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Sample records for induced map kinase

  1. 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 ...

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. [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.

  12. 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.

  13. 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.

  14. 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

  15. 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).

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  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. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling.

    PubMed

    Kim, Junwon; Rose, Mark D

    2015-12-01

    Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein β and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth

  18. Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling

    PubMed Central

    Kim, Junwon; Rose, Mark D.

    2015-01-01

    Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein β and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth

  19. 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.

  20. 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

  1. Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca{sup 2+}]{sub i} elevation

    SciTech Connect

    Chou, C.-T.; He Shiping; Jan, C.-R. . E-mail: crjan@isca.vghks.gov.tw

    2007-02-01

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH{sub 2}-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca{sup 2+}]{sub i} increases which involved the mobilization of intracellular Ca{sup 2+} stored in the endoplasmic reticulum and Ca{sup 2+} influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca{sup 2+} chelator, to prevent paroxetine-induced [Ca{sup 2+}]{sub i} increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca{sup 2+}-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation.

  2. 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

  3. 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

  4. Protective effects of green tea polyphenol extracts against ethanol-induced gastric mucosal damages in rats: stress-responsive transcription factors and MAP kinases as potential targets.

    PubMed

    Lee, Jeong-Sang; Oh, Tae-Young; Kim, Young-Kyung; Baik, Joo-Hyun; So, Sung; Hahm, Ki-Baik; Surh, Young-Joon

    2005-11-11

    There are multiple lines of compelling evidence from epidemiologic and laboratory studies supporting that frequent consumption of green tea is inversely associated with the risk of chronic human diseases including cancer. The chemopreventive and chemoprotective effects of green tea have been largely attributed to antioxidative and anti-inflammatory activities of its polyphenolic constituents, such as epigallocatechin gallate. The present study was designed to evaluate the efficacy of green tea polyphenols in protecting against alcohol-induced gastric damage and to elucidate the underlying mechanisms. Intragastric administration of ethanol to male Sprague-Dawley rats caused significant gastric mucosal damage, which was accompanied by elevated expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as transient activation of redox-sensitive transcription factors, such as NF-kappaB and AP-1, and mitogen-activated protein kinases (MAPKs). Oral administration of the green tea polyphenolic extract (GTE) significantly ameliorated mucosal damages induced by ethanol and also attenuated the ethanol-induced expression of COX-2 and iNOS. Inactivation of MAPKs, especially p38 and ERKl/2, by GTE might be responsible for inhibition of ethanol-induced expression of COX-2 and iNOS.

  5. Mangiferin, a Natural Xanthone, Protects Murine Liver in Pb(II) Induced Hepatic Damage and Cell Death via MAP Kinase, NF-κB and Mitochondria Dependent Pathways

    PubMed Central

    Pal, Pabitra Bikash; Sinha, Krishnendu; Sil, Parames C.

    2013-01-01

    One of the most well-known naturally occurring environmental heavy metals, lead (Pb) has been reported to cause liver injury and cellular apoptosis by disturbing the prooxidant-antioxidant balance via oxidative stress. Several studies, on the other hand, reported that mangiferin, a naturally occurring xanthone, has been used for a broad range of therapeutic purposes. In the present study, we, therefore, investigated the molecular mechanisms of the protective action of mangiferin against lead-induced hepatic pathophysiology. Lead [Pb(II)] in the form of Pb(NO3)2 (at a dose of 5 mg/kg body weight, 6 days, orally) induced oxidative stress, hepatic dysfunction and cell death in murine liver. Post treatment of mangiferin at a dose of 100 mg/kg body weight (6 days, orally), on the other hand, diminished the formation of reactive oxygen species (ROS) and reduced the levels of serum marker enzymes [alanine aminotranferase (ALT) and alkaline phosphatase (ALP)]. Mangiferin also reduced Pb(II) induced alterations in antioxidant machineries, restored the mitochondrial membrane potential as well as mutual regulation of Bcl-2/Bax. Furthermore, mangiferin inhibited Pb(II)-induced activation of mitogen-activated protein kinases (MAPKs) (phospho-ERK 1/2, phosphor-JNK phospho- p38), nuclear translocation of NF-κB and apoptotic cell death as was evidenced by DNA fragmentation, FACS analysis and histological assessment. In vitro studies using hepatocytes as the working model also showed the protective effect of mangiferin in Pb(II) induced cytotoxicity. All these beneficial effects of mangiferin contributes to the considerable reduction of apoptotic hepatic cell death induced by Pb(II). Overall results demonstrate that mangiferin exhibit both antioxidative and antiapoptotic properties and protects the organ in Pb(II) induced hepatic dysfunction. PMID:23451106

  6. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts.

    PubMed

    Knetsch, MLW.; Wang, M.; Snaar-Jagalska, B. E.; Heimovaara-Dijkstra, S.

    1996-06-01

    Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.

  7. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts.

    PubMed Central

    Knetsch, MLW.; Wang, M.; Snaar-Jagalska, B. E.; Heimovaara-Dijkstra, S.

    1996-01-01

    Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression. PMID:12239411

  8. 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.

  9. 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

  10. 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.

  11. Human p38{delta} MAP kinase mediates UV irradiation induced up-regulation of the gene expression of chemokine BRAK/CXCL14

    SciTech Connect

    Ozawa, Shigeyuki; Ito, Shin; Kato, Yasumasa; Kubota, Eiro; Hata, Ryu-Ichiro

    2010-06-11

    The mitogen-activated protein kinase (MAPK) family comprises ERK, JNK, p38 and ERK5 (big-MAPK, BMK1). UV irradiation of squamous cell carcinoma cells induced up-regulation of gene expression of chemokine BRAK/CXCL14, stimulated p38 phosphorylation, and down-regulated the phosphorylation of ERK. Human p38 MAPKs exist in 4 isoforms: p38{alpha}, {beta}, {gamma} and {delta}. The UV stimulation of p38 phosphorylation was not inhibited by the presence of SB203580 or PD169316, inhibitors of p38{alpha} and {beta}, suggesting p38 phosphorylation was not dependent on these 2 isoforms and that p38{gamma} and/or {delta} was responsible for the phosphorylation. In fact, inhibition of each of these 4 p38 isoforms by the introduction of short hairpin (sh) RNAs for respective isoforms revealed that only shRNA for p38{delta} attenuated the UV-induced up-regulation of BRAK/CXCL14 gene expression. In addition, over-expression of p38 isoforms in the cells showed the association of p38{delta} with ERK1 and 2, concomitant with down-regulation of ERK phosphorylation. The usage of p38{delta} isoform by UV irradiation is not merely due to the abundance of this p38 isoform in the cells. Because serum deprivation of the cells also induced an increase in BRAK/CXCL14 gene expression, and in this case p38{alpha} and/or {beta} isoform is responsible for up-regulation of BRAK/CXCL14 gene expression. Taken together, the data indicate that the respective stress-dependent action of p38 isoforms is responsible for the up-regulation of the gene expression of the chemokine BRAK/CXCL14.

  12. 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.

