activity against tumor necrosis factor-α (TNF-α) with 23-98% inhibition at 10μM concentration. Out of these seven compounds has shown potent inhibitory activity against p38 MAP kinase with IC50 values ranging from 12.97 to 223.5nM. In addition, the toxicity study against HepG2 cells was also carried out to confirm the safety profile of identified virtual hits.

  5. Fangchinoline suppresses the growth and invasion of human glioblastoma cells by inhibiting the kinase activity of Akt and Akt-mediated signaling cascades.

    PubMed

    Guo, Bingyu; Xie, Peng; Su, Jingyuan; Zhang, Tingting; Li, Xiaoming; Liang, Guobiao

    2016-02-01

    Glioblastoma multiforme (GBM) is one of the most palindromic and malignant central nervous system neoplasms, and the current treatment is not effectual for GBM. Research of specific medicine for GBM is significant. Fangchinoline possesses a wide range of pharmacological activities and attracts more attentions due to its anti-tumor effects. In this study, two WHO grade IV human GBM cell lines (U87 MG and U118 MG) were exposed to fangchinoline, and we found that fangchinoline specifically inhibits the kinase activity of Akt and markedly suppresses the phosphorylation of Thr308 and Ser473 of Akt in human GBM cells. We also observed that fangchinoline inhibits tumor cell proliferation and invasiveness and induces apoptosis through suppressing the Akt-mediated signaling cascades, including Akt/p21, Akt/Bad, and Akt/matrix metalloproteinases (MMPs). These data demonstrated that fangchinoline exerts its anti-tumor effects in human glioblastoma cells, at least partly by inhibiting the kinase activity of Akt and suppressing Akt-mediated signaling cascades. PMID:26408176

  6. p38 MAP kinase and MKK-1 co-operate in the generation of GM-CSF from LPS-stimulated human monocytes by an NF-κB-independent mechanism

    PubMed Central

    Meja, Koremu K; Seldon, Paul M; Nasuhara, Yasuyuki; Ito, Kazuhiro; Barnes, Peter J; Lindsay, Mark A; Giembycz, Mark A

    2000-01-01

    The extent to which the p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK)-1-signalling pathways regulate the expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) from LPS-stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor-α (TNFα).Lipopolysaccharide (LPS) evoked a concentration-dependent generation of GM-CSF from human monocytes. Temporally, this effect was preceded by an increase in GM-CSF mRNA transcripts and abolished by actinomycin D and cycloheximide.LPS-induced GM-CSF release and mRNA expression were associated with a rapid and time-dependent activation of p38 MAP kinase, ERK-1 and ERK-2.The respective MKK-1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS-induced GM-CSF generation by >90%, indicating that both of these signalling cascades co-operate in the generation of this cytokine.Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor κB (NF-κB) : DNA binding. SN50, an inhibitor of NF-κB translocation, abolished LPS-induced NF-κB : DNA binding and the elaboration of TNFα, a cytokine known to be regulated by NF-κB in monocytes. In contrast, SN50 failed to affect the release of GM-CSF from the same monocyte cultures.Collectively, these results suggest that the generation of GM-CSF by LPS-stimulated human monocytes is regulated in a co-operative fashion by p38 MAP kinase- and MKK-1-dependent signalling pathways independently of the activation of NF-κB. PMID:11082122

  7. The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2

    PubMed Central

    Roux, Milena Edna; Rasmussen, Magnus Wohlfahrt; Palma, Kristoffer; Lolle, Signe; Regué, Àngels Mateu; Bethke, Gerit; Glazebrook, Jane; Zhang, Weiping; Sieburth, Leslie; Larsen, Martin R; Mundy, John; Petersen, Morten

    2015-01-01

    Multi-layered defense responses are activated in plants upon recognition of invading pathogens. Transmembrane receptors recognize conserved pathogen-associated molecular patterns (PAMPs) and activate MAP kinase cascades, which regulate changes in gene expression to produce appropriate immune responses. For example, Arabidopsis MAP kinase 4 (MPK4) regulates the expression of a subset of defense genes via at least one WRKY transcription factor. We report here that MPK4 is found in complexes in vivo with PAT1, a component of the mRNA decapping machinery. PAT1 is also phosphorylated by MPK4 and, upon flagellin PAMP treatment, PAT1 accumulates and localizes to cytoplasmic processing (P) bodies which are sites for mRNA decay. Pat1 mutants exhibit dwarfism and de-repressed immunity dependent on the immune receptor SUMM2. Since mRNA decapping is a critical step in mRNA turnover, linking MPK4 to mRNA decay via PAT1 provides another mechanism by which MPK4 may rapidly instigate immune responses. PMID:25603932

  8. Porins from Salmonella enterica Serovar Typhimurium Activate the Transcription Factors Activating Protein 1 and NF-κB through the Raf-1-Mitogen-Activated Protein Kinase Cascade

    PubMed Central

    Galdiero, Massimiliano; Vitiello, Mariateresa; Sanzari, Emma; D’Isanto, Marina; Tortora, Annalisa; Longanella, Anna; Galdiero, Stefania

    2002-01-01

    In this study we examined the ability of Salmonella enterica serovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor κB (NF-κB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-κB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2′-amino-3′-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7β-acetoxy-1α,6β,9α-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-κB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-κB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-κB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-κB activation in cells treated with S. enterica serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation

  9. Stimulation of MAP-2 kinase activity in T lymphocytes by anti-CD3 or anti-Ti monoclonal antibody is partially dependent on protein kinase C.

    PubMed

    Nel, A E; Hanekom, C; Rheeder, A; Williams, K; Pollack, S; Katz, R; Landreth, G E

    1990-04-01

    Signaling via the alpha-beta T cell Ag receptor (Ti)-CD3 complex is a complicated event that implicates several protein kinases, most notably protein kinase C (PKC). We have recently identified a serine kinase in T lymphocytes with the following characteristics: molecular mass 43 kDa, in vitro substrate affinity for microtubule associated protein 2 (MAP-2) with a preference for Mn2+ during the catalytic reaction, and elution from DEAE resin over a salt range 100 to 200 mM NaCl. This kinase is activated in a rapidly reversible fashion during ligation of CD3/Ti by a process which involves prior phosphorylation; in vitro exposure of activated 43-kDa MAP-2 kinase (MAP-K) to an immobilized phosphatase abrogated its kinase activity. We now show that a MAP-2K response could also be obtained during treatment with mAb to Ti and the specific PKC agonist, PMA. Although the kinetics of the former response was rapidly reversible, PMA elicited a more prolonged response. The dose responsiveness for PMA was similar to the requirements for PKC activation in intact lymphocytes. Moreover, as with PKC, we found that the CD3-induced MAP-2K response could be further enhanced by using a second layer cross-linking antibody. The specificity of CD3/Ti in the Jurkat cell response is demonstrated by the fact that OKT-11(CD2) and anti-CD4 mAb did not stimulate a MAP-2K response. It was also not possible to elicit a response in a Jurkat cell mutant that lacks surface expression of CD3 and Ti. The specificity of PKC in these events was further explored with the cell permeant diacylglycerol, 1-oleoyl-2-acetylglycerol, and the nonagonist phorbol ester, 4 alpha-phorbol 12,13-didecanoate: whereas the former was an effective inducer of the MAP-2K response, the latter failed to yield any stimulation. Prior exposure of Jurkat cells to 100 mM PMA for 24 h eliminated greater than 60% of the MAP-2K response during anti-CD3 treatment. This response could also be inhibited in dose-dependent fashion by prior

  10. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

    PubMed Central

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

  11. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata.

    PubMed

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism.

  12. Resveratrol suppresses prostaglandin F(2α)-induced osteoprotegerin synthesis in osteoblasts: inhibition of the MAP kinase signaling.

    PubMed

    Kuroyanagi, Gen; Tokuda, Haruhiko; Matsushima-Nishiwaki, Rie; Kondo, Akira; Mizutani, Jun; Kozawa, Osamu; Otsuka, Takanobu

    2014-01-15

    Resveratrol, a natural polyphenol abundantly found in grape skins and red wine, possesses various beneficial properties for human health. In the present study, we investigated the mechanism underlying the effects of prostaglandin F2α (PGF2α) on osteoprotegerin (OPG) synthesis and of resveratrol on the OPG synthesis in osteoblast-like MC3T3-E1 cells. PGF2α stimulated both the release of the OPG protein and the expression of OPG mRNA. Treatment with PD98059, SB203580 and SP600125, specific inhibitors of MEK1/2, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) all suppressed the OPG release induced by PGF2α. Resveratrol also significantly reduced the PGF2α-stimulated OPG release and the mRNA levels of OPG. Similarly, treatment with SRT1720, an activator of SIRT1, also suppressed the PGF2α-stimulated OPG release. Resveratrol and SRT1720 both attenuated the phosphorylation of p44/p42 MAP kinase, MEK1/2, Raf-1, p38 MAP kinase and SAPK/JNK induced by PGF2α. These findings strongly suggest that resveratrol suppresses PGF2α-stimulated OPG synthesis by inhibiting the MAP kinase pathways in osteoblasts, and that the effect is mediated via SIRT1 activation.

  13. Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways.

    PubMed

    Rastogi, Ruchi; Jiang, Zhongliang; Ahmad, Nisar; Rosati, Rita; Liu, Yusen; Beuret, Laurent; Monks, Robert; Charron, Jean; Birnbaum, Morris J; Samavati, Lobelia

    2013-11-22

    Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

  14. Mitogen-activated Protein Kinase Kinase Kinase 1 Protects against Nickel-induced Acute Lung Injury

    PubMed Central

    Mongan, Maureen; Tan, Zongqing; Chen, Liang; Peng, Zhimin; Dietsch, Maggie; Su, Bing; Leikauf, George; Xia, Ying

    2008-01-01

    Nickel compounds are environmental and occupational hazards that pose serious health problems and are causative factors of acute lung injury. The c-jun N-terminal kinases (JNKs) are regulated through a mitogen-activated protein (MAP) 3 kinase-MAP2 kinase cascade and have been implicated in nickel toxicity. In this study, we used genetically modified cells and mice to investigate the involvement of two upstream MAP3Ks, MAP3K1 and 2, in nickel-induced JNK activation and acute lung injury. In mouse embryonic fibroblasts, levels of JNK activation and cytotoxicity induced by nickel were similar in the Map3k2-null and wild-type cells but were much lower in the Map3k1/Map3k2 double-null cells. Conversely, the levels of JNK activation and cytotoxicity were unexpectedly much higher in the Map3k1-null cells. In adult mouse tissue, MAP3K1 was widely distributed but was abundantly expressed in the bronchiole epithelium of the lung. Accordingly, MAP3K1 ablation in mice resulted in severe nickel-induced acute lung injury and reduced survival. Based on these findings, we propose a role for MAP3K1 in reducing JNK activation and protecting the mice from nickel-induced acute lung injury. PMID:18467339

  15. Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae

    PubMed Central

    Desfossés-Baron, Kristelle; Hammond-Martel, Ian; Simoneau, Antoine; Sellam, Adnane; Roberts, Stephen; Wurtele, Hugo

    2016-01-01

    The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes. PMID:27782169

  16. Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

    PubMed

    Badrinarayan, Preethi; Sastry, G Narahari

    2012-04-01

    In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. PMID:22306417

  17. Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase

    PubMed Central

    Jacobs, Dave; Glossip, Danielle; Xing, Heming; Muslin, Anthony J.; Kornfeld, Kerry

    1999-01-01

    MAP kinases phosphorylate specific groups of substrate proteins. Here we show that the amino acid sequence FXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to substrates in multiple protein families. FXFP and the D box, a different docking site, form a modular recognition system, as they can function independently or in combination. FXFP is specific for ERK, whereas the D box mediates binding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities of ERK and JNK result from recognition of shared and unique docking sites. These findings enabled us to predict new ERK substrates and design peptide inhibitors of ERK that functioned in vitro and in vivo. PMID:9925641

  18. FBGs cascade interrogation technique based on wavelength-to-delay mapping and KLT analysis

    NASA Astrophysics Data System (ADS)

    Hervás, J.; Barrera, D.; Fernández-Pousa, Carlos R.; Sales, S.

    2016-05-01

    The Karhunen-Loeve transform is applied to the coarsely sampled impulse response generated by an FBG cascade in order to calculate the temperature change suffered by the FBGs. Thanks to a dispersive media, the wavelength change performed by the temperature change produces a delay shift in the sample generated by an FBG, delay shift which is recorded in the eigenvalues calculated by the KLT routine, letting to measure the temperature variation. Although the FBGs samples are represented only by four points, a continuous temperature measurement can be performed thanks to the KLT algorithm. This means a three order reduction in the number of points giving this method a low computational complexity. Simulations are performed to validate the interrogation technique and estimate performance and an experimental example is provided to demonstrate real operation.

  19. Mapping the structural topology of IRS family cascades through computational biology.

    PubMed

    Chakraborty, Chiranjib; Doss, C George Priya; Bandyopadhyay, Sanghamitra; Sarkar, Bimal Kumar; Haneef, S A Syed

    2013-01-01

    Structural topologies of proteins play significant roles in analyzing their biological functions. Converting the amino acid data in a protein sequence into structural information to outline the function of a protein is a major challenge in post-genome research which can add an extra room in understanding the protein sequence-structure-function relationships. In this study, we performed a comprehensive bioinformatics analysis of structural topology of the IRS family members such as IRS-1, IRS-2, IRS-3, IRS-4, IRS-5 and IRS-6. Based on this assessment, we found that IRS-2 encloses the highest number of α helices, β sheets and β turns in the secondary structure topology compared to IRS-1 and IRS-6. IRS family members are rich in serine or leucine residues. Among the IRS family members, the highest percentage of serine and leucine was observed in IRS-1 (15%) and IRS-5 (10%), respectively. Notably, the highest number of disulphide bonds was observed in IRS-1 (10) which is responsible for structural stability of the protein. Hydrogen bond pattern in α helices and β sheet was recorded in IRS-1, IRS-2 and IRS-6. By conservation analysis, the longest protein IRS-3 was found to be highly conserved among the IRS family members. The cluster of sequence logo present in the N terminus of these cascades was noted, and highly conserved residues in N-terminal region help in the formation of the two highly conserved domains such as PH domain and PTB domain. Results generated from this analysis will be more beneficial to researchers in understanding more about insulin signalling mechanism(s) as well as insulin resistance pathway. We discuss here that bioinformatics tools utilized in this study can play a vital role in addressing the complexity of structural topology to understand structure-function relationships in insulin signalling cascades.

  20. The MAP kinase substrate MKS1 is a regulator of plant defense responses.

    PubMed

    Andreasson, Erik; Jenkins, Thomas; Brodersen, Peter; Thorgrimsen, Stephan; Petersen, Nikolaj H T; Zhu, Shijiang; Qiu, Jin-Long; Micheelsen, Pernille; Rocher, Anne; Petersen, Morten; Newman, Mari-Anne; Bjørn Nielsen, Henrik; Hirt, Heribert; Somssich, Imre; Mattsson, Ole; Mundy, John

    2005-07-20

    Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors.

  1. Transcriptional activation of MMP-13 by periodontal pathogenic LPS requires p38 MAP kinase.

    PubMed

    Rossa, Carlos; Liu, Min; Bronson, Paul; Kirkwood, Keith L

    2007-01-01

    Matrix metalloprotease-13 (MMP-13) is induced by pro-inflammatory cytokines and increased expression is associated with a number of pathological conditions such as tumor metastasis, osteoarthritis, rheumatoid arthritis and periodontal diseases. MMP-13 gene regulation and the signal transduction pathways activated in response to bacterial LPS are largely unknown. In these studies, the role of the mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-13 induced by lipopolysaccharide was investigated. Lipopolysaccharide from Escherichia coli and Actinobacillus actinomycetemcomitans significantly (P < 0.05) increased MMP-13 steady-state mRNA (average of 27% and 46% increase, respectively) in murine periodontal ligament fibroblasts. MMP-13 mRNA induction was significantly reduced by inhibition of p38 MAP kinase. Immunoblot analysis indicated that p38 signaling was required for LPS-induced MMP-13 expression. Lipopolysaccharide induced proximal promoter reporter (-660/+32 mMMP-13) gene activity required p38 signaling. Collectively, these results indicate that lipopolysaccharide-induced murine MMP-13 is regulated by p38 signaling through a transcriptional mechanism.

  2. Histone H3 as a novel substrate for MAP kinase phosphatase-1.

    PubMed

    Kinney, Corttrell M; Chandrasekharan, Unni M; Yang, Lin; Shen, Jianzhong; Kinter, Michael; McDermott, Michael S; DiCorleto, Paul E

    2009-02-01

    Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosphatase that has been shown to dephosphorylate MAP kinases. We used a "substrate-trap" technique involving a mutation in MKP-1 of the catalytically critical cysteine to a serine residue ("CS" mutant) to capture novel MKP-1 substrates. We transfected the MKP-1 (CS) mutant and control (wild-type, WT) constructs into phorbol 12-myristate 13-acetate (PMA)-activated COS-1 cells. MKP-1-substrate complexes were immunoprecipitated, which yielded four bands of 17, 15, 14, and 10 kDa with the CS MKP-1 mutant but not the WT MKP-1. The bands were identified by mass spectrometry as histones H3, H2B, H2A, and H4, respectively. Histone H3 was phosphorylated, and purified MKP-1 dephosphorylated histone H3 (phospho-Ser-10) in vitro; whereas, histone H3 (phospho-Thr-3) was unaffected. We have previously shown that thrombin and vascular endothelial growth factor (VEGF) upregulated MKP-1 in human endothelial cells (EC). We now show that both thrombin and VEGF caused dephosphorylation of histone H3 (phospho-Ser-10) and histone H3 (phospho-Thr-3) in EC with kinetics consistent with MKP-1 induction. Furthermore, MKP-1-specific small interfering RNA (siRNA) prevented VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation but had no effect on H3 (phospho-Thr-3 or Thr-11) dephosphorylation. In summary, histone H3 is a novel substrate of MKP-1, and VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation requires MKP-1. We propose that MKP-1-mediated H3 (phospho-Ser-10) dephosphorylation is a key regulatory step in EC activation by VEGF and thrombin. PMID:19020052

  3. Registered Report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

    PubMed Central

    Sharma, Vidhu; Young, Lisa; Cavadas, Miguel; Owen, Kate

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from “COT drives resistance to RAF inhibition through MAPK pathway reactivation” by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife. DOI: http://dx.doi.org/10.7554/eLife.11414.001 PMID:26999821

  4. Positive feedback regulation of maize NADPH oxidase by mitogen-activated protein kinase cascade in abscisic acid signalling

    PubMed Central

    Lin, Fan; Ding, Haidong; Wang, Jinxiang; Zhang, Hong; Zhang, Aying; Zhang, Yun; Tan, Mingpu; Dong, Wen; Jiang, Mingyi

    2009-01-01

    In maize (Zea mays), abscisic acid (ABA)-induced H2O2 production activates a 46 kDa mitogen-activated protein kinase (p46MAPK), and the activation of p46MAPK also regulates the production of H2O2. However, the mechanism for the regulation of H2O2 production by MAPK in ABA signalling remains to be elucidated. In this study, four reactive oxygen species (ROS)-producing NADPH oxidase (rboh) genes (ZmrbohA–D) were isolated and characterized in maize leaves. ABA treatment induced a biphasic response (phase I and phase II) in the expression of ZmrbohA–D and the activity of NADPH oxidase. Phase II induced by ABA was blocked by pretreatments with two MAPK kinase (MPKKK) inhibitors and two H2O2 scavengers, but phase I was not affected by these inhibitors or scavengers. Treatment with H2O2 alone also only induced phase II, and the induction was arrested by the MAPKK inhibitors. Furthermore, the ABA-activated p46MAPK was partially purified. Using primers corresponding to the sequences of internal tryptic peptides, the p46MAPK gene was cloned. Analysis of the tryptic peptides and the p46MAPK sequence indicate it is the known ZmMPK5. Treatments with ABA and H2O2 led to a significant increase in the activity of ZmMPK5, although ABA treatment only induced a slight increase in the expression of ZmMPK5. The data indicate that H2O2-activated ZmMPK5 is involved in the activation of phase II in ABA signalling, but not in phase I. The results suggest that there is a positive feedback loop involving NADPH oxidase, H2O2, and ZmMPK5 in ABA signalling. PMID:19592501

  5. Breast tumor kinase (protein tyrosine kinase 6) regulates heregulin-induced activation of ERK5 and p38 MAP kinases in breast cancer cells.

    PubMed

    Ostrander, Julie Hanson; Daniel, Andrea R; Lofgren, Kristopher; Kleer, Celina G; Lange, Carol A

    2007-05-01

    Total tyrosine kinase activity is often elevated in both cytosolic and membrane fractions of malignant breast tissue and correlates with a decrease in disease-free survival. Breast tumor kinase (Brk; protein tyrosine kinase 6) is a soluble tyrosine kinase that was cloned from a metastatic breast tumor and found to be overexpressed in a majority of breast tumors. Herein, we show that Brk is overexpressed in 86% of invasive ductal breast tumors and coexpressed with ErbB family members in breast cancer cell lines. Additionally, the ErbB ligand, heregulin, activates Brk kinase activity. Knockdown of Brk by stable expression of short hairpin RNA (shRNA) in T47D breast cancer cells decreases proliferation and blocks epidermal growth factor (EGF)- and heregulin-induced activation of Rac GTPase, extracellular signal-regulated kinase (ERK) 5, and p38 mitogen-activated protein kinase (MAPK) but not Akt, ERK1/2, or c-Jun NH(2)-terminal kinase. Furthermore, EGF- and heregulin-induced cyclin D1 expression is dependent on p38 signaling and inhibited by Brk shRNA knockdown. The myocyte enhancer factor 2 transcription factor target of p38 MAPK and ERK5 signaling is also sensitive to altered Brk expression. Finally, heregulin-induced migration of T47D cells requires p38 MAPK activity and is blocked by Brk knockdown. These results place Brk in a novel signaling pathway downstream of ErbB receptors and upstream of Rac, p38 MAPK, and ERK5 and establish the ErbB-Brk-Rac-p38 MAPK pathway as a critical mediator of breast cancer cell migration.

  6. Hormonal activation of a kinase cascade localized at the mitochondria is required for StAR protein activity.

    PubMed

    Poderoso, Cecilia; Maloberti, Paula; Duarte, Alejandra; Neuman, Isabel; Paz, Cristina; Cornejo Maciel, Fabiana; Podesta, Ernesto J

    2009-03-01

    It is known that ERK1/2 and MEK1/2 participate in the regulation of Star gene transcription. However, their role in StAR protein post-transcriptional regulation is not described yet. In this study we analyzed the relationship between the MAPK cascade and StAR protein phosphorylation and function. We have demonstrated that (a) steroidogenesis in MA-10 Leydig cells depends on the specific of ERK1/2 activation at the mitochondria; (b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in this organelle; (c) active ERK1/2 interacts with StAR protein, leads to StAR protein phosphorylation at Ser(232) only in the presence of cholesterol; (d) directed mutagenesis of Ser(232) (S232A) inhibited in vitro StAR protein phosphorylation by ERK1; (e) transient transfection of MA-10 cells with StAR S232A cDNA markedly reduced the yield of progesterone production. We show that StAR protein is a substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric complex that regulates cholesterol transport.

  7. Combined computational and experimental analysis reveals mitogen-activated protein kinase-mediated feedback phosphorylation as a mechanism for signaling specificity.

    PubMed

    Hao, Nan; Yildirim, Necmettin; Nagiec, Michal J; Parnell, Stephen C; Errede, Beverly; Dohlman, Henrik G; Elston, Timothy C

    2012-10-01

    Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity. PMID:22875986

  8. 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.

  9. Modeling and hazard mapping of complex cascading mass movement processes: the case of glacier lake 513, Carhuaz, Peru

    NASA Astrophysics Data System (ADS)

    Schneider, Demian; Huggel, Christian; García, Javier; Ludeña, Sebastian; Cochachin, Alejo

    2013-04-01

    that complex cascades of mass movement processes can realistically be modeled using different models and model parameters. The method to semi-automatically produce hazard maps is promising and should be applied in other case studies. Verification of model based results in the field remains an important requirement. Results from this study are important for the GLOF early warning system that is currently in an implementation phase, and for risk reduction efforts in general.

  10. The p21-activated kinase, PAK2, is important in the activation of numerous pancreatic acinar cell signaling cascades and in the onset of early pancreatitis events.

    PubMed

    Nuche-Berenguer, Bernardo; Ramos-Álvarez, Irene; Jensen, R T

    2016-06-01

    In a recent study we explored Group-1-p21-activated kinases (GP.1-PAKs) in rat pancreatic acini. Only PAK2 was present; it was activated by gastrointestinal-hormones/neurotransmitters and growth factors in a PKC-, Src- and small-GTPase-mediated manner. PAK2 was required for enzyme-secretion and ERK/1-2-activation. In the present study we examined PAK2's role in CCK and TPA-activation of important distal signaling cascades mediating their physiological/pathophysiological effects and analyzed its role in pathophysiological processes important in early pancreatitis. In rat pancreatic acini, PAK2-inhibition by the specific, GP.1.PAK-inhibitor, IPA-3-suppressed cholecystokinin (CCK)/TPA-stimulated activation of focal-adhesion kinases and mitogen-activated protein-kinases. PAK2-inhibition reversed the dual stimulatory/inhibitory effect of CCK/TPA on the PI3K/Akt/GSK-3β pathway. However, its inhibition did not affect PKC activation. PAK2-inhibition protected acini from CCK-induced ROS-generation; caspase/trypsin-activation, important in early pancreatitis; as well as from cell-necrosis. Furthermore, PAK2-inhibition reduced proteolytic-activation of PAK-2p34, which is involved in programmed-cell-death. To ensure that the study did not only rely in the specificity of IPA-3 as a PAK inhibitor, we used two other approaches for PAK inhibition, FRAX597 a ATP-competitive-GP.1-PAKs-inhibitor and infection with a PAK2-dominant negative(DN)-Advirus. Those two approaches confirmed the results obtained with IPA-3. This study demonstrates that PAK2 is important in mediating CCK's effect on the activation of signaling-pathways known to mediate its physiological/pathophysiological responses including several cellular processes linked to the onset of pancreatitis. Our results suggest that PAK2 could be a new, important therapeutic target to consider for the treatment of diseases involving deregulation of pancreatic acinar cells. PMID:26912410

  11. Geologic map of upper Eocene to Holocene volcanic and related rocks in the Cascade Range, Washington

    USGS Publications Warehouse

    Smith, James G.

    1993-01-01

    For geothermal reasons, the maps emphasize Quaternary volcanic rocks. Large igneous-related geothermal systems that have high temperatures are associated with Quaternary volcanic fields, and geothermal potential declines rapidly as age increases (Smith and Shaw, 1975). Most high-grade recoverable geothermal energy is likely to be associated with silicic volcanism less than 1 Ma. Lower grade (= lower temperature) geothermal resources may be associated with somewhat older rocks; however, volcanic rocks older than about 2 Ma are unlikely geothermal targets (Smith and Shaw, 1975).

  12. The mitogen-activated protein kinase (MAPK) cascade controls phosphatase and tensin homolog (PTEN) expression through multiple mechanisms.

    PubMed

    Ciuffreda, Ludovica; Di Sanza, Cristina; Cesta Incani, Ursula; Eramo, Adriana; Desideri, Marianna; Biagioni, Francesca; Passeri, Daniela; Falcone, Italia; Sette, Giovanni; Bergamo, Paola; Anichini, Andrea; Sabapathy, Kanaga; McCubrey, James A; Ricciardi, Maria Rosaria; Tafuri, Agostino; Blandino, Giovanni; Orlandi, Augusto; De Maria, Ruggero; Cognetti, Francesco; Del Bufalo, Donatella; Milella, Michele

    2012-06-01

    The mitogen-activated protein kinase (MAPK) and PI3K pathways are regulated by extensive crosstalk, occurring at different levels. In tumors, transactivation of the alternate pathway is a frequent "escape" mechanism, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity. Here we show that, in the M14 melanoma model, simultaneous inhibition of both MEK and mammalian target of rapamycin (mTOR) achieves synergistic effects at suboptimal concentrations, but becomes frankly antagonistic in the presence of relatively high concentrations of MEK inhibitors. This observation led to the identification of a novel crosstalk mechanism, by which either pharmacologic or genetic inhibition of constitutive MEK signaling restores phosphatase and tensin homolog (PTEN) expression, both in vitro and in vivo, and inhibits downstream signaling through AKT and mTOR, thus bypassing the need for double pathway blockade. This appears to be a general regulatory mechanism and is mediated by multiple mechanisms, such as MAPK-dependent c-Jun and miR-25 regulation. Finally, PTEN upregulation appears to be a major effector of MEK inhibitors' antitumor activity, as cancer cells in which PTEN is inactivated are consistently more resistant to the growth inhibitory and anti-angiogenic effects of MEK blockade. PMID:22215152

  13. Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain.

    PubMed

    Yang, Yang; Cao, Min-Jie; Alcocer, Marcos; Liu, Qing-Mei; Fei, Dan-Xia; Mao, Hai-Yan; Liu, Guang-Ming

    2015-06-01

    Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability. PMID:25728640

  14. Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography*

    PubMed Central

    Hoehenwarter, Wolfgang; Thomas, Martin; Nukarinen, Ella; Egelhofer, Volker; Röhrig, Horst; Weckwerth, Wolfram; Conrath, Uwe; Beckers, Gerold J. M.

    2013-01-01

    Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment. PMID:23172892

  15. Differential regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity phosphatase.

    PubMed Central

    Groom, L A; Sneddon, A A; Alessi, D R; Dowd, S; Keyse, S M

    1996-01-01

    The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location. Images PMID:8670865

  16. Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway.

    PubMed

    Kawada, M; Yamagoe, S; Murakami, Y; Suzuki, K; Mizuno, S; Uehara, Y

    1997-08-01

    While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenic transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When rat normal fibroblasts were cultured in suspension without substrate attachment, the cell cycle arrested in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulated. Conditional expression of oncogenic Ras induced the G1-S transition of the cell cycle and significantly shortened the half-life of p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevented the oncogenic Ras-induced degradation of p27Kip1. These results suggest that the loss of substrate attachment induces the cell cycle arrest through the up-regulation of p27Kip1 mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27Kip1 degradation through the activation of the MAP kinase signaling pathway. Furthermore, we have found that p27Kip1 is phosphorylated by MAP kinase in vitro and the phosphorylated p27Kip1 cannot bind to and inhibit cdk2.

  17. Characterization and mapping of the human rhodopsin kinase gene and screening of the gene for mutations in patients with retinitis pigmentosa

    SciTech Connect

    Khani, S.C.; Lin, D.; Magovcevic, I.

    1994-09-01

    Rhodopsin kinase (RK) is a cytosolic enzyme in rod photoreceptors that initiates the deactivation of the phototransductions cascade by phosphorylating photoactivated rhodopsin. Although the cDNA sequence of bovine RK has been determined previously, no human cDNA or genomic sequence has thus far been available for genetic studies. In order to investigate the possible role of this candidate gene in retinitis pigmentosa (RP) and allied diseases, we have isolated and characterized human cDNA and genomic clones derived from the RK locus. The coding sequence of the human gene is 1692 nucleotides in length and is split into seven exons. The human and the bovine sequence show 84% identity at the nucleotide level and 92% identity at the amino acid level. Thus far, the intronic sequences flanking each exon except for one have been determined. We have also mapped the human RK gene to chromosome 13q34 using fluorescence in situ hybridization. To our knowledge, no RP gene has as yet been linked to this region. However, since the substrate for RK (rhodopsin) and other members of the phototransduction cascade have been implicated in the pathogenesis of RP, it is conceivable that defects in RK can also cause some forms of this disease. We are evaluating this possibility by screening DNA from 173 patients with autosomal recessive RP and 190 patients with autosomal dominant RP. So far, we have found 11 patients with variant bands. In one patient with autosomal dominant RP we discovered the missense change Ser536Leu. Cosegregation studies and further sequencing of the variant bands are currently underway.

  18. Quantitative network mapping of the human kinome interactome reveals new clues for rational kinase inhibitor discovery and individualized cancer therapy.

    PubMed

    Cheng, Feixiong; Jia, Peilin; Wang, Quan; Zhao, Zhongming

    2014-06-15

    The human kinome is gaining importance through its promising cancer therapeutic targets, yet no general model to address the kinase inhibitor resistance has emerged. Here, we constructed a systems biology-based framework to catalogue the human kinome, including 538 kinase genes, in the broader context of the human interactome. Specifically, we constructed three networks: a kinase-substrate interaction network containing 7,346 pairs connecting 379 kinases to 36,576 phosphorylation sites in 1,961 substrates, a protein-protein interaction network (PPIN) containing 92,699 pairs, and an atomic resolution PPIN containing 4,278 pairs. We identified the conserved regulatory phosphorylation motifs (e.g., Ser/Thr-Pro) using a sequence logo analysis. We found the typical anticancer target selection strategy that uses network hubs as drug targets, might lead to a high adverse drug reaction risk. Furthermore, we found the distinct network centrality of kinases creates a high anticancer drug resistance risk by feedback or crosstalk mechanisms within cellular networks. This notion is supported by the systematic network and pathway analyses that anticancer drug resistance genes are significantly enriched as hubs and heavily participate in multiple signaling pathways. Collectively, this comprehensive human kinome interactome map sheds light on anticancer drug resistance mechanisms and provides an innovative resource for rational kinase inhibitor design.

  19. Gain-of-function mutations in the Caenorhabditis elegans lin-1 ETS gene identify a C-terminal regulatory domain phosphorylated by ERK MAP kinase.

    PubMed Central

    Jacobs, D; Beitel, G J; Clark, S G; Horvitz, H R; Kornfeld, K

    1998-01-01

    Genetic analysis of lin-1 loss-of-function mutations suggests that lin-1 controls multiple cell-fate decisions during Caenorhabditis elegans development and is negatively regulated by a conserved receptor tyrosine kinase-Ras-ERK mitogen-activated protein (MAP) kinase signal transduction pathway. LIN-1 protein contains an ETS domain and presumably regulates transcription. We identified and characterized six gain-of-function mutations that define a new class of lin-1 allele. These lin-1 alleles appeared to be constitutively active and unresponsive to negative regulation. Each allele has a single-base change that affects the predicted C terminus of LIN-1, suggesting this region is required for negative regulation. The C terminus of LIN-1 was a high-affinity substrate for Erk2 in vitro, suggesting that LIN-1 is directly regulated by ERK MAP kinase. Because mpk-1 ERK MAP kinase controls at least one cell-fate decision that does not require lin-1, our results suggest that MPK-1 contributes to the specificity of this receptor tyrosine kinase-Ras-MAP kinase signal transduction pathway by phosphorylating different proteins in different developmental contexts. These lin-1 mutations all affect a four-amino-acid motif, FQFP, that is conserved in vertebrate and Drosophila ETS proteins that are also phosphorylated by ERK MAP kinase. This sequence may be a substrate recognition motif for the ERK subfamily of MAP kinases. PMID:9691039

  20. Mapping Atmospheric Ammonia Emissions Using a Mobile Quantum Cascade Laser-based Open-path Sensor

    NASA Astrophysics Data System (ADS)

    Sun, K.; Tao, L.; Miller, D. J.; Khan, M. A.; Zondlo, M. A.

    2012-12-01

    Ammonia (NH3) is a key precursor to atmospheric fine particulate matter, with strong implications for regional air quality and global climate change. Despite the importance of atmospheric ammonia, its spatial/temporal variation is poorly characterized, and the knowledge of its sources, sinks, and transport is severely limited. Existing measurements suggest that traffic exhaust may provide significant amounts of ammonia in urban areas, which cause greater impacts on particulate matter formation and urban air quality. To capture the spatial and temporal variation of ammonia emissions, a portable, low power sensor with high time resolution is necessary. We have developed a portable open-path ammonia sensor with a detection limit of 0.5 ppbv ammonia for 1 s measurements. The sensor has a power consumption of about 60 W and is capable of running on a car battery continuously for 24 hours. An additional laser has been coupled to the sensor to yield concurrent N2O and CO measurements as tracers for determining various sources. The overall sensor prototype fits on a 60 cm × 20 cm aluminum breadboard. Roadside measurements indicated NH3/CO emission ratios of 4.1±5.4 ppbv/ppmv from a fleet of 320 vehicles, which agree with existing on-ramp measurements. Urban measurements in the Baltimore and Washington, DC metropolitan areas have shown significant ammonia mixing ratios concurrent with carbon monoxide levels from the morning and evening rush hours. On-road measurements of our open-path sensor have also been performed continuously from the Midwest to Princeton, NJ including urban areas such as Pittsburgh, tunnels, and relatively clean conditions. The emission ratios of ammonia against CO and/or CO2 help identify the sources and amounts of both urban and agricultural ammonia emissions. Preliminary data from both spatial mapping, monitoring, and vehicle exhaust measurements suggest that urban ammonia emissions from fossil fuel combustion are significant and may provide an

  1. IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases.

    PubMed

    Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Brender, Christine; Martin, Stephen R; Janzen, Julia; Kjaer, Sven; Smerdon, Stephen J; Ley, Steven C

    2014-06-10

    The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding.

  2. The MAPKKKs Ste11 and Bck1 jointly transduce the high oxidative stress signal through the cell wall integrity MAP kinase pathway

    PubMed Central

    Jin, Chunyan; Kim, Stephen K.; Willis, Stephen D.; Cooper, Katrina F.

    2016-01-01

    Oxidative stress stimulates the Rho1 GTPase, which in turn induces the cell wall integrity (CWI) MAP kinase cascade. CWI activation promotes stress-responsive gene expression through activation of transcription factors (Rlm1, SBF) and nuclear release and subsequent destruction of the repressor cyclin C. This study reports that, in response to high hydrogen peroxide exposure, or in the presence of constitutively active Rho1, cyclin C still translocates to the cytoplasm and is degraded in cells lacking Bck1, the MAPKKK of the CWI pathway. However, in mutants defective for both Bck1 and Ste11, the MAPKKK from the high osmolarity, pseudohyphal and mating MAPK pathways, cyclin C nuclear to cytoplasmic relocalization and destruction is prevented. Further analysis revealed that cyclin C goes from a diffuse nuclear signal to a terminal nucleolar localization in this double mutant. Live cell imaging confirmed that cyclin C transiently passes through the nucleolus prior to cytoplasmic entry in wild-type cells. Taken together with previous studies, these results indicate that under low levels of oxidative stress, Bck1 activation is sufficient to induce cyclin C translocation and degradation. However, higher stress conditions also stimulate Ste11, which reinforces the stress signal to cyclin C and other transcription factors. This model would provide a mechanism by which different stress levels can be sensed and interpreted by the cell. PMID:27135035

  3. Late Phase of the Endoplasmic Reticulum Stress Response Pathway Is Regulated by Hog1 MAP Kinase*

    PubMed Central

    Bicknell, Alicia A.; Tourtellotte, Joel; Niwa, Maho

    2010-01-01

    When unfolded proteins accumulate in the endoplasmic reticulum (ER) causing ER stress, the unfolded protein response (UPR) responds rapidly to induce a transcriptional program that functions to alleviate the stress. However, under extreme conditions, when UPR activation is not sufficient to alleviate ER stress, the stress may persist long term. Very little is known about how the cell responds to persistent ER stress that is not resolved by the immediate activation of the UPR. We show that Hog1 MAP kinase becomes phosphorylated during the late stage of ER stress and helps the ER regain homeostasis. Although Hog1 is well known to function in osmotic stress and cell wall integrity pathways, we show that the activation mechanism for Hog1 during ER stress is distinct from both of these pathways. During late stage ER stress, upon phosphorylation, Hog1 translocates into the nucleus and regulates gene expression. Subsequently, Hog1 returns to the cytoplasm, where its phosphorylation levels remain high. From its cytoplasmic location, Hog1 contributes to the activation of autophagy by enhancing the stability of Atg8, a critical autophagy protein. Thus, Hog1 coordinates a multifaceted response to persistent ER stress. PMID:20382742

  4. A C. elegans p38 MAP kinase pathway mutant protects from dopamine, methamphetamine, and MDMA toxicity

    PubMed Central

    Schreiber, Matthew A.; McIntire, Steven L.

    2011-01-01

    Biogenic amine systems are damaged by amphetamine abuse and in Parkinson's disease. The mechanisms mediating this damage are of high importance because of the public health impact of these problems. Here we have taken advantage of the C. elegans nematode model system to investigate genetic modifiers of biogenic amine toxicity. In a forward genetic screen, we identified a mutant resistant to the toxic effects of dopamine. This mutant was also resistant to toxic doses of methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA). In addition, this mutation conferred resistance to 6-hydroxydopamine damage to dopaminergic neurons in a Parkinson's disease model. Resistance was due to a mutation in the nsy-1 gene, orthologous to the mammalian ASK-1 MAPKKK. NSY-1 is in the highly conserved p38 MAP kinase pathway, which plays a crucial role in C. elegans innate immunity, suggesting that this pathway may play a role in biogenic amine toxicity system damage due to amphetamines and in the pathogenesis of Parkinson's disease in higher organisms. PMID:21565252

  5. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

    PubMed

    D'Souza, Serena A; Rajendran, Luckshika; Bagg, Rachel; Barbier, Louis; van Pel, Derek M; Moshiri, Houtan; Roy, Peter J

    2016-04-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. PMID:27123983

  6. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

    PubMed Central

    D’Souza, Serena A.; Rajendran, Luckshika; Bagg, Rachel; van Pel, Derek M.; Moshiri, Houtan; Roy, Peter J.