  13. 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

  14. Inhibition of the MAP3 kinase Tpl2 protects rodent and human β-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4

    PubMed Central

    Varin, E M; Wojtusciszyn, A; Broca, C; Muller, D; Ravier, M A; Ceppo, F; Renard, E; Tanti, J-F; Dalle, S

    2016-01-01

    Proinflammatory cytokines exert cytotoxic effects on β-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of β-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated β-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in β-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E β-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects β-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced β-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic β-cells. PMID:26794660

  15. 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

  16. 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

  17. 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

  18. Prolactin and growth hormone induce differential cytokine and chemokine profile in murine peritoneal macrophages in vitro: involvement of p-38 MAP kinase, STAT3 and NF-kappaB.

    PubMed

    Sodhi, Ajit; Tripathi, Anurag

    2008-02-01

    The role of immune-neuroendocrine interactions in the autoimmune diseases is well recognized. Autoimmune rheumatoid diseases in their active phase have been characterized by high levels of prolactin (PRL) as well as proinflammatory cytokines which suggest a co-relationship between them. In the present study, we have investigated the profile of cytokines secreted by macrophages on treatment with PRL and growth hormone (GH) in vitro. Significantly enhanced production of cytokines IL-1beta, IL-12p40 and IFN-gamma was observed on treatment of macrophages with PRL or GH. However, higher doses of PRL (1000 ng/ml) induced the production of anti-inflammatory cytokine IL-10, with significant abrogation in production of proinflammatory cytokines. It is further observed that PRL and GH induced the production of chemokines MIP-1alpha and RANTES. PRL but not GH selectively induced significantly enhanced production of MCP-1 and IP-10. It is further shown that p38 MAP kinase, STAT3 and NF-kappaB could play a differential regulatory role in PRL or GH induced production of cytokines by macrophages.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. DEPTOR promotes survival of cervical squamous cell carcinoma cells and its silencing induces apoptosis through downregulating PI3K/AKT and by up-regulating p38 MAP kinase

    PubMed Central

    Srinivas, Kalanghad Puthankalam; Viji, Remadevi; Dan, Vipin Mohan; Sajitha, Indira Sukumaran; Prakash, Rajappan; Rahul, Puthan Valappil; Santhoshkumar, Thankayyan R.; Lakshmi, Subhadra; Pillai, Madhavan Radhakrishna

    2016-01-01

    DEPTOR is an endogenous inhibitor of mTOR complexes, de-regulated in cancers. The present study reveals a vital role for DEPTOR in survival of cervical squamous cell carcinoma (SCC). DEPTOR was found to be overexpressed in both cervical SCC cells and tissues and it's silencing in cervical SCC cells induced apoptosis, mainly by up-regulation of p38 MAPK and by inhibiting PI3K/AKT pathway via a feed-back inhibition from mTORC1-S6K. DEPTOR silencing resulted in reduced expression of the nitric oxide synthases iNOS and eNOS, as well as increased activation of ERK1/2 and p38 MAP kinases. Activation of AKT signaling by overexpression of constitutively active-AKT (CA-AKT) failed to overcome the apoptosis caused by DEPTOR silencing. Similarly pharmacological inhibition of ERK also failed to control apoptosis. However pharmacological inhibition of p38 MAPK rescued the cells from apoptosis, indicating the major role of p38 MAPK in cell death induced by DEPTOR silencing. DEPTOR was also found to regulate ERK1/2 in an AKT dependent manner. DEPTOR knockdown induced cell death in SiHa cells overexpressing the anti-apoptotic Bcl-2 and Bcl-xL, indicating strong survival role of DEPTOR in these cells. DEPTOR overexpression activated PI3K/AKT by relieving the negative feed-back inhibition from mTORC1-S6K. DEPTOR regulation was also observed to be independent of HPV E6/E7 oncoproteins, but it might be a molecular co-factor contributing to cervical carcinogenesis. In summary, DEPTOR is found to promote survival of cervical SCC cells and its reduction induced apoptosis via differential effects on PI3K/AKT and p38 MAPK and can be a potential target in cervical SCC. PMID:26992219

  6. Basic fibroblast growth factor induces matrix metalloproteinase-13 via ERK MAP kinase-altered phosphorylation and sumoylation of Elk-1 in human adult articular chondrocytes

    PubMed Central

    Im, Hee-Jeong; Sharrocks, Andrew D; Lin, Xia; Yan, Dongyao; Kim, Jaesung; van Wijnen, Andre J; Hipskind, Robert A

    2009-01-01

    Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and release of basic fibroblast growth factor (bFGF) are principal aspects of the pathology of osteoarthritis (OA). ECM disruption leads to bFGF release, which activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway and its downstream target the Ets-like transcription factor Elk-1. Previously we demonstrated that the bFGF-ERK-Elk-1 signaling axis is responsible for the potent induction of MMP-13 in human primary articular chondrocytes. Here we report that, in addition to phosphorylation of Elk-1, dynamic posttranslational modification of Elk-1 by small ubiquitin-related modifier (SUMO) serves as an important mechanism through which MMP-13 gene expression is regulated. We show that bFGF activates Elk-1 mainly through the ERK pathway and that increased phosphorylation of Elk-1 is accompanied by decreased conjugation of SUMO to Elk-1. Reporter gene assays reveal that phosphorylation renders Elk-1 competent for induction of MMP-13 gene transcription, while sumoylation has the opposite effect. Furthermore, we demonstrate that the SUMO-conjugase Ubc9 acts as a key mediator for Elk-1 sumoylation. Taken together, our results suggest that sumoylation antagonizes the phosphorylation-dependent transactivation capacity of Elk-1. This attenuates transcription of its downstream target gene MMP-13 to maintain the integrity of cartilage ECM homeostasis.

  7. 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

  8. L-type calcium channels and MAP kinase contribute to thyrotropin-releasing hormone-induced depolarization in thalamic paraventricular nucleus neurons

    PubMed Central

    Kolaj, Miloslav; Zhang, Li

    2016-01-01

    In rat paraventricular thalamic nucleus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances neuronal excitability via concurrent decrease in a G protein-coupled inwardly rectifying K (GIRK)-like conductance and opening of a cannabinoid receptor-sensitive transient receptor potential canonical (TRPC)-like conductance. Here, we investigated the calcium (Ca2+) contribution to the components of this TRH-induced response. TRH-induced membrane depolarization was reduced in the presence of intracellular BAPTA, also in media containing nominally zero [Ca2+]o, suggesting a critical role for both intracellular Ca2+ release and Ca2+ influx. TRH-induced inward current was unchanged by T-type Ca2+ channel blockade, but was decreased by blockade of high-voltage-activated Ca2+ channels (HVACCs). Both the pharmacologically isolated GIRK-like and the TRPC-like components of the TRH-induced response were decreased by nifedipine and increased by BayK8644, implying Ca2+ influx via L-type Ca2+ channels. Only the TRPC-like conductance was reduced by either thapsigargin or dantrolene, suggesting a role for ryanodine receptors and Ca2+-induced Ca2+ release in this component of the TRH-induced response. In pituitary and other cell lines, TRH stimulates MAPK. In PVT neurons, only the GIRK-like component of the TRH-induced current was selectively decreased in the presence of PD98059, a MAPK inhibitor. Collectively, the data imply that TRH-induced depolarization and inward current in PVT neurons involve both a dependency on extracellular Ca2+ influx via opening of L-type Ca2+ channels, a sensitivity of a TRPC-like component to intracellular Ca2+ release via ryanodine channels, and a modulation by MAPK of a GIRK-like conductance component. PMID:27009047