    2016-01-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. PMID:27123983

  7. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

    PubMed

    D'Souza, Serena A; Rajendran, Luckshika; Bagg, Rachel; Barbier, Louis; van Pel, Derek M; Moshiri, Houtan; Roy, Peter J

    2016-04-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

  8. Aberrant Activation of p38 MAP Kinase-Dependent Innate Immune Responses Is Toxic to Caenorhabditis elegans.

    PubMed

    Cheesman, Hilary K; Feinbaum, Rhonda L; Thekkiniath, Jose; Dowen, Robert H; Conery, Annie L; Pukkila-Worley, Read

    2016-03-01

    Inappropriate activation of innate immune responses in intestinal epithelial cells underlies the pathophysiology of inflammatory disorders of the intestine. Here we examine the physiological effects of immune hyperactivation in the intestine of the nematode Caenorhabditis elegans. We previously identified an immunostimulatory xenobiotic that protects C. elegans from bacterial infection by inducing immune effector expression via the conserved p38 MAP kinase pathway, but was toxic to nematodes developing in the absence of pathogen. To investigate a possible connection between the toxicity and immunostimulatory properties of this xenobiotic, we conducted a forward genetic screen for C. elegans mutants that are resistant to the deleterious effects of the compound, and identified five toxicity suppressors. These strains contained hypomorphic mutations in each of the known components of the p38 MAP kinase cassette (tir-1, nsy-1, sek-1, and pmk-1), demonstrating that hyperstimulation of the p38 MAPK pathway is toxic to animals. To explore mechanisms of immune pathway regulation in C. elegans, we conducted another genetic screen for dominant activators of the p38 MAPK pathway, and identified a single allele that had a gain-of-function (gf) mutation in nsy-1, the MAP kinase kinase kinase that acts upstream of p38 MAPK pmk-1. The nsy-1(gf) allele caused hyperinduction of p38 MAPK PMK-1-dependent immune effectors, had greater levels of phosphorylated p38 MAPK, and was more resistant to killing by the bacterial pathogen Pseudomonas aeruginosa compared to wild-type controls. In addition, the nsy-1(gf) mutation was toxic to developing animals. Together, these data suggest that the activity of the MAPKKK NSY-1 is tightly regulated as part of a physiological mechanism to control p38 MAPK-mediated innate immune hyperactivation, and ensure cellular homeostasis in C. elegans. PMID:26818074

  9. A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase.

    PubMed Central

    Fox, T.; Coll, J. T.; Xie, X.; Ford, P. J.; Germann, U. A.; Porter, M. D.; Pazhanisamy, S.; Fleming, M. A.; Galullo, V.; Su, M. S.; Wilson, K. P.

    1998-01-01

    Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation. PMID:9827991

  10. Intestinal cell kinase, a MAP kinase-related kinase, regulates proliferation and G1 cell cycle progression of intestinal epithelial cells

    PubMed Central

    Kim, Jungeun; Vidrich, Alda; Sturgill, Thomas W.

    2009-01-01

    Intestinal cell kinase (ICK), originally cloned from the intestine and expressed in the intestinal crypt epithelium, is a highly conserved serine/threonine protein kinase that is similar to mitogen-activated protein kinases (MAPKs) in the catalytic domain and requires dual phosphorylation within a MAPK-like TDY motif for full activation. Despite these similarities to MAPKs, the biological functions of ICK remain unknown. In this study, we report that suppression of ICK expression in cultured intestinal epithelial cells by short hairpin RNA (shRNA) interference significantly impaired cellular proliferation and induced features of gene expression characteristic of colonic or enterocytic differentiation. Downregulation of ICK altered expression of cell cycle regulators (cyclin D1, c-Myc, and p21Cip1/WAF1) of G1-S transition, consistent with the G1 cell cycle delay induced by ICK shRNA. ICK deficiency also led to a significant decrease in the expression and/or activity of p70 ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E), concomitant with reduced expression of their upstream regulators, the mammalian target of rapamycin (mTOR) and the regulatory associated protein of mTOR (Raptor). Furthermore, ICK interacts with the mTOR/Raptor complex in vivo and phosphorylates Raptor in vitro. These results suggest that disrupting ICK function may downregulate protein translation of specific downstream targets of eIF4E and S6K1 such as cyclin D1 and c-Myc through the mTOR/Raptor signaling pathway. Taken together, our findings demonstrate an important role for ICK in proliferation and differentiation of intestinal epithelial cells. PMID:19696144

  11. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes.

    PubMed

    Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

    2015-11-26

    Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

  12. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

    PubMed Central

    Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

    2015-01-01

    Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. PMID:26703626

  13. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

    PubMed Central

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry

    2016-01-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  14. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels.

    PubMed

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry; Lee, C Justin

    2016-04-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  15. Genetic analysis of the Arabidopsis protein kinases MAP3Kε1 and MAP3Kε2 indicates roles in cell expansion and embryo development.

    PubMed

    Chaiwongsar, Suraphon; Strohm, Allison K; Su, Shih-Heng; Krysan, Patrick J

    2012-01-01

    MAP3Kε1 and MAP3Kε2 are a pair of Arabidopsis thaliana genes that encode protein kinases related to cdc7p from Saccharomyces cerevisiae. We have previously shown that the map3kε1;map3kε2 double-mutant combination causes pollen lethality. In this study, we have used an ethanol-inducible promoter construct to rescue this lethal phenotype and create map3kε1(-/-);map3kε2(-/-) double-mutant plants in order to examine the function of these genes in the sporophyte. These rescued double-mutant plants carry a yellow fluorescent protein (YFP)-MAP3Kε1 transgene under the control of the alcohol-inducible AlcA promoter from Aspergillus nidulans. The double-mutant plants were significantly smaller and had shorter roots than wild-type when grown in the absence of ethanol treatment. Microscopic analysis indicated that cell elongation was reduced in the roots of the double-mutant plants and cell expansion was reduced in rosette leaves. Treatment with ethanol to induce expression of YFP-MAP3Kε1 largely rescued the leaf phenotypes. The double-mutant combination also caused embryos to arrest in the early stages of development. Through the use of YFP reporter constructs we determined that MAP3Kε1 and MAP3Kε2 are expressed during embryo development, and also in root tissue. Our results indicate that MAP3Kε1 and MAP3Kε2 have roles outside of pollen development and that these genes affect several aspects of sporophyte development.

  16. The involvement of MAP kinases JNK and p38 in photodynamic injury of crayfish neurons and glial cells

    NASA Astrophysics Data System (ADS)

    Petin, Y. O.; Bibov, M. Y.; Uzdensky, A. B.

    2007-05-01

    The role of JNK and p38 MAP kinases in functional inactivation and necrosis of mechanoreceptor neurons as well as necrosis, apoptosis and proliferation of satellite glial cells induced by photodynamic treatment (10 -7 M Photosens, 30 min incubation, 670 nm laser irradiation at 0.4 W/cm2) in the isolated crayfish stretch receptor was studied using specific inhibitors SP600125 and SB202190, respectively. SP600125 enhanced PDT-induced apoptosis of photosensitized glial cells but did not influence PDT-induced changes in neuronal activity, density of glial nuclei around neuron body, and necrosis of receptor neurons and glial cells. SB202190 did not influence neuron activity and survival as well but reduced PDT-induced necrosis but not apoptosis of glial cells. Therefore, both MAP kinases influenced glial cells but not neurons. JNK protected glial cells from PDT-induced apoptosis but did not influence necrosis and proliferation of these cells. In contrast, p38 did not influence apoptosis but contributed into PDT-induced necrosis of glial cells and PDT-induced gliosis. These MAP kinase inhibitors may be used for modulation of photodynamic therapy of brain tumors.

  17. The putative lipid transporter, Arv1, is required for activating pheromone-induced MAP kinase signaling in Saccharomyces cerevisiae.

    PubMed

    Villasmil, Michelle L; Ansbach, Alison; Nickels, Joseph T

    2011-02-01

    Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P(2) and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5(Q59L), which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P(2) polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking.

  18. The Putative Lipid Transporter, Arv1, Is Required for Activating Pheromone-Induced MAP Kinase Signaling in Saccharomyces cerevisiae

    PubMed Central

    Villasmil, Michelle L.; Ansbach, Alison; Nickels, Joseph T.

    2011-01-01

    Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P2 and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5Q59L, which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P2 polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking. PMID:21098723

  19. Mapping the Dynamics Landscape of Conformational Transitions in Enzyme: The Adenylate Kinase Case

    PubMed Central

    Li, Dechang; Liu, Ming S.; Ji, Baohua

    2015-01-01

    Conformational transition describes the essential dynamics and mechanism of enzymes in pursuing their various functions. The fundamental and practical challenge to researchers is to quantitatively describe the roles of large-scale dynamic transitions for regulating the catalytic processes. In this study, we tackled this challenge by exploring the pathways and free energy landscape of conformational changes in adenylate kinase (AdK), a key ubiquitous enzyme for cellular energy homeostasis. Using explicit long-timescale (up to microseconds) molecular dynamics and bias-exchange metadynamics simulations, we determined at the atomistic level the intermediate conformational states and mapped the transition pathways of AdK in the presence and absence of ligands. There is clearly chronological operation of the functional domains of AdK. Specifically in the ligand-free AdK, there is no significant energy barrier in the free energy landscape separating the open and closed states. Instead there are multiple intermediate conformational states, which facilitate the rapid transitions of AdK. In the ligand-bound AdK, the closed conformation is energetically most favored with a large energy barrier to open it up, and the conformational population prefers to shift to the closed form coupled with transitions. The results suggest a perspective for a hybrid of conformational selection and induced fit operations of ligand binding to AdK. These observations, depicted in the most comprehensive and quantitative way to date, to our knowledge, emphasize the underlying intrinsic dynamics of AdK and reveal the sophisticated conformational transitions of AdK in fulfilling its enzymatic functions. The developed methodology can also apply to other proteins and biomolecular systems. PMID:26244746

  20. Activation of ERK/MAP kinase in the amygdala is required for memory consolidation of pavlovian fear conditioning.

    PubMed

    Schafe, G E; Atkins, C M; Swank, M W; Bauer, E P; Sweatt, J D; LeDoux, J E

    2000-11-01

    Although much has been learned about the neurobiological mechanisms underlying Pavlovian fear conditioning at the systems and cellular levels, relatively little is known about the molecular mechanisms underlying fear memory consolidation. The present experiments evaluated the role of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling cascade in the amygdala during Pavlovian fear conditioning. We first show that ERK/MAPK is transiently activated-phosphorylated in the amygdala, specifically the lateral nucleus (LA), at 60 min, but not 15, 30, or 180 min, after conditioning, and that this activation is attributable to paired presentations of tone and shock rather than to nonassociative auditory stimulation, foot shock sensitization, or unpaired tone-shock presentations. We next show that infusions of U0126, an inhibitor of ERK/MAPK activation, aimed at the LA, dose-dependently impair long-term memory of Pavlovian fear conditioning but leaves short-term memory intact. Finally, we show that bath application of U0126 impairs long-term potentiation in the LA in vitro. Collectively, these results demonstrate that ERK/MAPK activation is necessary for both memory consolidation of Pavlovian fear conditioning and synaptic plasticity in the amygdala.

  1. MAPK Cascades in Guard Cell Signal Transduction

    PubMed Central

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M.

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  2. MAPK Cascades in Guard Cell Signal Transduction.

    PubMed

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions.

  3. MAPK Cascades in Guard Cell Signal Transduction.

    PubMed

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  4. Induction of Macrophage Function in Human THP-1 Cells Is Associated with Rewiring of MAPK Signaling and Activation of MAP3K7 (TAK1) Protein Kinase

    PubMed Central

    Richter, Erik; Ventz, Katharina; Harms, Manuela; Mostertz, Jörg; Hochgräfe, Falko

    2016-01-01

    Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cyclin-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1) acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease. PMID:27066479

  5. Classification of Aurora-A Kinase Inhibitors Using Self-Organizing Map (SOM) and Support Vector Machine (SVM).

    PubMed

    Wang, Liyu; Wang, Zhi; Yan, Aixia; Yuan, Qipeng

    2011-01-17

    Two classification models of 148 Aurora-A kinase inhibitors were developed to separate active and weakly potent active inhibitors of Aurora-A kinase. Each molecule was represented by 12 selected molecular descriptors calculated by the ADRIANA.Code. Then the classification models were built using a Kohonen's Self-Organizing Map (SOM) and a Support Vector Machine (SVM) method, respectively, which could be used for virtual screening an existing database to find possible new lead compounds with higher activity. The prediction accuracy of the models for the training and test sets are 96.6 % and 90.0 % for SOM, 93.2 % and 93.3 % for SVM.

  6. Heterozygous Mutations in MAP3K7, Encoding TGF-β-Activated Kinase 1, Cause Cardiospondylocarpofacial Syndrome.

    PubMed

    Le Goff, Carine; Rogers, Curtis; Le Goff, Wilfried; Pinto, Graziella; Bonnet, Damien; Chrabieh, Maya; Alibeu, Olivier; Nistchke, Patrick; Munnich, Arnold; Picard, Capucine; Cormier-Daire, Valérie

    2016-08-01

    Cardiospondylocarpofacial (CSCF) syndrome is characterized by growth retardation, dysmorphic facial features, brachydactyly with carpal-tarsal fusion and extensive posterior cervical vertebral synostosis, cardiac septal defects with valve dysplasia, and deafness with inner ear malformations. Whole-exome sequencing identified heterozygous MAP3K7 mutations in six distinct CSCF-affected individuals from four families and ranging in age from 5 to 37 years. MAP3K7 encodes transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), which is involved in the mitogen-activated protein kinase (MAPK)-p38 signaling pathway. MAPK-p38 signaling was markedly altered when expression of non-canonical TGF-β-driven target genes was impaired. These findings support the loss of transcriptional control of the TGF-β-MAPK-p38 pathway in fibroblasts obtained from affected individuals. Surprisingly, although TAK1 is located at the crossroad of inflammation, immunity, and cancer, this study reports MAP3K7 mutations in a developmental disorder affecting mainly cartilage, bone, and heart. PMID:27426734

  7. Epidermal growth factor stimulates proliferation and migration of porcine trophectoderm cells through protooncogenic protein kinase 1 and extracellular-signal-regulated kinases 1/2 mitogen-activated protein kinase signal transduction cascades during early pregnancy.

    PubMed

    Jeong, Wooyoung; Kim, Jinyoung; Bazer, Fuller W; Song, Gwonhwa

    2013-12-01

    For successful implantation and establishment of early epitheliochorial placentation, porcine conceptuses require histotroph, including nutrients and growth factors, secreted by or transported into the lumen of the uterus. Epidermal growth factor (EGF), an essential component of histotroph, is known to have potential growth-promoting activities on the conceptus and uterine endometrium. However, little is known about its effects to transactivate cell signaling cascades responsible for proliferation, growth and differentiation of conceptus trophectoderm. In the present study, therefore, we determined that EGFR mRNA and protein were abundant in endometrial luminal and glandular epithelia, stratum compactum stroma and conceptus trophectoderm on days 13-14 of pregnancy, but not in any other cells of the uterus or conceptus. In addition, primary porcine trophectoderm (pTr) cells treated with EGF exhibited increased abundance of phosphorylated (p)-AKT1, p-ERK1/2 MAPK and p-P90RSK over basal levels within 5min, and effect that was maintained to between 30 and 120min. Immunofluorescence microscopy revealed abundant amounts of p-ERK1/2 MAPK and p-AKT1 proteins in the nucleus and, to a lesser extent, in the cytoplasm of pTr cells treated with EGF as compared to control cells. Furthermore, the abundance of p-AKT1 and p-ERK1/2 MAPK proteins was inhibited in control and EGF-treated pTr cells transfected with EGFR siRNA. Compared to the control siRNA transfected pTr cells, pTr cells transfected with EGFR siRNA exhibited an increase in expression of IFND and TGFB1, but there was no effect of expression of IFNG. Further, EGF stimulated proliferation and migration of pTr cells through activation of the PI3K-AKT1 and ERK1/2 MAPK-P90RSK cell signaling pathways. Collectively, these results support the hypothesis that EGF coordinately activates multiple cell signaling pathways critical to proliferation, migration and survival of trophectoderm cells that are critical to development of

  8. Apoptosis of cerebellar granule cells induced by organotin compounds found in drinking water: involvement of MAP kinases.

    PubMed

    Mundy, William R; Freudenrich, Theresa M

    2006-01-01

    Mono- and dialkyl organotin compounds are used primarily as heat stabilizers in polyvinyl chloride (PVC) plastics. Recently, monomethyltin (MMT), dimethyltin (DMT), monobutyltin (MBT), and dibutyltin (DBT) have been detected in water from homes and businesses served by PVC pipes. While trialkyl organotins such as trimethyltin (TMT) and triethyltin (TET) are well known neurotoxicants, the toxicity of the mono- and dialkyl organotins is not well described. The present study compared the cytotoxicity of organotins found in drinking water with the known neurotoxicant TMT in primary cultures of cerebellar granule cells, and examined the role of MAP kinase signaling in organotin-induced cell death. Twenty-four hour exposure to TMT resulted in a concentration-dependent decrease in cell viability with an EC(50) of 3 microM. Exposure to MMT, DMT, and MBT at concentrations up to 10 microM had no effect. DBT, however, was very potent, and decreased cell viability with an EC(50) of 0.3 microM. Staining of organotin-treated cerebellar granule cells with the nuclear dye Syto-13 revealed that TMT and DBT, but not MMT, DMT, or MBT, produced condensation and fragmentation of chromatin characteristic of apoptosis. TMT- and DBT-induced apoptosis was confirmed using TUNEL staining and measurement of PARP cleavage. Activation of MAP kinase pathways was examined after 6 h of exposure to the organotins which induced apoptosis. Both TMT and DBT activated ERK1/2, but only TMT activated the JNK/c-Jun and p38 pathways. Pharmacologic blockade of JNK/c-Jun and p38 activation significantly decreased apoptosis produced by TMT, but not by DBT. These results show that DBT is a potent neurotoxicant in vitro, but unlike TMT, does not induce cell death via activation of MAP kinase signaling.

  9. Interferon-Gamma Increases Endothelial Permeability by Causing Activation of p38 MAP Kinase and Actin Cytoskeleton Alteration.

    PubMed

    Ng, Chin Theng; Fong, Lai Yen; Sulaiman, Mohd Roslan; Moklas, Mohamad Aris Mohd; Yong, Yoke Keong; Hakim, Muhammad Nazrul; Ahmad, Zuraini

    2015-07-01

    Interferon-gamma (IFN-γ) is known to potentiate the progression of inflammatory diseases, such as inflammatory bowel disease and atherosclerosis. IFN-γ has been found to disrupt the barrier integrity of epithelial and endothelial cell both in vivo and in vitro. However, the mechanisms of IFN-γ underlying increased endothelial cell permeability have not been extensively elucidated. We reported that IFN-γ exhibits a biphasic nature in increasing endothelial permeability. The changes observed in the first phase (4-8 h) involve cell retraction and rounding in addition to condensed peripheral F-actin without a significant change in the F-/G-actin ratio. However, cell elongation, stress fiber formation, and an increased F-/G-actin ratio were noticed in the second phase (16-24 h). Consistent with our finding from the permeability assay, IFN-γ induced the formation of intercellular gaps in both phases. A delayed phase of increased permeability was observed at 12 h, which paralleled the onset of cell elongation, stress fiber formation, and increased F-/G-actin ratio. In addition, IFN-γ stimulated p38 mitogen-activated protein (MAP) kinase phosphorylation over a 24 h period. Inhibition of p38 MAP kinase by SB203580 prevented increases in paracellular permeability, actin rearrangement, and increases in the F-/G-actin ratio caused by IFN-γ. Our results suggest that p38 MAP kinase is activated in response to IFN-γ and causes actin rearrangement and altered cell morphology, which in turn mediates endothelial cell hyperpermeability. The F-/G-actin ratio might be involved in the regulation of actin distribution and cell morphology rather than the increased permeability induced by IFN-γ.

  10. Rac regulates collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets.

    PubMed

    Kageyama, Yasunari; Doi, Tomoaki; Akamatsu, Shigeru; Kuroyanagi, Gen; Kondo, Akira; Mizutani, Jun; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Ogura, Shinji

    2013-10-01

    We previously reported that the collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets. It has been shown that Rac, which belongs to the Rho family of small GTPases, is involved in the collagen-induced platelet aggregation. In this study, we investigated the role of Rac in the collagen-stimulated release of PDGF-AB and sCD40L in human platelets. Human blood was donated from healthy volunteers and platelet-rich plasma was obtained from the blood samples. The samples were then treated with 1.0 µg/ml collagen for 0, 1, 3, or 5 min and Rac1 activity was determined using the Rac1 Activation Assay kit. We found that collagen stimulated the activation of Rac in human platelets in a time-dependent manner. However, pre-treatment with NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, reduced the collagen-induced platelet aggregation. NSC23766 markedly attenuated not only the collagen-induced p44/p42 MAP kinase phosphorylation, but also the phosphorylation of HSP27 at three serine residues (Ser-15, Ser-78 and Ser-82). In addition, the collagen‑induced release of PDGF-AB and sCD40L was significantly suppressed by NSC23766 in a dose-dependent manner. These results strongly suggest that Rac regulates the collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets, resulting in the stimulation of PDGF-AB secretion and the release of sCD40L.

  11. The gene for creatine kinase, mitochondrial 2 (sarcomeric; CKMT2), maps to chromosome 5q13. 3

    SciTech Connect

    Richard, I.; Devaud, C. ); Cherif, D.; Cohen, D.; Beckmann, J.S. )

    1993-10-01

    YAC clones for the creatine kinase, mitochrondial 2 (sarcomeric; CKMT2), gene were isolated. One of these YACs was localized on chromosome 5q13.3 by fluorescence in situ hybridization. A polymorphic dinucleotide repeat (heterozygosity 0.77) was identified within the seventh intron of the CKMT2 gene. Genotyping of CEPH families allowed positioning of CKMT2 on the multipoint map of chromosome 5 between D5S424 and D5S428, distal to spinal muscular atrophy (SMA) (5q12-q14). 8 refs., 1 fig., 2 tabs.

  12. Intermittent Hypoxia-Induced Spinal Inflammation Impairs Respiratory Motor Plasticity by a Spinal p38 MAP Kinase-Dependent Mechanism.

    PubMed

    Huxtable, Adrianne G; Smith, Stephanie M C; Peterson, Timothy J; Watters, Jyoti J; Mitchell, Gordon S

    2015-04-29

    Inflammation is characteristic of most clinical disorders that challenge the neural control of breathing. Since inflammation modulates neuroplasticity, we studied the impact of inflammation caused by prolonged intermittent hypoxia on an important form of respiratory plasticity, acute intermittent hypoxia (three, 5 min hypoxic episodes, 5 min normoxic intervals) induced phrenic long-term facilitation (pLTF). Because chronic intermittent hypoxia elicits neuroinflammation and pLTF is undermined by lipopolysaccharide-induced systemic inflammation, we hypothesized that one night of intermittent hypoxia (IH-1) elicits spinal inflammation, thereby impairing pLTF by a p38 MAP kinase-dependent mechanism. pLTF and spinal inflammation were assessed in anesthetized rats pretreated with IH-1 (2 min hypoxia, 2 min normoxia; 8 h) or sham normoxia and allowed 16 h for recovery. IH-1 (1) transiently increased IL-6 (1.5 ± 0.2-fold; p = 0.02) and inducible nitric oxide synthase (iNOS) (2.4 ± 0.4-fold; p = 0.01) mRNA in cervical spinal homogenates, (2) elicited a sustained increase in IL-1β mRNA (2.4 ± 0.2-fold; p < 0.001) in isolated cervical spinal microglia, and (3) abolished pLTF (-1 ± 5% vs 56 ± 10% in controls; p < 0.001). pLTF was restored after IH-1 by systemic NSAID administration (ketoprofen; 55 ± 9%; p < 0.001) or spinal p38 MAP kinase inhibition (58 ± 2%; p < 0.001). IH-1 increased phosphorylated (activated) p38 MAP kinase immunofluorescence in identified phrenic motoneurons and adjacent microglia. In conclusion, IH-1 elicits spinal inflammation and impairs pLTF by a spinal p38 MAP kinase-dependent mechanism. By targeting inflammation, we may develop strategies to manipulate respiratory motor plasticity for therapeutic advantage when the respiratory control system is compromised (e.g., sleep apnea, apnea of prematurity, spinal injury, or motor neuron disease).

  13. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

    PubMed Central

    Miller, R D; Hogg, J; Ozaki, J H; Gell, D; Jackson, S P; Riblet, R

    1995-01-01

    The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs. Images Fig. 3 PMID:7479885

  14. Substituted N-aryl-6-pyrimidinones: A new class of potent, selective, and orally active p38 MAP kinase inhibitors

    SciTech Connect

    Devadas, Balekudru; Selness, Shaun R.; Xing, Li; Madsen, Heather M.; Marrufo, Laura D.; Shieh, Huey; Messing, Dean M.; Yang, Jerry Z.; Morgan, Heidi M.; Anderson, Gary D.; Webb, Elizabeth G.; Zhang, Jian; Devraj, Rajesh V.; Monahan, Joseph B.

    2012-02-28

    A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-{alpha} in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.

  15. Mitogen-activated protein kinase and abscisic acid signal transduction.

    PubMed

    Heimovaara-Dijkstra, S; Testerink, C; Wang, M

    2000-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C), calcium, potassium, pH and a transient activation of MAP kinase. The ABA signal transduction cascades have been shown to be tissue-specific, the transient activation of MAP kinase has until now only been found in barley aleurone cells. However, type 2C phosphatases are involved in the induction of most ABA responses, as shown by the PP2C-deficient abi-mutants. These phosphatases show high homology with phosphatases that regulate MAP kinase activity in yeast. In addition, the role of farnesyl transferase as a negative regulator of ABA responses also indicates towards involvement of MAP kinase in ABA signal transduction. Farnesyl transferase is known to regulate Ras proteins, Ras proteins in turn are known to regulate MAP kinase activation. Interestingly, Ras-like proteins were detected in barley aleurone cells. Further establishment of the involvement of MAP kinase in ABA signal transduction and its role therein, still awaits more study.

  16. Rewiring MAP kinases in Saccharomyces cerevisiae to regulate novel targets through ubiquitination

    PubMed Central

    Groves, Benjamin; Khakhar, Arjun; Nadel, Cory M; Gardner, Richard G; Seelig, Georg

    2016-01-01

    Evolution has often copied and repurposed the mitogen-activated protein kinase (MAPK) signaling module. Understanding how connections form during evolution, in disease and across individuals requires knowledge of the basic tenets that govern kinase-substrate interactions. We identify criteria sufficient for establishing regulatory links between a MAPK and a non-native substrate. The yeast MAPK Fus3 and human MAPK ERK2 can be functionally redirected if only two conditions are met: the kinase and substrate contain matching interaction domains and the substrate includes a phospho-motif that can be phosphorylated by the kinase and recruit a downstream effector. We used a panel of interaction domains and phosphorylation-activated degradation motifs to demonstrate modular and scalable retargeting. We applied our approach to reshape the signaling behavior of an existing kinase pathway. Together, our results demonstrate that a MAPK can be largely defined by its interaction domains and compatible phospho-motifs and provide insight into how MAPK-substrate connections form. DOI: http://dx.doi.org/10.7554/eLife.15200.001 PMID:27525484

  17. Rewiring MAP kinases in Saccharomyces cerevisiae to regulate novel targets through ubiquitination.

    PubMed

    Groves, Benjamin; Khakhar, Arjun; Nadel, Cory M; Gardner, Richard G; Seelig, Georg

    2016-01-01

    Evolution has often copied and repurposed the mitogen-activated protein kinase (MAPK) signaling module. Understanding how connections form during evolution, in disease and across individuals requires knowledge of the basic tenets that govern kinase-substrate interactions. We identify criteria sufficient for establishing regulatory links between a MAPK and a non-native substrate. The yeast MAPK Fus3 and human MAPK ERK2 can be functionally redirected if only two conditions are met: the kinase and substrate contain matching interaction domains and the substrate includes a phospho-motif that can be phosphorylated by the kinase and recruit a downstream effector. We used a panel of interaction domains and phosphorylation-activated degradation motifs to demonstrate modular and scalable retargeting. We applied our approach to reshape the signaling behavior of an existing kinase pathway. Together, our results demonstrate that a MAPK can be largely defined by its interaction domains and compatible phospho-motifs and provide insight into how MAPK-substrate connections form.

  18. INTEGRATING DETAILED SOIL SURVEY AND LANDTYPE MAPPING FOR WATERSHED SCALE ASSESSMENTS IN THE WESTERN OREGON CASCADE MOUNTAINS

    EPA Science Inventory

    Although the Western Oregon Cascades is one of the most intensely managed and economically important forest regions in North America, a lack of detailed soil information has hindered watershed-scale assessments of forest productivity, water supply, sensitive wildlife species, and...

  19. Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1.

    PubMed Central

    Hernández, M; Bayón, Y; Sánchez Crespo, M; Nieto, M L

    1997-01-01

    The release of [3H]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A2 (cPLA2) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA2 in SDS/polyacrylamide gels, which is a feature of the activation of cPLA2 by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA2, namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA2 more clearly than the activation of p42-MAK kinase did. Both cPLA2 and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-Jun kinase, also phosphorylated cPLA2 but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA2, and associates the release of arachidonate with nuclear signalling pathways. PMID:9359863

  20. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  1. Mapping the Putative G Protein-coupled Receptor (GPCR) Docking Site on GPCR Kinase 2

    PubMed Central

    Beautrait, Alexandre; Michalski, Kevin R.; Lopez, Thomas S.; Mannix, Katelynn M.; McDonald, Devin J.; Cutter, Amber R.; Medina, Christopher B.; Hebert, Aaron M.; Francis, Charnelle J.; Bouvier, Michel; Tesmer, John J. G.; Sterne-Marr, Rachel

    2014-01-01

    G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α2A-adrenergic receptors (α2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α2AAR with EC50 values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu4, Val7, Leu8, Val11, and Ser12, directly interacts with receptors, whereas residues such as Asp10, Tyr13, Ala16, Met17, Gly475, Val477, and Ile485 are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity. PMID:25049229

  2. Discriminating of ATP competitive Src kinase inhibitors and decoys using self-organizing map and support vector machine.

    PubMed

    Yan, Aixia; Hu, Xiaoying; Wang, Kai; Sun, Jing

    2013-02-01

    A data set containing 686 Src kinase inhibitors and 1,941 Src kinase non-binding decoys was collected and used to build two classification models to distinguish inhibitors from decoys. The data set was randomly split into a training set (458 inhibitors and 972 decoys) and a test set (228 inhibitors and 969 decoys). Each molecule was represented by five global molecular descriptors and 18 2D property autocorrelation descriptors calculated using the program ADRIANA.Code. Two machine learning methods, a Kohonen's self-organizing map (SOM) and a support vector machine (SVM), were utilized for the training and classification. For the test set, classification accuracy (ACC) of 99.92% and Matthews correlation coefficient (MCC) of 0.98 were achieved for the SOM model; ACC of 99.33% and MCC of 0.98 were obtained for the SVM model. Some molecular properties, such as molecular weight, number of atoms in a molecule, hydrogen bond properties, polarizabilities, electronegativities, and hydrophobicities, were found to be important for the inhibition of Src kinase.

  3. Identification of a 115kDa MAP-kinase activated by freezing and anoxic stresses in the marine periwinkle, Littorina littorea.

    PubMed

    MacDonald, Justin A; Storey, Kenneth B

    2006-06-15

    The mitogen-activated protein kinase (MAPK) cascade regulates changes in gene transcription by transmitting extracellular stimuli from the plasma membrane to the cell nucleus and has an important role to play in organismal responses to environmental stresses. The activities of MAPKs were investigated in the marine gastropod mollusk, Littorina littorea, a species that tolerates both extracellular freezing and long term oxygen deprivation. In-gel kinase assays revealed the presence of two MAPKs in foot muscle and hepatopancreas, a 42 and a 115kDa protein. Immunoblot analysis showed that both were MAPK proteins and that one was the periwinkle homologue of p42(ERK2). Size exclusion chromatography confirmed the 115kDa size of the novel snail MAPK and its role as the dominant MAPK activity in foot muscle. In-gel kinase assays, immunoblotting with phospho-specific ERK antibody, as well as kinase activity profiles from hydroxyapatite chromatography demonstrated that p115 MAPK kinase activity was increased in foot muscle in response to in vivo freezing or anoxia exposures. The results suggest a role for this novel kinase in environmental stress response. PMID:16620767

  4. Inducible activation of ERK5 MAP kinase enhances adult neurogenesis in the olfactory bulb and improves olfactory function.

    PubMed

    Wang, Wenbin; Lu, Song; Li, Tan; Pan, Yung-Wei; Zou, Junhui; Abel, Glen M; Xu, Lihong; Storm, Daniel R; Xia, Zhengui

    2015-05-20

    Recent discoveries have suggested that adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) may be required for at least some forms of olfactory behavior in mice. However, it is unclear whether conditional and selective enhancement of adult neurogenesis by genetic approaches is sufficient to improve olfactory function under physiological conditions or after injury. Furthermore, specific signaling mechanisms regulating adult neurogenesis in the SVZ/OB are not fully defined. We previously reported that ERK5, a MAP kinase selectively expressed in the neurogenic regions of the adult brain, plays a critical role in adult neurogenesis in the SVZ/OB. Using a site-specific knock-in mouse model, we report here that inducible and targeted activation of the endogenous ERK5 in adult neural stem/progenitor cells enhances adult neurogenesis in the OB by increasing cell survival and neuronal differentiation. This conditional ERK5 activation also improves short-term olfactory memory and odor-cued associative olfactory learning under normal physiological conditions. Furthermore, these mice show enhanced recovery of olfactory function and have more adult-born neurons after a zinc sulfate-induced lesion of the main olfactory epithelium. We conclude that ERK5 MAP kinase is an important endogenous signaling pathway regulating adult neurogenesis in the SVZ/OB, and that conditional activation of endogenous ERK5 is sufficient to enhance adult neurogenesis in the OB thereby improving olfactory function both under normal conditions and after injury.

  5. Inducible Activation of ERK5 MAP Kinase Enhances Adult Neurogenesis in the Olfactory Bulb and Improves Olfactory Function

    PubMed Central

    Wang, Wenbin; Lu, Song; Li, Tan; Pan, Yung-Wei; Zou, Junhui; Abel, Glen M.; Xu, Lihong; Storm, Daniel R.

    2015-01-01

    Recent discoveries have suggested that adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) may be required for at least some forms of olfactory behavior in mice. However, it is unclear whether conditional and selective enhancement of adult neurogenesis by genetic approaches is sufficient to improve olfactory function under physiological conditions or after injury. Furthermore, specific signaling mechanisms regulating adult neurogenesis in the SVZ/OB are not fully defined. We previously reported that ERK5, a MAP kinase selectively expressed in the neurogenic regions of the adult brain, plays a critical role in adult neurogenesis in the SVZ/OB. Using a site-specific knock-in mouse model, we report here that inducible and targeted activation of the endogenous ERK5 in adult neural stem/progenitor cells enhances adult neurogenesis in the OB by increasing cell survival and neuronal differentiation. This conditional ERK5 activation also improves short-term olfactory memory and odor-cued associative olfactory learning under normal physiological conditions. Furthermore, these mice show enhanced recovery of olfactory function and have more adult-born neurons after a zinc sulfate-induced lesion of the main olfactory epithelium. We conclude that ERK5 MAP kinase is an important endogenous signaling pathway regulating adult neurogenesis in the SVZ/OB, and that conditional activation of endogenous ERK5 is sufficient to enhance adult neurogenesis in the OB thereby improving olfactory function both under normal conditions and after injury. PMID:25995470

  6. QSAR Analysis of Some Antagonists for p38 map kinase Using Combination of Principal Component Analysis and Artificial Intelligence.

    PubMed

    Doosti, Elham; Shahlaei, Mohsen

    2015-01-01

    Quantitative relationships between structures of a set of p38 map kinase inhibitors and their activities were investigated by principal component regression (PCR) and principal componentartificial neural network (PC-ANN). Latent variables (called components) generated by principal component analysis procedure were applied as the input of developed Quantitative structure- activity relationships (QSAR) models. An exact study of predictability of PCR and PC-ANN showed that the later model has much higher ability to calculate the biological activity of the investigated molecules. Also, experimental and estimated biological activities of compounds used in model development step have indicated a good correlation. Obtained results show that a non-linear model explaining the relationship between the pIC50s and the calculated principal components (that extract from structural descriptors of the studied molecules) is superior than linear model. Some typical figures of merit for QSAR studies explaining the accuracy and predictability of the suggested models were calculated. Therefore, to design novel inhibitors of p38 map kinase with high potency and low undesired effects the developed QSAR models were used to estimate biological pIC50 of the studied compounds.

  7. Network analysis of sediment cascades derived from digital geomorphological maps - a comparative study of three catchments in the Austrian and Swiss Alps

    NASA Astrophysics Data System (ADS)

    Heckmann, Tobias; Hilger, Ludwig; Meßenzehl, Karoline; Hoffmann, Thomas; Schwanghart, Wolfgang; Götz, Joachim; Buckel, Johannes

    2014-05-01

    Sediment fluxes in alpine environments are strongly conditioned by sediment storage, resulting in non-linear feedbacks between sediment input and output. Recently, geomorphological mapping in alpine regions has focused on the static distribution of sediment storage units, neglecting the dynamic links of geomorphic processes units along the sediment pathways. Here we present an approach i) to analyse the sediment connectivity of a catchment based on coupling relationships of neighbouring storage units, and ii) to re-evaluate existing geomorphological maps in terms of the network structure. The approach is applied in three catchments in the European Alps, which were mapped in previous geomorphological studies: i) the Gradenbach catchment (Upper Tauern, Carinthia, Austria), ii) the (upper) Kaunertal (Ötztal Mountains, Tyrolia, Austria) and the Val Müschauns (Engadine, Switzerland). While the morphology of all study areas is predominantly controlled by former glaciations, only the two Austrian areas feature recent glaciers. The available geomorphic maps consist of non-overlapping polygons representing geomorphic process units of erosion, sediment transport and deposition. Mapping was conducted in the field, and supported by digital orthophotos and derivatives of LiDAR-based digital elevation models (slope, curvature, aspect, shaded relief, etc.). Based on the observation of diagnostic features, the geomorphic coupling state of every pair of adjacent landforms, i.e. the existence of sediment transfer across their common boundary, was assessed and mapped. Taking the landforms as nodes, and the inferred coupling relationships as edges, a graph model of the sediment transfer system is established. Graph theory offers a versatile toolbox for the analysis of the spatial structure of sediment cascades in different ways: Nodes are analysed for the number of incoming and outgoing edges, and classified as sediment source, sink, or link. Depending on the spatial and functional

  8. Extracellular simian virus 40 induces an ERK/MAP kinase-independent signalling pathway that activates primary response genes and promotes virus entry.

    PubMed

    Dangoria, N S; Breau, W C; Anderson, H A; Cishek, D M; Norkin, L C

    1996-09-01

    Simian virus 40 (SV40) binding to growth-arrested cells activated an intracellular signalling pathway that induced the up-regulation of the primary response genes c-myc, c-jun and c-sis within 30 min and of JE within 90 min. The up-regulation of the primary response genes occurred in the presence of cycloheximide and when UV-inactivated SV40 was adsorbed to cells. SV40 binding did not activate Raf or mitogen-activated protein kinase (MAP/ERK1), or mobilize intracellular Ca2+. The SV40-induced up-regulation of c-myc and c-jun was blocked by the tyrosine kinase inhibitor, genistein, and by the protein kinase C (PKC) inhibitor, calphostin C, but not by expression of the MAP kinase-specific phosphatase, MKP-1. These results suggest that the SV40-induced signalling pathway includes the activities of a tyrosine kinase and a Ca(2+)-independent isoform of PKC, but not of Raf or MAP kinase. Finally, SV40 infectious entry into cells was specifically and reversibly blocked by genistein.

  9. Regulation of gene expression by the small GTPase Rho through the ERK6 (p38γ) MAP kinase pathway

    PubMed Central

    Marinissen, Maria Julia; Chiariello, Mario; Gutkind, J. Silvio

    2001-01-01

    Small GTP-binding proteins of the Rho-family, Rho, Rac, and Cdc42, have been traditionally linked to the regulation of the cellular actin-based cytoskeleton. Rac and Cdc42 can also control the activity of JNK, thus acting in a molecular pathway transmitting extracellular signals to the nucleus. Interestingly, Rho can also regulate gene expression, albeit by a not fully understood mechanism. Here, we found that activated RhoA can stimulate c-jun expression and the activity of the c-jun promoter. As the complexity of the signaling pathways controlling the expression of c-jun has begun to be unraveled, this finding provided a unique opportunity to elucidate the biochemical routes whereby RhoA regulates nuclear events. We found that RhoA can initiate a linear kinase cascade leading to the activation of ERK6 (p38γ), a recently identified member of the p38 family of MAPKs. Furthermore, we present evidence that RhoA, PKN, MKK3/MKK6, and ERK6 (p38γ) are components of a novel signal transduction pathway involved in the regulation of gene expression and cellular transformation. PMID:11238375

  10. Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Huang, Y.; Kieber, J.; Luan, S.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

  11. Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases.

    PubMed

    Zeke, András; Bastys, Tomas; Alexa, Anita; Garai, Ágnes; Mészáros, Bálint; Kirsch, Klára; Dosztányi, Zsuzsanna; Kalinina, Olga V; Reményi, Attila

    2015-11-01

    Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles. PMID:26538579

  12. Functional Mapping of Protein Kinase A Reveals Its Importance in Adult Schistosoma mansoni Motor Activity

    PubMed Central

    de Saram, Paulu S. R.; Ressurreição, Margarida; Davies, Angela J.; Rollinson, David; Emery, Aidan M.; Walker, Anthony J.

    2013-01-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and ‘smart’ anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology

  13. Functional mapping of protein kinase A reveals its importance in adult Schistosoma mansoni motor activity.

    PubMed

    de Saram, Paulu S R; Ressurreição, Margarida; Davies, Angela J; Rollinson, David; Emery, Aidan M; Walker, Anthony J

    2013-01-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and 'smart' anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology.

  14. Digital data for preliminary geologic map of the Mount Hood 30- by 60-minute quadrangle, northern Cascade Range, Oregon

    USGS Publications Warehouse

    Lina Ma,; Sherrod, David R.; Scott, William E.

    2014-01-01

    This geodatabase contains information derived from legacy mapping that was published in 1995 as U.S. Geological Survey Open-File Report 95-219. The main component of this publication is a geologic map database prepared using geographic information system (GIS) applications. Included are pdf files to view or print the map sheet, the accompanying pamphlet from Open-File Report 95-219, and links to the original publication, which is available as scanned files in pdf format.

  15. Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-0-tetradecanoylphorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase.

    PubMed

    Kanemitsu, M Y; Lau, A F

    1993-08-01

    We previously reported that epidermal growth factor (EGF) induced the disruption of gap junctional communication (gjc) and serine phosphorylation of connexin43 (Cx43) in T51B rat liver epithelial cells. However, the cascade of events linking EGF receptor activation to these particular responses have not been fully characterized. Furthermore, the serine kinase(s) acting directly on Cx43 remain unidentified. In the current study, we demonstrate that downmodulation of 12-0-tetradecanoylphorbol 13-acetate (TPA)-sensitive protein kinase C (PKC) activity does not affect EGF's ability to reduce junctional permeability or phosphorylate Cx43 in T51B cells. EGF in the presence or absence of chronic TPA treatment stimulated marked increases in Cx43 phosphorylation on numerous sites as determined by two-dimensional tryptic phosphopeptide mapping. Computer-assisted sequence analysis of Cx43 identified several protein kinase phosphorylation consensus sites including two sites for mitogen-activated protein (MAP) kinase. EGF stimulated activation of MAP kinase in a time- and dose-dependent manner where the kinetics of kinase activity corroborated its possible involvement in mediating EGF's effects. Moreover, purified MAP kinase directly phosphorylated Cx43 on serine residues in vitro. Two-dimensional tryptic and chymotryptic phosphopeptide mapping demonstrated that the in vitro phosphopeptides represented a specific subset of the in vivo phosphopeptides produced in response to EGF after chronic TPA treatment. Therefore, EGF-induced disruption of gjc and phosphorylation of Cx43 may be mediated in part by MAP kinase in vivo.

  16. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter.

    PubMed Central

    Bruening, W; Giasson, B; Mushynski, W; Durham, H D

    1998-01-01

    The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in eukaryotic cells. In practice, the CMV promoter is often thought of as a constitutively active unregulated promoter. However, we have observed that transcription from the CMV promoter can be up-regulated by a variety of environmental stresses. Many forms of cellular stress stimulate MAP kinase signalling pathways, resulting in activation of stress-activated protein kinases [SAPKs, also called Jun N-terminal kinases (JNKs)] and p38 kinases. We have found that the same conditions that lead to activation of SAPK/JNKs and p38 kinases can also dramatically increase expression from the CMV promoter. Inhibitors of p38 kinases abolished basal transcription from the CMV promoter and completely blocked stress-induced up-regulation of the CMV promoter. Overexpression of a dominant negative JNK kinase had no effect on basal transcription, but significantly reduced up-regulation caused by stress. These results have grave implications for use of the CMV promoter. If the CMV promoter can be up-regulated by cellular stresses, inadvertent activation of the stress kinase pathways may complicate, if not invalidate, the interpretation of a wide range of experiments. PMID:9421504

  17. Selective regulation of MAP kinases and Chemokine expression after ligation of ICAM-1 on human airway epithelial cells

    PubMed Central

    Krunkosky, Thomas M; Jarrett, Carla L

    2006-01-01

    Background Intercellular adhesion molecule 1 (ICAM-1) is an immunoglobulin-like cell adhesion molecule expressed on the surface of multiple cell types, including airway epithelial cells. It has been documented that cross-linking ICAM-1 on the surface of leukocytes results in changes in cellular function through outside-inside signaling; however, the effect of cross-linking ICAM-1 on the surface of airway epithelial cells is currently unknown. The objective of this study was to investigate whether or not cross-linking ICAM-1 on the surface of airway epithelial cells phosphorylated MAP kinases or stimulated chemokine expression and secretion. Methods The human lung adenocarcinoma (A549) cells and primary cultures of normal human bronchial epithelial (NHBE) cells were used in these studies. To increase ICAM-1 surface expression, cultures were stimulated with TNFα to enhance ICAM-1 surface expression. Following ICAM-1 upregulation, ICAM-1 was ligated with a murine anti-human ICAM-1 antibody and subsequently cross-linked with a secondary antibody (anti-mouse IgG(ab')2) in the presence or absence of the MAP kinase inhibitors. Following treatments, cultures were assessed for MAPK activation and chemokine gene expression and secretion. Control cultures were treated with murine IgG1 antibody or murine IgG1 antibody and anti-mouse IgG(ab')2 to illustrate specificity. Data were analyzed for significance using a one-way analysis of variance (ANOVA) with Bonferroni post-test correction for multiple comparisons, and relative gene expression was analyzed using the 2-ΔΔCT method. Results ICAM-1 cross-linking selectively phosphorylated both ERK and JNK MAP kinases as detected by western blot analysis. In addition, cross-linking resulted in differential regulation of chemokine expression. Specifically, IL-8 mRNA and protein secretion was not altered by ICAM-1 cross-linking, in contrast, RANTES mRNA and protein secretion was induced in both epithelial cultures. These events were

  18. Arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan, inhibits MAP kinases and AP-1 activation via potent MKK inhibition: the role in TNF-alpha inhibition.

    PubMed

    Cho, Min Kyung; Jang, Young Pyo; Kim, Young Choong; Kim, Sang Geon

    2004-10-01

    Arctigenin, naturally occurring in Bardanae fructus, Saussurea medusa, Arctium lappa L., Torreya nucifera and Ipomea cairica, is a phenylpropanoid dibenzylbutyrolactone lignan with antioxidant and anti-inflammatory activities. Previously, we showed that arctigenin potently inhibited the induction of nitric oxide synthase (iNOS) by lipopolysaccharide (LPS), which involved suppression of NF-kappaB activation. In the present study, we examined the effects of arctigenin on mitogen-activated protein (MAP) kinase activation in Raw264.7 cells and MAP kinase kinase (MKK) activity. The effect of arctigenin on activator protein-1 (AP-1) activation was also studied in association with tumor necrosis factor-alpha (TNF-alpha) expression. Immunoblot analysis showed that arctigenin inhibited phosphorylation of MAP kinases ERK1/2, p38 kinase and JNK and their activities in Raw264.7 cells treated with LPS. Arctigenin potently inhibited the activity of MKK1 in vitro with the IC(50) value of 1 nM. Gel shift and reporter gene analyses revealed that arctigenin inhibited LPS-inducible AP-1 binding to the AP-1 consensus oligonucleotide and AP-1-mediated reporter gene expression. In view of the potential role of AP-1 in the induction of TNF-alpha, we next examined the inhibitory effects of arctigenin on the expression of TNF-alpha. Arctigenin blocked TNF-alpha production and decreased the level of TNF-alpha mRNA in the cells exposed to LPS. These results showed that arctigenin inhibited activation of MAP kinases including ERK1/2, p38 kinase and JNK through the inhibition of MKK activities, leading to AP-1 inactivation, which might, at least in part, contribute to the inhibition of TNF-alpha production.

  19. Systems Analysis of Adaptive Responses to MAP Kinase Pathway Blockade in BRAF Mutant Melanoma

    PubMed Central

    Capaldo, Brian J.; Roller, Devin; Axelrod, Mark J.; Koeppel, Alex F.; Petricoin, Emanuel F.; Slingluff, Craig L.; Weber, Michael J.; Mackey, Aaron J.; Gioeli, Daniel; Bekiranov, Stefan

    2015-01-01

    Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to mitogen-activated protein kinase (MAPK) pathway inhibition, we identified the combination of PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ErbB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment, we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and receptor tyrosine kinase (RTK) mutational status. Layered over base-line resistance was substantial upregulation of many ErbB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ErbB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes. PMID:26405815

  20. The p38 MAP kinase pathway modulates the hypoxia response and glutamate receptor trafficking in aging neurons.

    PubMed

    Park, Eun Chan; Rongo, Christopher

    2016-01-01

    Neurons are sensitive to low oxygen (hypoxia) and employ a conserved pathway to combat its effects. Here, we show that p38 MAP Kinase (MAPK) modulates this hypoxia response pathway in C. elegans. Mutants lacking p38 MAPK components pmk-1 or sek-1 resemble mutants lacking the hypoxia response component and prolyl hydroxylase egl-9, with impaired subcellular localization of Mint orthologue LIN-10, internalization of glutamate receptor GLR-1, and depression of GLR-1-mediated behaviors. Loss of p38 MAPK impairs EGL-9 protein localization in neurons and activates the hypoxia-inducible transcription factor HIF-1, suggesting that p38 MAPK inhibits the hypoxia response pathway through EGL-9. As animals age, p38 MAPK levels decrease, resulting in GLR-1 internalization; this age-dependent downregulation can be prevented through either p38 MAPK overexpression or removal of CDK-5, an antagonizing kinase. Our findings demonstrate that p38 MAPK inhibits the hypoxia response pathway and determines how aging neurons respond to hypoxia through a novel mechanism. PMID:26731517

  1. Cloning of a phosphatidylinositol 4-kinase gene based on fiber strength transcriptome QTL mapping in the cotton species Gossypium barbadense.

    PubMed

    Liu, H W; Shi, R F; Wang, X F; Pan, Y X; Zang, G Y; Ma, Z Y

    2012-09-25

    Sea Island cotton (Gossypium barbadense) is highly valued for its superior fiber qualities, especially fiber strength. Based on a transcript-derived fragment originated from transcriptome QTL mapping, a fiber strength related candidate gene of phosphatidylinositol 4-kinase cDNA, designated as GbPI4K, was first cloned, and its expression was characterized in the secondary cell wall thickening stage of G. barbadense fibers. The ORF of GbPI4K was found to be 1926 bp in length and encoded a predicted protein of 641 amino acid residues. The putative protein contained a clear PI3/4K kinase catalytic domain and fell into the plant type II PI4K cluster in phylogenetic analysis. In this study, the expression of cotton PI4K protein was also induced in Escherichia coli BL21 (DE3) as a fused protein. Semi-quantitative RT-PCR analysis showed that the gene expressed in the root, hypocotyl and leaf of the cotton plants. Real-time RT-PCR indicated that this gene in Sea Island cotton fibers expressed 10 days longer than that in Upland cotton fibers, and the main expression difference of PI4K between Sea Island cotton and Upland cotton in fibers was located in the secondary cell wall thickening stage of the fiber. Further analysis indicated that PI4K is a crucial factor in the ability of Rac proteins to regulate phospholipid signaling pathways.

  2. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

    SciTech Connect

    Menschikowski, Mario; Hagelgans, Albert; Eisenhofer, Graeme; Siegert, Gabriele

    2009-09-10

    The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}), but not interferon-{gamma} and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1{beta} and TNF-{alpha} correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-L-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1{beta} and TNF-{alpha}, and downstream by MAP kinase signaling pathways and metalloproteinases.

  3. Cell-permeable p38 MAP kinase promotes migration of adult neural stem/progenitor cells

    PubMed Central

    Hamanoue, Makoto; Morioka, Kazuhito; Ohsawa, Ikuroh; Ohsawa, Keiko; Kobayashi, Masaaki; Tsuburaya, Kayo; Akasaka, Yoshikiyo; Mikami, Tetsuo; Ogata, Toru; Takamatsu, Ken

    2016-01-01

    Endogenous neural stem/progenitor cells (NPCs) can migrate toward sites of injury, but the migration activity of NPCs is insufficient to regenerate damaged brain tissue. In this study, we showed that p38 MAP kinase (p38) is expressed in doublecortin-positive adult NPCs. Experiments using the p38 inhibitor SB203580 revealed that endogenous p38 participates in NPC migration. To enhance NPC migration, we generated a cell-permeable wild-type p38 protein (PTD-p38WT) in which the HIV protein transduction domain (PTD) was fused to the N-terminus of p38. Treatment with PTD-p38WT significantly promoted the random migration of adult NPCs without affecting cell survival or differentiation; this effect depended on the cell permeability and kinase activity of the fusion protein. These findings indicate that PTD-p38WT is a novel and useful tool for unraveling the roles of p38, and that this protein provides a reasonable approach for regenerating the injured brain by enhancing NPC migration. PMID:27067799

  4. The SLT2 (MPK1) MAP kinase homolog is involved in polarized cell growth in Saccharomyces cerevisiae

    PubMed Central

    1993-01-01

    Bud emergence, spindle pole body duplication and DNA replication are all dependent on the activation of the CDC28 protein kinase at the Start point in the G1 phase of the cell cycle. Bud emergence requires polarization of the cytoskeleton and secretory vesicles to a specific site on the cell surface. Cdc28p activated by G1-cyclins triggers polarization of actin to the site of bud emergence and favors apical bud growth (Lew, D. J., and S. I. Reed. 1993. J. Cell Biol. 120:1305- 1320). We isolated slt2-1 as a mutation that enhances the division defect of cdc28 mutants with defects at Start. Slt2p(Mpk1p) is a member of the MAP kinase family (Lee, K. S., K. Irie, Y. Gotoh, Y. Watanabe, H. Araki, E. Nishida, K. Matsumoto, and D. E. Levin. 1993. Mol. Cell. Biol. 13:3067-3075). We show that slt2 mutants exhibit phenotypes similar to those shown by mutants of the yeast actin cytoskeleton, including delocalization of chitin deposition and of actin cortical spots and the accumulation of secretory pathway membranes and vesicles. Furthermore, slt2::HIS3 act1-1 and slt2::HIS3 myo2-66 double mutants are inviable. We suggest that Slt2p functions downstream or in parallel with Cdc28p in promoting bud formation and apical growth. PMID:8276900

  5. Gene mapping in marsupials and monotremes, V. Synteny between hypoxanthine phosphoribosyltransferase and phosphoglycerate kinase in the platypus.

    PubMed

    Watson, J M; Graves, J A

    1988-01-01

    In order to extend comparative mapping studies to the monotreme mammals (subclass Prototheria), somatic-cell hybrids were obtained between Chinese-hamster cells deficient in hypoxanthine phosphoribosyltransferase (HPRT) and platypus fibroblasts. The characteristics of these hybrids closely resemble those of metatherian x eutherian hybrids, in that they are recovered at low frequency and they rapidly segregate and fragment platypus chromosomes. Biochemical and cytological studies of the hybrids, their subclones and HPRT-deficient revertants indicate that phosphoglycerate kinase is syntenic with HPRT in the platypus (as it is in other mammals); however, the studies do not permit chromosomal assignment of the syntenic group. The implications of the chromosomal location of this ancient synteny group for the evolution of the mammalian X chromosome are discussed.

  6. Glutamate-dependent transcriptional regulation in bergmann glia cells: involvement of p38 MAP kinase.

    PubMed

    Zepeda, Rossana C; Barrera, Iliana; Castelán, Francisco; Soto-Cid, Abraham; Hernández-Kelly, Luisa C; López-Bayghen, Esther; Ortega, Arturo

    2008-07-01

    Glutamate (Glu) is the major excitatory neurotransmitter in the Central Nervous System (CNS). Ionotropic and metabotropic glutamate receptors (GluRs) are present in neurons and glial cells and are involved in gene expression regulation. Mitogen-activated proteins kinases (MAPK) are critical for all the membrane to nuclei signaling pathways described so far. In cerebellar Bergmann glial cells, glutamate-dependent transcriptional regulation is partially dependent on p42/44 MAPK activity. Another member of this kinase family, p38 MAPK is activated by non-mitogenic stimuli through its Thr180/Tyr182 phosphorylation and phosphorylates cytoplasmic and nuclear protein targets involved in translational and transcriptional events. Taking into consideration that the role of p38MAPK in glial cells is not well understood, we demonstrate here that glutamate increases p38 MAPK phosphorylation in a time and dose dependent manner in cultured chick cerebellar Bergmann glial cells (BGC). Moreover, p38 MAPK is involved in the glutamate-induced transcriptional activation in these cells. Ionotropic as well as metabotropic glutamate receptors participate in p38 MAPK activation. The present findings demonstrate the involvement of p38 MAPK in glutamate-dependent gene expression regulation in glial cells.

  7. Pectin activation of MAP kinase and gene expression is WAK2 dependent.

    PubMed

    Kohorn, Bruce D; Johansen, Susan; Shishido, Akira; Todorova, Tanya; Martinez, Rhysly; Defeo, Elita; Obregon, Pablo

    2009-12-01

    The angiosperm extracellular matrix, or cell wall, is composed of a complex array of cellulose, hemicellulose, pectins and proteins, the modification and regulated synthesis of which are essential for cell growth and division. The wall associated kinases (WAKs) are receptor-like proteins that have an extracellular domain that bind pectins, the more flexible portion of the extracellular matrix, and are required for cell expansion as they have a role in regulating cellular solute concentrations. We show here that both recombinant WAK1 and WAK2 bind pectin in vitro. In protoplasts pectins activate, in a WAK2-dependent fashion, the transcription of vacuolar invertase, and a wak2 mutant alters the normal pectin regulation of mitogen-activated protein kinases. Microarray analysis shows that WAK2 is required for the pectin activation of numerous genes in protoplasts, many of which are involved in cell wall biogenesis. Thus, WAK2 plays a major role in signaling a diverse array of cellular events in response to pectin in the extracellular matrix.

  8. Inhibition of inflammation by a p38 MAP kinase targeted cell permeable peptide.

    PubMed

    Fu, Jing; Meng, Xianmei; He, Junyun; Gu, Jun

    2008-11-01

    p38 MAPK has been the key therapeutic target for multiple inflammation diseases. However, the clinical applications of p38 inhibitors, most of which target on the ATP binding groove in the kinase, have been held back, largely because of their limited specificity and severe side-effects. An alternative strategy to generate highly selective p38 inhibitor is to block the specific interaction in the p38 signal pathway. Based on the hypothesis that specific binding peptides targeting on the docking groove would interfere the intrinsic interaction between p38 and its partners, we have designed a fusion peptide containing 12aa p38 docking sequence derived from MKK3b and 11aa HIV-TAT transmembrane sequence to form a cell permeable peptide. The peptide specifically binds to p38, and aborts its interaction with upstream kinase as well as downstream substrates, and thus to inhibit p38 phosphorylation and its signaling. Furthermore, the induction and secretion of TNFalpha and other inflammatory factors by LPS are blocked in peptide treated cells and mice. Finally the peptide has been shown to significantly inhibit ear oedema in mice. Therefore, the peptide holds great potential as an anti-inflammation agent for the treatment of inflammation and its related diseases. PMID:18991745

  9. Menin uncouples Elk-1, JunD and c-Jun phosphorylation from MAP kinase activation.

    PubMed

    Gallo, Adriana; Cuozzo, Concetta; Esposito, Ilaria; Maggiolini, Marcello; Bonofiglio, Daniela; Vivacqua, Adele; Garramone, Maria; Weiss, Carsten; Bohmann, Dirk; Musti, Anna Maria

    2002-09-19

    Menin, a nuclear protein encoded by the tumor suppressor gene MEN1, interacts with the AP-1 transcription factor JunD and inhibits its transcriptional activity. In addition, overexpression of Menin counteracts Ras-induced tumorigenesis. We show that Menin inhibits ERK-dependent phosphorylation and activation of both JunD and the Ets-domain transcription factor Elk-1. We also show that Menin represses the inducible activity of the c-fos promoter. Furthermore, Menin expression inhibits Jun N-terminal kinase (JNK)-mediated phosphorylation of both JunD and c-Jun. Kinase assays show that Menin overexpression does not interfere with activation of either ERK2 or JNK1, suggesting that Menin acts at a level downstream of MAPK activation. An N-terminal deletion mutant of Menin that cannot inhibit JunD phosphorylation by JNK, can still repress JunD phosphorylation by ERK2, suggesting that Menin interferes with ERK and JNK pathways through two distinct inhibitory mechanisms. Taken together, our data suggest that Menin uncouples ERK and JNK activation from phosphorylation of their nuclear targets Elk-1, JunD and c-Jun, hence inhibiting accumulation of active Fos/Jun heterodimers. This study provides new molecular insights into the tumor suppressor function of Menin and suggests a mechanism by which Menin may interfere with Ras-dependent cell transformation and oncogenesis. PMID:12226747

  10. A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1.

    PubMed

    Kusari, Anasua B; Molina, Douglas M; Sabbagh, Walid; Lau, Chang S; Bardwell, Lee

    2004-01-19

    The Saccharomyces cerevisiae mitogen-activated protein kinases (MAPKs) Fus3 and Kss1 bind to multiple regulators and substrates. We show that mutations in a conserved docking site in these MAPKs (the CD/7m region) disrupt binding to an important subset of their binding partners, including the Ste7 MAPK kinase, the Ste5 adaptor/scaffold protein, and the Dig1 and Dig2 transcriptional repressors. Supporting the possibility that Ste5 and Ste7 bind to the same region of the MAPKs, they partially competed for Fus3 binding. In vivo, some of the MAPK mutants displayed reduced Ste7-dependent phosphorylation, and all of them exhibited multiple defects in mating and pheromone response. The Kss1 mutants were also defective in Kss1-imposed repression of Ste12. We conclude that MAPKs contain a structurally and functionally conserved docking site that mediates an overall positively acting network of interactions with cognate docking sites on their regulators and substrates. Key features of this interaction network appear to have been conserved from yeast to humans. PMID:14734536

  11. Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes.

    PubMed

    Grimsey, Neil J; Aguilar, Berenice; Smith, Thomas H; Le, Phillip; Soohoo, Amanda L; Puthenveedu, Manojkumar A; Nizet, Victor; Trejo, JoAnn

    2015-09-28

    Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β-activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2-mediated ubiquitination and TAB1-TAB2. TAB1-TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption.

  12. Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes

    PubMed Central

    Grimsey, Neil J.; Aguilar, Berenice; Smith, Thomas H.; Le, Phillip; Soohoo, Amanda L.; Puthenveedu, Manojkumar A.; Nizet, Victor

    2015-01-01

    Protease-activated receptor 1 (PAR1) is a G protein–coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β–activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2–mediated ubiquitination and TAB1–TAB2. TAB1–TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption. PMID:26391660

  13. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

    NASA Technical Reports Server (NTRS)

    Levonen, A. L.; Patel, R. P.; Brookes, P.; Go, Y. M.; Jo, H.; Parthasarathy, S.; Anderson, P. G.; Darley-Usmar, V. M.

    2001-01-01

    Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

  14. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

    PubMed

    Witte, Katrin; Koch, Egon; Volk, Hans-Dieter; Wolk, Kerstin; Sabat, Robert

    2015-01-01

    Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells.

  15. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

    PubMed

    Witte, Katrin; Koch, Egon; Volk, Hans-Dieter; Wolk, Kerstin; Sabat, Robert

    2015-01-01

    Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells. PMID:26406906

  16. Swertisin an Anti-Diabetic Compound Facilitate Islet Neogenesis from Pancreatic Stem/Progenitor Cells via p-38 MAP Kinase-SMAD Pathway: An In-Vitro and In-Vivo Study

    PubMed Central

    Dadheech, Nidheesh; Srivastava, Abhay; Paranjape, Neha; Gupta, Shivika; Dave, Arpita; Shah, Girish M.; Bhonde, Ramesh R.; Gupta, Sarita

    2015-01-01

    Transplanting islets serves best option for restoring lost beta cell mass and function. Small bio-chemical agents do have the potential to generate new islets mass, however lack of understanding about mechanistic action of these small molecules eventually restricts their use in cell-based therapies for diabetes. We recently reported “Swertisin” as a novel islet differentiation inducer, generating new beta cells mass more effectively. Henceforth, in the present study we attempted to investigate the molecular signals that Swertisin generate for promoting differentiation of pancreatic progenitors into islet cells. To begin with, both human pancreatic progenitors (PANC-1 cells) and primary cultured mouse intra-islet progenitor cells (mIPC) were used and tested for Swertisin induced islet neogenesis mechanism, by monitoring immunoblot profile of key transcription factors in time dependent manner. We observed Swertisin follow Activin-A mediated MEPK-TKK pathway involving role of p38 MAPK via activating Neurogenin-3 (Ngn-3) and Smad Proteins cascade. This MAP Kinase intervention in differentiation of cells was confirmed using strong pharmacological inhibitor of p38 MAPK (SB203580), which effectively abrogated this process. We further confirmed this mechanism in-vivo in partial pancreatectomised (PPx) mice model, where we could show Swertisin exerted potential increase in insulin transcript levels with persistent down-regulation of progenitor markers like Nestin, Ngn-3 and Pancreatic Duodenal Homeobox Gene-1 (PDX-1) expression, within three days post PPx. With detailed molecular investigations here in, we first time report the molecular mode of action of Swertisin for islet neogenesis mediated through MAP Kinase (MEPK-TKK) pathway involving Ngn-3 and Smad transcriptional regulation. These findings held importance for developing Swertisin as potent pharmacological drug candidate for effective and endogenous differentiation of islets in cell based therapy for diabetes

  17. Sequestration of Polo kinase to microtubules by phosphopriming-independent binding to Map205 is relieved by phosphorylation at a CDK site in mitosis

    PubMed Central

    Archambault, Vincent; D’Avino, Pier Paolo; Deery, Michael J.; Lilley, Kathryn S.; Glover, David M.

    2008-01-01

    The conserved Polo kinase controls multiple events in mitosis and cytokinesis. Although Polo-like kinases are regulated by phosphorylation and proteolysis, control of subcellular localization plays a major role in coordinating their mitotic functions. This is achieved largely by the Polo-Box Domain, which binds prephosphorylated targets. However, it remains unclear whether and how Polo might interact with partner proteins when priming mitotic kinases are inactive. Here we show that Polo associates with microtubules in interphase and cytokinesis, through a strong interaction with the microtubule-associated protein Map205. Surprisingly, this interaction does not require priming phosphorylation of Map205, and the Polo-Box Domain of Polo is required but not sufficient for this interaction. Moreover, phosphorylation of Map205 at a CDK site relieves this interaction. Map205 can stabilize Polo and inhibit its cellular activity in vivo. In syncytial embryos, the centrosome defects observed in polo hypomorphs are enhanced by overexpression of Map205 and suppressed by its deletion. We propose that Map205-dependent targeting of Polo to microtubules provides a stable reservoir of Polo that can be rapidly mobilized by the activity of Cdk1 at mitotic entry. PMID:18832073

  18. Sequence, structure, and active site analyses of p38 MAP kinase: exploiting DFG-out conformation as a strategy to design new type II leads.

    PubMed

    Badrinarayan, Preethi; Sastry, G Narahari

    2011-01-24

    A new knowledge, structure, and sequence based strategy involving the effective exploitation of the DFG-out conformation is delineated. A comprehensive analysis of the structure, sequence, cocrystals, and active sites of p38 MAP kinase crystal structures present in Protein Data Bank (PDB) and the FDA approved MAP kinase drugs has been done, and the information is used for the design of type II leads. The 98 crystal structures, 138 cocrystals, and 31 FDA drugs comprise of 7 different sequences of 2 organisms viz., Homo sapiens and Mus musculus differing in sequence length, constituting both homo- and heterochains. Multiple sequence alignment with ClustalW showed >95% sequence similarity with highly conserved domains and a high propensity for mutations in the activation loop. The bound ligands were extracted, and their interactions with DFG in and out conformations were studied. These cocrystals and FDA drugs were fragmented on the basis of their binding interactions and their affinity to ATP and allosteric sites. The fragment library thus generated contains 106 fragments with overlapping drug fragments. A blue print constituting three main parts viz., head (ATP region), linker (DFG region), and tail (allosteric region) has thus been formulated and used to design 64 type II p38 MAP kinase inhibitors. The above strategy has been employed to design potent type II p38 MAP kinase inhibitors, which are shown to be very promising. PMID:21141877

  19. Imidazolyl benzimidazoles and imidazo[4,5-b]pyridines as potent p38alpha MAP kinase inhibitors with excellent in vivo antiinflammatory properties.

    PubMed

    Mader, Mary; de Dios, Alfonso; Shih, Chuan; Bonjouklian, Rosanne; Li, Tiechao; White, Wesley; López de Uralde, Beatriz; Sánchez-Martinez, Concepción; del Prado, Miriam; Jaramillo, Carlos; de Diego, Eugenio; Martín Cabrejas, Luisa M; Dominguez, Carmen; Montero, Carlos; Shepherd, Timothy; Dally, Robert; Toth, John E; Chatterjee, Arindam; Pleite, Sehila; Blanco-Urgoiti, Jaime; Perez, Leticia; Barberis, Mario; Lorite, María José; Jambrina, Enrique; Nevill, C Richard; Lee, Paul A; Schultz, Richard C; Wolos, Jeffrey A; Li, Li C; Campbell, Robert M; Anderson, Bryan D

    2008-01-01

    Herein we report investigations into the p38alpha MAP kinase activity of trisubstituted imidazoles that led to the identification of compounds possessing highly potent in vivo activity. The SAR of a novel series of imidazopyridines is demonstrated as well, resulting in compounds possessing cellular potency and enhanced in vivo activity in the rat collagen-induced arthritis model of chronic inflammation.

  20. Virtual screening based on pharmacophore model followed by docking simulation studies in search of potential inhibitors for p38 map kinase.

    PubMed

    Shahlaei, Mohsen; Doosti, Elham

    2016-05-01

    P38 mitogen-activated protein (MAP) kinase inhibitors are closely involved in the production of inflammatory cytokines. These compounds are considered promising therapeutic agents for chronic inflammatory disorders. In this study, a ligand-based pharmacophore model of p38 map kinase inhibitors was developed. The best five features pharmacophore model includes two hydrogen bond acceptors, two hydrophobic and an aromatic hydrophobic features, which has the highest correlation coefficient (0.822), cost difference (134.158), low root mean square (RMS) of error (1.315). As well as the developed model shows a high goodness of fit and enrichment factor. The pharmacophore hypothesis has been validated by using a series of similar structures with varying affinities for the p38 map kinase. It also has been employed as a search query in different database searching with the ultimate goal of finding novel compounds which have the possibility to be modified into novel lead molecules. As a result, some hit compounds were introduced as final candidates by employing virtual screening and molecular docking procedure simultaneously. The results from pharmacophore modeling and molecular docking are complementary to each other and could serve as a useful approach for the discovery of potent small molecules as p38 map kinase inhibitors. PMID:27133076

  1. MAP KINASE SIGNALING IN PULMONARY FIBROBLASTS EXPOSED TO PARTICULATE MATTER (PM) AND BRONCHOAL VEOLAR LAVAGE FLUID (BALF) FROM HEALTHY AND HYPERTENSIVE RATS

    EPA Science Inventory

    MAP KINASE SIGNALING IN PULMONARY FIBROBLASTS EXPOSED TO PARTICULATE MATTER (PM) AND BRONCHOALVEOLAR LAVAGE FLUID (BALF) FROM HEALTHY AND HYPERTENSIVE RATS. 1P Zhang, UP Kodavanti. NHEERL, US EPA, Research Triangle Park, 1School of Vet Med, NCSU, Raleigh, NC
    Exposure to PM ma...

  2. Mapping the Hsp90 Genetic Network Reveals Ergosterol Biosynthesis and Phosphatidylinositol-4-Kinase Signaling as Core Circuitry Governing Cellular Stress

    PubMed Central

    O’Meara, Teresa R.; Valaei, Seyedeh Fereshteh; Diezmann, Stephanie; Cowen, Leah E.

    2016-01-01

    Candida albicans is a leading human fungal pathogen that causes life-threatening systemic infections. A key regulator of C. albicans stress response, drug resistance, morphogenesis, and virulence is the molecular chaperone Hsp90. Targeting Hsp90 provides a powerful strategy to treat fungal infections, however, the therapeutic utility of current inhibitors is compromised by toxicity due to inhibition of host Hsp90. To identify components of the Hsp90-dependent circuitry governing virulence and drug resistance that are sufficiently divergent for selective targeting in the pathogen, we pioneered chemical genomic profiling of the Hsp90 genetic network in C. albicans. Here, we screen mutant collections covering ~10% of the genome for hypersensitivity to Hsp90 inhibition in multiple environmental conditions. We identify 158 HSP90 chemical genetic interactors, most of which are important for growth only in specific environments. We discovered that the sterol C-22 desaturase gene ERG5 and the phosphatidylinositol-4-kinase (PI4K) gene STT4 are HSP90 genetic interactors under multiple conditions, suggesting a function upstream of Hsp90. By systematic analysis of the ergosterol biosynthetic cascade, we demonstrate that defects in ergosterol biosynthesis induce cellular stress that overwhelms Hsp90’s functional capacity. By analysis of the phosphatidylinositol pathway, we demonstrate that there is a genetic interaction between the PI4K Stt4 and Hsp90. We also establish that Stt4 is required for normal actin polarization through regulation of Wal1, and suggest a model in which defects in actin remodeling induces stress that creates a cellular demand for Hsp90 that exceeds its functional capacity. Consistent with this model, actin inhibitors are synergistic with Hsp90 inhibitors. We highlight new connections between Hsp90 and virulence traits, demonstrating that Erg5 and Stt4 enable activation of macrophage pyroptosis. This work uncovers novel circuitry regulating Hsp90

  3. Synergistic effect of vasoactive intestinal peptides on TNF-alpha-induced IL-6 synthesis in osteoblasts: amplification of p44/p42 MAP kinase activation.

    PubMed

    Natsume, Hideo; Tokuda, Haruhiko; Mizutani, Jun; Adachi, Seiji; Matsushima-Nishiwaki, Rie; Minamitani, Chiho; Kato, Kenji; Kozawa, Osamu; Otsuka, Takanobu

    2010-05-01

    We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of vasoactive intestinal peptide (VIP) on TNF-alpha-induced IL-6 synthesis in these cells. VIP, which by itself slightly stimulated IL-6 synthesis, synergistically enhanced the TNF-alpha-induced IL-6 synthesis in MC3T3-E1 cells. The synergistic effect of VIP on the TNF-alpha-induced IL-6 synthesis was concentration-dependent in the range between 1 and 70 nM. We previously reported that VIP stimulated cAMP production in MC3T3-E1 cells. Forskolin, a direct activator of adenylyl cyclase, or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP), a plasma membrane-permeable cAMP analogue, markedly enhanced the TNF-alpha-induced IL-6 synthesis as well as VIP. VIP markedly up-regulated the TNF-alpha-induced p44/p42 MAP kinase phosphorylation. The Akt phosphorylation stimulated by TNF-alpha was only slightly affected by VIP. PD98059, a specific inhibitor of MEK1/2, significantly suppressed the enhancement of TNF-alpha-induced IL-6 synthesis by VIP. The synergistic effect of a combination of VIP and TNF-alpha on the phosphorylation of p44/p42 MAP kinase was diminished by H-89, an inhibitor of cAMP-dependent protein kinase. These results strongly suggest that VIP synergistically enhances TNF-alpha-stimulated IL-6 synthesis via up-regulating p44/p42 MAP kinase through the adenylyl cyclase-cAMP system in osteoblasts.

  4. Combining Microinjection and Immunoblotting to Analyze MAP Kinase Phosphorylation in Single Starfish Oocytes and Eggs

    NASA Astrophysics Data System (ADS)

    Carroll, David J.; Hua, Wei

    The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 μm) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow for experiments testing the regulation of MAPK in single cells and for the manipulation of these cells by a quantitative microinjection technique.

  5. Wounding activates p38 map kinase and activation transcription factor 3 in leading keratinocytes.

    PubMed

    Harper, Erin G; Alvares, Stacy M; Carter, William G

    2005-08-01

    Quiescent epidermis anchors to laminin 5 in the basement membrane via integrin alpha6beta4. Wounding elevates expression of laminin 5, generating leading keratinocytes (LKs) that migrate via beta1 integrins. Laminin 5 was evaluated as a regulator of cell signaling, and mRNA and protein expression in LKs. An in vitro wound model was developed based on suspension and re-adhesion of quiescent human keratinocytes (HKs). DNA microarrays identified multiple mRNAs elevated 1.5 hours after suspension and re-adhesion including activation transcription factor 3 (ATF3). In vitro and in vivo, levels of ATF3 protein elevate in nuclei of LKs, but not in nuclei of the following cells, 2 hours after suspension or wounding but decline by 12-18 hours post injury. Significantly, null defects in laminin 5 or integrin beta4 that inhibit anchorage chronically elevate ATF3 in vivo. This suggests that adhesion to laminin 5, but not other ligands, suppresses activation. On suspension, ATF3 and other transcripts in the microarrays are elevated by phosphorylated p38 mitogen-activated protein kinase (P-p38), a stress kinase that regulates mRNA and cell motility. Inhibition of P-p38 with SB203580 prevents phosphorylation of ATF2, a transcription factor for ATF3 in LKs. Re-adhesion to laminin 5 via alpha6beta4 dephosphorylates P-p38 and suppresses ATF3 protein relative to cells in suspension. Thus, wounding of quiescent HKs disrupts laminin 5 adhesion to activate p38, generating mRNA transcripts that define LKs. Adhesion to deposits of laminin 5 via alpha6beta4 suppresses P-p38 and activation mRNAs including ATF3. Defects in laminin 5 and alpha6beta4 sustain P-p38 with probable pathological effects on transcription and migration.

  6. Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.

    PubMed

    Lopachev, Alexander V; Lopacheva, Olga M; Osipova, Ekaterina A; Vladychenskaya, Elizaveta A; Smolyaninova, Larisa V; Fedorova, Tatiana N; Koroleva, Olga V; Akkuratov, Evgeny E

    2016-07-01

    Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 μM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-μM and 10-μM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Molecular characterization of a potato MAP kinase transcriptionally regulated by multiple environmental stresses.

    PubMed

    Blanco, Flavio Antonio; Zanetti, María Eugenia; Casalongué, Claudia Anahí; Daleo, Gustavo Raúl

    2006-01-01

    The MAPK cascade is an evolutionary conserved signaling pathway that links external stimuli with cellular responses. Using polymerase chain reaction (PCR), a DNA fragment corresponding to a Solanum tuberosum MAPK, StMPK1, was isolated. StMPK1 amino acid sequence displayed over 90% identity with tomato MPK1 (LeMPK1) and tobacco SIPK. Southern blot analysis indicated that the gene encoding StMPK1 is present in a single copy in the potato genome. StMPK1 mRNA levels differentially accumulated in potato tuber in response to wounding and to wounding plus Fusarium solani f. sp. eumartii. Transcript accumulation after infection was transient and started earlier than what was observed in wounded tubers. StMPK1 mRNA levels also increased in potato tuber after 24 h of treatment with jasmonic acid (JA) and abscicic acid (ABA), but not in response to ethylene or salicylic acid. In addition, StMPK1 transcript levels increased after a heat-shock treatment at 42 degrees C. The results suggest that StMPK1 may participate in the cellular responses against multiple environmental stimuli in potato tubers.

  8. Differential expression profiles of poplar MAP kinase kinases in response to abiotic stresses and plant hormones, and overexpression of PtMKK4 improves the drought tolerance of poplar.

    PubMed

    Wang, Lei; Su, Hongyan; Han, Liya; Wang, Chuanqi; Sun, Yanlin; Liu, Fenghong

    2014-07-15

    Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play essential roles in plant growth, development and stress response. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), are integral in mediating various stress responses in plants. However, to date few data about the roles of poplar MAPKKs in stress signal transduction are available. In this study, we performed a systemic analysis of poplar MAPKK gene family expression profiles in response to several abiotic stresses and stress-associated hormones. Furthermore, Populus trichocarpa MAPKK4 (PtMKK4) was chosen for functional characterization. Transgenic analysis showed that overexpression of the PtMKK4 gene remarkably enhanced drought stress tolerance in the transgenic poplar plants. The PtMKK4-overexpressing plants also exhibited much lower levels of H2O2 and higher antioxidant enzyme activity after exposure to drought stress compared to the wide type lines. Besides, some drought marker genes including PtP5CS, PtSUS3, PtLTP3 and PtDREB8 exhibited higher expression levels in the transgenic lines than in the wide type under drought conditions. This study provided valuable information for understanding the putative functions of poplar MAPKKs involved in important signaling pathways under different stress conditions.

  9. Evaluate ERTS imagery for mapping and detection of changes of snowcover on land and on glaciers. [North Cascades, Washington and Tweedsmuir Glacier, Alaska

    NASA Technical Reports Server (NTRS)

    Meier, M. F. (Principal Investigator)

    1974-01-01

    The author has identified the following significant results. Snowlines on a small (6 sq km) drainage basin were accurately measured without use of digital processing, and snow patches as small as 150 m (maximum dimension) were correctly identified, proving that the resolution of ERTS is ample for snow mapping needs. The area of snow cover on 10 individual drainage basins in the North Cascades, Washington, has been determined at 12 different times; these data can be used for more accurate forecasts of streamflow. Progress has been made in distinguishing snow in trees using multispectral analysis. Motion of the surging Tweedsmuir Glacier was measured. Velocities ranged from 2 to 88 m per day; a zone of intense crevassing also appeared to spread up and down the glacier (at about 200 m per day upglacier). This tentative result may be of great importance to an understanding of surging glacier dynamics. ERTS images also show that the most recent debris flow (20-21 August 1973) from Mount Baker can be clearly discerned and mapped, in order to monitor this potential hazard.