  9. Inducement of mitogen-activated protein kinases in frozen shoulders

    PubMed Central

    Kanbe, Katsuaki; Inoue, Kazuhiko; Inoue, Yasuo; Chen, Qian

    2010-01-01

    Background Mitogen-activated protein (MAP) kinases are well-known molecules that play key roles in mechanical stress signals during skeletal development. To test our hypothesis that the synovium in frozen shoulders is induced by MAP kinases, immunohistochemical analyses for detecting expression and signal transduction of MAP kinases were performed in synovial tissue obtained from the rotator interval (RI) in frozen shoulders. Methods Synovial tissues were examined from 10 frozen shoulder patients with a mean age of 55.4 years (46–62 years). Synovial tissues between the long head of the biceps tendon (LHB) and the RI in frozen shoulders were stained with hematoxylin and eosin (H&E) and then examined with immunohistochemical staining. Extracellular signal-regulated (ERK), the Jun N-terminal (JNK), and p38 mitogen-activated protein (MAP) kinases, nuclear factor κB (NF-κB), p50, CD29 (β1-integrin), matrix metalloproteinase (MMP)-3, interleukin-6 (IL-6), CD56, CD68, S-100, and vascular endothelial growth factor (VEGF) were analyzed to detect expression patterns. Results H&E showed vascular proliferation with fibrin and fibrous tissue in the synovium of frozen shoulders. ERK was expressed in the epithelial cells of vascular tissue, and JNK was expressed strongly in the interstitial cells around vascular tissue; p38 MAPK was not expressed. NF-κB was expressed in vascular tissue, and IL-6 was expressed around vascular tissue. CD29 (β1-integrin) was expressed in vascular tissue and in superficial cells of synovial tissue. MMP-3 and VEGF were expressed on the surface layer of synovial tissue and vascular tissue, and CD68 was expressed on the surface layer. Nerve-related proteins, CD56 and S-100, were expressed weakly. Conclusions Mechanical stress on the LHB and RI in the shoulder may induce ERK and JNK to express NF-κB by CD29 to develop capsule contracture, producing MMP-3, IL-6, and VEGF. PMID:19214689

  10. 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

  11. 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

  12. 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

  13. In vitro studies on mangiferin protection against cadmium-induced human renal endothelial damage and cell death via the MAP kinase and NF-κB pathways.

    PubMed

    Rajendran, Peramaiyan; Rengarajan, Thamaraiselvan; Nishigaki, Yutaka; Palaniswami, Rajendran; Nishigaki, Ikuo

    2016-01-01

    The therapeutic effects of the natural antioxidant mangiferin (a xanthonoid and potent oxygen free radical scavenger), which is widely distributed in mango fruit, against CdCl(2)-induced toxicity in human renal glomerulus endothelial cells (HRGEC) were investigated. The viability of HREGCs that were treated with CdCl(2) (25 µ mol) and co-treated with mangiferin (75 µ mol) for 24 h was measured by crystal violet dye. The exposure of human glomerulus renal endothelial cells to cadmium promotes a polarized apical secretion of IL-6 and IL-8, two pivotal proinflammatory cytokines known to play a significant role in renal inflammation. Proinflammatory cytokine secretion by human renal glomerulus endothelial cells could be the result of cadmium-induced IL-6 secretion via an NF-κB-dependent pathway. However, IL-8 secretion involves the phosphor-JNK phospho-p38 signaling pathway. The results of the current study reveal that mangiferin could prevent both cadmium-induced IL-6 and IL-8 secretion by human glomerulus endothelial cells and be used to prevent renal inflammation. PMID:25798666

  14. Inhibition of MAP kinase/NF-kB mediated signaling and attenuation of lipopolysaccharide induced severe sepsis by cerium oxide nanoparticles

    PubMed Central

    Selvaraj, Vellaisamy; Nepal, Niraj; Rogers, Steven; Manne, Nandini D.P.K.; Arvapalli, Ravikumar; Rice, Kevin M.; Asano, Shinichi; Fankhanel, Erin; Ma, Jane J.; Shokuhfar, Tolou; Maheshwari, Mani; Blough, Eric R.

    2015-01-01

    Sepsis is a life threatening disease that is associated with high mortality. Existing treatments have failed to improve survivability in septic patients. The purpose of this present study is to evaluate whether cerium oxide nanoparticles (CeO2NPs) can prevent lipopolysaccharide (LPS) induced severe sepsis mortality by preventing hepatic dysfunction in male Sprague Dawley rats. Administration of a single dose (0.5 mg/kg) of CeO2NPs intravenously to septic rats significantly improved survival rates and functioned to restore body temperature, respiratory rate and blood pressure towards baseline. Treatment-induced increases in animal survivability were associated with decreased hepatic damage along with reductions in serum cytokines/chemokines, and diminished inflammatory related signaling. Kupffer cells and macrophage cells exposed to CeO2NPs exhibited decreases in LPS-induced cytokine release (TNF-α, IL-1β, IL-6, HMGB1) which were associated with diminished cellular ROS, reduced levels of nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and decreased nuclear factor-kappa light chain enhancer of activated B cells (NF-kB) transcriptional activity. The findings of this study indicate that CeO2NPs may be useful as a therapeutic agent for sepsis. PMID:25968464

  15. Coordinate regulation of IkappaB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-kappaB-inducing kinase.

    PubMed

    Nemoto, S; DiDonato, J A; Lin, A

    1998-12-01

    IkappaB kinases (IKKalpha and IKKbeta) are key components of the IKK complex that mediates activation of the transcription factor NF-kappaB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-kappaB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 and can potentiate the stimulatory effect of TNF-alpha on IKK and NF-kappaB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK by TNF-alpha. MEKK1 interacts with and stimulates the activities of both IKKalpha and IKKbeta in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals.

  16. Nuclear versus cytosolic activity of the yeast Hog1 MAP kinase in response to osmotic and tunicamycin-induced ER stress.

    PubMed

    García-Marqués, Sara; Randez-Gil, Francisca; Prieto, Jose A

    2015-07-22

    We examined the physiological significance of the nuclear versus cytosolic localization of the MAPK Hog1p in the ability of yeast cells to cope with osmotic and ER (endoplasmic reticulum) stress. Our results indicate that nuclear import of Hog1p is not critical for osmoadaptation. Plasma membrane-anchored Hog1p is still able to induce increased expression of GPD1 and glycerol accumulation. This is a key osmoregulatory event, although a small production of the osmolyte coupled with the nuclear import of Hog1p is sufficient to provide osmoresistance. On the contrary, the nuclear activity of Hog1p is dispensable for ER stress adaptation.