  10. A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission.

    PubMed

    Bardwell, A J; Flatauer, L J; Matsukuma, K; Thorner, J; Bardwell, L

    2001-03-30

    The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo. PMID:11134045

  11. ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes.

    PubMed

    Ellederová, Zdenka; Cais, Ondrej; Susor, Andrej; Uhlírová, Katka; Kovárová, Hana; Jelínková, Lucie; Tomek, Wolfgang; Kubelka, Michal

    2008-02-01

    Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.

  12. Phosphorylation of trihelix transcriptional repressor ASR3 by MAP KINASE4 negatively regulates Arabidopsis immunity.

    PubMed

    Li, Bo; Jiang, Shan; Yu, Xiao; Cheng, Cheng; Chen, Sixue; Cheng, Yanbing; Yuan, Joshua S; Jiang, Daohong; He, Ping; Shan, Libo

    2015-03-01

    Proper control of immune-related gene expression is crucial for the host to launch an effective defense response. Perception of microbe-associated molecular patterns (MAMPs) induces rapid and profound transcriptional reprogramming via unclear mechanisms. Here, we show that ASR3 (ARABIDOPSIS SH4-RELATED3) functions as a transcriptional repressor and plays a negative role in regulating pattern-triggered immunity (PTI) in Arabidopsis thaliana. ASR3 belongs to a plant-specific trihelix transcription factor family for which functional studies are lacking. MAMP treatments induce rapid phosphorylation of ASR3 at threonine 189 via MPK4, a mitogen-activated protein kinase that negatively regulates PTI responses downstream of multiple MAMP receptors. ASR3 possesses transcriptional repressor activity via its ERF-associated amphiphilic repression motifs and negatively regulates a large subset of flg22-induced genes. Phosphorylation of ASR3 by MPK4 enhances its DNA binding activity to suppress gene expression. Importantly, the asr3 mutant shows enhanced disease resistance to virulent bacterial pathogen infection, whereas transgenic plants overexpressing the wild-type or phospho-mimetic form of ASR3 exhibit compromised PTI responses. Our studies reveal a function of the trihelix transcription factors in plant innate immunity and provide evidence that ASR3 functions as a transcriptional repressor regulated by MAMP-activated MPK4 to fine-tune plant immune gene expression.

  13. Opposing actions of TGF{beta} and MAP kinase signaling in undifferentiated hen granulosa cells

    SciTech Connect

    Woods, Dori C.; Haugen, Morgan J.; Johnson, A.L. . E-mail: johnson.128@nd.edu

    2005-10-21

    The present studies were conducted to establish interactions between transforming growth factor (TGF)-{beta} and the epidermal growth factor (EGF) family members, TGF{alpha} and betacellulin (BTC), relative to proliferation and differentiation of granulosa cells in hen ovarian follicles. Results presented demonstrate expression of TGF{beta} isoforms, plus TGF{alpha}, BTC, and ErbB receptors in prehierarchal follicles, thus establishing the potential for autocrine/paracrine signaling and cross-talk within granulosa cells at the onset of differentiation. Treatment with TGF{alpha} or BTC increases levels of TGF{beta}1 mRNA in undifferentiated granulosa cells, while the selective inhibitor of mitogen activated protein kinase signaling, U0126, reverses these effects. Moreover, TGF{beta}1 attenuates c-myc mRNA expression and granulosa cell proliferation, while TGF{alpha} blocks both these inhibitory effects. Collectively, these data provide evidence that EGF family ligands regulate both the expression and biological actions of TGF{beta}1 in hen granulosa cells, and indicate that the timely interaction of these opposing factors is an important modulator of both granulosa cell proliferation and differentiation.

  14. MAP kinase subcellular localization controls both pattern and proliferation in the developing Drosophila wing

    PubMed Central

    Marenda, Daniel R.; Vrailas, Alysia D.; Rodrigues, Aloma B.; Cook, Summer; Powers, Maureen A.; Lorenzen, James A.; Perkins, Lizabeth A.; Moses, Kevin

    2007-01-01

    Mitogen-activated protein kinases (MAPKs) phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK phosphorylation was always followed by rapid nuclear translocation. However, we and others have found that MAPK phosphorylation is not always sufficient for nuclear translocation in vivo. In the developing Drosophila wing, MAPK-mediated signaling is required both for patterning and for cell proliferation, although the mechanism of this differential control is not fully understood. Here, we show that phosphorylated MAPK (pMAPK) is held in the cytoplasm in differentiating larval and pupal wing vein cells, and we show that this cytoplasmic hold is required for vein cell fate. At the same time, we show that MAPK does move into the nucleus of other wing cells where it promotes cell proliferation. We propose a novel Ras pathway bifurcation in Drosophila and our results suggest a mechanism by which MAPK phosphorylation can signal two different cellular outcomes (differentiation versus proliferation) based on the subcellular localization of MAPK. PMID:16308331

  15. Distinct and combined roles of the MAP kinases of Cochliobolus heterostrophus in virulence and stress responses.

    PubMed

    Igbaria, Aeid; Lev, Sophie; Rose, Mark S; Lee, Bee Na; Hadar, Ruthi; Degani, Ofir; Horwitz, Benjamin A

    2008-06-01

    Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essential for virulence in fungi, including Cochliobolus heterostrophus, a necrotrophic pathogen causing Southern corn leaf blight. We compared the phenotypes of mutants in three MAPK genes: HOG1, MPS1, and CHK1. The chk1 and mps1 mutants show autolytic appearance, light pigmentation, and dramatic reduction in virulence and conidiation. Similarity of mps1 and chk1 mutants is reflected by coregulation by these two MAPKs of several genes. Unlike chk1, mps1 mutants are female-fertile and form normal-looking appressoria. HOG1 mediates resistance to hyperosmotic and, to a lesser extent, oxidative stress, and is required for stress upregulation of glycerol-3-phosphate phosphatase, transaldolase, and a monosaccharide transporter. Hog1, but not Mps1 or Chk1, was rapidly phosphorylated in response to increased osmolarity. The hog1 mutants have smaller appressoria and cause decreased disease symptoms on maize leaves. Surprisingly, loss of MPS1 in a wild-type or hog1 background improved resistance to some stresses. All three MAPKs contribute to the regulation of central developmental functions under normal and stress conditions, and full virulence cannot be achieved without appropriate input from all three pathways. PMID:18473669

  16. Therapeutic potential of p38 MAP kinase inhibition in the management of cardiovascular disease.

    PubMed

    Fisk, Marie; Gajendragadkar, Parag R; Mäki-Petäjä, Kaisa M; Wilkinson, Ian B; Cheriyan, Joseph

    2014-06-01

    p38 mitogen-activated protein kinases (p38 MAPKs) are key signalling molecules that regulate cellular behavior in response to environmental stresses. They regulate pro-inflammatory cytokines and therefore p38 MAPKs are implicated in the pathogenesis of many inflammatory-driven conditions, including atherosclerosis. Therapeutic inhibition of p38 MAPKs to attenuate inflammation has been the focus of comprehensive research in the last 2 decades, following the discovery of p38α as the molecular target of pyrindinyl imidazole compounds, which suppress the cytokines tumor necrosis factor-α and interleukin-1. The potential of p38 MAPK inhibitors was initially explored within archetypal inflammatory conditions such as rheumatoid arthritis and Crohn's disease, but early studies demonstrated poor clinical efficacy and unacceptable side effects. Subsequent clinical trials evaluating different p38 MAPK inhibitor compounds in disease models such as chronic obstructive pulmonary disease (COPD) and atherosclerosis have shown potential clinical efficacy. This review aims to provide succinct background information regarding the p38 MAPK signaling pathway, a focus of p38 MAPKs in disease, and a brief summary of relevant pre-clinical studies. An update of human clinical trial experience encompassing a clinically orientated approach, dedicated to cardiovascular disease follows. It provides a current perspective of the therapeutic potential of p38 MAPK inhibitors in the cardiovascular domain, including safety, tolerability, and pharmacokinetics.

  17. The wheat MAP kinase phosphatase 1 alleviates salt stress and increases antioxidant activities in Arabidopsis.

    PubMed

    Zaidi, Ikram; Ebel, Chantal; Belgaroui, Nibras; Ghorbel, Mouna; Amara, Imène; Hanin, Moez

    2016-04-01

    Mitogen-activated protein kinase phosphatases (MKPs) are important negative regulators in the MAPK signaling pathways, which play crucial roles in plant growth, development and stress responses. We have previously shown that the heterologous expression of a durum wheat MKP, TMKP1, results in increased tolerance to salt stress in yeast but its particular contribution in salt stress tolerance in plants was not investigated. Here, TMKP1 was overexpressed in Arabidopsis thaliana and physiological changes were assessed in transgenic plants exposed to stress conditions. Under salt stress and especially LiCl, the TMKP1 overexpressors displayed higher germination rates in comparison to wild type plants. The enhancement of salt stress tolerance was accompanied by increased antioxidant enzyme activities, namely superoxide dismutase, catalase and peroxydases. Such increases in antioxidant activities were concomitant with lower malondialdehyde, superoxide anion O2(-) and hydrogen peroxide levels in the TMKP1 transgenic seedlings. Moreover, we provide evidence that, in contrast to the Arabidopsis ortholog AtMKP1, TMKP1 acts as a positive regulator of salt stress tolerance via its ectopic expression in the Arabidopsis mkp1 mutant. PMID:26927025

  18. Network Modeling Reveals Cross Talk of MAP Kinases during Adaptation to Caspofungin Stress in Aspergillus fumigatus

    PubMed Central

    Baldin, Clara; Weber, Jakob; Guthke, Reinhard; Kniemeyer, Olaf; Brakhage, Axel A.; Linde, Jörg

    2015-01-01

    Mitogen activated protein kinases (MAPKs) are highly conserved in eukaryotic organisms. In pathogenic fungi, their activities were assigned to different physiological functions including drug adaptation and resistance. Aspergillus fumigatus is a human pathogenic fungus, which causes life-threatening invasive infections. Therapeutic options against invasive mycoses are still limited. One of the clinically used drugs is caspofungin, which specifically targets the fungal cell wall biosynthesis. A systems biology approach, based on comprehensive transcriptome data sets and mathematical modeling, was employed to infer a regulatory network and identify key interactions during adaptation to caspofungin stress in A. fumigatus. Mathematical modeling and experimental validations confirmed an intimate cross talk occurring between the cell wall-integrity and the high osmolarity-glycerol signaling pathways. Specifically, increased concentrations of caspofungin promoted activation of these signalings. Moreover, caspofungin affected the intracellular transport, which caused an additional osmotic stress that is independent of glucan inhibition. High concentrations of caspofungin reduced this osmotic stress, and thus decreased its toxic activity. Our results demonstrated that MAPK signaling pathways play a key role during caspofungin adaptation and are contributing to the paradoxical effect exerted by this drug. PMID:26356475

  19. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

    PubMed Central

    Gell, D; Jackson, S P

    1999-01-01

    In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80. PMID:10446239

  20. MAP kinase phosphatase 1 harbors a novel PTS1 and is targeted to peroxisomes following stress treatments.

    PubMed

    Kataya, Amr R A; Schei, Edit; Lillo, Cathrine

    2015-05-01

    In Arabidopsis thaliana, twenty mitogen-activated protein kinases (MAPKs/MPKs) are regulated by five MAP kinase phosphatases (MKPs). Arabidopsis MKP1 has an important role in biotic, abiotic and genotoxic stresses and has been shown to interact with and negatively regulate specifically MPK3 and MPK6. MKP1 has been reported to have a role in negative regulation of reactive oxygen species (ROS) and salicylic acid (SA) production. As essential organelles involved in production of ROS and SA, peroxisomes could possibly be an important compartment for MKP1 activity, however MKP1 was previously reported to be cytosolic. By screening Arabidopsis protein phosphatases for peroxisomal targeting signal 1 (PTS1), we identified MKP1 as a putative peroxisomal protein. Arabidopsis MKP1 was found to harbor a non-canonical PTS1-like tripeptide (Ser-Ala-Leu>) that is conserved in MKP1 orthologs. We show experimentally that the C-terminal Ser-Ala-Leu> can function as a novel PTS1, and alanine in position -2, adds more relaxation to the plant PTS1 motif. The full-length MKP1 remained in the cytosol when transiently expressed in Arabidopsis mesophyll protoplasts under standard conditions. When different biotic and abiotic stresses were applied to mesophyll protoplasts, the full length protein changed its targeting to unidentified organelle-like structures that subsequently fused with peroxisomes. Our results identify MKP1 as a protein dually targeted to cytosol and peroxisomes. The finding that MKP1 targets peroxisomes by a non-canonical PTS1 under stressful conditions highlights the complexity of peroxisomal targeting mechanism.

  1. Overlapping functions of the MAP4K family kinases Hppy and Msn in Hippo signaling

    PubMed Central

    Li, Shuangxi; Cho, Yong Suk; Yue, Tao; Ip, Y Tony; Jiang, Jin

    2015-01-01

    The Hippo (Hpo) tumor suppressor pathway is an evolutionarily conserved signaling pathway that controls tissue growth and organ size in species ranging from Drosophila to human, and its malfunction has been implicated in many types of human cancer. In this study, we conducted a kinome screen and identified Happyhour (Hppy)/MAP4K3 as a novel player in the Hpo pathway. Our biochemical study showed that Hppy binds and phosphorylates Wts. Our genetic experiments suggest that Hppy acts in parallel and partial redundantly with Misshapen (Msn)/MAP4K4 to regulate Yki nuclear localization and Hpo target gene expression in Drosophila wing imaginal discs. Furthermore, we showed that cytoskeleton stress restricts Yki nuclear localization through Hppy and Msn when Hpo activity is compromised, thus providing an explanation for the Wts-dependent but Hpo-independent regulation of Yki in certain contexts. Our study has unraveled an additional layer of complexity in the Hpo signaling pathway and laid down a foundation for exploring how different upstream regulators feed into the core Hpo pathway. PMID:27462435

  2. Role played by Disabled-2 in albumin induced MAP Kinase signalling

    SciTech Connect

    Diwakar, Ramaswamy Pearson, Alexander L.; Colville-Nash, Paul; Baines, Deborah L.; Dockrell, Mark E.C.

    2008-02-15

    Albumin has been shown to activate the mitogen activated protein kinase (MAPK) pathway in proximal tubular cells (PTECs) of the kidney. Megalin, the putative receptor for albumin has potential signalling properties. However, the mechanisms by which megalin signals are unclear. The adaptor phosphoprotein Disabled-2 (Dab2) is known to interact with the cytoplasmic tail of megalin and may be involved in albumin-mediated MAPK signalling. In this study, we investigated the role of Dab2 in albumin-mediated MAPK signalling and further studied the role of Dab2 in albumin-induced TGF{beta}-1 secretion, a MAPK dependent event. We used RNA interference to knockdown Dab2 protein abundance in HKC-8 cells a model of human PTECs. Albumin activated ERK1,2 and Elk-1 in a MEK-1 dependent manner and resulted in secretion of TGF{beta}-1. In the absence of albumin, knockdown of Dab2 resulted in a trend towards increase in pERK1,2 consistent with its putative role as an inhibitor of cell proliferation. However albumin-induced ERK1,2 activation was completely abolished by Dab2 knockdown. Dab2 knockdown did not however result in inhibition of albumin-induced TGF{beta}-1 secretion. These results suggest that Dab2 is a ligand dependent bi-directional regulator of ERK1,2 activity by demonstrating that in addition to its more traditional role as an inhibitor of ERK1,2 it may also activate ERK1,2.

  3. The Complexity of the ERK/MAP-Kinase Pathway and the Treatment of Melanoma Skin Cancer

    PubMed Central

    Wellbrock, Claudia; Arozarena, Imanol

    2016-01-01

    The central role played by the ERK/MAPK pathway downstream of RAS in human neoplasias is best exemplified in the context of melanoma skin cancer. Signaling through the MAPK pathway is crucial for the proliferation of melanocytes, the healthy pigment cells that give rise to melanoma. However, hyper-activation of the MAPK-pathway is found in over 90% of melanomas with approximately 50% of all patients displaying mutations in the kinase BRAF, and approximately 28% of all patients harboring mutations in the MAPK-pathway up-stream regulator NRAS. This finding has led to the development of BRAF and MEK inhibitors whose application in the clinic has shown unprecedented survival responses. Unfortunately the responses to MAPK pathway inhibitors are transient with most patients progressing within a year and a median progression free survival of 7–10 months. The disease progression is due to the development of drug-resistance based on various mechanisms, many of them involving a rewiring of the MAPK pathway. In this article we will review the complexity of MAPK signaling in melanocytic cells as well as the mechanisms of action of different MAPK-pathway inhibitors and their correlation with clinical response. We will reflect on mechanisms of innate and acquired resistance that limit patient's response, with a focus on the MAPK signaling network. Because of the resurgence of antibody-based immune-therapies there is a growing feeling of failure in the targeted therapy camp. However, recent studies have revealed new windows of therapeutic opportunity for melanoma sufferers treated with drugs targeting the MAPK pathway, and these opportunities will be discussed. PMID:27200346

  4. Topical alpha-selective p38 MAP kinase inhibition reduces acute skin inflammation in guinea pig

    PubMed Central

    Medicherla, Satyanarayana; Ma, Jing Ying; Reddy, Mamtha; Esikova, Irina; Kerr, Irene; Movius, Fabiola; Higgins, Linda S; Protter, Andrew A

    2010-01-01

    Certain skin pathologies, including psoriasis, are thought to be immune-mediated inflammatory diseases. Available literature clearly indicates the involvement of inflammatory cells (neutrophils, T cells, and macrophages), their cytokines, and the p38 mitogen-activated protein kinase (MAPK) signaling pathway in the pathophysiology of psoriasis. Neutrophils play an important role in the formation of acute inflammatory changes in psoriasis. Acute inflammation or acute flares in psoriasis remain poorly addressed in clinical medicine. In this communication, we first establish a simple and reproducible model for studying neutrophil-mediated acute skin inflammation. Using the hairless guinea pig, due to the similarity of skin architecture to that of human, acute inflammation was induced with an intradermal injection of 50 μg/mL lipopolysaccharide (LPS) in 50 μL solution. Myeloperoxidase (MPO) activity was measured by MPO-positive neutrophils and shown to increase for 24-hours post-injection. Simultaneously, the level of phosphorylated p38 MAPK was documented for 48-hours post-LPS injection in the skin. Next, we used this model to examine the therapeutic potential of an α-selective p38 MAPK inhibitor, SCIO-469. A comparison of topical application of SCIO-469 at 5 mg/mL or 15 mg/mL to vehicle revealed that SCIO-469 dose-dependently reduces acute skin inflammation and that this effect is statistically significant at the higher dose. Further examination of tissues that received this dose also revealed statistically significant reduction of MPO activity, phosphorylated p38 MAPK, interleukin-6, and cyclooxygenase-2. These data suggest that the α-selective p38 MAPK inhibitor, SCIO-469, acts as a topical anti-inflammatory agent via the p38 MAPK pathway to reduce neutrophil induced acute inflammation in the skin. These observations suggest that α-selective p38 MAPK inhibition may be an effective therapeutic strategy to manage acute skin inflammation PMID:22096353

  5. Mitochondrial DNA is released by shock and activates neutrophils via p38 map kinase.

    PubMed

    Zhang, Qin; Itagaki, Kiyoshi; Hauser, Carl J

    2010-07-01

    Bacterial DNA (bDNA) can activate an innate-immune stimulatory "danger" response via toll-like receptor 9 (TLR9). Mitochondrial DNA (mtDNA) is unique among endogenous molecules in that mitochondria evolved from prokaryotic ancestors. Thus, mtDNA retains molecular motifs similar to bDNA. It is unknown, however, whether mtDNA is released by shock or is capable of eliciting immune responses like bDNA. We hypothesized shock-injured tissues might release mtDNA and that mtDNA might act as a danger-associated molecular pattern (or "alarmin") that can activate neutrophils (PMNs) and contribute to systemic inflammatory response syndrome. Standardized trauma/hemorrhagic shock caused circulation of mtDNA as well as nuclear DNA. Human PMNs were incubated in vitro with purified mtDNA or nuclear DNA, with or without pretreatment by chloroquine (an inhibitor of endosomal receptors like TLR9). Neutrophil activation was assessed as matrix metalloproteinase (MMP) 8 and MMP-9 release as well as p38 and p44/42 mitogen-activated protein kinase (MAPK) phosphorylation. Mitochondrial DNA induced PMN MMP-8/MMP-9 release and p38 phosphorylation but did not activate p44/42. Responses were inhibited by chloroquine. Nuclear DNA did not induce PMN activation. Intravenous injection of disrupted mitochondria (mitochondrial debris) into rats induced p38 MAPK activation and IL-6 and TNF-alpha accumulation in the liver. In summary, mtDNA is released into the circulation by shock. Mitochondrial DNA activates PMN p38 MAPK, probably via TLR9, inducing an inflammatory phenotype. Mitochondrial DNA may act as a danger-associated molecular pattern or alarmin after shock, contributing to the initiation of systemic inflammatory response syndrome.

  6. Effect of selective inhibition of the p38 MAP kinase pathway on platelet aggregation.

    PubMed

    Kuliopulos, Athan; Mohanlal, Ramon; Covic, Lidija

    2004-12-01

    Systemic inflammation has been shown to be a contributing factor to the instability of atherosclerotic plaques in patients with acute coronary syndromes (ACS). VX-702, a novel p38 mitogen-activated protein kinase (MAPK) inhibitor, is currently under investigation in ACS patients with unstable angina to evaluate its safety and efficacy during percutaneous coronary intervention (PCI). The role of p38 MAPK in platelet aggregation of normal individuals was examined using the selective second generation p38 MAPK inhibitor VX-702. Treatment of platelets with thrombin (activates PAR1 and PAR4 thrombin receptors), SFLLRN (PAR1), AYPGKF (PAR4), collagen (alpha2beta1 and GPVI/FCgammaIIR receptors) and U46619 (TXA(2)) resulted in strong activation of p38 MAPK. Activation of the GPIb von Willebrand factor receptor with ristocetin did not stimulate p38 MAPK. Pre-treatment of platelets with 1 microM VX-702 completely inhibited activation of p38 MAPK by thrombin, SFLLRN, AYPGKF, U46619, and collagen. There was no effect of VX-702 on platelet aggregation induced by any of the agonists in the presence or absence of aspirin, heparin or apyrase. It has been postulated that a potential role of p38 MAPK is to activate phospholipase A(2) (cPLA(2)) which catalyses formation of arachidonic acid leading to production of thromboxane. Interestingly, we show contrasting effects of p38 MAPK inhibition as compared to aspirin inhibition on platelet aggregation in response to collagen. Blockade of TXA(2) production by aspirin results in significant inhibition of collagen activation. However,VX-702 has no effect on collagen-mediated platelet aggregation, suggesting that blocking p38 MAPK does not effect thromboxane production in human platelets. Therefore, unlike aspirin blockade of thromboxane production in platelets, p38 MAPK inhibitors such as VX-702 do not significantly affect platelet function and would not be expected to contribute to an elevated risk of bleeding side-effects in treated

  7. Induction of MAP kinase phosphatase 3 through Erk/MAP kinase activation in three oncogenic Ras (H-, K- and N-Ras)-expressing NIH/3T3 mouse embryonic fibroblast cell lines

    PubMed Central

    Koo, JaeHyung; Wang, Sen; Kang, NaNa; Hur, Sun Jin; Bahk, Young Yil

    2016-01-01

    Ras oncoproteins are small molecular weight GTPases known for their involvement in oncogenesis, which operate in a complex signaling network with multiple effectors. Approximately 25% of human tumors possess mutations in a member of this family. The Raf1/MEK/Erk1/2 pathway is one of the most intensively studied signaling mechanisms. Different levels of regulation account for the inactivation of MAP kinases by MAPK phosphatases in a cell type- and stimuli-dependent manner. In the present study, using three inducible Ras-expressing NIH/3T3 cell lines, we demonstrated that MKP3 upregulation requires the activation of the Erk1/2 pathway, which correlates with the shutdown of this pathway. We also demonstrated, by applying pharmacological inhibitors and effector mutants of Ras, that induction of MKP3 at the protein level is positively regulated by the oncogenic Ras/Raf/MEK/Erk1/2 signaling pathway. [BMB Reports 2016; 49(7): 370-375] PMID:26818088

  8. Phospholipase C{gamma}1 stimulates transcriptional activation of the matrix metalloproteinase-3 gene via the protein kinase C/Raf/ERK cascade

    SciTech Connect

    Shin, Soon Young; Choi, Ha Young; Ahn, Bong-Hyun; Son, Sang Wook; Lee, Young Han . E-mail: younghan@hanyang.ac.kr

    2007-02-16

    The phospholipid hydrolase phospholipase C{gamma}1 (PLC{gamma}1) plays a major role in regulation of cell proliferation, development, and cell motility. Overexpression of PLC{gamma}1 is associated with tumor development, and it is overexpressed in some tumors. Matrix metalloproteinase-3 (MMP-3) is a protein involved in tumor invasion and metastasis. Here, we demonstrate that overexpression of PLC{gamma}1 stimulates MMP-3 expression at the transcriptional level via the PKC-mediated Raf/MEK1/ERK signaling cascade. We propose that modulation of PLC{gamma}1 activity might be of value in controlling the activity of MMPs, which are important regulators of invasion and metastasis in malignant tumors.

  9. An Atlas of the Human Kinome Reveals the Mutational Landscape Underlying Dysregulated Phosphorylation Cascades in Cancer.

    PubMed

    Olow, Aleksandra; Chen, Zhongzhong; Niedner, R Hannes; Wolf, Denise M; Yau, Christina; Pankov, Aleksandr; Lee, Evelyn Pei Rong; Brown-Swigart, Lamorna; van 't Veer, Laura J; Coppé, Jean-Philippe

    2016-04-01

    Kinase inhibitors are used widely to treat various cancers, but adaptive reprogramming of kinase cascades and activation of feedback loop mechanisms often contribute to therapeutic resistance. Determining comprehensive, accurate maps of kinase circuits may therefore help elucidate mechanisms of response and resistance to kinase inhibitor therapies. In this study, we identified and validated phosphorylatable target sites across human cell and tissue types to generate PhosphoAtlas, a map of 1,733 functionally interconnected proteins comprising the human phospho-reactome. A systematic curation approach was used to distill protein phosphorylation data cross-referenced from 38 public resources. We demonstrated how a catalog of 2,617 stringently verified heptameric peptide regions at the catalytic interface of kinases and substrates could expose mutations that recurrently perturb specific phospho-hubs. In silico mapping of 2,896 nonsynonymous tumor variants identified from thousands of tumor tissues also revealed that normal and aberrant catalytic interactions co-occur frequently, showing how tumors systematically hijack, as well as spare, particular subnetworks. Overall, our work provides an important new resource for interrogating the human tumor kinome to strategically identify therapeutically actionable kinase networks that drive tumorigenesis. Cancer Res; 76(7); 1733-45. ©2016 AACR. PMID:26921330

  10. Evaluate ERTS imagery for mapping and detection of changes of snowcover on land and on glaciers. [Cascade Mountains

    NASA Technical Reports Server (NTRS)

    Meier, M. F. (Principal Investigator)

    1974-01-01

    The author has identified the following significant results. Snowlines on a small drainage basin were accurately identified on bulk ERTS-1 images without use of digital processing, and results checked with high altitude and ground-based photography. The area and approximate shape of snow patches as small as 20,000 sq m could be correctly identified with a magnifying scanning densitometer. The resolution of ERTS is more than ample for most snow mapping needs. Mount Baker, Washington, has a large crater south of the summit and an area north of the summit which emit considerable geothermal heat in the form of fumaroles and hot ground. Temperatures are being monitored using an ERTS DCS. Debris flows are occassionally released from the crater due to water saturation at the base of a heavy snowpack lying on hydrothermally altered hot ground. These debris flows present a possible hazard to life and property, as they are discharged down the Boulder Glacier toward Baker Lake, the upper of two major hydroelectric power reservoirs which are situated above the populated Skagit River Valley. ERTS-1 images show that the most recent debris flow (20-21 August 1973) can be clearly discerned and mapped. ERTS images provide another important tool for monitoring this potential hazard.

  11. Arsenic induces apoptosis in rat cerebellar neurons via activation of JNK3 and p38 MAP kinases.

    PubMed

    Namgung, U; Xia, Z

    2001-07-15

    Primary cultures of rat cerebellar neurons were used to study mechanisms of arsenic neurotoxicity. Exposure to 5, 10, or 15 microM sodium arsenite reduced cerebellar neuron viability and induced nuclear fragmentation and condensation as well as DNA degradation to oligonucleosome fragments. Exposure to 1 or 5 mM dimethylarsinic acid caused similar changes. Therefore, both inorganic arsenite and organic dimethylarsinic acid induce apoptosis in cerebellar neurons, with the inorganic form being more toxic. Cotreatment with cycloheximide or actinomycin D, inhibitors of protein or RNA synthesis, respectively, or with the caspase inhibitor zVAD, completely blocked arsenite-induced cerebellar neuron apoptosis. This implies that arsenite-induced cerebellar neuron apoptosis requires new gene expression and caspase activation. Interestingly, sodium arsenite selectively activated p38 and JNK3, but not JNK1 or JNK2 in cerebellar neurons. Blocking the p38 or JNK signaling pathways using the inhibitors SB203580 or CEP-1347 protected cerebellar neurons against arsenite-induced apoptosis. These data suggest that arsenite neurotoxicity may be due to apoptosis caused by activation of p38 and JNK3 MAP kinases.

  12. TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.

    PubMed

    Borghi, Alice; Verstrepen, Lynn; Beyaert, Rudi

    2016-09-15

    Tumor Necrosis Factor (TNF) is a potent inflammatory cytokine that exerts its functions through the activation of two distinct receptors, TNFR1 and TNFR2. Both receptors can activate canonical NF-κB and JNK MAP kinase signaling, while TNFR2 can also activate non-canonical NF-κB signaling, leading to numerous changes in gene expression that drive inflammation, cell proliferation and cell survival. On the other hand, TNFR1 also activates signaling pathways leading to cell death by either apoptosis or necroptosis, depending on the cellular context. A key player in TNFR1- and TNFR2-induced signaling is the RING finger protein TRAF2, which is recruited to both receptors upon their stimulation. TRAF2 exerts multiple receptor-specific functions but also mediates cross-talk between TNFR1 and TNFR2, dictating the outcome of TNF stimulation. In this review, we provide an overview of the positive and negative regulatory role of TRAF2 in different TNFR1 and TNFR2 signaling pathways. We discuss the underlying molecular mechanism of action, distinguishing between TRAF2 scaffold and E3 ubiquitin ligase functions, and the regulation of TRAF2 by specific post-translational modifications. Finally, we elaborate on some possible strategies to modulate TRAF2 function in the context of therapeutic targeting in autoimmunity and cancer.

  13. Conditional deletion of ERK5 MAP kinase in the nervous system impairs pheromone information processing and pheromone-evoked behaviors.

    PubMed

    Zou, Junhui; Storm, Daniel R; Xia, Zhengui

    2013-01-01

    ERK5 MAP kinase is highly expressed in the developing nervous system but absent in most regions of the adult brain. It has been implicated in regulating the development of the main olfactory bulb and in odor discrimination. However, whether it plays an essential role in pheromone-based behavior has not been established. Here we report that conditional deletion of the Mapk7 gene which encodes ERK5 in mice in neural stem cells impairs several pheromone-mediated behaviors including aggression and mating in male mice. These deficits were not caused by a reduction in the level of testosterone, by physical immobility, by heightened fear or anxiety, or by depression. Using mouse urine as a natural pheromone-containing solution, we provide evidence that the behavior impairment was associated with defects in the detection of closely related pheromones as well as with changes in their innate preference for pheromones related to sexual and reproductive activities. We conclude that expression of ERK5 during development is critical for pheromone response and associated animal behavior in adult mice.

  14. Enteropathogenic Escherichia coli outer membrane proteins induce iNOS by activation of NF-kappaB and MAP kinases.

    PubMed

    Malladi, Vasantha; Puthenedam, Manjula; Williams, Peter H; Balakrishnan, Arun

    2004-12-01

    Enteropathogenic Escherichia coli (EPEC) infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. Nitric oxide (NO) is an important modulator of intestinal inflammatory response. The aim of the present study was to investigate whether EPEC outer membrane proteins (OMPs) up regulate epithelial cell expression of inducible nitric oxide synthase (iNOS) and to examine the role of NF-kappaB and MAP kinases (MAPK) on nitrite production. iNOS mRNA expression was assessed by RT-PCR. Nitrite levels were measured by Griess reaction. NF-kappaB activation by OMPs was evaluated by EMSA and immunoblotting was done to detect MAPK activation. EPEC OMP up regulated iNOS, induced nitrite production and NF-kappaB and MAPK were activated in caco-2 cells. The nitrite levels decreased when NF-kappaB and MAPK inhibitors were used. Thus, EPEC OMPs induce iNOS expression and NO production through activation of NF-kappaB and MAPK.

  15. Genetic activation of ERK5 MAP kinase enhances adult neurogenesis and extends hippocampus-dependent long-term memory.

    PubMed

    Wang, Wenbin; Pan, Yung-Wei; Zou, Junhui; Li, Tan; Abel, Glen M; Palmiter, Richard D; Storm, Daniel R; Xia, Zhengui

    2014-02-01

    Recent studies have shown that inhibition of adult neurogenesis impairs the formation of hippocampus-dependent memory. However, it is not known whether increasing adult neurogenesis affects the persistence of hippocampus-dependent long-term memory. Furthermore, signaling mechanisms that regulate adult neurogenesis are not fully defined. We recently reported that the conditional and targeted knock-out of ERK5 MAP kinase in adult neurogenic regions of the mouse brain attenuates adult neurogenesis in the hippocampus and disrupts several forms of hippocampus-dependent memory. Here, we developed a gain-of-function knock-in mouse model to specifically activate endogenous ERK5 in the neurogenic regions of the adult brain. We report that the selective and targeted activation of ERK5 increases adult neurogenesis in the dentate gyrus by enhancing cell survival, neuronal differentiation, and dendritic complexity. Conditional ERK5 activation also improves the performance of challenging forms of spatial learning and memory and extends hippocampus-dependent long-term memory. We conclude that enhancing signal transduction of a single signaling pathway within adult neural stem/progenitor cells is sufficient to increase adult neurogenesis and improve the persistence of hippocampus-dependent memory. Furthermore, activation of ERK5 may provide a novel therapeutic target to improve long-term memory.

  16. Regulating Global Sumoylation by a MAP Kinase Hog1 and Its Potential Role in Osmo-Tolerance in Yeast

    PubMed Central

    Panahi, Mahmoud; Zhu, Ming; Wang, Yuqi

    2014-01-01

    Sumoylation, a post-translational protein modification by small ubiquitin-like modifier (SUMO), has been implicated in many stress responses. Here we analyzed the potential role of sumoylation in osmo-response in yeast. We find that osmotic stress induces rapid accumulation of sumoylated species in normal yeast cells. Interestingly, disruption of MAP kinase Hog1 leads to a much higher level of accumulation of sumoylated conjugates that are independent of new protein synthesis. We also find that the accumulation of sumoylated species is dependent on a SUMO ligase Siz1. Notably, overexpression of SIZ1 in HOG1-disruption mutants (hog1Δ) but not in wild type cells leads to a markedly increased and prolonged accumulation of sumoylated species. Examination of osmo-tolerance of yeast mutants that display either an increase or a decrease in the global sumoylation level revealed an inverse relationship between accumulation of sumoylated conjugates and osmo-tolerance. Further investigation has shown that many of the sumoylated species induced by hyperosmotic stress are actually poly-sumoylated. Together, these findings indicate that abnormal accumulation of poly-sumoylated conjugates is harmful for osmo-tolerance in yeast, and suggest that Hog1 promotes adaptation to hyperosmotic stress partially via regulation of global sumoylation level. PMID:24498309

  17. Rck1 up-regulates pseudohyphal growth by activating the Ras2 and MAP kinase pathways independently in Saccharomyces cerevisiae.

    PubMed

    Chang, Miwha; Kang, Chang-Min; Park, Yong-Sung; Yun, Cheol-Won

    2014-02-21

    Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD+1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae. PMID:24491552

  18. TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.

    PubMed

    Borghi, Alice; Verstrepen, Lynn; Beyaert, Rudi

    2016-09-15

    Tumor Necrosis Factor (TNF) is a potent inflammatory cytokine that exerts its functions through the activation of two distinct receptors, TNFR1 and TNFR2. Both receptors can activate canonical NF-κB and JNK MAP kinase signaling, while TNFR2 can also activate non-canonical NF-κB signaling, leading to numerous changes in gene expression that drive inflammation, cell proliferation and cell survival. On the other hand, TNFR1 also activates signaling pathways leading to cell death by either apoptosis or necroptosis, depending on the cellular context. A key player in TNFR1- and TNFR2-induced signaling is the RING finger protein TRAF2, which is recruited to both receptors upon their stimulation. TRAF2 exerts multiple receptor-specific functions but also mediates cross-talk between TNFR1 and TNFR2, dictating the outcome of TNF stimulation. In this review, we provide an overview of the positive and negative regulatory role of TRAF2 in different TNFR1 and TNFR2 signaling pathways. We discuss the underlying molecular mechanism of action, distinguishing between TRAF2 scaffold and E3 ubiquitin ligase functions, and the regulation of TRAF2 by specific post-translational modifications. Finally, we elaborate on some possible strategies to modulate TRAF2 function in the context of therapeutic targeting in autoimmunity and cancer. PMID:26993379

  19. Disassembly of microtubules and inhibition of neurite outgrowth, neuroblastoma cell proliferation, and MAP kinase tyrosine dephosphorylation by dibenzyl trisulphide.

    PubMed

    Rösner, H; Williams, L A; Jung, A; Kraus, W

    2001-08-22

    Dibenzyl trisulphide (DTS), a main lipophilic compound in Petiveria alliacea L. (Phytolaccaceae), was identified as one of the active immunomodulatory compounds in extracts of the plant. To learn more about its biological activities and molecular mechanisms, we conducted one-dimensional NMR interaction studies with bovine serum albumin (BSA) and tested DTS and related compounds in two well-established neuronal cell-and-tissue culture systems. We found that DTS preferentially binds to an aromatic region of BSA which is rich in tyrosyl residues. In SH-SY5Y neuroblastoma cells, DTS attenuates the dephosphorylation of tyrosyl residues of MAP kinase (erk1/erk2). In the same neuroblastoma cell line and in Wistar 38 human lung fibroblasts, DTS causes a reversible disassembly of microtubules, but it did not affect actin dynamics. Probably due to the disruption of the microtubule dynamics, DTS also inhibits neuroblastoma cell proliferation and neurite outgrowth from spinal cord explants. Related dibenzyl compounds with none, one, or two sulphur atoms were found to be significantly less effective. These data confirmed that the natural compound DTS has a diverse spectrum of biological properties, including cytostatic and neurotoxic actions in addition to immunomodulatory activities.

  20. Roles of mitogen activated protein kinases and EGF receptor in arsenite-stimulated matrix metalloproteinase-9 production

    SciTech Connect

    Cooper, Karen L.; Myers, Terrance Alix; Rosenberg, Martina; Chavez, Miquella; Hudson, Laurie G. . E-mail: lghudson@unm.edu

    2004-11-01

    The dermatotoxicity of arsenic is well established and epidemiological studies identify an increased incidence of keratinocytic tumors (basal cell and squamous cell carcinoma) associated with arsenic exposure. Little is known about the underlying mechanisms of arsenic-mediated skin carcinogenesis, but activation of mitogen-activated protein (MAP) kinases and subsequent regulation of downstream target genes may contribute to tumor promotion and progression. In this study, we investigated activation of the extracellular signal regulated kinase (ERK) and the stress-associated kinase p38 by arsenite in HaCat cells, a spontaneously immortalized human keratinocyte cell line. Arsenite concentrations {>=}100 {mu}M stimulate rapid activation of p38 and ERK MAP kinases. However, upon extended exposure (24 h), persistent stimulation of p38 and ERK MAP kinases was detected at low micromolar concentrations of arsenite. Although ERK and p38 were activated with similar time and concentration dependence, the mechanism of activation differed for these two MAP kinases. ERK activation by arsenite was fully dependent on the catalytic activity of the epidermal growth factor (EGF) receptor and partially dependent on Src-family kinase activity. In contrast, p38 activation was independent of EGF receptor or Src-family kinase activity. Arsenite-stimulated MAP kinase signal transduction resulted in increased production of matrix metalloproteinase (MMP)-9, an AP-1 regulated gene product. MMP-9 induction by arsenite was prevented when EGF receptor or MAP kinase signaling was inhibited. These studies indicate that EGF receptor activation is a component of arsenite-mediated signal transduction and gene expression in keratinocytes and that low micromolar concentrations of arsenite stimulate key signaling pathways upon extended exposure. Stimulation of MAP kinase cascades by arsenic and subsequent regulation of genes including c-fos, c-jun, and the matrix degrading proteases may play an important

  1. An inside job: hacking into Janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii.

    PubMed

    Denkers, Eric Y; Bzik, David J; Fox, Barbara A; Butcher, Barbara A

    2012-02-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype. PMID:22104110

  2. An inside job: hacking into Janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii.

    PubMed

    Denkers, Eric Y; Bzik, David J; Fox, Barbara A; Butcher, Barbara A

    2012-02-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype.

  3. An Inside Job: Hacking into Janus Kinase/Signal Transducer and Activator of Transcription Signaling Cascades by the Intracellular Protozoan Toxoplasma gondii

    PubMed Central

    Bzik, David J.; Fox, Barbara A.; Butcher, Barbara A.

    2012-01-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype. PMID:22104110

  4. Cdc37p is involved in osmoadaptation and controls high osmolarity-induced cross-talk via the MAP kinase Kss1p.

    PubMed

    Yang, Xiao-Xian; Hawle, Patricija; Bebelman, Jan Paul; Meenhuis, Annemarie; Siderius, Marco; van der Vies, Saskia M

    2007-09-01

    Cdc37p, the p50 homolog of Saccharomyces cerevisiae, is an Hsp90 cochaperone involved in the targeting of protein kinases to Hsp90. Here we report a role for Cdc37p in osmoadaptive signalling in this yeast. The osmosensitive phenotype that is displayed by the cdc37-34 mutant strain appears not to be the consequence of deficient signalling through the high osmolarity glycerol (HOG) MAP kinase pathway. Rather, Cdc37p appears to play a role in the filamentous growth (FG) pathway, which mediates adaptation to high osmolarity parallel to the HOG pathway. The osmosensitive phenotype of the cdc37-34 mutant strain is aggravated upon the deletion of the HOG gene. We report that the hyper-osmosensitive phenotype of the cdc37-34, hog1 mutant correlates to a reduced of activity of the FG pathway. We utilized this phenotype to isolate suppressor genes such as KSS1 that encodes a MAP kinase that functions in the FG pathway. We report that Kss1p interacts physically with Cdc37p. Like Kss1p, the second suppressor that we isolated, Dse1p, is involved in cell wall biogenesis or maintenance, suggesting that Cdc37p controls osmoadapation by regulating mitogen-activated protein kinase signalling aimed at adaptive changes in cell wall organization. PMID:17451450

  5. S-allyl cysteine protects against 6-hydroxydopamine-induced neurotoxicity in the rat striatum: involvement of Nrf2 transcription factor activation and modulation of signaling kinase cascades.

    PubMed

    Tobón-Velasco, Julio César; Vázquez-Victorio, Genaro; Macías-Silva, Marina; Cuevas, Elvis; Ali, Syed F; Maldonado, Perla D; González-Trujano, María Eva; Cuadrado, Antonio; Pedraza-Chaverrí, José; Santamaría, Abel

    2012-09-01

    Pharmacological activation at the basal ganglia of the transcription factor Nrf2, guardian of redox homeostasis, holds a strong promise for the slow progression of Parkinson's disease (PD). However, a potent Nrf2 activator in the brain still must be found. In this study, we have investigated the potential use of the antioxidant compound S-allyl cysteine (SAC) in the activation of Nrf2 in 6-hydoxydopamine (6-OHDA)-intoxicated rats. In the rat striatum, SAC by itself promoted the Nrf2 dissociation of Keap-1, its nuclear translocation, the subsequent association with small MafK protein, and further binding of the Nrf2/MafK complex to ARE sequence, as well as the up-regulation of Nrf2-dependent genes encoding the antioxidant enzymes HO-1, NQO-1, GR, and SOD-1. In vivo and in vitro experiments to identify signaling pathways activated by SAC pointed to Akt as the most likely kinase participating in Nrf2 activation by SAC. In PC12 cells, SAC stimulated the activation of Akt and ERK1/2 and inhibited JNK1/2/3 activation. In the rat striatum, the SAC-induced activation of Nrf2 is likely to contribute to inhibit the toxic effects of 6-OHDA evidenced by phase 2 antioxidant enzymes up-regulation, glutathione recovery, and attenuation of reactive oxygen species (ROS), nitric oxide (NO), and lipid peroxides formation. These early protective effects correlated with the long-term preservation of the cellular redox status, the striatal dopamine (DA) and tyrosine hydroxylase (TH) levels, and the improvement of motor skills. Therefore, this study indicates that, in addition to direct scavenging actions, the activation of Nrf2 by SAC might confer neuroprotective responses through the modulation of kinase signaling pathways in rodent models of PD, and suggests that this antioxidant molecule may have a therapeutic value in this human pathology.

  6. Cascade Chaotic System With Applications.

    PubMed

    Zhou, Yicong; Hua, Zhongyun; Pun, Chi-Man; Chen, C L Philip

    2015-09-01

    Chaotic maps are widely used in different applications. Motivated by the cascade structure in electronic circuits, this paper introduces a general chaotic framework called the cascade chaotic system (CCS). Using two 1-D chaotic maps as seed maps, CCS is able to generate a huge number of new chaotic maps. Examples and evaluations show the CCS's robustness. Compared with corresponding seed maps, newly generated chaotic maps are more unpredictable and have better chaotic performance, more parameters, and complex chaotic properties. To investigate applications of CCS, we introduce a pseudo-random number generator (PRNG) and a data encryption system using a chaotic map generated by CCS. Simulation and analysis demonstrate that the proposed PRNG has high quality of randomness and that the data encryption system is able to protect different types of data with a high-security level.

  7. [Roles of the cross talk between MAP kinases and Keap1-Nrf2-ARE signaling pathways in chronic obstructive pulmonary disease].

    PubMed

    Wang, Shu-Jun; Chen, Ya-Jun; Wang, Shan-Shan; Wang, Dian-Lei; Wang, Chen-Yin; Yang, Li-Li; Chen, Jin-Pei

    2015-02-01

    Chronic obstructive pulmonary disease (COPD), a common preventable and treatable disease, is characterized by airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways. Its main pathological manifestations include airway inflammation, mucus hypersecretion, oxidative stress and apoptotic epithelial cells. Recent research suggests that MAP kinases and Keap1-Nrf2-ARE signaling pathway are involved in the pathological process of inflammation and oxidative stress. This review explores the potential role of the cross talk of these signaling pathways in airway inflammation, mucus hypersecretion, oxidative stress and apoptotic epithelial cells. To clarify the roles of cross talk between MAP kinases and Keap1-Nrf2-ARE signaling pathway, we also focus on the drugs related to that in the treatment of COPD, and it provides ideas for more drug research in the treatment of COPD.

  8. Receptor tyrosine and MAP kinase are involved in effects of H(2)O(2) on interstitial cells of Cajal in murine intestine.

    PubMed

    Choi, Seok; Yeum, Cheol Ho; Kim, Young Dae; Park, Chan Guk; Kim, Man Yoo; Park, Jong-Seong; Jeong, Han-Seong; Kim, Byung Joo; So, Insuk; Kim, Ki Whan

    2010-01-01

    Hydrogen peroxide (H(2)O(2)) is involved in intestinal motility through changes of smooth muscle activity. However, there is no report as to the modulatory effects of H(2)O(2) on interstitial cells of Cajal (ICC). We investigated the H(2)O(2) effects and signal transductions to determine whether the intestinal motility can be modulated through ICC. We performed whole-cell patch clamp in cultured ICC from murine intestine and molecular analyses. H(2)O(2) hyperpolarized the membrane and inhibited pacemaker currents. These effects were inhibited by glibenclamide, an inhibitor of ATP-sensitive K+ (K(ATP)) channels. The free-radical scavenger catalase inhibited the H(2)O(2)-induced effects. MAFP and AACOCF3 (a cytosolic phospholipase A2 inhibitors) or SC-560 and NS-398 (a selective COX-1 and 2 inhibitor) or AH6809 (an EP2 receptor antagonist) inhibited the H(2)O(2)-induced effects. PD98059 (a mitogen activated/ERK-activating protein kinase inhibitor) inhibited the H(2)O(2)-induced effects, though SB-203580 (a p38 MAPK inhibitor) or a JNK inhibitor did not affect. H(2)O(2)-induced effects could not be inhibited by LY-294002 (an inhibitor of PI3-kinases), calphostin C (a protein kinase C inhibitor) or SQ-22536 (an adenylate cyclase inhibitor). Adenoviral infection analysis revealed H2O2 stimulated tyrosine kinase activity and AG 1478 (an antagonist of epidermal growth factor receptor tyrosine kinase) inhibited the H(2)O(2)-induced effects. These results suggest H(2)O(2) can modulate ICC pacemaker activity and this occur by the activation of K(ATP) channels through PGE(2) production via receptor tyrosine kinase-dependent MAP kinase activation.

  9. Localization of the human stress responsive MAP kinase-like CSAIDs binding protein (CSBP) gene to chromosome 6p21.3/21.2

    SciTech Connect

    McDonnell, P.C.; Young, P.R.; DiLella, A.G.

    1995-09-01

    The proinflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) play a pivotal role in the initiation of inflammatory responses. Soluble protein antagonists of IL-1 and TNF, such as IL-1ra, sTNFR-Fc fusion, and monoclonal antibodies to TNF have proven to be effective at blocking acute and chronic responses in a number of animal models of inflammatory diseases such as rheumatoid arthritis, septic shock, and inflammatory bowel disease. Consequently, there has been considerable interest in discovering compounds that could inhibit the production of these cytokines and might therefore become treatments. Recently, a structurally related series of pyridinyl imidazoles was found to block IL-1 and TNF production from LPS-stimulated human monocytes and to ameliorate inflammatory diseases significantly in vivo, leading to their being named CSAIDs (cytokine suppressive anti-inflammatory drugs). The protein target of these compounds, termed CSBP (CSAID binding protein), was discovered to be a new member of the MAP kinase family of serine-threonine protein kinases whose kinase activity is activated by LPS in human monocytes. Independently, the same kinase, or its rodent homologues, was found to respond also to chemical, thermal, and osmotic stress and IL-1 treatment. Inhibition of this kinase correlated with reduction in inflammatory cytokine production from LPS-activated monocytes. 15 refs., 1 fig.

  10. PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

    PubMed

    Teichert, Ines; Steffens, Eva Katharina; Schnaß, Nicole; Fränzel, Benjamin; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

    2014-09-01

    Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

  11. The effects of cardamonin on lipopolysaccharide-induced inflammatory protein production and MAP kinase and NFκB signalling pathways in monocytes/macrophages

    PubMed Central

    Hatziieremia, S; Gray, A I; Ferro, V A; Paul, A; Plevin, R

    2006-01-01

    Background and purpose: In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)-induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action. Experimental approaches: Cardamonin was isolated from the Greek plant A. absinthium L. Its effects were assessed on LPS-induced nitrite release and iNOS and COX-2 protein expression in two macrophage cell lines. Western blotting was used to investigate its effects on phosphorylation of the mitogen activated protein (MAP) kinases, ERK, JNK and p38 MAP kinase, and activation of the NFκB pathway, at the level of IκBα degradation and phosphorylation of NFκB. Also its effects on NFκB and GAS/GAF-DNA binding were assessed by EMSA. Key results: Cardamonin concentration-dependently inhibited both NO release and iNOS expression but had no effect on COX-2 expression. It did not affect phosphorylation of the MAP kinases, degradation of IκBα or phosphorylation of NFκB. However, it inhibited NFκB DNA-binding in both LPS-stimulated cells and nuclear extracts of the cells (in vitro). It also inhibited IFNγ-stimulated iNOS induction and GAS/GAF-DNA binding. Conclusions and Implications: These results show that the inhibitory effect of cardamonin on LPS-induced iNOS induction is not mediated via effects on the initial activation of the NFκB or MAP kinase pathways but is due to a direct effect on transcription factor binding to DNA. However, although some selectivity in cardamonin's action is implicated by its inability to affect COX-2 expression, its exact mechanism(s) of action has yet to be identified. PMID:16894344

  12. High humidity suppresses ssi4-mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expression.

    PubMed

    Zhou, Fasong; Menke, Frank L H; Yoshioka, Keiko; Moder, Wolfgang; Shirano, Yumiko; Klessig, Daniel F

    2004-09-01

    The Arabidopsis ssi4 mutant, which exhibits spontaneous lesion formation, constitutive expression of pathogenesis-related (PR) genes and enhanced resistance to virulent bacterial and oomycete pathogens, contains a gain-of-function mutation in a TIR-NBS-LRR type R gene. Epistatic analyses revealed that both PR gene expression and disease resistance are activated via a salicylic acid (SA)- and EDS1-dependent, but NPR1- and NDR1-independent signaling pathway. In this study, we demonstrate that in moderate relative humidity (RH; 60%), the ssi4 mutant accumulates H(2)O(2) and SA prior to lesion formation and displays constitutive activation of the MAP kinases AtMPK6 and AtMPK3. It also constitutively expresses a variety of defense-associated genes, including those encoding the WRKY transcription factors AtWRKY29 and AtWRKY6, the MAP kinases AtMPK6 and AtMPK3, the powdery mildew R proteins RPW8.1 and RPW8.2, EDS1 and PR proteins. All of these ssi4-induced responses, as well as the chlorotic, stunted morphology and enhanced disease resistance phenotype, are suppressed by high RH (95%) growth conditions. Thus, a humidity sensitive factor (HSF) appears to function at an early point in the ssi4 signaling pathway. All ssi4 phenotypes, except for MAP kinase activation, also were suppressed by the eds1-1 mutation. Thus, ssi4-induced MAP kinase activation occurs downstream of the HSF but either upstream of EDS1 or on a separate branch of the ssi4 signaling pathway. SA is a critical signaling component in ssi4-mediated defense responses. However, exogenously supplied SA failed to restore lesion formation in high RH-grown ssi4 plants, although it induced defense gene expression. Thus, additional signals also are involved.

  13. The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola.

    PubMed

    Cho, Yangrae; Cramer, Robert A; Kim, Kwang-Hyung; Davis, Josh; Mitchell, Thomas K; Figuli, Patricia; Pryor, Barry M; Lemasters, Emily; Lawrence, Christopher B

    2007-06-01

    Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway. PMID:17280842

  14. Calmodulin-dependent protein kinase II/cAMP response element-binding protein/Wnt/β-catenin signaling cascade regulates angiotensin II-induced podocyte injury and albuminuria.

    PubMed

    Jiang, Lei; Xu, Lingling; Song, Yuxian; Li, Jianzhong; Mao, Junhua; Zhao, Allan Zijian; He, Weichun; Yang, Junwei; Dai, Chunsun

    2013-08-01

    Angiotensin II (Ang II) plays a pivotal role in promoting podocyte dysfunction and albuminuria, however, the underlying mechanisms have not been fully delineated. In this study, we found that Ang II induced Wnt1 expression and β-catenin nuclear translocation in cultured mouse podocytes. Blocking Wnt signaling with Dickkopf-1 (Dkk1) or β-catenin siRNA attenuated Ang II-induced podocyte injury. Ang II could also induce the phosphorylation of calmodulin-dependent protein kinase (CaMK) II and cAMP response element-binding protein (CREB) in cultured podocytes. Blockade of this pathway with CK59 or CREB siRNA could significantly inhibit Ang II-induced Wnt/β-catenin signaling and podocyte injury. In in vivo studies, administration of Ang II promoted Wnt/β-catenin signaling, aggregated podocyte damage, and albuminuria in mice. CK59 could remarkably ameliorate Ang II-induced podocyte injury and albuminuria. Furthermore, ectopic expression of exogenous Dkk1 also attenuated Ang II-induced podocytopathy in mice. Taken together, this study demonstrates that the CaMK II/CREB/Wnt/β-catenin signaling cascade plays an important role in regulating Ang II-induced podocytopathy. Targeting this signaling pathway may offer renal protection against the development of proteinuric kidney diseases. PMID:23803607

  15. A superoxide anion-scavenger, 1,3-selenazolidin-4-one suppresses serum deprivation-induced apoptosis in PC12 cells by activating MAP kinase

    SciTech Connect

    Nishina, Atsuyoshi; Kimura, Hirokazu; Kozawa, Kunihisa; Sommen, Geoffroy; Nakamura, Takao; Heimgartner, Heinz; Koketsu, Mamoru; Furukawa, Shoei

    2011-12-15

    Synthetic organic selenium compounds, such as ebselen, may show glutathione peroxidase-like antioxidant activity and have a neurotrophic effect. We synthesized 1,3-selenazolidin-4-ones, new types of synthetic organic selenium compounds (five-member ring compounds), to study their possible applications as antioxidants or neurotrophic-like molecules. Their superoxide radical scavenging effects were assessed using the quantitative, highly sensitive method of real-time kinetic chemiluminescence. At 166 {mu}M, the O{sub 2}{sup -} scavenging activity of 1,3-selenazolidin-4-ones ranged from 0 to 66.2%. 2-[3-(4-Methoxyphenyl)-4-oxo-1,3-selenazolidin-2-ylidene]malononitrile (compound b) showed the strongest superoxide anion-scavenging activity among the 6 kinds of 2-methylene-1,3-selenazolidin-4-ones examined. Compound b had a 50% inhibitory concentration (IC{sub 50}) at 92.4 {mu}M and acted as an effective and potentially useful O{sub 2}{sup -} scavenger in vitro. The effect of compound b on rat pheochromocytome cell line PC12 cells was compared with that of ebselen or nerve growth factor (NGF) by use of the MTT [3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay. When ebselen was added at 100 {mu}M or more, toxicity toward PC12 cells was evident. On the contrary, compound b suppressed serum deprivation-induced apoptosis in PC12 cells more effectively at a concentration of 100 {mu}M. The activity of compound b to phosphorylate mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) 1/2 (MAP kinase) in PC12 cells was higher than that of ebselen, and the former at 100 {mu}M induced the phosphorylation of MAP kinase to a degree similar to that induced by NGF. From these results, we conclude that this superoxide anion-scavenger, compound b, suppressed serum deprivation-induced apoptosis by promoting the phosphorylation of MAP kinase. -- Highlights: Black-Right-Pointing-Pointer We newly synthesized 1,3-selenazolidin-4-ones to

  16. Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins.

    PubMed

    Oh, Chang-Sik; Martin, Gregory B

    2011-04-22

    Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction. PMID:21378171

  17. DLK-1/p38 MAP Kinase Signaling Controls Cilium Length by Regulating RAB-5 Mediated Endocytosis in Caenorhabditis elegans.

    PubMed

    van der Vaart, Aniek; Rademakers, Suzanne; Jansen, Gert

    2015-12-01

    Cilia are sensory organelles present on almost all vertebrate cells. Cilium length is constant, but varies between cell types, indicating that cilium length is regulated. How this is achieved is unclear, but protein transport in cilia (intraflagellar transport, IFT) plays an important role. Several studies indicate that cilium length and function can be modulated by environmental cues. As a model, we study a C. elegans mutant that carries a dominant active G protein α subunit (gpa-3QL), resulting in altered IFT and short cilia. In a screen for suppressors of the gpa-3QL short cilium phenotype, we identified uev-3, which encodes an E2 ubiquitin-conjugating enzyme variant that acts in a MAP kinase pathway. Mutation of two other components of this pathway, dual leucine zipper-bearing MAPKKK DLK-1 and p38 MAPK PMK-3, also suppress the gpa-3QL short cilium phenotype. However, this suppression seems not to be caused by changes in IFT. The DLK-1/p38 pathway regulates several processes, including microtubule stability and endocytosis. We found that reducing endocytosis by mutating rabx-5 or rme-6, RAB-5 GEFs, or the clathrin heavy chain, suppresses gpa-3QL. In addition, gpa-3QL animals showed reduced levels of two GFP-tagged proteins involved in endocytosis, RAB-5 and DPY-23, whereas pmk-3 mutant animals showed accumulation of GFP-tagged RAB-5. Together our results reveal a new role for the DLK-1/p38 MAPK pathway in control of cilium length by regulating RAB-5 mediated endocytosis.

  18. MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

    SciTech Connect

    Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala; Glas, Rickard

    2010-08-27

    Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.

  19. Microglial Signaling in Chronic Pain with a Special Focus on Caspase 6, p38 MAP Kinase, and Sex Dependence.

    PubMed

    Berta, T; Qadri, Y J; Chen, G; Ji, R R

    2016-09-01

    Microglia are the resident immune cells in the spinal cord and brain. Mounting evidence suggests that activation of microglia plays an important role in the pathogenesis of chronic pain, including chronic orofacial pain. In particular, microglia contribute to the transition from acute pain to chronic pain, as inhibition of microglial signaling reduces pathologic pain after inflammation, nerve injury, and cancer but not baseline pain. As compared with inflammation, nerve injury induces much more robust morphologic activation of microglia, termed microgliosis, as shown by increased expression of microglial markers, such as CD11b and IBA1. However, microglial signaling inhibitors effectively reduce inflammatory pain and neuropathic pain, arguing against the importance of morphologic activation of microglia in chronic pain sensitization. Importantly, microglia enhance pain states via secretion of proinflammatory and pronociceptive mediators, such as tumor necrosis factor α, interleukins 1β and 18, and brain-derived growth factor. Mechanistically, these mediators have been shown to enhance excitatory synaptic transmission and suppress inhibitory synaptic transmission in the pain circuits. While early studies suggested a predominant role of microglia in the induction of chronic pain, further studies have supported a role of microglia in the maintenance of chronic pain. Intriguingly, recent studies show male-dominant microglial signaling in some neuropathic pain and inflammatory pain states, although both sexes show identical morphologic activation of microglia after nerve injury. In this critical review, we provide evidence to show that caspase 6-a secreted protease that is expressed in primary afferent axonal terminals surrounding microglia-is a robust activator of microglia and induces profound release of tumor necrosis factor α from microglia via activation of p38 MAP kinase. The authors also show that microglial caspase 6/p38 signaling is male dominant in some

  20. DLK-1/p38 MAP Kinase Signaling Controls Cilium Length by Regulating RAB-5 Mediated Endocytosis in Caenorhabditis elegans

    PubMed Central

    van der Vaart, Aniek; Rademakers, Suzanne; Jansen, Gert

    2015-01-01

    Cilia are sensory organelles present on almost all vertebrate cells. Cilium length is constant, but varies between cell types, indicating that cilium length is regulated. How this is achieved is unclear, but protein transport in cilia (intraflagellar transport, IFT) plays an important role. Several studies indicate that cilium length and function can be modulated by environmental cues. As a model, we study a C. elegans mutant that carries a dominant active G protein α subunit (gpa-3QL), resulting in altered IFT and short cilia. In a screen for suppressors of the gpa-3QL short cilium phenotype, we identified uev-3, which encodes an E2 ubiquitin-conjugating enzyme variant that acts in a MAP kinase pathway. Mutation of two other components of this pathway, dual leucine zipper-bearing MAPKKK DLK-1 and p38 MAPK PMK-3, also suppress the gpa-3QL short cilium phenotype. However, this suppression seems not to be caused by changes in IFT. The DLK-1/p38 pathway regulates several processes, including microtubule stability and endocytosis. We found that reducing endocytosis by mutating rabx-5 or rme-6, RAB-5 GEFs, or the clathrin heavy chain, suppresses gpa-3QL. In addition, gpa-3QL animals showed reduced levels of two GFP-tagged proteins involved in endocytosis, RAB-5 and DPY-23, whereas pmk-3 mutant animals showed accumulation of GFP-tagged RAB-5. Together our results reveal a new role for the DLK-1/p38 MAPK pathway in control of cilium length by regulating RAB-5 mediated endocytosis. PMID:26657059

  1. Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced.

    PubMed

    Onnockx, Sheela; Xie, Jingwei; Degraef, Chantal; Erneux, Christophe; Pirson, Isabelle

    2009-09-10

    Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

  2. Inhibition of MAP kinase promotes the recruitment of corepressor SMRT by tamoxifen-bound estrogen receptor alpha and potentiates tamoxifen action in MCF-7 cells

    SciTech Connect

    Hong, Wei; Chen, Linfeng; Li, Juan; Yao, Zhi

    2010-05-28

    Estrogen receptor alpha (ER{alpha}), a ligand controlled transcription factor, plays an important role in breast cancer growth and endocrine therapy. Tamoxifen (TAM) antagonizes ER{alpha} activity and has been applied in breast cancer treatment. TAM-bound ER{alpha} associates with nuclear receptor-corepressors. Mitogen-activated protein kinase (MAPK) has been elucidated to result in cross-talk between growth factor and ER{alpha} mediated signaling. We show that activated MAPK represses interaction of TAM-bound ER{alpha} with silencing mediator for retinoid and thyroid hormone receptors (SMRT) and inhibits the recruitment of SMRT by ER{alpha} to certain estrogen target genes. Blockade of MAPK signaling cascade with MEK inhibitor U0126 promotes the interaction and subsequently inhibits ER{alpha} activity via enhanced recruitment of SMRT, leading to reduced expression of ER{alpha} target genes. The growth rate of MCF-7 cells was decelerated when treated with both TAM and U0126. Moreover, the growth of MCF-7 cells stably expressing SMRT showed a robust repression in the presence of TAM and U0126. These results suggest that activated MAPK signaling cascade attenuates antagonist-induced recruitment of SMRT to ER{alpha}, suggesting corepressor mediates inhibition of ER{alpha} transactivation and breast cancer cell growth by antagonist. Taken together, our finding indicates combination of antagonist and MAPK inhibitor could be a helpful approach for breast cancer therapy.

  3. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2014-01-01

    Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation. PMID:25421240

  4. uPAR peptide antagonist alters regulation of MAP kinases and Bcl-2 family members in favor of apoptosis in MDA-MB-231 cell line

    PubMed Central

    Tarighi, P.; Montazeri, H.; Khorramizadeh, M.R.; sobhani, A. Madadkar; Ostad, S.N.; Ghahremani, M.H.

    2015-01-01

    Urokinase plasminogen activator receptor (uPAR) and its ligands play a major role in many tumors by mediating extracellular matrix degradation and signaling cascades leading to tumor growth, invasion and metastasis. Recently we introduced uPAR decapeptide antagonist with cytotoxic effect on MDA-MB-231 cell line. In this study we assessed the alteration in uPAR downstream signaling following treatment with the peptide antagonist. In this regard, extracellular-signal-regulated kinase (ERK) and p38 from mitogen-activated protein kinase family and Bcl-2, Bim and Bax from Bcl-2 protein family were investigated. Our data revealed that the peptide caused p38 activation and low ERK activation. On the other hand, the peptide induced down-regulation of Bcl-2 and up-regulation of Bim without Bax modulation. Changes in target protein expression/activation explain the apoptotic property of the peptide and highlight its potential to be used as a therapeutic agent in cancerous cells expressing high levels of uPAR. PMID:26600846

  5. A novel PPAR{gamma} agonist, KR62776, suppresses RANKL-induced osteoclast differentiation and activity by inhibiting MAP kinase pathways

    SciTech Connect

    Park, Ju-Young; Bae, Myung-Ae; Cheon, Hyae Gyeong; Kim, Sung Soo; Hong, Jung-Min; Kim, Tae-Ho; Choi, Je-Yong; Kim, Sang-Hyun; Lim, Jiwon; Choi, Chang-Hyuk; Shin, Hong-In; Kim, Shin-Yoon Park, Eui Kyun

    2009-01-16

    We investigated the effects of a novel peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, KR62776, on osteoclast differentiation and function, and on the underlying signaling pathways. KR62776 markedly suppressed differentiation into osteoclasts in various osteoclast model systems, including bone marrow mononuclear (BMM) cells and a co-culture of calvarial osteoblasts and BMM cells. KR62776 suppressed the activation of tartrate-resistant acid phosphatase (TRAP) and the expression of genes associated with osteoclast differentiation, such as TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), and osteoclast-associated receptor (OSCAR). Furthermore, KR62776 reduced resorption pit formation in osteoclasts, and down-regulated genes essential for osteoclast activity, such as Src and {alpha}v{beta}3 integrin. An analysis of a signaling pathway showed that KR62776 inhibited the receptor activator of nuclear factor-{kappa}B ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and nuclear factor-{kappa}B (NF-{kappa}B). Together, these results demonstrate that KR62776 negatively affects osteoclast differentiation and activity by inhibiting the RANKL-induced activation of MAP kinases and NF-{kappa}B.

  6. Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

    SciTech Connect

    Calabrese, G.; Sallese, M.; Stornaiuolo, A.

    1994-09-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.

  7. BGP-15, a PARP-inhibitor, prevents imatinib-induced cardiotoxicity by activating Akt and suppressing JNK and p38 MAP kinases.

    PubMed

    Sarszegi, Zsolt; Bognar, Eszter; Gaszner, Balazs; Kónyi, Attila; Gallyas, Ferenc; Sumegi, Balazs; Berente, Zoltan

    2012-06-01

    In this study, we investigate the cardiotoxic effects of the well-known cytostatic agent imatinib mesylate (Gleevec), and presented evidence for the cardioprotective effect of BGP-15 which is a novel insulin sensitizer. The cardiotoxic effect of imatinib mesylate was assessed in Langendorff rat heart perfusion system. The cardiac high-energy phosphate levels (creatine phosphate (PCr) and ATP) were monitored in situ by (31)P NMR spectroscopy. The protein oxidation, lipid peroxidation, and the activation of signaling pathways were determined from the freeze-clamped hearts. Prolonged treatment of the heart with imatinib mesylate (20 mg/kg) resulted in cardiotoxicity, which were characterized by the depletion of high-energy phosphates (PCr and ATP), and significantly increased protein oxidation and lipid peroxidation. Imatinib mesylate treatment-induced activation of MAP kinases (including ERK1/2, p38, and JNK) and the phosphorylation of Akt and GSK-3beta. BGP-15 (200 μM) prevented the imatinib mesylate-induced oxidative damages, attenuated the depletion of high-energy phosphates, altered the signaling effect of imatinib mesylate by preventing p38 MAP kinase and JNK activation, and induced the phosphorylation of Akt and GSK-3beta. The suppressive effect of BGP-15 on p38 and JNK activation could be significant because these kinases contribute to the cell death and inflammation in the isolated perfused heart.

  8. ERK1/2 MAP kinases promote cell cycle entry by rapid, kinase-independent disruption of retinoblastoma-lamin A complexes.

    PubMed

    Rodríguez, Javier; Calvo, Fernando; González, José M; Casar, Berta; Andrés, Vicente; Crespo, Piero

    2010-11-29

    As orchestrators of essential cellular processes like proliferation, ERK1/2 mitogen-activated protein kinase signals impact on cell cycle regulation. A-type lamins are major constituents of the nuclear matrix that also control the cell cycle machinery by largely unknown mechanisms. In this paper, we disclose a functional liaison between ERK1/2 and lamin A whereby cell cycle progression is regulated. We demonstrate that lamin A serves as a mutually exclusive dock for ERK1/2 and the retinoblastoma (Rb) protein. Our results reveal that, immediately after their postactivation entrance in the nucleus, ERK1/2 dislodge Rb from its interaction with lamin A, thereby facilitating its rapid phosphorylation and consequently promoting E2F activation and cell cycle entry. Interestingly, these effects are independent of ERK1/2 kinase activity. We also show that cellular transformation and tumor cell proliferation are dependent on the balance between lamin A and nuclear ERK1/2 levels, which determines Rb accessibility for phosphorylation/inactivation.

  9. Lipopolysaccharide induces activation of the Raf-1/MAP kinase pathway. A putative role for Raf-1 in the induction of the IL-1 beta and the TNF-alpha genes.

    PubMed

    Reimann, T; Büscher, D; Hipskind, R A; Krautwald, S; Lohmann-Matthes, M L; Baccarini, M

    1994-12-15

    Bacterial LPS is a potent macrophage activator. The early steps in LPS signal transduction involve the tyrosine phosphorylation and activation of a number of kinases of the src family, and inhibition of this pathway causes a severe impairment in the production of the cytokines TNF-alpha and IL-1 beta. We find that LPS-induced macrophages activation also involves the Raf-1 kinase, a key component in mitogenic signal transduction. Treatment of BAC-1.2F5 macrophages with LPS causes phosphorylation and activation of Raf-1. This is paralleled by the stimulation of MEK-1 and MAP-kinase activity and by the phosphorylation of the transcription factor Elk-1, a nuclear target of MAP-kinase. Activation of the Raf/MAP-kinase pathway was inhibited upon pretreatment of the cells with genistein, a tyrosine kinase inhibitor. Raf-1 must thus lie downstream of tyrosine kinase in LPS signal transduction. However, Raf-1 is not a direct substrate of a LPS-induced tyrosine kinase, because Raf-1 immunoisolated from LPS-induced cells contains only phosphoserine. This resembles the situation after CSF-1-stimulation of macrophages, in which Raf-1 clearly transduces a signal generated by the CSF-1 receptor kinase, but is phosphorylated exclusively in serine. Phosphopeptide maps of Raf-1 immunoprecipitated from LPS- or CSF-1-treated cells are indistinguishable, suggesting that these agents activate Raf-1 by similar mechanisms. Finally, v-raf-infected BAC-1.2F5 macrophages were found to constitutively express low levels of IL-1 beta and TNF-alpha. These data argue that Raf-1 functions downstream of tyrosine kinases in LPS-mediated macrophage activation and cytokine production. PMID:7989771

  10. Lipopolysaccharide induces activation of the Raf-1/MAP kinase pathway. A putative role for Raf-1 in the induction of the IL-1 beta and the TNF-alpha genes.

    PubMed

    Reimann, T; Büscher, D; Hipskind, R A; Krautwald, S; Lohmann-Matthes, M L; Baccarini, M

    1994-12-15

    Bacterial LPS is a potent macrophage activator. The early steps in LPS signal transduction involve the tyrosine phosphorylation and activation of a number of kinases of the src family, and inhibition of this pathway causes a severe impairment in the production of the cytokines TNF-alpha and IL-1 beta. We find that LPS-induced macrophages activation also involves the Raf-1 kinase, a key component in mitogenic signal transduction. Treatment of BAC-1.2F5 macrophages with LPS causes phosphorylation and activation of Raf-1. This is paralleled by the stimulation of MEK-1 and MAP-kinase activity and by the phosphorylation of the transcription factor Elk-1, a nuclear target of MAP-kinase. Activation of the Raf/MAP-kinase pathway was inhibited upon pretreatment of the cells with genistein, a tyrosine kinase inhibitor. Raf-1 must thus lie downstream of tyrosine kinase in LPS signal transduction. However, Raf-1 is not a direct substrate of a LPS-induced tyrosine kinase, because Raf-1 immunoisolated from LPS-induced cells contains only phosphoserine. This resembles the situation after CSF-1-stimulation of macrophages, in which Raf-1 clearly transduces a signal generated by the CSF-1 receptor kinase, but is phosphorylated exclusively in serine. Phosphopeptide maps of Raf-1 immunoprecipitated from LPS- or CSF-1-treated cells are indistinguishable, suggesting that these agents activate Raf-1 by similar mechanisms. Finally, v-raf-infected BAC-1.2F5 macrophages were found to constitutively express low levels of IL-1 beta and TNF-alpha. These data argue that Raf-1 functions downstream of tyrosine kinases in LPS-mediated macrophage activation and cytokine production.

  11. Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics

    PubMed Central

    1995-01-01

    We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule- associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be

  12. Neuronal apoptosis induced by selective inhibition of Rac GTPase versus global suppression of Rho family GTPases is mediated by alterations in distinct mitogen-activated protein kinase signaling cascades.

    PubMed

    Stankiewicz, Trisha R; Ramaswami, Sai Anandi; Bouchard, Ron J; Aktories, Klaus; Linseman, Daniel A

    2015-04-10

    Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad.

  13. Glycyrrhetinic acid induces cytoprotective autophagy via the inositol-requiring enzyme 1α-c-Jun N-terminal kinase cascade in non-small cell lung cancer cells.

    PubMed

    Tang, Zheng-Hai; Zhang, Le-Le; Li, Ting; Lu, Jia-Hong; Ma, Dik-Lung; Leung, Chung-Hang; Chen, Xiu-Ping; Jiang, Hu-Lin; Wang, Yi-Tao; Lu, Jin-Jian

    2015-12-22

    Glycerrhetinic acid (GA), one of the main bioactive constituents of Glycyrrhiza uralensis Fisch, exerts anti-cancer effects on various cancer cells. We confirmed that GA inhibited cell proliferation and induced apoptosis in non-small cell lung cancer A549 and NCI-H1299 cells. GA also induced expression of autophagy marker phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II) and punta formation of green fluorescent protein microtubule-associated protein light-chain 3. We further proved that expression of GA-increased autophagy marker was attributed to activation instead of suppression of autophagic flux. The c-jun N-terminal kinase (JNK) pathway was activated after incubation with GA. Pretreatment with the JNK inhibitor SP600125 or silencing of the JNK pathway by siRNA of JNK or c-jun decreased GA-induced autophagy. The endoplasmic reticulum (ER) stress responses were also apparently stimulated by GA by triggering the inositol-requiring enzyme 1α (IRE1α) pathway. The GA-induced JNK pathway activation and autophagy were decreased by IRE1α knockdown, and inhibition of autophagy or the JNK cascade increased GA-stimulated IRE1α expression. In addition, GA-induced cell proliferative inhibition and apoptosis were increased by inhibition of autophagy or the JNK pathway. Our study was the first to demonstrate that GA induces cytoprotective autophagy in non-small cell lung cancer cells by activating the IRE1α-JNK/c-jun pathway. The combined treatment of autophagy inhibitors markedly enhances the anti-neoplasmic activity of GA. Such combination shows potential as a strategy for GA or GA-contained prescriptions in cancer therapy.

  14. Neuronal Apoptosis Induced by Selective Inhibition of Rac GTPase versus Global Suppression of Rho Family GTPases Is Mediated by Alterations in Distinct Mitogen-activated Protein Kinase Signaling Cascades*

    PubMed Central

    Stankiewicz, Trisha R.; Ramaswami, Sai Anandi; Bouchard, Ron J.; Aktories, Klaus; Linseman, Daniel A.

    2015-01-01

    Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad. PMID:25666619

  15. Mapping

    ERIC Educational Resources Information Center

    Kinney, Douglas M.; McIntosh, Willard L.

    1978-01-01

    Geologic mapping in the United States increased by about one-quarter in the past year. Examinations of mapping trends were in the following categories: (1) Mapping at scales of 1:100, 000; (2) Metric-scale base maps; (3) International mapping, and (4) Planetary mapping. (MA)

  16. Staphylococcus aureus α-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells.

    PubMed

    Eiffler, Ina; Behnke, Jane; Ziesemer, Sabine; Müller, Christian; Hildebrandt, Jan-Peter

    2016-09-01

    Membrane potential (Vm)-, Na(+)-, or Ca(2+)-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with Staphylococcus aureus α-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel-forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla pore, were used to investigate the permeability of the pore. Na(+) as well as Ca(2+) ions were able to pass the Hla pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by α-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense. PMID:27496896

  17. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

    PubMed

    Adderley, Shaquria P; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O; Breslin, Jerome W

    2015-07-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity. PMID:25948734

  18. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers

    PubMed Central

    Adderley, Shaquria P.; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O.

    2015-01-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity. PMID:25948734

  19. Staphylococcus aureus α-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells.

    PubMed

    Eiffler, Ina; Behnke, Jane; Ziesemer, Sabine; Müller, Christian; Hildebrandt, Jan-Peter

    2016-09-01

    Membrane potential (Vm)-, Na(+)-, or Ca(2+)-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with Staphylococcus aureus α-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel-forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla pore, were used to investigate the permeability of the pore. Na(+) as well as Ca(2+) ions were able to pass the Hla pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by α-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense.

  20. N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

    PubMed

    Modarresi, Rozbeh; Lafond, Toulouse; Roman-Blas, Jorge A; Danielson, Keith G; Tuan, Rocky S; Seghatoleslami, M Reza

    2005-05-01

    We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin. PMID:15723280

  1. The transcription factor Ste12 mediates the regulatory role of the Tmk1 MAP kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus Trichoderma atroviride.

    PubMed

    Gruber, Sabine; Zeilinger, Susanne

    2014-01-01

    Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12.

  2. The transcription factor Ste12 mediates the regulatory role of the Tmk1 MAP kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus Trichoderma atroviride.

    PubMed

    Gruber, Sabine; Zeilinger, Susanne

    2014-01-01

    Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12. PMID:25356841

  3. The Transcription Factor Ste12 Mediates the Regulatory Role of the Tmk1 MAP Kinase in Mycoparasitism and Vegetative Hyphal Fusion in the Filamentous Fungus Trichoderma atroviride

    PubMed Central

    Gruber, Sabine; Zeilinger, Susanne

    2014-01-01

    Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12. PMID:25356841

  4. Inhibition of p38 MAP kinase in the early posttransplantation phase redistributes blood vessels from the surrounding stroma into the transplanted endocrine tissue.

    PubMed

    Johansson, Asa; Sandvik, Daniel; Carlsson, Per-Ola

    2006-01-01

    Transplanted pancreatic islets attain a chronically decreased vascular density following transplantation, despite the increased concentrations of vascular endothelial growth factor (VEGF) secreted from beta-cells in response to hypoxia during culture and in the immediate posttransplantation phase. VEGF, however, exerts dual effects on endothelial cells, and in islet endothelial cells of the adult, the vascular permeability-inducing effects of VEGF seem normally more pronounced than those to induce angiogenesis. p38 MAP kinase activity has recently been shown to serve as a switch to separate these properties of VEGF; inhibition of p38 MAP kinase activity enhances VEGF-induced angiogenesis and, at the same time, abrogates VEGF-induced vascular permeability. We hypothesized that the revascularization of transplanted islets may be hampered by a predisposition of adult islet endothelial cells to react to VEGF by forming fenestrae rather than migrating and proliferating. We therefore administered the p38 MAP kinase inhibitor SB203580 by daily IP injections for the first 14 days following transplantation, and then studied the influence of this treatment on the oxygen tension, blood perfusion, and vascular density of the islet grafts 1 month posttransplantation. SB203580 treatment redistributed islet graft blood vessels from the stroma into the endocrine tissue, and this redistribution of blood vessels into the endocrine tissue was accompanied by an increased oxygenation of the islet cells. However, the total number of blood vessels in the tissue was not affected. The blood perfusion of the islet grafts was also similar in control and SB203580-treated animals. Our results suggest that effects of VEGF to preferentially induce vascular permeability may partially contribute to, but is not the main cause of, low revascularization of transplanted islets.