  17. 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

  18. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

    PubMed Central

    Lizcano, Jose M; Alrubaie, Saif; Kieloch, Agnieszka; Deak, Maria; Leevers, Sally J; Alessi, Dario R

    2003-01-01

    An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well d

  19. Role of the stress-activated protein kinases in endothelin-induced cardiomyocyte hypertrophy.

    PubMed Central

    Choukroun, G; Hajjar, R; Kyriakis, J M; Bonventre, J V; Rosenzweig, A; Force, T

    1998-01-01

    The signal transduction pathways governing the hypertrophic response of cardiomyocytes are not well defined. Constitutive activation of the stress-activated protein kinase (SAPK) family of mitogen-activated protein (MAP) kinases or another stress-response MAP kinase, p38, by overexpression of activated mutants of various components of the pathways is sufficient to induce a hypertrophic response in cardiomyocytes, but it is not clear what role these pathways play in the response to physiologically relevant hypertrophic stimuli. To determine the role of the SAPKs in the hypertrophic response, we used adenovirus-mediated gene transfer of SAPK/ERK kinase-1 (KR) [SEK-1(KR)], a dominant inhibitory mutant of SEK-1, the immediate upstream activator of the SAPKs, to block signal transmission down the SAPK pathway in response to the potent hypertrophic agent, endothelin-1 (ET-1). SEK-1(KR) completely inhibited ET-1-induced SAPK activation without affecting activation of the other MAP kinases implicated in the hypertrophic response, p38 and extracellular signal-regulated protein kinases (ERK)-1/ERK-2. Expression of SEK-1(KR) markedly inhibited the ET-1-induced increase in protein synthesis. In contrast, the MAPK/ERK kinase inhibitor, PD98059, which blocks ERK activation, and the p38 inhibitor, SB203580, had no effect on ET-1-induced protein synthesis. ET-1 also induced a significant increase in atrial natriuretic factor mRNA expression as well as in the percentage of cells with highly organized sarcomeres, responses which were also blocked by expression of SEK-1(KR). In summary, inhibiting activation of the SAPK pathway abrogated the hypertrophic response to ET-1. These data are the first demonstration that the SAPKs are necessary for the development of agonist-induced cardiomyocyte hypertrophy, and suggest that in response to ET-1, they transduce critical signals governing the hypertrophic response. PMID:9769323

  20. [Protein kinase C activation induces platelet apoptosis].

    PubMed

    Zhao, Li-Li; Chen, Meng-Xing; Zhang, Ming-Yi; Dai, Ke-Sheng

    2013-10-01

    Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. T cell antigen receptor engagement stimulates c-raf phosphorylation and induces c-raf-associated kinase activity via a protein kinase C-dependent pathway.

    PubMed

    Siegel, J N; Klausner, R D; Rapp, U R; Samelson, L E

    1990-10-25

    The c-raf kinase has been shown to be activated following stimulation of several tyrosine kinase growth factor receptors. We examined changes in c-raf following engagement of the T cell receptor for antigen (TCR), a stimulus which activates both a non-receptor tyrosine kinase and protein kinase C (PKC). We found that activation of the T-cell receptor on the T cell hybridoma 2B4 causes a rapid and stoichiometric hyperphosphorylation of c-raf and an increase in c-raf-associated kinase activity. Phosphoamino acid analysis showed that the phosphorylation was entirely on serine residues. High-resolution phosphopeptide mapping showed the appearance of a single major new phosphopeptide with TCR stimulation. That phosphopeptide was shown to comigrate with the major new phosphopeptide induced in response to phorbol ester. When cells were depleted of PKC by pretreatment with high concentrations of phorbol ester, TCR stimulation was no longer capable of inducing c-raf-associated kinase activity. To determine whether activation of the tyrosine kinase alone would activate c-raf, we examined the 2B4 variant cell line FL.8. In response to Thy-1 stimulation, these cells activate the tyrosine kinase but not protein kinase C due to a deficiency in TCR eta chain expression. We found that in contrast to Thy-1 stimulation of 2B4 cells, stimulation of FL.8 cells does not lead to the induction of c-raf-associated kinase activity, although phorbol ester activates the kinase to an equivalent degree in both cells. We conclude that T cell receptor activation of c-raf occurs via phosphorylation by the serine/threonine kinase PKC. Activation of c-raf through PKC represents a mechanism distinct from that reported for tyrosine kinase growth factor receptors.

  8. 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.

  9. 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.

  10. Integrative analysis of kinase networks in TRAIL-induced apoptosis provides a source of potential targets for combination therapy.

    PubMed

    So, Jonathan; Pasculescu, Adrian; Dai, Anna Y; Williton, Kelly; James, Andrew; Nguyen, Vivian; Creixell, Pau; Schoof, Erwin M; Sinclair, John; Barrios-Rodiles, Miriam; Gu, Jun; Krizus, Aldis; Williams, Ryan; Olhovsky, Marina; Dennis, James W; Wrana, Jeffrey L; Linding, Rune; Jorgensen, Claus; Pawson, Tony; Colwill, Karen

    2015-04-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous secreted peptide and, in preclinical studies, preferentially induces apoptosis in tumor cells rather than in normal cells. The acquisition of resistance in cells exposed to TRAIL or its mimics limits their clinical efficacy. Because kinases are intimately involved in the regulation of apoptosis, we systematically characterized kinases involved in TRAIL signaling. Using RNA interference (RNAi) loss-of-function and cDNA overexpression screens, we identified 169 protein kinases that influenced the dynamics of TRAIL-induced apoptosis in the colon adenocarcinoma cell line DLD-1. We classified the kinases as sensitizers or resistors or modulators, depending on the effect that knockdown and overexpression had on TRAIL-induced apoptosis. Two of these kinases that were classified as resistors were PX domain-containing serine/threonine kinase (PXK) and AP2-associated kinase 1 (AAK1), which promote receptor endocytosis and may enable cells to resist TRAIL-induced apoptosis by enhancing endocytosis of the TRAIL receptors. We assembled protein interaction maps using mass spectrometry-based protein interaction analysis and quantitative phosphoproteomics. With these protein interaction maps, we modeled information flow through the networks and identified apoptosis-modifying kinases that are highly connected to regulated substrates downstream of TRAIL. The results of this analysis provide a resource of potential targets for the development of TRAIL combination therapies to selectively kill cancer cells.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. Dietary turmeric modulates DMBA-induced p21{sup ras}, MAP kinases and AP-1/NF-{kappa}B pathway to alter cellular responses during hamster buccal pouch carcinogenesis

    SciTech Connect

    Garg, Rachana; Ingle, Arvind; Maru, Girish

    2008-11-01

    The chemopreventive efficacy of turmeric has been established in experimental systems. However, its mechanism(s) of action are not fully elucidated in vivo. The present study investigates the mechanism of turmeric-mediated chemoprevention in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis at 2, 4, 6, 10 and 12 weeks. Dietary turmeric (1%) led to decrease in DMBA-induced tumor burden and multiplicity, and enhanced the latency period in parallel, to its modulatory effects on oncogene products and various cellular responses during HBP tumorigenesis. DMBA-induced expression of ras oncogene product, p21 and downstream target, the mitogen-activated protein kinases were significantly decreased by turmeric during HBP carcinogenesis. Turmeric also diminished the DMBA-induced mRNA expression of proto-oncogenes (c-jun, c-fos) and NF-{kappa}B, leading to decreased protein levels and in further attenuation of DMBA-induced AP-1/NF-{kappa}B DNA-binding in the buccal pouch nuclear extracts. Besides, buccal pouch of hamsters receiving turmeric diet showed significant alterations in DMBA-induced effects: (a) decrease in cell proliferation (diminished PCNA and Bcl2 expression), (b) enhanced apoptosis (increased expression of Bax, caspase-3 and apoptotic index), (c) decrease in inflammation (levels of Cox-2, the downstream target of AP-1/NF-{kappa}B, and PGE2) and (d) aberrant expression of differentiation markers, the cytokeratins (1, 5, 8, and 18). Together, the protective effects of dietary turmeric converge on augmenting apoptosis of the initiated cells and decreasing cell proliferation in DMBA-treated animals, which in turn, is reflected in decreased tumor burden, multiplicity and enhanced latency period. Some of these biomarkers are likely to be helpful in monitoring clinical trials and evaluating drug effect measurements.