  5. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

    PubMed

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  6. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

    PubMed

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

  7. [6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin.

    PubMed

    Kim, Sue Ok; Kundu, Joydeb Kumar; Shin, Young Kee; Park, Jin-Hong; Cho, Myung-Haing; Kim, Tae-Yoon; Surh, Young-Joon

    2005-04-01

    [6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has a wide array of pharmacologic effects. The present study was aimed at unraveling the molecular mechanisms underlying previously reported antitumor promoting effects of [6]-gingerol in mouse skin in vivo. One of the well-recognized molecular targets for chemoprevention is cyclooxygenase-2 (COX-2) that is abnormally upregulated in many premalignant and malignant tissues and cells. In our present study, topical application of [6]-gingerol inhibited COX-2 expression in mouse skin stimulated with a prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Since the transcription factor nuclear factor-kappaB (NF-kappaB) is known to regulate COX-2 induction, we attempted to determine the effect of [6]-gingerol on TPA-induced activation of NF-kappaB. Pretreatment with [6]-gingerol resulted in a decrease in both TPA-induced DNA binding and transcriptional activities of NF-kappaB through suppression of IkappaBalpha degradation and p65 nuclear translocation. Phosphorylation of both IkappaBalpha and p65 was substantially blocked by [6]-gingerol. In addition, [6]-gingerol inhibited TPA-stimulated interaction of phospho-p65-(Ser-536) with cAMP response element binding protein-binding protein, a transcriptional coactivator of NF-kappaB. Moreover, [6]-gingerol prevented TPA-induced phosphorylation and catalytic activity of p38 mitogen-activated protein (MAP) kinase that regulates COX-2 expression in mouse skin. The p38 MAP kinase inhibitor SB203580 attenuated NF-kappaB activation and subsequent COX-2 induction in TPA-treated mouse skin. Taken together, our data suggest that [6]-gingerol inhibits TPA-induced COX-2 expression in mouse skin in vivo by blocking the p38 MAP kinase-NF-kappaB signaling pathway. PMID:15735738

  8. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

    PubMed Central

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  9. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

    PubMed

    Adderley, Shaquria P; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O; Breslin, Jerome W

    2015-07-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity.

  10. Altered Dendritic Cell Phenotype in Response to Leishmania amazonensis Amastigote Infection Is Mediated by MAP Kinase, ERK

    PubMed Central

    Boggiatto, Paola Mercedes; Jie, Fei; Ghosh, Mousumi; Gibson-Corley, Katherine Nicole; Ramer-Tait, Amanda Ellen; Jones, Douglas Elliot; Petersen, Christine Anne

    2009-01-01

    Initiation of productive immune responses against Leishmania depends on the successful transition of dendritic cells (DC) from an immature to a mature phenotype. This process is characterized by high CD40 surface expression as well as interleukin-12 production, which are frequently seen in response to L. major infection. In vivo footpad infection of C3HeB/FeJ mice for 7 days with L. amazonensis promoted an immature CD11c+ DC phenotype characterized by both significantly low CD40 surface expression and significantly decreased interleukin-12p40 production compared with L. major infection of these same mice. In vitro infection of bone marrow-derived dendritic cells with L. amazonensis amastigotes resulted in rapid and significant phosphorylation of the mitogen activated protein kinase, extracellular signal-regulated kinase 1/2, observed within minutes of exposure to the parasite. Infection with L. amazonensis promastigotes led to increased 1/2 phosphorylation after 4 hours of infection compared with L. major infection, which correlated with promastigote transformation into amastigotes. Treatment of bone marrow-derived dendritic cells with a mitogen activated protein kinase kinase-specific inhibitor, PD98059, led to regained surface CD40 expression and interleukin-12p40 production following L. amazonensis amastigote infection compared with non-treated, infected DC. Treatment of L. amazonensis-infected mice with the highly-specific mitogen activated protein kinase kinase inhibitor, CI-1040, enhanced surface CD40 expression on CD11c+ DC obtained from the draining lymph node. L. amazonensis amastigotes, through activation of extracellular signal-regulated kinase 1/2, inhibit the ability of DC to undergo proper maturation both in vitro and in vivo. PMID:19349356

  11. The ubiquitous mitochondrial creatine kinase gene maps to a conserved region on human chromosome 15q15 and mouse chromosome 2 bands F1-F3

    SciTech Connect

    Steeghs, K.; Wieringa, B.; Merkx, G.

    1994-11-01

    Members of the creatine kinase isoenzyme family (CKs; EC 2.7.3.2) are found in mitochondria and specialized subregions of the cytoplasm and catalyze the reversible exchange of high-energy phosphoryl between ATP and phosphocreatine. At least four functionally active genes, which encode the distinct CK subunits CKB, CKM, CKMT1 (ubiquitous), and CKMT2 (sarcomeric), and a variable number of CKB pseudogenes have been identified. Here, we report the use of a CKMT1 containing phage to map the CKMT1 gene by in situ hybridization on both human and mouse chromosomes.

  12. [INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

    PubMed

    Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

    2015-01-01

    To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only

  13. Fragment-Based Screening Maps Inhibitor Interactions in the ATP-Binding Site of Checkpoint Kinase 2

    PubMed Central

    Silva-Santisteban, M. Cris; Westwood, Isaac M.; Boxall, Kathy; Brown, Nathan; Peacock, Sam; McAndrew, Craig; Barrie, Elaine; Richards, Meirion; Mirza, Amin; Oliver, Antony W.; Burke, Rosemary; Hoelder, Swen; Jones, Keith; Aherne, G. Wynne; Blagg, Julian; Collins, Ian; Garrett, Michelle D.; van Montfort, Rob L. M.

    2013-01-01

    Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragment-based screening campaign using a combination of a high-concentration AlphaScreen™ kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors. PMID:23776527

  14. The NO-cGMP-PKG signaling pathway regulates synaptic plasticity and fear memory consolidation in the lateral amygdala via activation of ERK/MAP kinase.

    PubMed

    Ota, Kristie T; Pierre, Vicki J; Ploski, Jonathan E; Queen, Kaila; Schafe, Glenn E

    2008-10-01

    Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and long-term potentiation (LTP) at thalamic and cortical input pathways to the LA. In behavioral experiments, rats given intra-LA infusions of either the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibited dose-dependent impairments or enhancements of fear memory consolidation, respectively. In slice electrophysiology experiments, bath application of Rp-8-Br-PET-cGMPS or the guanylyl cyclase inhibitor LY83583 impaired LTP at thalamic, but not cortical inputs to the LA, while bath application of 8-Br-cGMP or the guanylyl cyclase activator YC-1 resulted in enhanced LTP at thalamic inputs to the LA. Interestingly, YC-1-induced enhancement of LTP in the LA was reversed by concurrent application of the MEK inhibitor U0126, suggesting that the NO-cGMP-PKG signaling pathway may promote synaptic plasticity and fear memory formation in the LA, in part by activating the ERK/MAPK signaling cascade. As a test of this hypothesis, we next showed that rats given intra-LA infusion of the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibit impaired or enhanced activation, respectively, of ERK/MAPK in the LA after fear conditioning. Collectively, our findings suggest that an NO-cGMP-PKG-dependent form of synaptic plasticity at thalamic input synapses to the LA may underlie memory consolidation of Pavlovian fear conditioning, in part, via activation of the ERK/MAPK signaling cascade.

  15. Suppression of growth factor-mediated MAP kinase activation by v-raf in macrophages: a putative role for the MKP-1 phosphatase.

    PubMed

    Krautwald, S; Büscher, D; Dent, P; Ruthenberg, K; Baccarini, M

    1995-03-16

    Many tyrosine kinase growth factor receptors activate the MAP Kinase (MAPK) pathway by stimulating the activity of the RAF kinase. In some, but not all cell types, the expression of activated RAF is sufficient to induce constitutive MAPK activation. In BAC-1.2F5 macrophages the expression of virally activated RAF does not correlate with constitutive MAPK activation; on the contrary, growth factor-mediated stimulation of MAPK activity is suppressed in these cells. Suppression correlates with v-RAF expression, as MAPK activation is normal in a revertant cell line that stopped expressing v-RAF. Inhibition of MAPK activation is associated with lack of ERK-2 tyrosine phosphorylation, and is not due to the suppression of CSF-1-mediated MEK activation. Pretreatment with vanadate restores growth factor-stimulated activation and tyrosine phosphorylation of MAPK in v-RAF-expressing macrophages, indicating the involvement of a tyrosine phosphatase. Interestingly, v-RAF-expressing macrophages contain low constitutive levels of MKP-1 mRNA, an immediate early gene that encodes a MAPK-specific phosphatase and is induced in the parental cell line by CSF-1 treatment. The restoration of MAPK activation by vanadate pretreatment and the presence of MKP-1 mRNA in v-RAF-expressing macrophages raise the intriguing possibility that in macrophages RAF may be feeding back on the MAPK pathway by participating in the control of MKP-1 expression. PMID:7700643

  16. FvBck1, a component of cell wall integrity MAP kinase pathway, is required for virulence and oxidative stress response in sugarcane Pokkah Boeng pathogen

    PubMed Central

    Zhang, Chengkang; Wang, Jianqiang; Tao, Hong; Dang, Xie; Wang, Yang; Chen, Miaoping; Zhai, Zhenzhen; Yu, Wenying; Xu, Liping; Shim, Won-Bo; Lu, Guodong; Wang, Zonghua

    2015-01-01

    Fusarium verticillioides (formerly F. moniliforme) is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a possibly carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane. PMID:26500635

  17. Mapping of the NEP receptor tyrosine kinase gene to human chromosome 6p21.3 and mouse chromosome 17C

    SciTech Connect

    Edelhoff, S.; Disteche, C.M.; Sweetser, D.A.

    1995-01-01

    The mouse receptor tyrosine kinase (RTK) NEP, also called Ptk-3, is widely expressed, with high levels in proliferating neuroepithelia of mouse embryos. The recently described human discoidin domain receptor (DDR) has a predicted amino acid sequence 93% identical to that of murine NEP and may be its human homologue. We have mapped the gene encoding NEP in human and mouse by fluorescence in situ hybridization using a mouse cDNA probe. The NEP/Nep gene maps to human chromosome 6p21.3 and mouse chromosome 17C, respectively. This places the NEP/Nep gene at, or near, the major histocompatibility (MHC) locus-HLA in human and H2 in mouse, respectively. Based on its pattern of expression during development, NEP and Nep represent candidate genes for several MHC-linked developmental abnormalities in human and mouse. 19 refs., 1 fig.

  18. Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38α MAP kinase: a combined computational and experimental study.

    PubMed

    Zhu, Shun; Travis, Sue M; Elcock, Adrian H

    2013-07-01

    A major current challenge for drug design efforts focused on protein kinases is the development of drug resistance caused by spontaneous mutations in the kinase catalytic domain. The ubiquity of this problem means that it would be advantageous to develop fast, effective computational methods that could be used to determine the effects of potential resistance-causing mutations before they arise in a clinical setting. With this long-term goal in mind, we have conducted a combined experimental and computational study of the thermodynamic effects of active-site mutations on a well-characterized and high-affinity interaction between a protein kinase and a small-molecule inhibitor. Specifically, we developed a fluorescence-based assay to measure the binding free energy of the small-molecule inhibitor, SB203580, to the p38α MAP kinase and used it measure the inhibitor's affinity for five different kinase mutants involving two residues (Val38 and Ala51) that contact the inhibitor in the crystal structure of the inhibitor-kinase complex. We then conducted long, explicit-solvent thermodynamic integration (TI) simulations in an attempt to reproduce the experimental relative binding affinities of the inhibitor for the five mutants; in total, a combined simulation time of 18.5 μs was obtained. Two widely used force fields - OPLS-AA/L and Amber ff99SB-ILDN - were tested in the TI simulations. Both force fields produced excellent agreement with experiment for three of the five mutants; simulations performed with the OPLS-AA/L force field, however, produced qualitatively incorrect results for the constructs that contained an A51V mutation. Interestingly, the discrepancies with the OPLS-AA/L force field could be rectified by the imposition of position restraints on the atoms of the protein backbone and the inhibitor without destroying the agreement for other mutations; the ability to reproduce experiment depended, however, upon the strength of the restraints' force constant

  19. Nitric Oxide Affects ERK Signaling through Down-Regulation of MAP Kinase Phosphatase Levels during Larval Development of the Ascidian Ciona intestinalis

    PubMed Central

    Palumbo, Anna

    2014-01-01

    In the ascidian Ciona intestinalis larval development and metamorphosis require a complex interplay of events, including nitric oxide (NO) production, MAP kinases (ERK, JNK) and caspase-3 activation. We have previously shown that NO levels affect the rate of metamorphosis, regulate caspase activity and promote an oxidative stress pathway, resulting in protein nitration. Here, we report that NO down-regulates MAP kinase phosphatases (mkps) expression affecting positively ERK signaling. By pharmacological approach, we observed that the reduction of endogenous NO levels caused a decrease of ERK phosphorylation, whereas increasing levels of NO induced ERK activation. We have also identified the ERK gene network affected by NO, including mpk1, mpk3 and some key developmental genes by quantitative gene expression analysis. We demonstrate that NO induces an ERK-independent down-regulation of mkp1 and mkp3, responsible for maintaining the ERK phosphorylation levels necessary for transcription of key metamorphic genes, such as the hormone receptor rev-erb and the van willebrand protein vwa1c. These results add new insights into the role played by NO during larval development and metamorphosis in Ciona, highlighting the cross-talk between different signaling pathways. PMID:25058405

  20. The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway

    PubMed Central

    Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

    2014-01-01

    OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation. PMID:25411776

  1. Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases

    PubMed Central

    Farhat, M; Poissonnier, A; Hamze, A; Ouk-Martin, C; Brion, J-D; Alami, M; Feuillard, J; Jayat-Vignoles, C

    2014-01-01

    Defects in apoptosis are frequently the cause of cancer emergence, as well as cellular resistance to chemotherapy. These phenotypes may be due to mutations of the tumor suppressor TP53 gene. In this study, we examined the effect of various mitotic spindle poisons, including the new isocombretastatin derivative isoNH2CA-4 (a tubulin-destabilizing molecule, considered to bind to the colchicine site by analogy with combretastatin A-4), on BL (Burkitt lymphoma) cells. We found that resistance to spindle poison-induced apoptosis could be reverted in tumor protein p53 (TP53)-mutated cells by EBV (Epstein Barr virus) infection. This reversion was due to restoration of the intrinsic apoptotic pathway, as assessed by relocation of the pro-apoptotic molecule Bax to mitochondria, loss of mitochondrial integrity and activation of the caspase cascade with PARP (poly ADP ribose polymerase) cleavage. EBV sensitized TP53-mutated BL cells to all spindle poisons tested, including vincristine and taxol, an effect that was systematically downmodulated by pretreatment of cells with inhibitors of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Exogenous activation of p38 and JNK pathways by dihydrosphingosine reverted resistance of TP53-mutated BL cells to spindle poisons. Dihydrosphingosine treatment of TP53-deficient Jurkat and K562 cell lines was also able to induce cell death. We conclude that activation of p38 and JNK pathways may revert resistance of TP53-mutated cells to spindle poisons. This opens new perspectives for developing alternative therapeutic strategies when the TP53 gene is inactivated. PMID:24787013

  2. Preventing Phosphorylation of Sterol Regulatory Element-Binding Protein 1a by MAP-Kinases Protects Mice from Fatty Liver and Visceral Obesity

    PubMed Central

    Haas, Jutta; Kremer, Lorena; Jacob, Sylvia; Hartwig, Sonja; Nitzgen, Ulrike; Muller–Wieland, Dirk

    2012-01-01

    The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP–1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP–1a mice the phosphorylation–deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo. PMID:22384276

  3. MAP Kinase Phosphatase 1 (MKP-1/DUSP1) is Neuroprotective in Huntington’s Disease Via Additive Effects of JNK and p38 Inhibition

    PubMed Central

    Taylor, David M.; Moser, Roger; Régulier, Etienne; Breuillaud, Lionel; Dixon, Meredith; Beesen, Ayshe Ana; Elliston, Linda; Silva Santos, Mariana de Fatima; Kim, Jinho; Jones, Lesley; Goldstein, Darlene R.; Ferrante, Robert J.; Luthi-Carter, Ruth

    2013-01-01

    We previously demonstrated that sodium butyrate is neuroprotective in Huntington’s disease (HD) mice and that this therapeutic effect is associated with increased expression of mitogen-activated protein kinase/dual-specificity phosphatase 1 (MKP-1/DUSP1). Here we show that enhancing MKP-1 expression is sufficient to achieve neuroprotection in lentiviral models of HD. Wild-type MKP-1 overexpression inhibited apoptosis in primary striatal neurons exposed to an N-terminal fragment of polyglutamine-expanded huntingtin (Htt171-82Q), blocking caspase-3 activation and significantly reducing neuronal cell death. This neuroprotective effect of MKP-1 was demonstrated to be dependent on its enzymatic activity, being ablated by mutation of its phosphatase domain and being attributed to inhibition of specific MAP kinases (MAPKs). Overexpression of MKP-1 prevented the polyglutamine-expanded huntingtin-induced activation of c-Jun N-terminal kinases (JNKs) and p38 MAPKs, whereas extracellular signal-regulated kinase 1/2 (ERK1/2) activation was not altered by either polyglutamine-expanded Htt or MKP-1. Moreover, mutants of MKP-1 that selectively prevented p38 or JNK binding confirmed the important dual contributions of p38 and JNK regulation to MKP-1-mediated neuroprotection. These results demonstrate additive effects of p38 and JNK MAPK inhibition by MKP-1 without consequence to ERK activation in this striatal neuron-based paradigm. MKP-1 also provided neuroprotection in vivo in a lentiviral model of HD neuropathology in rat striatum. Taken together, these data extend previous evidence that JNK- and p38-mediated pathways contribute to HD pathogenesis and, importantly, show that therapies simultaneously inhibiting both JNK and p38 signalling pathways may lead to improved neuroprotective outcomes. PMID:23392662

  4. Multiperiod quantum-cascade nanoheterostructures: Epitaxy and diagnostics

    SciTech Connect

    Egorov, A. Yu. Brunkov, P. N.; Nikitina, E. V.; Pirogov, E. V.; Sobolev, M. S.; Lazarenko, A. A.; Baidakova, M. V.; Kirilenko, D. A.; Konnikov, S. G.

    2014-12-15

    Advances in the production technology of multiperiod nanoheterostructures of quantum-cascade lasers with 60 cascades by molecular-beam epitaxy (MBE) on an industrial multiple-substrate MBE machine are discussed. The results obtained in studying the nanoheterostructures of quantum-cascade lasers by transmission electron microscopy, high-resolution X-ray diffraction analysis, and photoluminescence mapping are presented.

  5. Analysis of signal transduction pathways during anoxia exposure in a marine snail: a role for p38 MAP kinase and downstream signaling cascades.

    PubMed

    Larade, Kevin; Storey, Kenneth B

    2006-01-01

    The responses of members of the three main MAPK families (ERK, JNK/SAPK, p38 MAPK), as well as selected peripheral pathways, were examined in hepatopancreas of the marine periwinkle, Littorina littorea, to determine if anoxia exposure influenced the total protein content or the phosphorylation status of any key components. The content of active phospho-p38 MAPK was 2-fold higher in hepatopancreas from anoxic snails relative to controls. A 1.7-fold increase in the amount of phospho-Hsp27 and a 1.3-fold increase in phospho-CREB correlated well with the changes in p38 MAPK phosphorylation. Activation of these factors via p38 MAPK may be vital to the reorganization of metabolic responses to anoxia in hepatopancreas. No changes in components of the JNK/SAPK and ERK pathways occurred and transcription factors involved in lipid metabolism did not appear to be affected by anoxia. The present analysis of a variety of signaling pathways has implicated the p38 MAPK pathway as a key anoxia-responsive signal transduction pathway in L. littorea. PMID:16326124

  6. MCPIP1 regulates fibroblast migration in 3D collagen matrices downstream of MAP kinases and NF-κB

    PubMed Central

    Chao, Jie; Dai, Xiaoniu; Peña, Tiffany; Doyle, David A.; Guenther, Timothy M.; Carlson, Mark A.

    2015-01-01

    The fibroblast-populated 3D collagen matrix has been used to model matrix contraction, cell motility, and general fibroblast biology. MCPIP1 (monocyte chemotactic protein-induced protein 1) has been shown to regulate inflammation, angiogenesis, and cellular motility. In the present study, we demonstrated induction of MCPIP1 in human fibroblasts embedded in the stress-released 3D collagen matrix, which occurred through activation of mitogen-activated protein kinases, phosphoinositide 3-kinase, and NF-κB. Furthermore, MCPIP1 induction was associated with inhibition of fibroblast migration out of the nested collagen matrix. MCPIP1 induction or ectopic expression also upregulated p53. RNA interference of p53 prevented the inhibition of migration produced by induction or ectopic expression of MCPIP1. Our findings suggest a new role for MCPIP1 as a molecular switch that regulates fibroblast migration in the nested collagen matrix model. PMID:26399696

  7. GRAVITY STUDIES IN THE CASCADE RANGE.

    USGS Publications Warehouse

    Finn, Carol; Williams, David

    1983-01-01

    A compatible set of gravity data has been compiled for the entire Cascade Range. From this data set a series of interpretive color gravity maps have been prepared, including a free air anomaly map, Bouguer anomaly map at a principle, and an alternate reduction density, and filtered and derivative versions of the Bouguer anomaly map. The regional anomaly pattern and gradients outline the various geological provinces adjacent to the Cascade Range and delineate major structural elements in the range. The more local anomalies and gradients may delineate low density basin and caldera fill, faults, and shallow plutons. Refs.

  8. A MAP kinase gene, BMK1, is required for conidiation and pathogenicity in the rice leaf spot pathogen Bipolaris oryzae.

    PubMed

    Moriwaki, Akihiro; Kihara, Junichi; Mori, Chie; Arase, Sakae

    2007-01-01

    We isolated and characterized BMK1, a gene encoding a mitogen-activated protein kinase (MAPK), from the rice leaf spot pathogen Bipolaris oryzae. The deduced amino acid sequence showed significant homology with Fus3/Kss1 MAPK homologues from other phytopathogenic fungi. The BMK1 disruptants showed impaired hyphal growth, no conidial production, and loss of virulence against rice leaves, indicating that the BMK1 is essential for conidiation and pathogenicity in B. oryzae. PMID:16546358

  9. A Solution of the Direct and Inverse Potential Problems for Arbitrary Cascades of Airfoils

    NASA Technical Reports Server (NTRS)

    Mutterperl, William

    1944-01-01

    Methods are given of determining the potential flow plast an arbitrary cascade of airfoils and the inverse problem of determining an airfoil having a prescribed velocity distribution in cascade. Results indicated that Cartesian mapping function method may be satisfactorily extended to include cascades. Numerical calculation for computing cascades by Cartesian mapping function method is considerably greater than for single airfoils but much less than hitherto required for cascades. Detailed results are presented graphically.

  10. Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors.

    PubMed

    Li, Geoffrey C; Srivastava, Atul K; Kim, Jonggul; Taylor, Susan S; Veglia, Gianluigi

    2015-07-01

    Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme's conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI5-24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme's fold. PMID:26030372

  11. Bakuchiol suppresses proliferation of skin cancer cells by directly targeting Hck, Blk, and p38 MAP kinase

    PubMed Central

    Lee, Younghyun; Yang, Hee; Heo, Yong-Seok; Bode, Ann M.; Lee, Ki Won; Dong, Zigang

    2016-01-01

    Bakuchiol is a meroterpene present in the medicinal plant Psoralea corylifolia, which has been traditionally used in China, India, Japan and Korea for the treatment of premature ejaculation, knee pain, alopecia spermatorrhea, enuresis, backache, pollakiuria, vitiligo, callus, and psoriasis. Here, we report the chemopreventive properties of bakuchiol, which acts by inhibiting epidermal growth factor (EGF)-induced neoplastic cell transformation. Bakuchiol also decreased viability and inhibited anchorage-independent growth of A431 human epithelial carcinoma cells. Bakuchiol reduced A431 xenograft tumor growth in an in vivo mouse model. Using kinase profiling, we identified Hck, Blk and p38 mitogen activated protein kinase (MAPK) as targets of bakuchiol, which directly bound to each kinase in an ATP-competitive manner. Bakuchiol also inhibited EGF-induced signaling pathways downstream of Hck, Blk and p38 MAPK, including the MEK/ERKs, p38 MAPK/MSK1 and AKT/p70S6K pathways. This report is the first mechanistic study identifying molecular targets for the anticancer activity of bakuchiol and our findings indicate that bakuchiol exhibits potent anticancer activity by targeting Hck, Blk and p38 MAPK. PMID:26910280

  12. Phosphorylation of the tubulin-binding protein, stathmin, by Cdk5 and MAP kinases in the brain.

    PubMed

    Hayashi, Kanehiro; Pan, Yong; Shu, Hongjun; Ohshima, Toshio; Kansy, Janice W; White, Charles L; Tamminga, Carol A; Sobel, André; Curmi, Patrick A; Mikoshiba, Katsuhiko; Bibb, James A

    2006-10-01

    Regulation of cytoskeletal dynamics is essential to neuronal plasticity during development and adulthood. Dysregulation of these mechanisms may contribute to neuropsychiatric and neurodegenerative diseases. The neuronal protein kinase, cyclin-dependent kinase 5 (Cdk5), is involved in multiple aspects of neuronal function, including regulation of cytoskeleton. A neuroproteomic search identified the tubulin-binding protein, stathmin, as a novel Cdk5 substrate. Stathmin was phosphorylated by Cdk5 in vitro at Ser25 and Ser38, previously identified as mitogen-activated protein kinase (MAPK) and p38 MAPKdelta sites. Cdk5 predominantly phosphorylated Ser38, while MAPK and p38 MAPKdelta predominantly phosphorylated Ser25. Stathmin was phosphorylated at both sites in mouse brain, with higher levels in cortex and striatum. Cdk5 knockout mice exhibited decreased phospho-Ser38 levels. During development, phospho-Ser25 and -Ser38 levels peaked at post-natal day 7, followed by reduction in total stathmin. Inhibition of protein phosphatases in striatal slices caused an increase in phospho-Ser25 and a decrease in total stathmin. Interestingly, the prefrontal cortex of schizophrenic patients had increased phospho-Ser25 levels. In contrast, total and phospho-Ser25 stoichiometries were decreased in the hippocampus of Alzheimer's patients. Thus, microtubule regulatory mechanisms involving the phosphorylation of stathmin may contribute to developmental synaptic pruning and structural plasticity, and may be involved in neuropsychiatric and neurodegenerative disorders.

  13. Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways

    PubMed Central

    Li, Ko-Jen; Wu, Cheng-Han; Shen, Chieh-Yu; Kuo, Yu-Min; Yu, Chia-Li; Hsieh, Song-Chou

    2016-01-01

    The biological significance of membrane transfer (trogocytosis) between polymorphonuclear neutrophils (PMNs) and mononuclear cells (MNCs) remains unclear. We investigated the biological/immunological effects and molecular basis of trogocytosis among various immune cells in healthy individuals and patients with active systemic lupus erythematosus (SLE). By flow cytometry, we determined that molecules in the immunological synapse, including HLA class-I and-II, CD11b and LFA-1, along with CXCR1, are exchanged among autologous PMNs, CD4+ T cells, and U937 cells (monocytes) after cell-cell contact. Small interfering RNA knockdown of the integrin adhesion molecule CD11a in U937 unexpectedly enhanced the level of total membrane transfer from U937 to PMN cells. Functionally, phagocytosis and IL-8 production by PMNs were enhanced after co-culture with T cells. Total membrane transfer from CD4+ T to PMNs delayed PMN apoptosis by suppressing the extrinsic apoptotic molecules, BAX, MYC and caspase 8. This enhancement of activities of PMNs by T cells was found to be mediated via p38- and P44/42-Akt-MAP kinase pathways and inhibited by the actin-polymerization inhibitor, latrunculin B, the clathrin inhibitor, Pitstop-2, and human immunoglobulin G, but not by the caveolin inhibitor, methyl-β-cyclodextrin. In addition, membrane transfer from PMNs enhanced IL-2 production by recipient anti-CD3/anti-CD28 activated MNCs, and this was suppressed by inhibitors of mitogen-activated protein kinase (PD98059) and protein kinase C (Rottlerin). Of clinical significance, decreased total membrane transfer from PMNs to MNCs in patients with active SLE suppressed mononuclear IL-2 production. In conclusion, membrane transfer from MNCs to PMNs, mainly at the immunological synapse, transduces survival and activation signals to enhance PMN functions and is dependent on actin polymerization, clathrin activation, and Fcγ receptors, while membrane transfer from PMNs to MNCs depends on MAP kinase and

  14. Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin

    SciTech Connect

    Pratheeshkumar, Poyil; Son, Young-Ok; Wang, Xin; Divya, Sasidharan Padmaja; Joseph, Binoy; Hitron, John Andrew; Wang, Lei; Kim, Donghern; Yin, Yuanqin; Roy, Ram Vinod; Lu, Jian; Zhang, Zhuo; Wang, Yitao; and others

    2014-10-01

    Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE{sub 2} and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways. - Highlights: • C3G inhibited UVB-induced oxidative damage and inflammation. • C3G inhibited UVB-induced COX-2, iNOS and PGE{sub 2} production. • C3G

  15. Mapping.

    ERIC Educational Resources Information Center

    Kinney, Douglas M.; McIntosh, Willard L.

    1979-01-01

    The area of geological mapping in the United States in 1978 increased greatly over that reported in 1977; state geological maps were added for California, Idaho, Nevada, and Alaska last year. (Author/BB)

  16. LPLUNC1 Inhibits Nasopharyngeal Carcinoma Cell Growth via Down-Regulation of the MAP Kinase and Cyclin D1/E2F Pathways

    PubMed Central

    Li, Xiaoling; Zhang, Wenling; Fan, Songqing; Shi, Lei; Li, Xiayu; Gong, Zhaojian; Ma, Jian; Zhou, Ming; Xiang, Juanjuan; Peng, Shuping; Xiang, Bo; Deng, Hao; Yang, Yunbo; Li, Yong; Xiong, Wei; Zeng, Zhaoyang; Li, Guiyuan

    2013-01-01

    Long-palate, lung and nasal epithelium clone 1 (LPLUNC1) gene expression is relatively tissue specific. It is highly expressed in nontumor nasopharyngeal epithelial tissues, but its expression is reduced in nasopharyngeal carcinoma (NPC), indicating that LPLUNC1 may be associated with the tumorigenesis of NPC. To study the effects of LPLUNC1 on NPC tumorigenesis, a full-length LPLUNC1 expression plasmid was stably transfected into the NPC cell line, 5-8F. Our data indicated that LPLUNC1 inhibited NPC cell proliferation in vitro and tumor formation in vivo. LPLUNC1 also delayed cell cycle progression from G1 to S phase and inhibited the expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and phosphorylated Rb. To further investigate the molecular mechanisms underlying the suppressive effects of LPLUNC1 on NPC tumorigenesis, cDNA microarray was performed. These studies revealed that LPLUNC1 inhibited the expression of certain mitogen-activated protein (MAP) kinases (MAPK) kinases and cell cycle-related molecules. Western blotting confirmed that the expression of MEK1, phosphorylated ERK1/2, phosphorylated JNK1/2, c-Myc and c-Jun were inhibited by LPLUNC1. Furthermore, the transcriptional activity of AP-1 was down-regulated by LPLUNC1, suggesting that the MAPK signaling pathway is regulated by LPLUNC1. Taken together, the present study indicates that LPLUNC1 delays NPC cell growth by inhibiting the MAPK and cyclin D1/E2F pathways and suggests that LPLUNC1 may represent a promising candidate tumor suppressor gene associated with NPC. PMID:23650533

  17. The three-dimensional structure of MAP kinase p38[beta]: different features of the ATP-binding site in p38[beta] compared with p38[alpha

    SciTech Connect

    Patel, Sangita B.; Cameron, Patricia M.; O'Keefe, Stephen J.; Frantz-Wattley, Betsy; Thompson, Jed; O'Neill, Edward A.; Tennis, Trevor; Liu, Luping; Becker, Joseph W.; Scapin, Giovanna; Merck

    2010-10-18

    The p38 mitogen-activated protein kinases are activated in response to environmental stress and cytokines and play a significant role in transcriptional regulation and inflammatory responses. Of the four p38 isoforms known to date, two (p38{alpha} and p38{beta}) have been identified as targets for cytokine-suppressive anti-inflammatory drugs. Recently, it was reported that specific inhibition of the p38{alpha} isoform is necessary and sufficient for anti-inflammatory efficacy in vivo, while further inhibition of p38{beta} may not provide any additional benefit. In order to aid the development of p38{alpha}-selective compounds, the three-dimensional structure of p38{beta} was determined. To do so, the C162S and C119S,C162S mutants of human MAP kinase p38{beta} were cloned, expressed in Escherichia coli and purified. Initial screening hits in crystallization trials in the presence of an inhibitor led upon optimization to crystals that diffracted to 2.05 {angstrom} resolution and allowed structure determination (PDB codes 3gc8 and 3gc9 for the single and double mutant, respectively). The structure of the p38{alpha} C162S mutant in complex with the same inhibitor is also reported (PDB code 3gc7). A comparison between the structures of the two kinases showed that they are highly similar overall but that there are differences in the relative orientation of the N- and C-terminal domains that causes a reduction in the size of the ATP-binding pocket in p38{beta}. This difference in size between the two pockets could be exploited in order to achieve selectivity.

  18. Single Muscle Immobilization Decreases Single-Fibre Myosin Heavy Chain Polymorphism: Possible Involvement of p38 and JNK MAP Kinases

    PubMed Central

    Derbré, Frédéric; Droguet, Mickaël; Léon, Karelle; Troadec, Samuel; Pennec, Jean-Pierre; Giroux-Metges, Marie-Agnès; Rannou, Fabrice

    2016-01-01

    Purpose Muscle contractile phenotype is affected during immobilization. Myosin heavy chain (MHC) isoforms are the major determinant of the muscle contractile phenotype. We therefore sought to evaluate the effects of muscle immobilization on both the MHC composition at single-fibre level and the mitogen-activated protein kinases (MAPK), a family of intracellular signaling pathways involved in the stress-induced muscle plasticity. Methods The distal tendon of female Wistar rat Peroneus Longus (PL) was cut and fixed to the adjacent bone at neutral muscle length. Four weeks after the surgery, immobilized and contralateral PL were dissociated and the isolated fibres were sampled to determine MHC composition. Protein kinase 38 (p38), extracellular signal-regulated kinases (ERK1/2), and c-Jun- NH2-terminal kinase (JNK) phosphorylations were measured in 6- and 15-day immobilized and contralateral PL. Results MHC distribution in immobilized PL was as follows: I = 0%, IIa = 11.8 ± 2.8%, IIx = 53.0 ± 6.1%, IIb = 35.3 ± 7.3% and I = 6.1 ± 3.9%, IIa = 22.1 ± 3.4%, IIx = 46.6 ± 4.5%, IIb = 25.2 ± 6.6% in contralateral muscle. The MHC composition in immobilized muscle is consistent with a faster contractile phenotype according to the Hill’s model of the force-velocity relationship. Immobilized and contralateral muscles displayed a polymorphism index of 31.1% (95% CI 26.1–36.0) and 39.3% (95% CI 37.0–41.5), respectively. Significant increases in p38 and JNK phosphorylation were observed following 6 and 15 days of immobilization. Conclusions Single muscle immobilization at neutral length induces a shift of MHC composition toward a faster contractile phenotype and decreases the polymorphic profile of single fibres. Activation of p38 and JNK could be a potential mechanism involved in these contractile phenotype modifications during muscle immobilization. PMID:27383612

  19. Differences in cell death induction by Phytophthora Elicitins are determined by signal components downstream of MAP kinase kinase in different species of Nicotiana and cultivars of Brassica rapa and Raphanus sativus.

    PubMed

    Takemoto, Daigo; Hardham, Adrienne R; Jones, David A

    2005-07-01

    Elicitins are small, secreted proteins produced by species of the plant-pathogenic oomycete Phytophthora. They induce hypersensitive cell death in most Nicotiana species and in some cultivars of Brassica rapa and Raphanus sativus. In this study, two true-breeding Fast Cycling B. rapa lines were established that showed severe necrosis (line 7-R) or no visible response (line 18-NR) after treatment with elicitin. Unexpectedly, microscopic examination revealed localized cell death in line 18-NR plants, and expression levels of various defense-marker genes were comparable in both lines. These results suggested that both "responsive" and "nonresponsive" plants responded to elicitin but differed in the extent of the cell death response. Expression of a constitutively active form of Arabidopsis (Arabidopsis thaliana) MAP kinase kinase 4 (AtMEK4(DD)) also induced rapid development of confluent cell death in line 7-R, whereas line 18-NR showed no visible cell death. Similarly, elicitin-responsive Nicotiana species and R. sativus cultivars showed significantly stronger cell death responses following expression of AtMEK4(DD) compared with nonresponsive species/cultivars. Line 7-R also showed higher sensitivity to toxin-containing culture filtrates produced by Alternaria brassicicola, and toxin sensitivity cosegregated with elicitin responsiveness, suggesting that the downstream responses induced by elicitin and Alternaria toxin share factors that control the extent of cell death. Interestingly, elicitin responsiveness was shown to correlate with greater susceptibility to A. brassicicola (a necrotroph) in B. rapa but less susceptibility to Phytophthora nicotianae (a hemibiotroph) in Nicotiana, suggesting a more extensive cell death response could cause opposite effects on the outcomes of biotrophic versus necrotrophic plant-pathogen interactions.

  20. Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases.

    PubMed

    Engstrom, Anna; Wang, Hao; Xia, Zhengui

    2015-08-01

    Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults.

  1. p38 MAP kinase inhibitor suppresses transforming growth factor-β2-induced type 1 collagen production in trabecular meshwork cells.

    PubMed

    Inoue-Mochita, Miyuki; Inoue, Toshihiro; Fujimoto, Tomokazu; Kameda, Takanori; Awai-Kasaoka, Nanako; Ohtsu, Naoki; Kimoto, Kenichi; Tanihara, Hidenobu

    2015-01-01

    Glaucoma is an age-related neurodegenerative disease of retinal ganglion cells, and appropriate turnover of the extracellular matrix in the trabecular meshwork is important in its pathology. Here, we report the effects of Rho-associated kinase (ROCK) and p38 MAP kinase on transforming growth factor (TGF)-β2-induced type I collagen production in human trabecular meshwork cells. TGF-β2 increased RhoA activity, actin polymerization, and myosin light chain 2 phosphorylation. These effects were significantly inhibited by Y-27632, but not SB203580. TGF-β2 also increased promoter activity, mRNA synthesis, and protein expression of COL1A2. These effects were significantly inhibited by SB203580, but not Y-27632. Additionally, Y-27632 did not significantly inhibit TGF-β2-induced promoter activation, or phosphorylation or nuclear translocation of Smad2/3, whereas SB203580 partially suppressed these processes. Collectively, TGF-β2-induced production of type 1 collagen is suppressed by p38 inhibition and accompanied by partial inactivation of Smad2/3, in human trabecular meshwork cells.

  2. Conditional Inhibition of Adult Neurogenesis by Inducible and Targeted Deletion of ERK5 MAP Kinase Is Not Associated with Anxiety/Depression-Like Behaviors1,2

    PubMed Central

    Zou, Junhui; Wang, Wenbin; Pan, Yung-Wei; Abel, Glen M.; Storm, Daniel R.

    2015-01-01

    Abstract Although there is evidence that adult neurogenesis contributes to the therapeutic efficacy of chronic antidepressant treatment for anxiety and depression disorders, the role of adult neurogenesis in the onset of depression-related symptoms is still open to question. To address this issue, we utilized a transgenic mouse strain in which adult neurogenesis was specifically and conditionally impaired by Nestin-CreER-driven, inducible knockout (icKO) of erk5 MAP kinase in Nestin-expressing neural progenitors of the adult mouse brain upon tamoxifen administration. Here, we report that inhibition of adult neurogenesis by this mechanism is not associated with an increase of the baseline anxiety or depression in non-stressed animals, nor does it increase the animal’s susceptibility to depression after chronic unpredictable stress treatment. Our findings indicate that impaired adult neurogenesis does not lead to anxiety or depression. PMID:26464972

  3. Role of the p70 S6 kinase cascade in neutrophilic differentiation and proliferation of HL-60 cells-a study of transferrin receptor-positive and -negative cells obtained from dimethyl sulfoxide- or retinoic acid-treated HL-60 cells.

    PubMed

    Kanayasu-Toyoda, Toshie; Yamaguchi, Teruhide; Oshizawa, Tadashi; Kogi, Mieko; Uchida, Eriko; Hayakawa, Takao

    2002-09-01

    Previously, we suggested that p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells; this conclusion was based on our analysis of transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells that appeared after treatment with dimethyl sulfoxide (Me(2)SO). In this study, we analyzed the upstream of p70 S6K in relation to the differentiation and proliferation of both cell types. The granulocyte colony-stimulating factor (G-CSF)-induced enhancement of phosphatidylinositol 3-kinase (PI3K) activity in Trf-R(+) cells was markedly higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited these activities. The wortmannin-dependent enhancement of neutrophilic differentiation was similar to that induced by rapamycin. From these results, we conclude that the PI3K/p70 S6K cascade may play an important role in negative regulation of neutrophilic differentiation in HL-60 cells. For the G-CSF-dependent proliferation, however, p70 S6K appears to be a highly important pathway through not only a PI3K-dependent but also possibly an independent cascade.

  4. Aloe-Emodin Induces Chondrogenic Differentiation of ATDC5 Cells via MAP Kinases and BMP-2 Signaling Pathways.

    PubMed

    Yang, Ming; Li, Liang; Heo, Seok-Mo; Soh, Yunjo

    2016-07-01

    Endochondral bone formation is the process by which mesenchymal cells condense into chondrocytes, which are ultimately responsible for new bone formation. The processes of chondrogenic differentiation and hypertrophy are critical for bone formation and are therefore highly regulated. The present study was designed to investigate the effect of aloe-emodin on chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Aloe-emodin treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. ATDC5 cells were treated with aloe-emodin and stained with alcian blue. Compared with the control cells, the ATDC5 cells showed more intense alcian blue staining. This finding suggested that aloe-emodin induced the synthesis of matrix proteoglycans and increased the activity of alkaline phosphatase. Aloe-emodin also enhanced the expressions of chondrogenic marker genes such as collagen II, collagen X, BSP and RunX2 in a time-dependent manner. Furthermore, examination of the MAPK signaling pathway showed that aloe-emodin increased the activation of extracellular signal-regulated kinase (ERK), but had no effect on p38 and c-jun N-terminal kinase (JNK). Aloe-emodin also enhanced the protein expression of BMP-2 in a time-dependent manner. Thus, these results showed that aloe-emodin exhibited chodromodulating effects via the BMP-2 or ERK signaling pathway. Aloe-emodin may have potential future applications for the treatment of growth disorders. PMID:27350340

  5. BDNF-mediated regulation of ethanol consumption requires the activation of the MAP kinase pathway and protein synthesis.

    PubMed

    Jeanblanc, Jerome; Logrip, Marian L; Janak, Patricia H; Ron, Dorit

    2013-02-01

    We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494-13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK, but not PLC-γ or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS.

  6. BDNF-mediated regulation of ethanol consumption requires the activation of the MAP kinase pathway and protein synthesis

    PubMed Central

    Jeanblanc, Jerome; Logrip, Marian L.; Janak, Patricia H.; Ron, Dorit

    2013-01-01

    We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494–13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK, but not PLC-γ or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS. PMID:23189980

  7. IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components.

    PubMed

    Ariel, Amiram; Novick, Daniela; Rubinstein, Menachem; Dinarello, Charles A; Lider, Ofer; Hershkoviz, Rami

    2002-07-01

    Cytokines and chemokines play an essential role in recruiting leukocytes from the circulation to the peripheral sites of inflammation by modulating cellular interactions with endothelial cell ligands and extracellular matrix (ECM). Herein, we examined regulation of T cell adhesion to ECM ligands by two major proinflammatory cytokines, interleukin (IL)-12 and IL-18. IL-12 and IL-18 induced T cell adhesion to fibronectin (FN) and hyaluronic acid at low (pM) concentrations that were mediated by specific adhesion molecules expressed on the T cell surface, namely, beta(1) integrins and CD44, respectively. The induction of adhesion by IL-12 and IL-18 was inhibited by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase inhibitors (PD098059 and SB203580, respectively). In contrast, IL-12- and IL-18-induced interferon-gamma (INF-gamma) secretion from T cells was inhibited by SB203580, but not by PD098059. It is interesting that low concentrations of IL-12 and IL-18 induced T cell adhesion to FN in a synergistic manner. Thus, in addition to the regulation of late inflammatory functions such as INF-gamma production, IL-12 and IL-18, alone or in combination, regulate early inflammatory events such as T cell adhesion to inflamed sites. PMID:12101280

  8. The Ste5 scaffold directs mating signaling by catalytically unlocking the Fus3 MAP kinase for activation.

    PubMed

    Good, Matthew; Tang, Grace; Singleton, Julie; Reményi, Attila; Lim, Wendell A

    2009-03-20

    The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3's activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsically poor substrate for Ste7, although the related filamentation MAPK, Kss1, is an excellent substrate. We identify and structurally characterize a domain in Ste5 that catalytically unlocks Fus3 for phosphorylation by Ste7. This domain selectively increases the k(cat) of Ste7-->Fus3 phosphorylation but has no effect on Ste7-->Kss1 phosphorylation. The dual requirement for both Ste7 and this Ste5 domain in Fus3 activation explains why Fus3 is selectively activated by the mating pathway and not by other pathways that also utilize Ste7. PMID:19303851

  9. Aloe-Emodin Induces Chondrogenic Differentiation of ATDC5 Cells via MAP Kinases and BMP-2 Signaling Pathways

    PubMed Central

    Yang, Ming; Li, Liang; Heo, Seok-Mo; Soh, Yunjo

    2016-01-01

    Endochondral bone formation is the process by which mesenchymal cells condense into chondrocytes, which are ultimately responsible for new bone formation. The processes of chondrogenic differentiation and hypertrophy are critical for bone formation and are therefore highly regulated. The present study was designed to investigate the effect of aloe-emodin on chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Aloe-emodin treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. ATDC5 cells were treated with aloe-emodin and stained with alcian blue. Compared with the control cells, the ATDC5 cells showed more intense alcian blue staining. This finding suggested that aloe-emodin induced the synthesis of matrix proteoglycans and increased the activity of alkaline phosphatase. Aloe-emodin also enhanced the expressions of chondrogenic marker genes such as collagen II, collagen X, BSP and RunX2 in a time-dependent manner. Furthermore, examination of the MAPK signaling pathway showed that aloe-emodin increased the activation of extracellular signal-regulated kinase (ERK), but had no effect on p38 and c-jun N-terminal kinase (JNK). Aloe-emodin also enhanced the protein expression of BMP-2 in a time-dependent manner. Thus, these results showed that aloe-emodin exhibited chodromodulating effects via the BMP-2 or ERK signaling pathway. Aloe-emodin may have potential future applications for the treatment of growth disorders. PMID:27350340

  10. Retinotopic map plasticity in adult cat visual cortex is accompanied by changes in Ca2+/calmodulin-dependent protein kinase II alpha autophosphorylation.

    PubMed

    Van den Bergh, G; Eysel, U T; Vandenbussche, E; Vandesande, F; Arckens, L

    2003-01-01

    In adult cats, the induction of homonymous binocular central retinal lesions causes a dramatic reorganization of the topographic map in the sensory-deprived region of the primary visual cortex. To investigate the possible involvement of the alpha-subunit of the calcium/calmodulin dependent protein kinase type II (alphaCaMKII) in this form of brain plasticity, we performed in situ hybridization and Western blotting experiments to analyze mRNA, protein and autophosphorylation levels of this multifunctional kinase. No differences in the mRNA or protein levels were observed between the central, sensory-deprived and the peripheral, non-deprived regions of area 17 of retinal lesion animals or between corresponding cortical regions of normal control animals. Western blotting with an alphaCaMKII threonine-286 phosphorylation-state specific antiserum consistently showed a small, albeit not significant, increase of alphaCaMKII autophosphorylation in the central versus the peripheral region of cortical area 17, and this both in normal subjects as well as in retinal lesion animals with a 3-day post-lesion survival time. In contrast, a post-lesion survival time of 14 days resulted in a alphaCaMKII autophosphorylation level that was four times higher in visually-deprived area 17 than in the non-deprived cortical region. This increased phosphorylation state is not a direct consequence of the decrease in visual activity in these neurons, because we would have expected to see a similar change at shorter or longer post-lesion survival times or in the visually deprived visual cortex of animals in which the left optic tract and the corpus callosum were surgically cut. No such changes were observed, leading to the conclusion that the phosphorylation changes observed at 14 days are related to a delayed reorganization of the retinotopic map of the striate cortex. PMID:12849747

  11. Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

    SciTech Connect

    Black, Adrienne T.; Joseph, Laurie B.; Casillas, Robert P.; Heck, Diane E.; Gerecke, Donald R.; Sinko, Patrick J.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-06-15

    Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 {mu}M) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A{sub 4} (LTA{sub 4}) hydrolase and leukotriene C{sub 4} (LTC{sub 4}) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA{sub 4} hydrolase and LTC{sub 4} synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.

  12. 22(R)-hydroxycholesterol induces HuR-dependent MAP kinase phosphatase-1 expression via mGluR5-mediated Ca(2+)/PKCα signaling.

    PubMed

    Kim, Hyunmi; Woo, Joo Hong; Lee, Jee Hoon; Joe, Eun-Hye; Jou, Ilo

    2016-08-01

    MAP kinase phosphatase (MKP)-1 plays a pivotal role in controlling MAP kinase (MAPK)-dependent (patho) physiological processes. Although MKP-1 gene expression is tightly regulated at multiple levels, the underlying mechanistic details remain largely unknown. In this study, we demonstrate that MKP-1 expression is regulated at the post-transcriptional level by 22(R)-hydroxycholesterol [22(R)-HC] through a novel mechanism. 22(R)-HC induces Hu antigen R (HuR) phosphorylation, cytoplasmic translocation and binding to MKP-1 mRNA, resulting in stabilization of MKP-1 mRNA. The resulting increase in MKP-1 leads to suppression of JNK-mediated inflammatory responses in brain astrocytes. We further demonstrate that 22(R)-HC-induced phosphorylation of nuclear HuR is mediated by PKCα, which is activated in the cytosol by increases in intracellular Ca(2+) levels mediated by the phospholipase C/inositol 1,4,5-triphosphate receptor (PLC/IP3R) pathway and translocates from cytoplasm to nucleus. In addition, pharmacological interventions reveal that metabotropic glutamate receptor5 (mGluR5) is responsible for the increases in intracellular Ca(2+) that underlie these actions of 22(R)-HC. Collectively, our findings identify a novel anti-inflammatory mechanism of 22(R)-HC, which acts through PKCα-mediated cytoplasmic shuttling of HuR to post-transcriptionally regulate MKP-1 expression. These findings provide an experimental basis for the development of a RNA-targeted therapeutic agent to control MAPK-dependent inflammatory responses.

  13. Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana.

    PubMed

    Huang, Shuaishuai; He, Zhangjiang; Zhang, Shiwei; Keyhani, Nemat O; Song, Yulin; Yang, Zhi; Jiang, Yahui; Zhang, Wenli; Pei, Yan; Zhang, Yongjun

    2015-10-01

    The entomopathogenic fungus, Beauveria bassiana, is of environmental and economic importance as an insect pathogen, currently used for the biological control of a number of pests. Cell wall integrity and conidiation are critical parameters for the ability of the fungus to infect insects and for production of the infectious propagules. The contribution of calcineurin and the Slt2 MAP kinase to cell wall integrity and development in B. bassiana was investigated. Gene knockouts of either the calcineurin CNA1 subunit or the Slt2 MAP kinase resulted in decreased tolerance to calcofluor white and high temperature. In contrast, the Δcna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δslt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δslt2 and Δcna1 strains during growth under cell wall stress as compared to the wild type. Both Δslt2 and Δcna1 strains showed significant alterations in growth, conidiation, and viability. Elevation of intracellular ROS levels, and decreased conidial hydrophobicity and adhesion to hydrophobic surfaces, were also seen for both mutants, as well as decreased virulence. Under cell wall stress conditions, inactivation of Slt2 significantly repressed CN-mediated phosphatase activity suggesting some level of cross talk between the two pathways. Comparative transcriptome profiling of the Δslt2 and Δcna1 strains revealed alterations in the expression of distinct gene sets, with overlap in transcripts involved in cell wall integrity, stress response, conidiation and virulence. These data illustrate convergent and divergent phenotypes and targets of the calcineurin and Slt2 pathways in B. bassiana.

  14. Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signaling pathways in SKH-1 mice skin.

    PubMed

    Divya, Sasidharan Padmaja; Wang, Xin; Pratheeshkumar, Poyil; Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Hitron, John Andrew; Wang, Lei; Asha, Padmaja; Shi, Xianglin; Zhang, Zhuo

    2015-04-01

    Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm(2)) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicate that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways.

  15. Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signalling pathways in SKH-1 mice skin

    PubMed Central

    Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Hitron, John Andrew; Wang, Lei; Asha, Padmaja; Shi, Xianglin; Zhang, Zhuo

    2015-01-01

    Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm2) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicates that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways. PMID:25680589

  16. cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation.

    PubMed

    Rodriguez, Pedro; Rojas, Juan

    2016-03-01

    cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation. PMID:26335579

  17. cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation.

    PubMed

    Rodriguez, Pedro; Rojas, Juan

    2016-03-01

    cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation.

  18. Rational approaches towards lead optimization of kinase inhibitors: the issue of specificity.

    PubMed

    Badrinarayan, Preethi; Sastry, G Narahari

    2013-01-01

    Kinases are one of the most popular classes of drug targets as they are involved in signal transduction pathways, which are wired through a phosphotransfer cascade and elicit a number of important and essential physiological responses. Kinase specificity has emerged as one of the major issues to be addressed in drug discovery approaches. In most kinases the active site is the ATP binding site and finding suitable hits which maximize the affinity of binding has been traditionally important to obtain the type I inhibitors. While type I inhibitors have effective binding affinity more often than not they encounter side-effects usually associated with specificity. Therefore in recent times it has become indispensable to optimize specificity for developing effective kinase inhibitors. The review presents an overview of kinase drug discovery and the different strategies used to date for the design of kinase leads accounting for their success and failure. A number of strategies exploiting different aspects of kinases like allosteric site, size of the gatekeeper residue, DFG-loop, chemotype selectivity, non-covalent interactions, salt-bridge, solvation, etc. have been explored to circumvent the specificity problem in kinases. The probable hot-spots in kinases having a propensity to bring in specificity have been delineated with special emphasis on the design of type II inhibitors with increased specificity from existing type I using fragment tailoring approach. In this review we illustrate the current strategies by taking p38 MAP kinase as a model and expect that such strategies are general and can be extended to the other members of the kinase family. PMID:23260022

  19. TGF-β1 inhibition of IFN-γ-induced signaling and Th1 gene expression in CD4+ T cells is Smad3 independent but MAP kinase dependent

    PubMed Central

    Park, Il-Kyoo; Letterio, John J.; Gorham, James D.

    2007-01-01

    In addition to classic Smad signaling pathways, the pleiotropic immunoregulatory cytokine TGF-β1 can activate MAP kinases, but a role for TGF-β1-MAP kinase pathways in T cells has not been defined heretofore. We have shown previously that TGF-β1 inhibits Th1 development by inhibiting IFN-γ’s induction of T-bet and other Th1 differentiation genes, and that TGF-β1 inhibits receptor-proximal IFN-γ-Jak-Stat signaling responses. We now show that these effects of TGF-β1 are independent of the canonical TGF-β1 signaling module Smad3, but involve a specific MAP kinase pathway. In primary T cells, TGF-β1 activated the MEK/ERK and p38 MAP kinase pathways, but not the JNK pathway. Inhibition of the MEK/ERK pathway completely eliminated the inhibitory effects of TGF-β1 on IFN-γ responses in T cells, whereas inhibition of the p38 pathway had no effect. Thus, TGF-β1’s inhibition of IFN-γ signaling in T cells is mediated through a highly specific Smad3-independent, MEK/ERK-dependent signaling pathway. PMID:17403540

  20. p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38

    PubMed Central

    Hori, Takeshi; Moore, Rick

    2016-01-01

    Nuclear receptor constitutive androstane receptor (CAR, NR1I3), which regulates hepatic drug and energy metabolisms as well as cell growth and death, is sequestered in the cytoplasm as its inactive form phosphorylated at threonine 38. CAR activators elicit dephosphorylation, and nonphosphorylated CAR translocates into the nucleus to activate its target genes. CAR was previously found to require p38 mitogen-activated protein kinase (MAPK) to transactivate the cytochrome P450 2B (CYP2B) genes. Here we have demonstrated that p38 MAPK forms a complex with CAR, enables it to bind to the response sequence, phenobarbital-responsive enhancer module (PBREM), within the CYP2B promoter, and thus recruits RNA polymerase II to activate transcription. Subsequently, p38 MAPK elicited rephosphorylation of threonine 38 to inactivate CAR and exclude it from the nucleus. Thus, nuclear p38 MAPK exerted dual regulation by sequentially activating and inactivating CAR-mediated transcription through phosphorylation of threonine 38. PMID:27074912

  1. Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes.

    PubMed

    Pinilla-Macua, Itziar; Watkins, Simon C; Sorkin, Alexander

    2016-02-23

    Signaling from epidermal growth factor receptor (EGFR) to extracellular-stimuli-regulated protein kinase 1/2 (ERK1/2) is proposed to be transduced not only from the cell surface but also from endosomes, although the role of endocytosis in this signaling pathway is controversial. Ras is the only membrane-anchored component in the EGFR-ERK signaling axis, and therefore, its location determines intracellular sites of downstream signaling. Hence, we labeled endogenous H-Ras (HRas) with mVenus fluorescent protein using gene editing in HeLa cells. mVenus-HRas was primarily located at the plasma membrane, and in small amounts in tubular recycling endosomes and associated vesicles. EGF stimulation resulted in fast but transient activation of mVenus-HRas. Although EGF:EGFR complexes were rapidly accumulated in endosomes together with the Grb2 adaptor, very little, if any, mVenus-HRas was detected in these endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 remained high beyond the point of the physical separation of HRas from EGF:EGFR complexes and down-regulation of Ras activity. Paradoxically, this sustained MEK1/2 and ERK1/2 activation was dependent on the active EGFR kinase. Cell surface biotinylation and selective inactivation of surface EGFRs suggested that a small fraction of active EGFRs remaining in the plasma membrane is responsible for continuous signaling to MEK1/2 and ERK1/2. We propose that, under physiological conditions of cell stimulation, EGFR endocytosis serves to spatially separate EGFR-Grb2 complexes and Ras, thus terminating Ras-mediated signaling. However, sustained minimal activation of Ras by a small pool of active EGFRs in the plasma membrane is sufficient for extending MEK1/2 and ERK1/2 activities.

  2. Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes

    PubMed Central

    Pinilla-Macua, Itziar; Watkins, Simon C.; Sorkin, Alexander

    2016-01-01

    Signaling from epidermal growth factor receptor (EGFR) to extracellular-stimuli–regulated protein kinase 1/2 (ERK1/2) is proposed to be transduced not only from the cell surface but also from endosomes, although the role of endocytosis in this signaling pathway is controversial. Ras is the only membrane-anchored component in the EGFR–ERK signaling axis, and therefore, its location determines intracellular sites of downstream signaling. Hence, we labeled endogenous H-Ras (HRas) with mVenus fluorescent protein using gene editing in HeLa cells. mVenus-HRas was primarily located at the plasma membrane, and in small amounts in tubular recycling endosomes and associated vesicles. EGF stimulation resulted in fast but transient activation of mVenus-HRas. Although EGF:EGFR complexes were rapidly accumulated in endosomes together with the Grb2 adaptor, very little, if any, mVenus-HRas was detected in these endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 remained high beyond the point of the physical separation of HRas from EGF:EGFR complexes and down-regulation of Ras activity. Paradoxically, this sustained MEK1/2 and ERK1/2 activation was dependent on the active EGFR kinase. Cell surface biotinylation and selective inactivation of surface EGFRs suggested that a small fraction of active EGFRs remaining in the plasma membrane is responsible for continuous signaling to MEK1/2 and ERK1/2. We propose that, under physiological conditions of cell stimulation, EGFR endocytosis serves to spatially separate EGFR–Grb2 complexes and Ras, thus terminating Ras-mediated signaling. However, sustained minimal activation of Ras by a small pool of active EGFRs in the plasma membrane is sufficient for extending MEK1/2 and ERK1/2 activities. PMID:26858456

  3. Haemophilus parasuis induces activation of NF-κB and MAP kinase signaling pathways mediated by toll-like receptors.

    PubMed

    Chen, Yushan; Liu, Ting; Langford, Paul; Hua, Kexin; Zhou, Shanshan; Zhai, Yajun; Xiao, Hongde; Luo, Rui; Bi, Dingren; Jin, Hui; Zhou, Rui

    2015-06-01

    Glässer's disease in pigs caused by Haemophilus parasuis is characterized by a severe membrane inflammation. In our previous study, we have identified activation of the transcription factor NF-κB after H. parasuis infection of porcine epithelial cells. In this study, we found that H. parasuis infection also contributed to the activation of p38/JNK MAPK pathway predominantly linked to inflammation, but not the ERK MAPK pathway associated with growth, differentiation and development. Inhibition of NF-κB, p38 and JNK but not ERK activity significantly reduced IL-8 and CCL4 expression by H. parasuis. We also found TLR1, TLR2, TLR4 and TLR6 were required for NF-κB, p38 and JNK MAPK activation. Furthermore, MyD88 and TRIF signaling cascades were essential for H. parasuis-induced NF-κB activation. These results provided new insights into the molecular pathways underlying the inflammatory response induced by H. parasuis.

  4. Rapid activation of sodium-proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades.

    PubMed Central

    Garnovskaya, M N; Mukhin, Y; Raymond, J R

    1998-01-01

    These experiments tested the hypothesis that signalling elements involved in the activation of the extracellular signal-regulated protein kinase (ERK) mediate rapid activation of sodium-proton exchange (NHE) in fibroblasts when both signals are initiated by a single G protein-coupled receptor, the 5-HT1A receptor. Similarities between the two processes were comparable concentration-response curves and time-courses, and overlapping sensitivity to some pharmacological inhibitors of tyrosine kinases (staurosporine and genistein), and phosphoinositide 3'-kinase (wortmannin and LY204002). Activation of NHE was much more sensitive to the phosphatidylcholine-specific phospholipase inhibitor (D609) than was ERK. Neither pathway was sensitive to manoeuvres designed to block PKC. In contrast, Src or related kinases appear to be required to activate ERK, but not NHE. Transfection of cDNA constructs encoding inactive mutant phosphoinositide 3'-kinase, Grb2, Sos, Ras, and Raf molecules were successful in attenuating ERK, but had essentially no effect upon NHE activation. Finally, PD98059, an inhibitor of mitogen activated/extracellular signal regulated kinase kinase, blocked ERK but not NHE activation. Thus, in CHO fibroblast cells, activation by the 5-HT1A receptor of ERK and NHE share a number of overlapping features. However, our studies do not support a major role for ERK, when activated by the 5-HT1A receptor, as a short-term upstream regulator of NHE activity. PMID:9461547

  5. Mitochondrial DAMPs from femoral reamings activate neutrophils via formyl peptide receptors and P44/42 MAP Kinase

    PubMed Central

    Hauser, Carl J.; Sursal, Tolga; Rodriguez, Edward K.; Appleton, Paul T.; Zhang, Qin; Itagaki, Kiyoshi

    2010-01-01

    Hypothesis Fractures and femoral reaming are associated with lung injury. The mechanisms linking fractures and inflammation are unclear; but tissue disruption might release mitochondria. Mitochondria are evolutionarily derived from bacteria and contain “Damage Associated Molecular Patterns” (DAMPs) like formylated peptides that can activate immunocytes. We therefore studied whether fracture reaming releases mitochondrial DAMPs (MTD) and how MTD act on immune cells. Methods Femur fracture reamings (FFx) from 10 patients were spun to remove bone particulates. Supernatants were assayed for mitochondrial DNA (mtDNA). Mitochondria were isolated from the residual reaming slurry, sonicated and spun at 12,000g. The resultant MTD were assayed for their ability to cause neutrophil (PMN) Ca2+ transient production, p44/42 MAPK phosphorylation, IL-8 release and matrix metalloproteinase-9 (MMP9) release with and without formyl peptide receptor-1 (FPR1) blockade. Rats were injected with MTD and whole lung assayed for p44/42 activation. Results mtDNA appears at many thousand fold normal plasma levels in FFx and at intermediate levels in patients’ plasma, suggesting release from fracture to plasma. FFx MTD caused brisk PMN Ca2+ flux, activated PMN p44/42 MAPK and caused PMN release of IL-8 and MMP9. Responses to MTD were inhibited by FPR1 blockade using Cyclosporin H and anti-FPR1. MTD injection caused P44/42 phosphorylation in rat lung. Conclusions FFx reaming releases mitochondria into the wound and circulation. MTD then activates PMN. Release of damage signals like MTD from FFx may underlie activation of the cytokine cascades known to be associated with facture fixation and lung injury. PMID:20736789

  6. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  7. Hydrogen Peroxide–Mediated Activation of MAP Kinase 6 Modulates Nitric Oxide Biosynthesis and Signal Transduction in Arabidopsis[w

    PubMed Central

    Wang, Pengcheng; Du, Yanyan; Li, Yuan; Ren, Dongtao; Song, Chun-Peng

    2010-01-01

    Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological and developmental processes in plants, including lateral root development. In this study, we used biochemical and genetic approaches to analyze the function of Arabidopsis thaliana mitogen-activated protein kinase 6 (MPK6) in the regulation of NO synthesis in response to hydrogen peroxide (H2O2) during lateral root development. In both mpk6 mutants studied, H2O2-induced NO synthesis and nitrate reductase (NR) activity were decreased dramatically. Furthermore, one NR isoform, NIA2, was required for the MPK6-mediated production of NO induced by H2O2. Notably, NIA2 interacted physically with MPK6 in vitro and in vivo and also served as a substrate of MPK6. Phosphorylation of NIA2 by MPK6 led to an increase in NR activity, and Ser-627 was identified as the putative phosphorylation site on NIA2. Phenotypical analysis revealed that mpk6-2 and mpk6-3 seedlings produce more and longer lateral roots than wild-type plants did after application of the NO donor sodium nitroprusside or H2O2. These data support strongly a function of MPK6 in modulating NO production and signal transduction in response to H2O2 during Arabidopsis root development. PMID:20870959

  8. Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4.

    PubMed

    Brodersen, Peter; Petersen, Morten; Bjørn Nielsen, Henrik; Zhu, Shijiang; Newman, Mari-Anne; Shokat, Kevan M; Rietz, Steffen; Parker, Jane; Mundy, John

    2006-08-01

    Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective in defense gene induction in response to ethylene (ET), and that they are more susceptible than wild-type (WT) to Alternaria brassicicola that induces the ET/JA defense pathway(s). Both SA-repressing and ET/JA-(co)activating functions depend on MPK4 kinase activity and involve the defense regulators EDS1 and PAD4, as mutations in these genes suppress de-repression of the SA pathway and suppress the block of the ET/JA pathway in mpk4. EDS1/PAD4 thus affect SA-ET/JA signal antagonism as activators of SA but as repressors of ET/JA defenses, and MPK4 negatively regulates both of these functions. We also show that the MPK4-EDS1/PAD4 branch of ET defense signaling is independent of the ERF1 transcription factor, and use comparative microarray analysis of ctr1, ctr1/mpk4, mpk4 and WT to show that MPK4 is required for induction of a small subset of ET-regulated genes. The regulation of some, but not all, of these genes involves EDS1 and PAD4.

  9. Design and synthesis of 5-[(2-chloro-6-fluorophenyl)acetylamino]-3-(4-fluorophenyl)-4-(4-pyrimidinyl)isoxazole (AKP-001), a novel inhibitor of p38 MAP kinase with reduced side effects based on the antedrug concept.

    PubMed

    Hasumi, Koichi; Sato, Shuichiro; Saito, Takahisa; Kato, Jun-ya; Shirota, Kazuhiko; Sato, Jun; Suzuki, Hiroyuki; Ohta, Shuji

    2014-08-01

    Inhibitors of p38 mitogen-activated protein (MAP) kinase, which are closely involved in the production of inflammatory cytokines, are considered promising curative drugs for chronic inflammatory disorders. However, there is also a growing concern regarding its systemic side effects. To reduce the occurrence of side effects, we have identified a novel p38 MAP kinase inhibitor that shows properties of an antedrug, which imparts its effect solely on the inflammatory site and is metabolically inactivated right after. We have designed isoxazole derivatives through the addition of a fresh interacting fourth site to the structure of the prototypical p38 MAP kinase inhibitor that harbors three point interactive sites. The derivative 26d (AKP-001) shows excellent p38 MAP kinase inhibitory activity and a high selectivity for various kinases. Its rapid metabolism has been confirmed in rats. Moreover, 26d has been shown to be effective in animal models of inflammatory bowel disease.

  10. Insulin inhibits glucocorticoid-stimulated L-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression by activation of the c-Jun N-terminal kinase pathway.

    PubMed Central

    De Los Pinos E; Fernández De Mattos S; Joaquin, M; Tauler, A

    2001-01-01

    The hepatic isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K/Fru-2,6-BPase) is transcriptionally stimulated by glucocorticoids, whereas insulin blocks this stimulatory effect. Although this inhibitory effect has been extensively reported, nothing is known about the signalling pathway responsible. We have used well-characterized inhibitors for proteins involved in different signalling cascades to assess the involvement of these pathways on the transcriptional regulation of glucocorticoid-stimulated PF2K/Fru-2,6-BPase by insulin. Our results demonstrate that the phosphoinositide 3-kinase, p70/p85 ribosomal S6 kinase, extracellular signal-regulated protein kinase (ERK)1/2 and p38 mitogen-activated protein (MAP) kinase pathways are not involved in the inhibitory effect of insulin on glucocorticoid-stimulated PF2K/Fru-2,6-BPase. To evaluate the implication of the MAP kinase/ERK kinase (MEK)-4-stress-activated protein kinase-c-Jun-N-terminal protein kinase ('JNK-SAPK') pathway we overexpressed the N-terminal JNK-binding domain of the JNK-interacting protein 1 ('JIP-1'), demonstrating that activation of JNK is necessary for the insulin inhibitory effect. Moreover, overexpression of MEK kinase 1 and JNK-haemagglutinin resulted in the inhibition of the glucocorticoid-stimulated PF2K/Fru-2,6-BPase. These results provide clear and specific evidence for the role of JNK in the insulin inhibition of glucocorticoid-stimulated PF2K/Fru-2,6-BPase gene expression. In addition, we performed experiments with a mutant of the glucocorticoid receptor in which the JNK phosphorylation target Ser-246 had been mutated to Ala. Our results demonstrate that the phosphorylation of the glucocorticoid receptor on Ser-246 is not responsible for the JNK repression of glucocorticoid-stimulated PF2K/Fru-2,6-BPase gene expression. PMID:11139390

  11. Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signaling pathways in SKH-1 mice skin

    SciTech Connect

    Divya, Sasidharan Padmaja; Wang, Xin; Pratheeshkumar, Poyil; Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Hitron, John Andrew; Wang, Lei; Asha, Padmaja; Shi, Xianglin; Zhang, Zhuo

    2015-04-01

    Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm{sup 2}) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E{sub 2} (PGE{sub 2}), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicate that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways. - Highlights: • Blackberry extract inhibits UVB-induced glutathione depletion.

  12. Dual modulation of ERK1/2 and p38 MAP kinase activities induced by minocycline reverses the neurotoxic effects of the prion protein fragment 90-231.

    PubMed

    Corsaro, Alessandro; Thellung, Stefano; Chiovitti, Katia; Villa, Valentina; Simi, Alessandro; Raggi, Federica; Paludi, Domenico; Russo, Claudio; Aceto, Antonio; Florio, Tullio

    2009-02-01

    Several in vitro and in vivo studies addressed the identification of molecular determinants of the neuronal death induced by PrP(Sc) or related peptides. We developed an experimental model to assess PrP(Sc) neurotoxicity using a recombinant polypeptide encompassing amino acids 90-231 of human PrP (hPrP90-231) that corresponds to the protease-resistant core of PrP(Sc) identified in prion-infected brains. By means of mild thermal denaturation, we can convert hPrP90-231 from a PrP(C)-like conformation into a PrP(Sc)-like structure. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing caspase 3 and p38-dependent apoptosis, while in the native alpha-helix-rich conformation, hPrP90-231 did not induce cell toxicity. The aim of this study was to identify drugs able to block hPrP90-231 neurotoxic effects, focusing on minocycline, a tetracycline with known neuroprotective activity. hPrP90-231 caused a caspase 3-dependent apoptosis via the blockade of ERK1/2 activation and the subsequent activation of p38 MAP kinase. We propose that hPrP90-231-induced apoptosis is dependent on the inhibition of ERK1/2 responsiveness to neurotrophic factors, removing a tonic inhibition of p38 activity and resulting in caspase 3 activation. Minocycline prevented hPrP90-231-induced toxicity interfering with this mechanism: the pretreatment with this tetracycline restored ERK1/2 activity and reverted p38 and caspase 3 activities. The effects of minocycline were not mediated by the prevention of hPrP90-231 structural changes or cell internalization (differently from Congo Red). In conclusion, minocycline elicits anti-apoptotic effects against the neurotoxic activity of hPrP90-231 and these effects are mediated by opposite modulation of ERK1/2 and p38 MAP kinase activities.

  13. Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein.

    PubMed

    Ohtsuka, T; Shimizu, K; Yamamori, B; Kuroda, S; Takai, Y

    1996-01-19

    Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.

  14. Activation of p38 Mitogen-Activated Protein Kinase Promotes Epidermal Growth Factor Receptor Internalization

    PubMed Central

    Vergarajauregui, Silvia; Miguel, Anitza San; Puertollano, Rosa

    2006-01-01

    Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions. PMID:16683917

  15. Cascade Mtns. Oregon

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The ground near one of the long-dormant Three Sisters volcanoes in the Cascade Mountains of west-central Oregon has risen approximately 10centimeters in a 10-by-20-km parcel since 1996, meaning that magma or underground lava is slowly flowing into the area, according to a research team from the U.S. Geological Survey. The Three Sisters area -- which contains five volcanoes -- is only about 170 miles from Mount St. Helens, which erupted in 1980. Both are part of the Cascades Range, a line of 27volcanoes stretching from British Columbia in Canada to northern California. This perspective view was created by draping a simulated natural color ASTER image over digital topography from the U.S. Geological Survey National Elevation Dataset.

    This image was acquired on May 28, 2000 by the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) on NASA's Terra satellite. With its 14 spectral bands from the visible to the thermal infrared wavelength region, and its high spatial resolution of 15 to 90 meters (about 50 to 300 feet), ASTER will image Earth for the next 6 years to map and monitor the changing surface of our planet.

    ASTER is one of five Earth-observing instruments launched December 18,1999, on NASA's Terra satellite. The instrument was built by Japan's Ministry of Economy, Trade and Industry. A joint U.S./Japan science team is responsible for validation and calibration of the instrument and the data products. Dr. Anne Kahle at NASA's Jet Propulsion Laboratory, Pasadena, California, is the U.S. Science team leader; Bjorn Eng of JPL is the project manager. ASTER is the only high resolution imaging sensor on Terra. The Terra mission is part of NASA's Earth Science Enterprise, along-term research and technology program designed to examine Earth's land, oceans, atmosphere, ice and life as a total integrated system.

    The broad spectral coverage and high spectral resolution of ASTER will provide scientists in numerous disciplines with critical

  16. Anti-apoptotic signaling of pleiotrophin through its receptor, anaplastic lymphoma kinase.

    PubMed

    Bowden, Emma T; Stoica, Gerald E; Wellstein, Anton

    2002-09-27

    The secreted growth factor pleiotrophin (PTN) can induce mitogenesis in cells that express the receptor for this growth factor, anaplastic lymphoma kinase (ALK). Here we examine the ability of PTN to produce anti-apoptotic signals. We demonstrate that PTN is a survival factor for SW-13 epithelial cells and show that ribozyme-mediated depletion of ALK from SW-13 cells abolishes this effect of PTN. Furthermore, in serum-starved NIH3T3 fibroblasts PTN prevents apoptosis (measured by annexin V staining) with an EC(50) of 0.2 ng/ml and induces cell growth at higher concentrations of PTN. A polyclonal antibody against the PTN ligand-binding domain of the ALK receptor (alpha-LBD) was a partial agonist for ALK in NIH3T3 cells. This alpha-LBD antibody showed high agonist activity for anti-apoptosis (56 +/- 9% relative to PTN), low agonist activity for cell growth (21 +/- 1% relative to PTN), and was an antagonist of PTN-induced cell growth (61 +/- 2% inhibition). Both MAP kinase and phosphatidylinositol (PI) 3-kinase cascades in NIH3T3 cells were activated by PTN, and this effect persisted for up to 3 h. Surprisingly, the anti-apoptotic effect of PTN was completely blocked by the MAP kinase inhibitor UO126, but was not affected by the PI 3-kinase inhibitor LY294002. In contrast, PTN-dependent cell growth required both MAPK and PI 3-kinase activity. We conclude that anti-apoptotic signaling of PTN through ALK in NIH3T3 fibroblasts is via the MAP kinase pathway.

  17. p38 MAP kinase-dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER-mitochondria association and mitochondria motility.

    PubMed

    Li, Lei; Gao, Guang; Shankar, Jay; Joshi, Bharat; Foster, Leonard J; Nabi, Ivan R

    2015-11-01

    Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8-amino acid motif (533-541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41-amino acid region containing 14-3-3- and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER-mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER-mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER-mitochondria association and mitochondria motility.

  18. Axodendritic contacts onto calcium/calmodulin-dependent protein kinase type II-expressing neurons in the barn owl auditory space map.

    PubMed

    Rodriguez-Contreras, Adrian; Liu, Xiao-Bo; DeBello, William M

    2005-06-01

    In the owl midbrain, a map of auditory space is synthesized in the inferior colliculus (IC) and conveyed to the optic tectum (OT). Ascending auditory information courses through these structures via topographic axonal projections. Little is known about the molecular composition of projection neurons or their postsynaptic targets. To visualize axodendritic contacts between identified cell types, we used double-label immunohistochemistry, in vivo retrograde tracing, in vitro anterograde tracing, high-resolution confocal microscopy, three-dimensional reconstruction and fly-through visualization. We discovered a major class of IC neurons that strongly expressed calcium/calmodulin-dependent protein kinase type II, alpha subunit (CaMKII). The distribution of these cells within the IC was mostly restricted to the external nucleus of the IC (ICX), in which the auditory space map is assembled. A large proportion of ICX-OT projection neurons were CaMKII positive. In addition to being the principal outputs, CaMKII cells were in direct contact with axonal boutons emanating from the main source of input to ICX, the lateral shell of the central nucleus of the inferior colliculus (ICCls). Numerous sites of putative synaptic contact were found on the somata, proximal dendrites, and distal dendrites. Double-label immunoelectron microscopy confirmed the existence of synapses between ICCls axons and the dendrites of CaMKII cells. Collectively, our data indicate that CaMKII ICX neurons are a cellular locus for the computation of auditory space-specific responses. Because the ICCls-ICX projection is physically altered during experience-dependent plasticity, these results lay the groundwork for probing microanatomical rearrangements that may underlie plasticity and learning. PMID:15944389

  19. Evaluate ERTS imagery for mapping and detection of changes of snowcover on land and on glaciers. [Cascade Range, Washington and Tweedsmuir Glacier, Alaska

    NASA Technical Reports Server (NTRS)

    Meier, M. F. (Principal Investigator)

    1974-01-01

    The author has identified the following significant results. The area of snowcover on 10 individual drainage basins in the North Cascades, Washington, has been determined by use of a semi-automatic radiance threshold technique. The result is a unique record of the changing water storage as snow in these important hydrologic units, the runoff of which is utilized for hydroelectric power, dilution of wastes and heat, support of salmon migration, and irrigation. These data allow a new type of hydrologic modelling to proceed which should permit more accurate forecasts of streamflow. A new technique has been developed for measuring snow-covered area or snowline altitude semi-automatically. This variable contour overlay method permits the snowcover to be matched efficiently to the best fit contour of altitude. The motion of the Yentna Glacier during the concluding phase of its surge was successfully measured by a flicker technique using images of two dates. It appears that displacements as small as 100 to 200 m can be measured. Motion of the Tweedsmuir Glacier in Alaska was measured using ERTS-1 images enlarged to 1:50,000. Changes detected included a shock wave moving down the glacier, the margin expanding, the moraine pattern deforming, and the marginal valley deepening.

  20. Are free energy calculations useful in practice? A comparison with rapid scoring functions for the p38 MAP kinase protein system.

    PubMed

    Pearlman, D A; Charifson, P S

    2001-10-11

    Precise thermodynamic integration free energy simulations have been applied to a congeneric series of 16 inhibitors to the p38 MAP kinase protein for which the experimental binding data (IC(50)) is known. The relative free energy of binding for each compound has been determined. For comparison, the same series of compounds have also scored using the best rapid scoring functions used in database screening. From the results of these calculations, we find (1) that precise free energy simulations allow predictions that are reliable and in good agreement with experiment; (2) that predictions of lower reliability, but still in good qualitative agreement with experiment, can be obtained using the OWFEG free energy grid method, at a much lower computational cost; (3) and that other methods, not based on free energy simulations yield results in much poorer agreement with experiment. A new predictive index, which measures the reliability of a prediction method in the context of normal use, is defined and calculated for each scoring method. Predictive indices of 0.84, 0.56, 0.04, -0.05, and 0.25 are calculated for thermodynamic integration, OWFEG, ChemScore, PLPScore, and Dock Energy Score, respectively, where +1.0 is perfect correct prediction, -1.0 is perfect incorrect prediction, and 0.0 is random.