  1. 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.

  2. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    SciTech Connect

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-10-17

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK){zeta}, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGK{zeta} siRNA transfection decreased H{sub 2}O{sub 2}-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGK{zeta} also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGK{zeta} rapidly translocated to the cytoplasm following H{sub 2}O{sub 2} treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGK{zeta}, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.

  3. 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

  4. Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

    PubMed Central

    Tejeda-Muñoz, Nydia; González-Aguilar, Héctor; Santoyo-Ramos, Paula; Castañeda-Patlán, M. Cristina

    2015-01-01

    The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells. PMID:26711256

  5. Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

    SciTech Connect

    Geiss, Brian J.; Cano, Gina L.; Tavis, John E.; Morrison, Lynda A. . E-mail: morrisla@slu.edu

    2004-12-05

    Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.

  6. 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

  7. 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

  8. FES kinase participates in KIT-ligand induced chemotaxis

    SciTech Connect

    Voisset, Edwige; Lopez, Sophie; Chaix, Amandine; Vita, Marina; George, Coralie; Dubreuil, Patrice; De Sepulveda, Paulo

    2010-02-26

    FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicated in cell migration.

  9. FES kinase participates in KIT-ligand induced chemotaxis.

    PubMed

    Voisset, Edwige; Lopez, Sophie; Chaix, Amandine; Vita, Marina; George, Coralie; Dubreuil, Patrice; De Sepulveda, Paulo

    2010-02-26

    FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicated in cell migration.

  10. 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

  11. 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

  12. 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.

  13. Choline phosphate potentiates sphingosine-1-phosphate-induced Raf-1 kinase activation dependent of Ras--phosphatidylinositol-3-kinase pathway.

    PubMed

    Lee, Michael; Han, Sang Seop

    2002-04-01

    In NIH3T3 cells, sphingosine-1-phosphate (S1P) caused a significant increase of Raf-1 kinase activity as early as 2 min. Interestingly, choline phosphate (ChoP) produced synergistic increase of S1P-stimulated Raf-1 kinase activation in the presence of ATP while showing additive effect in the absence of ATP. However, Raf-1 kinase activation induced by S1P decreased in the presence of ATP when applied alone. The overexpression of N-terminal fragment of Raf-1 (RfI) to inhibit Raf--Ras interaction caused the inhibition of S1P-induced Raf-1 kinase activation. Also, wortmannin, phosphatidylinositol-3-kinase (PI3K) inhibitor, exhibited inhibitory effects on S1P-induced activation of Raf-1 kinase. In addition, we demonstrated that the chemical antioxidant, N-acetylcysteine attenuated Raf-1 activation induced by S1P, suggesting that H(2)O(2) may be required for the signalling pathway leading to Raf-1 activation. This H(2)O(2)-induced Raf-1 kinase activation was also blocked by inhibition of Ras--PI3K signalling pathway using alpha-hydroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H(2)O(2) stimulation of Ras--PI3K pathway, and is enhanced by ChoP in the presence of ATP.

  14. Cosmic string induced CMB maps

    SciTech Connect

    Landriau, M.; Shellard, E. P. S.

    2011-02-15

    We compute maps of CMB temperature fluctuations seeded by cosmic strings using high resolution simulations of cosmic strings in a Friedmann-Robertson-Walker universe. We create full-sky, 18 deg. and 3 deg. CMB maps, including the relevant string contribution at each resolution from before recombination to today. We extract the angular power spectrum from these maps, demonstrating the importance of recombination effects. We briefly discuss the probability density function of the pixel temperatures, their skewness, and kurtosis.

  15. Participation of mitogen-activated protein kinase in thapsigargin- and TPA-induced histamine production in murine macrophage RAW 264.7 cells

    PubMed Central

    Shiraishi, Muneshige; Hirasawa, Noriyasu; Kobayashi, Yuriko; Oikawa, Shinji; Murakami, Akira; Ohuchi, Kazuo

    2000-01-01

    Stimulation of the murine macrophage cell line RAW 264.7 with thapsigargin, an endomembrane Ca2+-ATPase inhibitor, induced histamine production in a time- and concentration-dependent manner. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), also enhanced histamine production. α-Fluoromethylhistidine, a suicide substrate of L-histidine decarboxylase (HDC), suppressed the thapsigargin (30 nM)- and TPA (30 nM)-induced histamine production. Both thapsigargin (30 nM) and TPA (30 nM) induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059, a specific inhibitor of MEK-1 which phosphorylates p44/p42 MAP kinase, strongly suppressed both the thapsigargin (30 nM)- and TPA (30 nM)-induced histamine production, whereas SB203580, a specific inhibitor of p38 MAP kinase, inhibited them only partially. The other MEK-1 inhibitor, U-0126, also inhibited both the thapsigargin- and TPA-induced histamine production in a concentration-dependent manner. Thapsigargin (30 nM) and TPA (30 nM) increased the levels of HDC mRNA at 4 h, but PD98059 suppressed both the thapsigargin- and TPA-induced increases in the HDC mRNA level. These findings indicate that thapsigargin and TPA induce histamine production in RAW 264.7 cells by increasing the level of HDC mRNA, and that both the thapsigargin- and TPA-induced histamine production are regulated largely by p44/p42 MAP kinase and partially by p38 MAP kinase.. PMID:10711350

  16. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    SciTech Connect

    Martin, Claire; Lafosse, Jean-Michel; Malavaud, Bernard; Cuvillier, Olivier

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. Sphingosine kinase 1 deficiency exacerbates LPS-induced neuroinflammation.