  1. Involvement of the H1 histamine receptor, p38 MAP kinase, MLCK, and Rho/ROCK in histamine-induced endothelial barrier dysfunction

    PubMed Central

    Adderley, Shaquria P.; Zhang, Xun E.; Breslin, Jerome W.

    2015-01-01

    Objective The mechanisms by which histamine increases microvascular permeability remain poorly understood. We tested the hypothesis that H1 receptor activation disrupts the endothelial barrier and investigated potential downstream signals. Methods We used confluent endothelial cell (EC) monolayers, assessing transendothelial electrical resistance (TER) as an index of barrier function. Human umbilical vein EC (HUVEC), cardiac microvascular EC (HCMEC), and dermal microvascular EC (HDMEC) were compared. Receptor expression was investigated using Western blotting, immunofluorescence (IF) confocal microscopy and RT-PCR. Receptor function and downstream signaling pathways were tested using pharmacologic antagonists and inhibitors, respectively. Results We identified H1-H4 receptors on all three EC types. H1 antagonists did not affect basal TER but prevented the histamine-induced decrease in TER. Blockade of H2 or H3 attenuated the histamine response only in HDMEC, while inhibition of H4 attenuated the response only in HUVEC. Combined inhibition of both PKC and PI3K caused exaggerated histamine-induced barrier dysfunction in HDMEC, whereas inhibition of p38 MAP kinase attenuated the histamine response in all three EC types. Inhibition of RhoA, ROCK, or MLCK also prevented the histamine-induced decrease in TER in HDMEC. Conclusion The data suggest that multiple signaling pathways contribute to histamine-induced endothelial barrier dysfunction via the H1 receptor. PMID:25582918

  2. Targeting the MAP kinase pathway in astrocytoma cells using a recombinant anthrax lethal toxin as a way to inhibit cell motility and invasion.

    PubMed

    Al-Dimassi, Saleh; Salloum, Gilbert; Saykali, Bechara; Khoury, Oula; Liu, Shihui; Leppla, Stephen H; Abi-Habib, Ralph; El-Sibai, Mirvat

    2016-05-01

    Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Understanding and targeting cancer cell invasion is an important therapeutic approach. Cell invasion is a complex process that replies on many signaling pathways including the mitogen-activated protein (MAP) kinase (MAPK). In many cell lines, the use of MAPK-targeted drugs proved to be a potential method to inhibit cancer cell motility. In the present study, we use a recombinant anthrax lethal toxin (LeTx), which selectively inhibits the MAPK pathway, in order to target invasion. LeTx proved ineffective on cell survival in astrocytoma (as well as normal cells). However, astrocytoma cells that were treated with LeTx showed a significant decrease in cell motility as seen by wound healing as well as random 2D motility in serum. The cells also showed a decrease in invasion across a collagen matrix. The effect of LeTx on cell migration was mediated though the deregulation of Rho GTPases, which play a role in cell motility. Finally, the effect of LeTx on cell migration and Rho GTPases was mimicked by the inhibition of the MAPK pathway. In this study, we describe for the first time the effect of the LeTx on cancer cell motility and invasion not cell survival making it a potentially selective brain tumor invasion inhibitor. PMID:26984023

  3. Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8

    SciTech Connect

    Sipley, J.D.; Anderson, C.W.; Menninger, J.C.

    1995-08-01

    The DNA-activated serine/threonine protein kinase (DNA-PK) is composed for a large ({approximately}460 kDa) catalytic polypeptide (DNA-PK{sub cs}) and Ku, a heterodimeric DNA-binding component (p70/p80) that targets DNA-PK{sub cs} to DNA. A 41-kbp segment of the DNA-PK{sub cs} gene was isolated, and a 7902-bp segment was sequenced. The sequence contains a polymorphic Pvu II restriction enzyme site, and comparing the sequence with that of the cDNA revealed the positions of nine exons. The DNA-PK{sub cs} gene was mapped to band q11 of chromosome 8 by in situ hybridization. This location is coincident with that of XRCC7, the gene that complements the DNA double-strand break repair and V(D)J recombination defects (where V is variable, D is diversity, and J is joining) of hamster V3 and murine severe combined immunodeficient (scid) cells. 50 refs., 4 figs., 2 tabs.

  4. Dynamic control of yeast MAP kinase network by induced association and dissociation between the Ste50 scaffold and the Opy2 membrane anchor.

    PubMed

    Yamamoto, Katsuyoshi; Tatebayashi, Kazuo; Tanaka, Keiichiro; Saito, Haruo

    2010-10-01

    Membrane localization of the Ste11 MAPKKK is essential for activation of both the filamentous growth/invasive growth (FG/IG) MAP kinase (MAPK) pathway and the SHO1 branch of the osmoregulatory HOG MAPK pathway, and is mediated by binding of the Ste50 scaffold protein to the Opy2 membrane anchor. We found that Opy2 has two major (CR-A and CR-B), and one minor (CR-D), binding sites for Ste50. CR-A binds Ste50 constitutively and can transmit signals to both the Hog1 and Fus3/Kss1 MAPKs. CR-B, in contrast, binds Ste50 only when Opy2 is phosphorylated by Yck1/Yck2 under glucose-rich conditions and transmits the signal preferentially to the Hog1 MAPK. Ste50 phosphorylation by activated Hog1/Fus3/Kss1 MAPKs downregulates the HOG MAPK pathway by dissociating Ste50 from Opy2. Furthermore, Ste50 phosphorylation, together with MAPK-specific protein phosphatases, reduces the basal activity of the HOG and the mating MAPK pathways. Thus, dynamic regulation of Ste50-Opy2 interaction fine-tunes the MAPK signaling network. PMID:20932477

  5. The FUS3/KSS1-type MAP kinase gene FPK1 is involved in hyphal growth, conidiation and plant infection of Fusarium proliferatum.

    PubMed

    Zhao, Pei-Bao; Ren, Ai-Zhi; Li, Duo-Chuan

    2011-01-01

    Fusarium proliferatum is an important pathogen of maize that is responsible for ear rots, stalk rots and seeding blight worldwide. During the past decade, F. proliferatum has caused several severe epidemics of maize seedling blight in many areas of China, which led to significant losses in maize. To understand the molecular mechanisms in the fungal developmental regulation and pathogenicity, we isolated and characterized the FPK1 gene (GenBank accession No. HQ844224) encoding a MAP kinase homolog of FUS3/KSS1 in yeast. The gene includes a 1,242-bp DNA sequence from ATG to TAA, with a coding region of 1,068 bp, 3 introns (58 bp, 56 bp and 60 bp) and a predicted protein of 355 aa.The mutant ΔFPK1, which has a disruption of the FPK1 gene, showed reduced vegetative growth, fewer and shorter aerial mycelia, strongly impaired conidiation and spore germination, as well as deviant germ tube outgrowth. When the strain was inoculated in susceptible maize varieties, the infection of the mutant ΔFPK1 was delayed, and the infection efficiency was reduced compared to the wild-type strain. Complementation of the disruptions within the FPK1 open reading frame restored wild-type levels of conidiation, growth rate and virulence to maize seedlings. Our results indicated that the FPK1 gene functioned in hyphal growth, conidiation, spore germination and virulence in F. proliferatum. PMID:22286038

  6. Repression of ergosterol biosynthesis is essential for stress resistance and is mediated by the Hog1 MAP kinase and the Mot3 and Rox1 transcription factors.

    PubMed

    Montañés, Fernando Martínez; Pascual-Ahuir, Amparo; Proft, Markus

    2011-02-01

    Hyperosmotic stress triggers a complex adaptive response that is dominantly regulated by the Hog1 MAP kinase in yeast. Here we characterize a novel physiological determinant of osmostress tolerance, which involves the Hog1-dependent transcriptional downregulation of ergosterol biosynthesis genes (ERG). Yeast cells considerably lower their sterol content in response to high osmolarity. The transcriptional repressors Mot3 and Rox1 are essential for this response. Both factors together with Hog1 are required to rapidly and transiently shut down transcription of ERG2 and ERG11 upon osmoshock. Mot3 abundance and its binding to the ERG2 promoter is stimulated by osmostress in a Hog1-dependent manner. As an additional layer of control, the expression of the main transcriptional activator of ERG gene expression, Ecm22, is negatively regulated by Hog1 and Mot3/Rox1 upon salt shock. Oxidative stress also triggers repression of ERG2, 11 transcription and a profound decrease in total sterol levels. However, this response was only partially dependent on Mot3/Rox1 and Hog1. Finally, we show that the upc2-1 mutation confers stress insensitive hyperaccumulation of ergosterol, overexpression of ERG2, 11 and severe sensitivity to salt and oxidative stress. Our results indicate that transcriptional control of ergosterol biosynthesis is an important physiological target of stress signalling.

  7. The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger.

    PubMed

    Priegnitz, Bert-Ewald; Brandt, Ulrike; Pahirulzaman, Khomaizon A K; Dickschat, Jeroen S; Fleißner, André

    2015-06-01

    Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.

  8. Network effects, cascades and CCP interoperability

    NASA Astrophysics Data System (ADS)

    Feng, Xiaobing; Hu, Haibo; Pritsker, Matthew

    2014-03-01

    To control counterparty risk, financial regulations such as the Dodd Frank Act are increasingly requiring standardized derivatives trades to be cleared by central counterparties (CCPs). It is anticipated that in the near-term future, CCPs across the world will be linked through interoperability agreements that facilitate risk-sharing but also serve as a conduit for transmitting shocks. This paper theoretically studies a network with CCPs that are linked through interoperability arrangements, and studies the properties of the network that contribute to cascading failures. The magnitude of the cascading is theoretically related to the strength of network linkages, the size of the network, the logistic mapping coefficient, a stochastic effect and CCP's defense lines. Simulations indicate that larger network effects increase systemic risk from cascading failures. The size of the network N raises the threshold value of shock sizes that are required to generate cascades. Hence, the larger the network, the more robust it will be.

  9. Defining a key receptor-CheA kinase contact and elucidating its function in the membrane-bound bacterial chemosensory array: a disulfide mapping and TAM-IDS Study.

    PubMed

    Piasta, Kene N; Ulliman, Caleb J; Slivka, Peter F; Crane, Brian R; Falke, Joseph J

    2013-06-01

    The three core components of the ubiquitous bacterial chemosensory array - the transmembrane chemoreceptor, the histidine kinase CheA, and the adaptor protein CheW - assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate kinase activity. Both the regulatory domain of the kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor-CheA kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium, and other bacteria. Here we employ disulfide mapping and tryptophan and alanine mutation to identify docking sites (TAM-IDS) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA kinase binding in the array and a regulatory function central to transmission of the

  10. Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

    PubMed

    Jin, Shan-Xue; Arai, Junko; Tian, Xuejun; Kumar-Singh, Rajendra; Feig, Larry A

    2013-07-26

    RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

  11. Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

    PubMed Central

    Denhardt, D T

    1996-01-01

    The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families

  12. A hierarchical phosphorylation cascade that regulates the timing of PERIOD nuclear entry reveals novel roles for proline-directed kinases and GSK-3β/SGG in circadian clocks

    PubMed Central

    Ko, Hyuk Wan; Kim, Eun Young; Chiu, Joanna; Vanselow, Jens T.; Kramer, Achim; Edery, Isaac

    2010-01-01

    The daily timing of when PERIOD (PER) proteins translocate from the cytoplasm to the nucleus is a critical step in clock mechanisms underpinning circadian rhythms in animals. Numerous lines of evidence indicate that phosphorylation plays a prominent role in regulating various aspects of PER function and metabolism, including changes in its daily stability and subcellular distribution. In this report we show that phosphorylation of serine 661 (Ser661) by a proline-directed kinase(s) is a key phospho-signal on the Drosophila PER protein (dPER) that regulates the timing of its nuclear accumulation. Mutations that block phosphorylation at Ser661 do not affect dPER stability but delay its nuclear entry in key pacemaker neurons, yielding longer behavioral rhythms. Intriguingly, abolishing phosphorylation at Ser661 also attenuates the extent of dPER hyperphosphorylation in vivo, suggesting the phosphorylated state of Ser661 regulates phosphorylation at other sites on dPER. Indeed, we identify Ser657 as a site that is phosphorylated by GSK-3β (SHAGGY; SGG) in a manner dependent on priming at Ser661. Although not as dramatic as mutating Ser661, mutations that abolish phosphorylation at Ser657 also lead to longer behavioral periods, suggesting that a multi-kinase hierarchical phosphorylation module regulates the timing of dPER nuclear entry. Together with evidence in mammalian systems, our findings implicate Pro-directed kinases in clock mechanisms and suggest that PER proteins are key downstream targets of lithium therapy, a potent inhibitor of GSK-3β used to treat manic depression, a disorder associated with clock malfunction in humans. PMID:20861372

  13. 31P NMR 2D Mapping of Creatine Kinase Forward Flux Rate in Hearts with Postinfarction Left Ventricular Remodeling in Response to Cell Therapy

    PubMed Central

    Gao, Ling; Cui, Weina; Zhang, Pengyuan; Jang, Albert; Zhu, Wuqiang; Zhang, Jianyi

    2016-01-01

    Utilizing a fast 31P magnetic resonance spectroscopy (MRS) 2-dimensional chemical shift imaging (2D-CSI) method, this study examined the heterogeneity of creatine kinase (CK) forward flux rate of hearts with postinfarction left ventricular (LV) remodeling. Immunosuppressed Yorkshire pigs were assigned to 4 groups: 1) A sham-operated normal group (SHAM, n = 6); 2) A 60 minutes distal left anterior descending coronary artery ligation and reperfusion (MI, n = 6); 3) Open patch group; ligation injury plus open fibrin patch over the site of injury (Patch, n = 6); and 4) Cell group, hiPSCs-cardiomyocytes, -endothelial cells, and -smooth muscle cells (2 million, each) were injected into the injured myocardium pass through a fibrin patch (Cell+Patch, n = 5). At 4 weeks, the creatine phosphate (PCr)/ATP ratio, CK forward flux rate (Flux PCr→ATP), and k constant of CK forward flux rate (kPCr→ATP) were severely decreased at border zone myocardium (BZ) adjacent to MI. Cell treatment results in significantly increase of PCr/ATP ratio and improve the value of kPCr→ATP and Flux PCr→ATP in BZ myocardium. Moreover, the BZ myocardial CK total activity and protein expression of CK mitochondria isozyme and CK myocardial isozyme were significantly reduced, but recovered in response to cell treatment. Thus, cell therapy results in improvement of BZ bioenergetic abnormality in hearts with postinfarction LV remodeling, which is accompanied by significantly improvements in BZ CK activity and CK isozyme expression. The fast 2D 31P MR CSI mapping can reliably measure the heterogeneity of bioenergetics in hearts with post infarction LV remodeling. PMID:27606901

  14. Inhibition of lipid phosphate phosphatase activity by VPC32183 suppresses the ability of diacylglycerol pyrophosphate to activate ERK(1/2) MAP kinases.

    PubMed

    Violet, Pierre-Christian; Billon-Denis, Emmanuelle; Robin, Philippe

    2012-11-01

    The lipidic metabolite, diacylglycerol pyrophosphate (DGPP), in its dioctanoyl form (DGPP 8:0), has been described as an antagonist for mammalian lysophosphatidic acid (LPA) receptors LPA1 and LPA3. In this study we show that DGPP 8:0 does not antagonize LPA dependent activation of ERK(1/2) MAP kinases but strongly stimulated them in various mammalian cell lines. LPA and DGPP 8:0 stimulation of ERK(1/2) occurred through different pathways. The DGPP 8:0 effect appeared to be dependent on PKC, Raf and MEK but was insensitive to pertussis toxin and did not involve G protein activation. Finally we showed that DGPP 8:0 effect on ERK(1/2) was dependent on its dephosphorylation by a phosphatase activity sharing lipid phosphate phosphatase properties. The inhibition of this phosphatase activity by VPC32183, a previously characterized LPA receptor antagonist, blocked the DGPP 8:0 effect on ERK(1/2) activation. Moreover, down-regulation of lipid phosphate phosphatase 1 (LPP1) expression by RNA interference technique also reduced DGPP 8:0-induced ERK(1/2) activation. Consistently, over expression of LPP1 in HEK293 cells increases DGPP 8:0 hydrolysis and this increased activity was inhibited by VPC32183. In conclusion, DGPP 8:0 does not exert its effect by acting on a G protein coupled receptor, but through its dephosphorylation by LPP1, generating dioctanoyl phosphatidic acid which in turn activates PKC. These results suggest that LPP1 could have a positive regulatory function on cellular signaling processes such as ERK(1/2) activation.

  15. 31P NMR 2D Mapping of Creatine Kinase Forward Flux Rate in Hearts with Postinfarction Left Ventricular Remodeling in Response to Cell Therapy.

    PubMed

    Gao, Ling; Cui, Weina; Zhang, Pengyuan; Jang, Albert; Zhu, Wuqiang; Zhang, Jianyi

    2016-01-01

    Utilizing a fast 31P magnetic resonance spectroscopy (MRS) 2-dimensional chemical shift imaging (2D-CSI) method, this study examined the heterogeneity of creatine kinase (CK) forward flux rate of hearts with postinfarction left ventricular (LV) remodeling. Immunosuppressed Yorkshire pigs were assigned to 4 groups: 1) A sham-operated normal group (SHAM, n = 6); 2) A 60 minutes distal left anterior descending coronary artery ligation and reperfusion (MI, n = 6); 3) Open patch group; ligation injury plus open fibrin patch over the site of injury (Patch, n = 6); and 4) Cell group, hiPSCs-cardiomyocytes, -endothelial cells, and -smooth muscle cells (2 million, each) were injected into the injured myocardium pass through a fibrin patch (Cell+Patch, n = 5). At 4 weeks, the creatine phosphate (PCr)/ATP ratio, CK forward flux rate (Flux PCr→ATP), and k constant of CK forward flux rate (kPCr→ATP) were severely decreased at border zone myocardium (BZ) adjacent to MI. Cell treatment results in significantly increase of PCr/ATP ratio and improve the value of kPCr→ATP and Flux PCr→ATP in BZ myocardium. Moreover, the BZ myocardial CK total activity and protein expression of CK mitochondria isozyme and CK myocardial isozyme were significantly reduced, but recovered in response to cell treatment. Thus, cell therapy results in improvement of BZ bioenergetic abnormality in hearts with postinfarction LV remodeling, which is accompanied by significantly improvements in BZ CK activity and CK isozyme expression. The fast 2D 31P MR CSI mapping can reliably measure the heterogeneity of bioenergetics in hearts with post infarction LV remodeling. PMID:27606901

  16. p38 MAP kinase-mediated NMDA receptor-dependent suppression of hippocampal hypersynchronicity in a mouse model of Alzheimer's disease.

    PubMed

    Ittner, Arne A; Gladbach, Amadeus; Bertz, Josefine; Suh, Lisa S; Ittner, Lars M

    2014-01-01

    Hypersynchronicity of neuronal brain circuits is a feature of Alzheimer's disease (AD). Mouse models of AD expressing mutated forms of the amyloid-β precursor protein (APP), a central protein involved in AD pathology, show cortical hypersynchronicity. We studied hippocampal circuitry in APP23 transgenic mice using telemetric electroencephalography (EEG), at the age of onset of memory deficits. APP23 mice display spontaneous hypersynchronicity in the hippocampus including epileptiform spike trains. Furthermore, spectral contributions of hippocampal theta and gamma oscillations are compromised in APP23 mice, compared to non-transgenic controls. Using cross-frequency coupling analysis, we show that hippocampal gamma amplitude modulation by theta phase is markedly impaired in APP23 mice. Hippocampal hypersynchronicity and waveforms are differentially modulated by injection of riluzole and the non-competitive N-methyl-D-aspartate (NMDA) receptor inhibitor MK801, suggesting specific involvement of voltage-gated sodium channels and NMDA receptors in hypersynchronicity thresholds in APP23 mice. Furthermore, APP23 mice show marked activation of p38 mitogen-activated protein (MAP) kinase in hippocampus, and injection of MK801 but not riluzole reduces activation of p38 in the hippocampus. A p38 inhibitor induces hypersynchronicity in APP23 mice to a similar extent as MK801, thus supporting suppression of hypersynchronicity involves NMDA receptors-mediated p38 activity. In summary, we characterize components of hippocampal hypersynchronicity, waveform patterns and cross-frequency coupling in the APP23 mouse model by pharmacological modulation, furthering the understanding of epileptiform brain activity in AD.

  17. CXCL13/CXCR5 enhances sodium channel Nav1.8 current density via p38 MAP kinase in primary sensory neurons following inflammatory pain

    PubMed Central

    Wu, Xiao-Bo; Cao, De-Li; Zhang, Xin; Jiang, Bao-Chun; Zhao, Lin-Xia; Qian, Bin; Gao, Yong-Jing

    2016-01-01

    CXCL13 is a B lymphocyte chemoattractant and activates CXCR5 receptor in the immune system. Here we investigated whether CXCL13/CXCR5 mediates inflammatory pain in dorsal root ganglia (DRG) and the underlying mechanisms. Peripheral injection of complete Freund’s Adjuvant (CFA) increased the expression of CXCL13 and CXCR5 in DRG neurons. In Cxcr5−/− mice, CFA-induced pain hypersensitivity were attenuated. Whole-cell patch-clamp recording showed that the excitability of dissociated DRG neurons was increased after CFA injection or CXCL13 incubation from wild-type (WT) mice, but not from Cxcr5−/− mice. Additionally, sodium channel Nav1.8 was co-expressed with CXCR5 in dissociated DRG neurons, and the increased neuronal excitability induced by CFA or CXCL13 was reduced by Nav1.8 blocker. Intrathecal injection of Nav1.8 blocker also attenuated intrathecal injection of CXCL13-induced pain hypersensitivity. Furthermore, CXCL13 increased Nav1.8 current density in DRG neurons, which was inhibited by p38 MAP kinase inhibitor. CFA and CXCL13 increased p38 phosphorylation in the DRG of WT mice but not Cxcr5−/− mice. Finally, intrathecal p38 inhibitor alleviated CXCL13-induced pain hypersensitivity. Taken together, these results demonstrated that CXCL13, upregulated by peripheral inflammation, acts on CXCR5 on DRG neurons and activates p38, which increases Nav1.8 current density and further contributes to the maintenance of inflammatory pain. PMID:27708397

  18. Cascade photo production at CLAS

    SciTech Connect

    Goetz, John; Hicks, Kenneth H.

    2014-09-01

    The famous discovery of the Omega in 1964 put the quark model on firm ground and since then a lot of effort has been spent on mapping out the baryonic and mesonic states. Over the following decades, many excited baryons with light quarks (up, down and strange) have been measured, but by most predictions, only a small percentage of those expected have been found. In this talk, I will discuss a newly developing technique using an (unflavored) photon beam to excite protons to doubly-strange "Cascade" (Xi) states. Advantages of such an experiment and associated difficulties will be presented, along with recent results from the CLAS detector at Jefferson Lab in Virginia.

  19. Mitogen-Activated Protein Kinase Phosphatase 2 Regulates the Inflammatory Response in Sepsis▿

    PubMed Central

    Cornell, Timothy T.; Rodenhouse, Paul; Cai, Qing; Sun, Lei; Shanley, Thomas P.

    2010-01-01

    Sepsis results from a dysregulation of the regulatory mechanisms of the pro- and anti-inflammatory response to invading pathogens. The mitogen-activated protein (MAP) kinase cascades are key signal transduction pathways involved in the cellular production of cytokines. The dual-specific phosphatase 1 (DUSP 1), mitogen-activated protein kinase phosphatase-1 (MKP-1), has been shown to be an important negative regulator of the inflammatory response by regulating the p38 and Jun N-terminal protein kinase (JNK) MAP kinase pathways to influence pro- and anti-inflammatory cytokine production. MKP-2, also a dual-specific phosphatase (DUSP 4), is a phosphatase highly homologous with MKP-1 and is known to regulate MAP kinase signaling; however, its role in regulating the inflammatory response is not known. We hypothesized a regulatory role for MKP-2 in the setting of sepsis. Mice lacking the MKP-2 gene had a survival advantage over wild-type mice when challenged with intraperitoneal lipopolysaccharide (LPS) or a polymicrobial infection via cecal ligation and puncture. The MKP-2−/− mice also exhibited decreased serum levels of both pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], IL-6) and anti-inflammatory cytokines (IL-10) following endotoxin challenge. Isolated bone marrow-derived macrophages (BMDMs) from MKP-2−/− mice showed increased phosphorylation of the extracellular signal-regulated kinase (ERK), decreased phosphorylation of JNK and p38, and increased induction of MKP-1 following LPS stimulation. The capacity for cytokine production increased in MKP-2−/− BMDMs following MKP-1 knockdown. These data support a mechanism by which MKP-2 targets ERK deactivation, thereby decreasing MKP-1 and thus removing the negative inhibition of MKP-1 on cytokine production. PMID:20351138

  20. Phosphorylation of ERK/MAP Kinase Is Required for Long-Term Potentiation in Anatomically Restricted Regions of the Lateral Amygdala in Vivo

    ERIC Educational Resources Information Center

    Schafe, Glenn E.; Swank, Michael W.; Rodriguez, Sarina M.; Debiec, Jacek; Doyere, Valerie

    2008-01-01

    We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/ MAPK) is transiently activated in anatomically restricted regions of the lateral amygdala (LA) following Pavlovian fear conditioning and that blockade of ERK/MAPK activation in the LA impairs both fear memory consolidation and long-term…

  1. Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events

    PubMed Central

    1995-01-01

    Lipopolysaccharide (LPS), a highly conserved component of the outer membrane of gram-negative bacteria, stimulates macrophages to release various cytokine and eicosanoid mediators of the immune response. The mechanism by which LPS stimulates these cells is poorly characterized. One of the most rapid LPS-stimulated events is the phosphorylation and activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase. We wished to examine the role of MAP kinase in LPS- induced signaling in murine macrophages by activating MAP kinase independently of LPS. An expression vector encoding a Raf-1:estrogen receptor (ER) chimeric protein was transfected into the murine macrophage cell line RAW 264.7. Activation of this chimeric protein (delta Raf-1:ER) by estradiol resulted in rapid and prolonged activation of MAP kinase, as expected from previous results implicating Raf-1 as an upstream activator of this signaling cascade. LPS stimulation induced accumulation of MAP kinase phosphatase 1 messenger RNA, whereas delta Raf-1:ER activation did not, perhaps accounting for the more prolonged activation of MAP kinase seen in response to delta Raf-1:ER activation. Similarly, activation of DNA binding by the transcription factor, nuclear factor (NF) kappa B, as assessed by electrophoretic mobility shift assay, occurred in response to LPS stimulation but not in response to delta Raf-1:ER activation or phorbol myristate acetate (PMA) stimulation. Using an enzyme-linked immunosorbent assay for murine tumor necrosis factor alpha (TNF-alpha), we found that LPS and PMA stimulation and delta Raf-1:ER activation induced secretion of TNF-alpha, although the amount of TNF-alpha secreted in response to delta Raf-1:ER activation and PMA stimulation was approximately 20-fold less than that secreted in response to LPS. Correspondingly, accumulation of TNF-alpha messenger RNA was weakly induced by delta Raf-1:ER activation or PMA stimulation, whereas strong induction was noted in

  2. Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events.

    PubMed

    Hambleton, J; McMahon, M; DeFranco, A L

    1995-07-01

    Lipopolysaccharide (LPS), a highly conserved component of the outer membrane of gram-negative bacteria, stimulates macrophages to release various cytokine and eicosanoid mediators of the immune response. The mechanism by which LPS stimulates these cells is poorly characterized. One of the most rapid LPS-stimulated events is the phosphorylation and activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase. We wished to examine the role of MAP kinase in LPS-induced signaling in murine macrophages by activating MAP kinase independently of LPS. An expression vector encoding a Raf-1:estrogen receptor (ER) chimeric protein was transfected into the murine macrophage cell line RAW 264.7. Activation of this chimeric protein (delta Raf-1:ER) by estradiol resulted in rapid and prolonged activation of MAP kinase, as expected from previous results implicating Raf-1 as an upstream activator of this signaling cascade. LPS stimulation induced accumulation of MAP kinase phosphatase 1 messenger RNA, whereas delta Raf-1:ER activation did not, perhaps accounting for the more prolonged activation of MAP kinase seen in response to delta Raf-1:ER activation. Similarly, activation of DNA binding by the transcription factor, nuclear factor (NF) kappa B, as assessed by electrophoretic mobility shift assay, occurred in response to LPS stimulation but not in response to delta Raf-1:ER activation or phorbol myristate acetate (PMA) stimulation. Using an enzyme-linked immunosorbent assay for murine tumor necrosis factor alpha (TNF-alpha), we found that LPS and PMA stimulation and delta Raf-1:ER activation induced secretion of TNF-alpha, although the amount of TNF-alpha secreted in response to delta Raf-1:ER activation and PMA stimulation was approximately 20-fold less than that secreted in response to LPS. Correspondingly, accumulation of TNF-alpha messenger RNA was weakly induced by delta Raf-1:ER activation or PMA stimulation, whereas strong induction was noted in

  3. Involvement of Protein Kinase C in the Suppression of Apoptosis and in Polarity Establishment in Aspergillus nidulans under Conditions of Heat Stress

    PubMed Central

    Katayama, Takuya; Uchida, Hirotaka; Ohta, Akinori; Horiuchi, Hiroyuki

    2012-01-01

    The pkcA gene, which encodes a protein kinase C (PKC) in the filamentous fungus Aspergillus nidulans, is essential for its viability. However, little is known about its functions. To address this issue, we constructed and characterized temperature-sensitive mutants of pkcA. The conidia of these mutants swelled slightly and exhibited apoptotic phenotypes at 42°C. The apoptotic phenotypes were suppressed by an osmotic stabilizer. Under these conditions, the conidia swelled extensively and did not form germ tubes. Moreover, polarized distribution of F-actin was not observed. We then utilized deletion mutants of bckA, an ortholog of Saccharomyces cerevisiae bck1 that encodes a mitogen-activated protein (MAP) kinase kinase kinase and functions downstream of PKC in the cell wall integrity pathway. These mutants exhibited apoptotic phenotypes at 42°C, but they did not show defects in polarity establishment under osmotically stabilized conditions. These results suggest that PkcA plays multiple roles during germination under conditions of heat stress. The first of these roles is the suppression of apoptosis induction, while the other involves polarity establishment. The former depends on the MAP kinase cascade, whereas the latter does not. In addition, repolarization, which was observed after depolarization in the wild-type strain and the bckA deletion mutant under conditions of heat stress, was not observed in the pkcA-ts mutant. This suggests that PkcA also plays role in polarity establishment during hyphal growth independent of the MAP kinase cascade under these conditions. PMID:23209763

  4. In vivo Evaluation of Two Thiazolidin-4-one Derivatives in High Sucrose Diet Fed Pre-diabetic Mice and Their Modulatory Effect on AMPK, Akt and p38 MAP Kinase in L6 Cells

    PubMed Central

    Mudgal, Jayesh; Shetty, Priya; Reddy, Neetinkumar D.; Akhila, H. S.; Gourishetti, Karthik; Mathew, Geetha; Nayak, Pawan G.; Kumar, Nitesh; Kishore, Anoop; Kutty, Nampurath G.; Nandakumar, Krishnadas; Shenoy, Rekha R.; Rao, Chamallamudi M.; Joseph, Alex

    2016-01-01

    We had previously demonstrated the anti-diabetic potential and pancreatic protection of two thiazolidin-4-one derivatives containing nicotinamide moiety (NAT-1 and NAT-2) in STZ-induced diabetic mice. However, due to the limitations of the STZ model, we decided to undertake a detailed evaluation of anti-diabetic potential of the molecules on a high sucrose diet (HSD) fed diabetic mouse model. Further, in vitro mechanistic studies on the phosphorylation of AMPK, Akt and p38 MAP kinase in L6 myotubes and anti-inflammatory studies in RAW264.7 mouse monocyte macrophage cells were performed. 15 months of HSD induced fasting hyperglycaemia and impaired glucose tolerance in mice. Treatment with NAT-1 and NAT-2 (100 mg/kg) for 45 days significantly improved the glucose tolerance and lowered fasting blood glucose levels compared to untreated control. An improvement in the elevated triglycerides and total cholesterol levels, and favorable rise in HDL cholesterol were also observed with test drug treatment. Also, no major changes were observed in the liver (albumin, AST and ALT) and kidney (creatinine and urea) parameters. This was further confirmed in their respective histology profiles which revealed no gross morphological changes. In L6 cells, significant phosphorylation of Akt and p38 MAP kinase proteins were observed with 100 μM of NAT-1 and NAT-2 with no significant changes in phosphorylation of AMPK. The molecules failed to exhibit anti-inflammatory activity as observed by their effect on the generation of ROS and nitrite, and nuclear levels of NF-κB in LPS-stimulated RAW264.7 cells. In summary, the molecules activated Akt and p38 MAP kinase which could have partly contributed to their anti-hyperglycaemic and hypolipidemic activities in vivo. PMID:27790148

  5. Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

    SciTech Connect

    Arai, Roberto J.; Debbas, Victor; Stern, Arnold; Monteiro, Hugo P.

    2008-12-01

    Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.

  6. Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. Possible requirement of Gq-mediated p21ras activation coupled to a Ca2+/calmodulin-sensitive tyrosine kinase.

    PubMed

    Eguchi, S; Matsumoto, T; Motley, E D; Utsunomiya, H; Inagami, T

    1996-06-14

    In cultured rat vascular smooth muscle cells, angiotensin II (Ang II) induced a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor, which was insensitive to pertussis toxin but was abolished by the phospholipase C inhibitor, U73122. The Ang II-induced MAPK activation was not affected by the protein kinase C inhibitor, GF109203X, and was only partially impaired by pretreatment with a phorbol ester, whereas both treatments completely prevented MAPK activation by the phorbol ester. Intracellular Ca2+ chelation by TMB-8, but not extracellular Ca2+ chelation or inhibition of Ca2+ influx, abolished Ang II-induced MAPK activation. The calmodulin inhibitor, calmidazolium, and the tyrosine kinase inhibitor, genistein, completely blocked MAPK activation by Ang II as well as by the Ca2+ ionophore A23187. Ang II caused a rapid increase in the binding of GTP to p21(ras), and this was inhibited by genistein, TMB-8, and calmidazolium but not by pertussis toxin or GF109203X. These data suggest that Ang II-induced MAPK activation through the Ang II type 1 receptor could be mediated by p21(ras)activation through a currently unidentified tyrosine kinase that lies downstream of Gq-coupled Ca2+/calmodulin signals.

  7. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  8. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

    SciTech Connect

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L.; Hood, Molly M.; Lord, John W.; Lu, Wei-Ping; Miller, David F.; Patt, William C.; Smith, Bryan D.; Vogeti, Lakshminarayana; Kaufman, Michael D.; Petillo, Peter A.; Wise, Scott C.; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L.

    2012-01-20

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

  9. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

    PubMed

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L; Hood, Molly M; Lord, John W; Lu, Wei-Ping; Miller, David F; Patt, William C; Smith, Bryan D; Vogeti, Lakshminarayana; Kaufman, Michael D; Petillo, Peter A; Wise, Scott C; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L

    2010-10-01

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

  10. The nitrogen cascade

    SciTech Connect

    Galloway J.N.; Aber J.D.; Erisman J.W.; Seitzinger S.P.; Howarth R.W.; Cowling E.B.; Cosby B.J.

    2003-04-01

    Human production of food and energy is the dominant continental process that breaks the triple bond in molecular nitrogen (N{sub 2}) and creates reactive nitrogen (Nr) species. Circulation of anthropogenic Nr in Earth's atmosphere, hydrosphere, and biosphere has a wide variety of consequences, which are magnified with time as Nr moves along its biogeochemical pathway. The same atom of Nr can cause multiple effects in the atmosphere, in terrestrial ecosystems, in freshwater and marine systems, and on human health. We call this sequence of effects the nitrogen cascade. As the cascade progresses, the origin of Nr becomes unimportant. Reactive nitrogen does not cascade at the same rate through all environmental systems; some systems have the ability to accumulate Nr, which leads to lag times in the continuation of the cascade. These lags slow the cascade and result in Nr accumulation in certain reservoirs, which in turn can enhance the effects of Nr on that environment. The only way to eliminate Nr accumulation and stop the cascade is to convert Nr back to nonreactive N{sub 2}.

  11. Ras-mutant cancer cells display B-Raf binding to Ras that activates extracellular signal-regulated kinase and is inhibited by protein kinase A phosphorylation.

    PubMed

    Li, Yanping; Takahashi, Maho; Stork, Philip J S

    2013-09-20

    The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.

  12. Geothermal research, Oregon Cascades: Final technical report

    SciTech Connect

    Priest, G.R.; Black, G.L.

    1988-10-27

    Previous USDOE-funded geothermal studies have produced an extensive temperature gradient and heat flow data base for the State of Oregon. One of the important features identified as a result of these studies is a rapid transition from heat flow values on the order of 40 mW/m/sup 2/ in the Willamette Valley and Western Cascades to values of greater than or equal to100 mW/m/sup 2/ in the High Cascades and the eastern portion of the Western Cascades. These data indicate that the Cascade Range in Oregon has potential as a major geothermal province and stimulated much of the later work completed by government agencies and private industry. Additional data generated as a result of this grant and published in DOGAMI Open-File Report 0-86-2 further define the location and magnitude of this transition zone. In addition, abundant data collected from the vicinity of Breitenbush and Austin Hot Springs have permitted the formulation of relatively detailed models of these hydrothermal systems. These models are published in DOGAMI Open-File Report 0-88-5. Task 1.2 of the Deliverables section of Amendment M001 is fulfilled by DOGAMI publication GMS-48, Geologic map of the McKenzie Bridge quadrangle, Lane County, Oregon. This map was printed in October, 1988, and is part of the final submission to USDOE. 8 refs.

  13. Lifespans of Cascade Arc volcanoes

    NASA Astrophysics Data System (ADS)

    Calvert, A. T.

    2015-12-01

    Compiled argon ages reveal inception, eruptive episodes, ages, and durations of Cascade stratovolcanoes and their ancestral predecessors. Geologic mapping and geochronology show that most Cascade volcanoes grew episodically on multiple scales with periods of elevated behavior lasting hundreds of years to ca. 100 kyr. Notable examples include the paleomag-constrained, few-hundred-year-long building of the entire 15-20 km3 Shastina edifice at Mt. Shasta, the 100 kyr-long episode that produced half of Mt. Rainier's output, and the 30 kyr-long episode responsible for all of South and Middle Sister. Despite significant differences in timing and rates of construction, total durations of active and ancestral volcanoes at discrete central-vent locations are similar. Glacier Peak, Mt. Rainier, Mt. Adams, Mt. Hood, and Mt. Mazama all have inception ages of 400-600 ka. Mt. St. Helens, Mt. Jefferson, Newberry Volcano, Mt. Shasta and Lassen Domefield have more recent inception ages of 200-300 ka. Only the Sisters cluster and Mt. Baker have established eruptive histories spanning less than 50 kyr. Ancestral volcanoes centered 5-20 km from active stratocones appear to have similar total durations (200-600 kyr), but are less well exposed and dated. The underlying mechanisms governing volcano lifecycles are cryptic, presumably involving tectonic and plumbing changes and perhaps circulation cycles in the mantle wedge, but are remarkably consistent along the arc.

  14. The NO-cGMP-PKG Signaling Pathway Regulates Synaptic Plasticity and Fear Memory Consolidation in the Lateral Amygdala via Activation of ERK/MAP Kinase

    ERIC Educational Resources Information Center

    Ota, Kristie T.; Pierre, Vicki J.; Ploski, Jonathan E.; Queen, Kaila; Schafe, Glenn E.

    2008-01-01

    Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and…

  15. Cascading Off Continental Shelves

    NASA Astrophysics Data System (ADS)

    Huthnance, J.

    Cascading is the motion of dense water that is formed by cooling, evaporation or freezing in the surface layer, along a sloping sea bottom to a greater depth. It is in- fluential in water-mass formation and particularly in ventilation of intermediate and abyssal layers, hence affecting thermohaline circulation and global climate. Cascad- ing is intermittent in time and space, takes place in the bottom layers and cannot be traced via satellites. Hence it is rarely observed while in progress, and there is a dearth of knowledge of the statistical and main individual characteristics of cascading: most favourable locations, frequency of occurrence, density difference, speed of sinking, off-shore volume fluxes etc. The INTAS 99-1600 project "Dense water overflows off continental shelves (cascading)" aims to (i) improve understanding and modelling of dense water overflows, ie. cascading as a meso-scale process (ii) extrapolate from spe- cific observations (focused on the edge of the continental shelf) using generic models and (iii) estimate its influence on fluxes of dissolved and particulate matter between the shelf and open ocean in the bottom boundary layer. An overview of the project to date will be given: - collation of existing confirmed observations of cascading, and data of relevant laboratory experiments, to provide a common data base for modelling; - search of oceanographic data banks and collation of wider relevant data; - systematic analysis; inter-comparison, identifying factors and mechanisms in pre-conditioning, initiation, the evolving form and the end-stages of dense water overflow. - developing a linked set of new or modified models (1.5-layer to 3-D full-physics) capable of sim- ulating the main driving mechanisms and predicting the characteristics of cascading; - developing a model to study the generation and movement of mudslides that the cas- cading process can trigger if sediment material becomes unstable over a steep bottom slope.

  16. Pseudomonas aeruginosa pyocyanin activates NRF2-ARE-mediated transcriptional response via the ROS-EGFR-PI3K-AKT/MEK-ERK MAP kinase signaling in pulmonary epithelial cells.