    PubMed

    Grin'kina, Natalia M; Karnabi, Eddy E; Damania, Dushyant; Wadgaonkar, Sunil; Muslimov, Ilham A; Wadgaonkar, Raj

    2012-01-01

    The pathogenesis of inflammation in the central nervous system (CNS), which contributes to numerous neurodegenerative diseases and results in encephalopathy and neuroinflammation, is poorly understood. Sphingolipid metabolism plays a crucial role in maintaining cellular processes in the CNS, and thus mediates the various pathological consequences of inflammation. For a better understanding of the role of sphingosine kinase activation during neuroinflammation, we developed a bacterial lipopolysaccharide (LPS)-induced brain injury model. The onset of the inflammatory response was observed beginning 4 hours after intracerebral injection of LPS into the lateral ventricles of the brain. A comparison of established neuroinflammatory parameters such as white matter rarefactions, development of cytotoxic edema, astrogliosis, loss of oligodendrocytes, and major cytokines levels in wild type and knockout mice suggested that the neuroinflammatory response in SphK1-/- mice was significantly upregulated. At 6 hours after intracerebroventricular injection of LPS in SphK1-/- mice, the immunoreactivity of the microglia markers and astrocyte marker glial fibrillary acidic protein (GFAP) were significantly increased, while the oligodendrocyte marker O4 was decreased compared to WT mice. Furthermore, western blotting data showed increased levels of GFAP. These results suggest that SphK1 activation is involved in the regulation of LPS induced brain injury. RESEARCH HIGHLIGHTS: • Lipopolysaccharide (LPS) intracerebral injection induces severe neuroinflammation. • Sphingosine kinase 1 deletion worsens the effect of the LPS. • Overexpression of SphK1 might be a potential new treatment approach to neuroinflammation.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. The Arabidopsis MAP kinase kinase 7: A crosstalk point between Auxin signaling and defense responses?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant-pathogen interaction induces a complex host response that coordinates various signaling pathways through multiple signal molecules. Besides the well-documented signal molecules salicylic acid (SA), ethylene and jasmonic acid, auxin is emerging as an important player in this response. We recent...

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. Serum- and glucocorticoid-inducible kinases in microglia.

    PubMed

    Inoue, Koichi; Sakuma, Eisuke; Morimoto, Hiroyuki; Asai, Hayato; Koide, Yoshinori; Leng, Tiandong; Wada, Ikuo; Xiong, Zhi-Gang; Ueki, Takatoshi

    2016-09-01

    Microglia are derived from myelogenous cells and contribute to immunological and inflammatory responses in central nervous system. They play important roles not only in infectious diseases and inflammation after stroke, but also in psychiatric diseases such as schizophrenia. While recent studies suggest the significances of serum- and glucocorticoid-inducible kinases (SGKs) in other immune cells such as macrophages, T cells and dendritic cells, their role in microglia remains unknown. Here we, for the first time, report that SGK1 and SGK3 are expressed in multiple microglial cell lines. An SGK inhibitor, gsk650394, inhibits cell viability. In addition, lipopolysaccharide-induced expression of inflammatory regulators iNOS and TNFα was enhanced by gsk650394. Furthermore, translocation of NF-κB was enhanced by gsk650394. Taken together, these findings suggest that SGKs may play an important role in regulating microglial viability and inflammatory responses. PMID:27457803

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  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.

  16. 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

  17. Role of serum- and glucocorticoid-inducible kinases in stroke.

    PubMed

    Inoue, Koichi; Leng, Tiandong; Yang, Tao; Zeng, Zhao; Ueki, Takatoshi; Xiong, Zhi-Gang

    2016-07-01

    Increased expression of serum- and glucocorticoid-inducible kinase 1 (SGK1) can be induced by stress and growth factors in mammals, and plays an important role in cancer, diabetes, and hypertension. A recent work suggested that SGK1 activity restores damage in a stroke model. To further investigate the role of SGKs in ischemic brain injury, we examined how SGK inhibitors influence stroke outcome in vivo and neurotoxicity in vitro. Infarct volumes were compared in adult mice with middle cerebral artery occlusion, followed by 24 h reperfusion, in the absence or presence of SGK inhibitors. Neurotoxicity assay, electrophysiological recording, and fluorescence Ca(2+) imaging were carried out using cultured cortical neurons to evaluate the underlying mechanisms. Contrary to our expectation, infarct volume by stroke decreased significantly when SGK inhibitor, gsk650394, or EMD638683, was administrated 30 min before middle cerebral artery occlusion under normal and diabetic conditions. SGK inhibitors reduced neurotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors, a leading factor responsible for cell death in stroke. SGK inhibitors also ameliorated Ca(2+) increase and peak amplitude of NMDA current in cultured neurons. In addition, SGK inhibitor gsk650394 decreased phosphorylation of Nedd4-2 and inhibited voltage-gated sodium currents. These observations suggest that SGK activity exacerbates stroke damage and that SGK inhibitors may be useful candidates for therapeutic intervention. To investigate the role of serum- and glucocorticoid-inducible kinases (SGKs) in ischemic brain injury, we examined how SGK inhibitors influence stroke outcome. Infarct volumes induced by middle cerebral artery occlusion were decreased significantly by SGK inhibitors. The inhibitors also reduced glutamate toxicity, at least partly, by attenuation of NMDA and voltage-gated sodium currents. Thus, SGK inhibition attenuates stroke damage. PMID:27123541

  18. Loss of Raf Kinase Inhibitory Protein Induces Radioresistance in Prostate Cancer

    SciTech Connect

    Woods Ignatoski, Kathleen M.; Grewal, Navdeep K.; Markwart, Sonja M.; Vellaichamy, Adaikkalam; Chinnaiyan, Arul M.; Yeung, Kam; Ray, Michael E.; Keller, Evan T.

    2008-09-01

    Purpose: External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. Methods and Materials: The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. Results: RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. Conclusion: The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.

  19. Reactive oxygen species and p38 mitogen-activated protein kinase activate Bax to induce mitochondrial cytochrome c release and apoptosis in response to malonate.

    PubMed

    Gomez-Lazaro, M; Galindo, M F; Melero-Fernandez de Mera, R M; Fernandez-Gómez, F J; Concannon, C G; Segura, M F; Comella, J X; Prehn, J H M; Jordan, J

    2007-03-01

    Malonate, an inhibitor of mitochondrial complex II, is a widely used toxin to study neurodegeneration in Huntington's disease and ischemic stroke. We have shown previously that malonate increased reactive oxygen species (ROS) production in human SH-SY5Y neuroblastoma cells, leading to oxidative stress, cytochrome c release, and apoptotic cell death. Expression of a green fluorescent protein-Bax fusion protein in SH-SY5Y neuroblastoma cells demonstrated a Bax redistribution from the cytosol to mitochondria after 12 to 24 h of malonate treatment that coincided with mitochondrial potential collapse and chromatin condensation. Inhibition of Bax translocation using furosemide, as well as Bax gene deletion, afforded significant protection against malonate-induced apoptosis. Further experiments revealed that malonate induced a prominent increase in the level of activated p38 mitogen-activated protein (MAP) kinase and that treatment with the p38 MAP kinase inhibitor SKF86002 potently blocked malonate-induced Bax translocation and apoptosis. Treatment with vitamin E diminished ROS production, reduced the activation status of p38 MAP kinase, inhibited Bax translocation, and protected against malonate-induced apoptosis. Our data suggest that malonate-induced ROS production and subsequent p38 MAP kinase activation mediates the activation of the pro-apoptotic Bax protein to induce mitochondrial membrane permeabilization and neuronal apoptosis.

  20. Ligand-induced receptor-like kinase complex regulates floral organ abscission in Arabidopsis

    PubMed Central

    Meng, Xiangzong; Zhou, Jinggeng; Tang, Jiao; Li, Bo; de Oliveira, Marcos V. V.; Chai, Jijie; He, Ping; Shan, Libo

    2016-01-01

    SUMMARY Abscission is a developmental process that enables plants to shed unwanted organs. In Arabidopsis, the floral organ abscission is regulated by a signaling pathway consisting of the peptide ligand IDA, the receptor-like kinases (RLKs) HAE and HSL2, and a downstream MAP kinase (MAPK) cascade. However, little is known about the molecular link between ligand-receptor pairs and intracellular signaling. Here, we report that the SERK family RLKs function redundantly in regulating floral organ abscission downstream of IDA and upstream of the MAPK cascade. IDA induces heterodimerization of HAE/HSL2 and SERKs, which transphosphorylate each other. The SERK3 residues mediating its interaction with the immune receptor FLS2 and the brassinosteroid receptor BRI1 are also required for IDA-induced HAE/HSL2-SERK3 interaction, suggesting SERKs serve as co-receptors of HAE/HSL2 in perceiving IDA. Thus, our study reveals the signaling activation mechanism in floral organ abscission by IDA-induced HAE/HSL2-SERK complex formation accompanied by transphosphorylation. PMID:26854226

  1. 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

  2. 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

  3. Attenuation of BPDE-induced p53 accumulation by TPA is associated with a decrease in stability and phosphorylation of p53 and down-regulation of NF-κB activation: Role of p38 MAP kinase

    PubMed Central

    Mukherjee, Jagat J.; Sikka, Harish C.

    2005-01-01

    DNA damage caused by benzo[a]pyrene (BP) or other PAHs induce p53 protein as a protective measure to eliminate the possibility of mutagenic fixation of the DNA damage. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits p53 response induced by BP and other DNA-damaging agents and may cause tumor promotion. The molecular mechanism of attenuation of BP-induced p53 response by TPA is not known. We investigated the effect of TPA on p53 response in BPDE-treated mouse epidermal JB6(P+) Cl 41 cells. BPDE treatment induced p53 accumulation which was attenuated significantly by TPA. Cells treated with BPDE and TPA showed increased ratio of Mdm2 to p53 proteins in p53 immunoprecipitate and decreased p53 life span compared to BPDE-treated cells indicating p53 destabilization by TPA. TPA also inhibited BPDE-induced p53 phosphorylation at serine15. Activation of both ERKs and p38 MAPK by BPDE and attenuation of BPDE-induced p53 accumulation by U0126 or SB202190, specific inhibitor of MEK1/2 or p38 MAPK, indicate the role of ERKs and p38 MAPK in p53 accumulation. Interestingly, TPA potentiated BPDE-induced activation of ERKs whereas p38 MAPK activation was significantly inhibited by TPA, suggesting that inhibition of p38 MAPK is involved in p53 attenuation by TPA. Furthermore SB202190 treatment caused decreased p53 stability and inhibition of phosphorylation of p53 at serine 15 in BPDE-treated cells. We also observed that TPA or SB202190 attenuated BPDE-induced NF-κB activation in JB6 (Cl 41) cells harboring NF-κB reporter plasmid. To our knowledge this is the first report that TPA inhibits chemical carcinogen-induced NF-κB activation. Interference of TPA with BPDE-induced NF-κB activation implicates abrogation of p53 function which has been discussed. Overall our data suggest that abrogation of BPDE-induced p53 response and of NF-κB activation by TPA is mediated by impairment of signaling pathway involving p38 MAPK. PMID:16244358

  4. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization

    PubMed Central

    Lavoie, Hugo; Thevakumaran, Neroshan; Gavory, Gwenaëlle; Li, John; Padeganeh, Abbas; Guiral, Sébastien; Duchaine, Jean; Mao, Daniel Y. L.; Bouvier, Michel; Sicheri, Frank; Therrien, Marc

    2016-01-01

    RAF kinases play a prominent role in cancer. Their mode of activation is complex, but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and as a result undesirably stimulate RAS/ERK-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe BRET-based biosensors for the extended RAF family enabling the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization as well as for probing structural determinants of RAF dimerization in vivo. Our findings, which appear generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain. PMID:23685672

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

    DOE PAGESBeta

    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

  6. 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.

  7. Overexpression of a kinase-deficient form of the EDR1 gene enhances powdery mildew resistance and ethylene-induced senescence in Arabidopsis.

    PubMed

    Tang, Dingzhong; Innes, Roger W

    2002-12-01

    The EDR1 gene of Arabidopsis has previously been reported to encode a Raf-like mitogen-activated protein kinase kinase (MAPKK) kinase, and to function as a negative regulator of disease resistance. A phylogenetic analysis of plant and animal protein kinases revealed, however, that plant Raf-like kinases are more closely related to animal mixed lineage kinases (MLKs) than Raf-like kinases, and are deeply divergent from both classes of animal kinases, making inferences of substrate specificity questionable. We, therefore, assayed the kinase activity of recombinant EDR1 protein in vitro. The EDR1 kinase domain displayed autophosphorylation activity and phosphorylated the common MAP kinase substrate myelin basic protein. The EDR1 kinase domain also phosphorylated a kinase-deficient EDR1 protein, indicating that EDR1 autophosphorylation can occur via an intermolecular mechanism. Overexpression of a kinase-deficient full-length EDR1 gene (35S::dnEDR1) in wild-type Arabidopsis plants caused a dominant negative phenotype, conferring resistance to powdery mildew (Erysiphe cichoracearum) and enhancing ethylene-induced senescence. RNA-gel blot analyses showed that the 35S::dnEDR1 transgene was highly transcribed in transgenic plants. Western blot analysis, however, revealed that neither the wild-type nor mutant EDR1 protein could be detected in these lines, indicating that the dominant negative phenotype may be caused by a translational inhibition mechanism rather than by a protein level effect. Overexpression of orthologous dnEDR1 constructs may provide a novel strategy for controlling powdery mildew disease in crops.

  8. 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.

  9. 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

  10. Osmotic Stress Induces Rapid Activation of a Salicylic Acid–Induced Protein Kinase and a Homolog of Protein Kinase ASK1 in Tobacco Cells

    PubMed Central

    Mikołajczyk, Monika; Awotunde, Olubunmi S.; Muszyńska, Grażyna; Klessig, Daniel F.; Dobrowolska, Grażyna

    2000-01-01

    In tobacco cells, osmotic stress induced the rapid activation of two protein kinases that phosphorylate myelin basic protein. Immunological studies demonstrated that the 48-kD kinase is the salicylic acid–induced protein kinase (SIPK), a member of the mitogen-activated protein kinase family. SIPK was activated 5 to 10 min after the cells were exposed to osmotic stresses, and its activity persisted for ∼30 min. In contrast, the 42-kD kinase was activated within 1 min after osmotic stress, and its activity was maintained for ∼2 hr. Moreover, in addition to myelin basic protein, the 42-kD kinase phosphorylated casein and two transcription factors, c-Jun and ATF-2. This latter enzyme was inactivated by a serine/threonine–specific phosphatase but, unlike SIPK, was not affected by a tyrosine-specific phosphatase. After the 42-kD kinase was purified to apparent homogeneity, tryptic peptide analysis indicated that it is a homolog of Arabidopsis serine/threonine kinase1 (ASK1). PMID:10634915

  11. S6 Kinase Reflects and Regulates Ethanol-Induced Sedation

    PubMed Central

    Acevedo, Summer F.; Peru y Colón de Portugal, Raniero L.; Gonzalez, Dante A.; Rodan, Aylin R.

    2015-01-01

    Alcohol use disorders (AUDs) affect people at great individual and societal cost. Individuals at risk for AUDs are sensitive to alcohol's rewarding effects and/or resistant to its aversive and sedating effects. The molecular basis for these traits is poorly understood. Here, we show that p70 S6 kinase (S6k), acting downstream of the insulin receptor (InR) and the small GTPase Arf6, is a key mediator of ethanol-induced sedation in Drosophila. S6k signaling in the adult nervous system determines flies' sensitivity to sedation. Furthermore, S6k activity, measured via levels of phosphorylation (P-S6k), is a molecular marker for sedation and overall neuronal activity: P-S6k levels are decreased when neurons are silenced, as well as after acute ethanol sedation. Conversely, P-S6k levels rebound upon recovery from sedation and are increased when neuronal activity is enhanced. Reducing neural activity increases sensitivity to ethanol-induced sedation, whereas neuronal activation decreases ethanol sensitivity. These data suggest that ethanol has acute silencing effects on adult neuronal activity, which suppresses InR/Arf6/S6k signaling and results in behavioral sedation. In addition, we show that activity of InR/Arf6/S6k signaling determines flies' behavioral sensitivity to ethanol-induced sedation, highlighting this pathway in acute responses to ethanol. SIGNIFICANCE STATEMENT Genetic factors play a major role in the development of addiction. Identifying these genes and understanding their molecular mechanisms is a necessary first step in the development of targeted therapeutic intervention. Here, we show that signaling from the insulin receptor in Drosophila neurons determines flies' sensitivity to ethanol-induced sedation. We show that this signaling cascade includes the small GTPase Arf6 and S6 kinase (S6k). In addition, activity of S6k is regulated by acute ethanol exposure and by neuronal activity. S6k activity is therefore both an acute target of ethanol exposure and

  12. Trypanosoma cruzi: Oxidative stress induces arginine kinase expression.

    PubMed

    Miranda, Mariana R; Canepa, Gaspar E; Bouvier, Leon A; Pereira, Claudio A

    2006-12-01

    Trypanosoma cruzi arginine kinase is a key enzyme in cell energy management and is also involved in pH and nutritional stress response mechanisms. T. cruzi epimastigotes treated with hydrogen peroxide presented a time-dependent increase in arginine kinase expression, up to 10-fold, when compared with untreated parasites. Among other oxidative stress-generating compounds tested, only nifurtimox produced more than 2-fold increase in arginine kinase expression. Moreover, parasites overexpressing arginine kinase showed significantly increased survival capability during hydrogen peroxide exposure. These findings suggest the participation of arginine kinase in oxidative stress response systems. PMID:16725140

  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. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    PubMed Central

    Mahajan, S; Fargnoli, J; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-01-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation. PMID:7565679

  15. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    PubMed

    Mahajan, S; Fargnoli, J; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-10-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation. PMID:7565679

  16. The NPM-ALK tyrosine kinase mimics TCR signalling pathways, inducing NFAT and AP-1 by RAS-dependent mechanisms.

    PubMed

    Turner, Suzanne D; Yeung, Debra; Hadfield, Kathryn; Cook, Simon J; Alexander, Denis R

    2007-04-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expression is associated with the lymphoid malignancy anaplastic large cell lymphoma (ALCL) and results from a t(2;5) chromosomal translocation. We show that NPM-ALK induces Ras activation and phosphorylation of the ERK MAP Kinase consistent with activation of the Ras-MAP Kinase pathway. Furthermore, we demonstrate that activation of Ras is necessary for inducing transcription via NFAT/AP-1 composite transcriptional binding sites. This activity is dependent on NPM-ALK forming complexes with proteins that bind to autophosphorylated tyrosine residues at positions 156, 567 and 664, associated with binding to IRS-1, Shc and PLCgamma, respectively. Specifically, NPM-ALK activates transcription from the TRE promoter element, an AP-1 binding region, an activity dependent on both Ras and Shc activity. Our results show that NPM-ALK mimics activated T-cell receptor signalling by inducing pathways associated with the activation of NFAT/AP-1 transcription factors that bind to promoter elements found in a broad array of cytokine genes.

  17. 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

  18. UVB exposure enhanced benzanthrone-induced inflammatory responses in SKH-1 mouse skin by activating the expression of COX-2 and iNOS through MAP kinases/NF-κB/AP-1 signalling pathways.

    PubMed

    Abbas, Sabiya; Alam, Shamshad; Pal, Anu; Kumar, Mahadeo; Singh, Dhirendra; Ansari, Kausar Mahmood

    2016-10-01

    This study was conducted to explore the role of UVB on benzanthrone (BA)-induced skin inflammation and its mechanism/s. SKH-1 hairless mice were topically exposed with BA (25 and 50 mg/kg b.wt) either alone or along with UVB (50 mJ/cm(2)) for 24 h and estimation of ROS, histopathological analysis, myeloperoxidase (MPO) activity, mast cell staining, immunohistochemistry for COX-2 and iNOS as well as western blotting for MAPKs, p-NF-κB, c-jun, c-fos COX-2 and iNOS were carried out. Enhanced ROS generation, increased epidermal thickness, mast cell number, MPO activity, enhanced expression of COX-2 and iNOS, MAPKs, c-jun, c-fos, NF-κB were found in BA either alone or when followed by UVB treatment, compared to the control groups. Expression of COX-2, iNOS and phosphorylation of ERK1/2 were found to be more enhanced in BA and UVB- exposed group compared to BA and UVB only group, while phosphorylation of JNK1/2, p38, NF-κB and expression of c-jun and c-fos were comparable with BA and UVB only groups. In summary, we suggest that UVB exposure enhanced BA-induced SKH-1 skin inflammation possibly via oxidative stress-mediated activation of MAPKs-NF-κB/AP-1 signalling, which subsequently increased the expression of COX-2 and iNOS and led to inflammation in SKH-1 mouse skin.

  19. 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

  20. 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.

  1. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

    PubMed

    Luz, Simão; Kongsuphol, Patthara; Mendes, Ana Isabel; Romeiras, Francisco; Sousa, Marisa; Schreiber, Rainer; Matos, Paulo; Jordan, Peter; Mehta, Anil; Amaral, Margarida D; Kunzelmann, Karl; Farinha, Carlos M

    2011-11-01

    Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

  2. 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.

  3. 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

  4. Berberine induces dedifferentiation by actin cytoskeleton reorganization via phosphoinositide 3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes

    PubMed Central

    Yu, Seon-Mi; Cho, Hongsik; Kim, Gwang-Hoon; Chung, Ki-Wha; Seo, Sung-Yum

    2016-01-01

    Osteoarthritis is a nonrheumatologic joint disease characterized by progressive degeneration of the cartilage extracellular matrix. Berberine (BBR) is an isoquinoline alkaloid used in traditional Chinese medicine, the majority of which is extracted from Huang Lian (Coptis chinensis). Although numerous studies have revealed the anticancer activity of BBR, its effects on normal cells, such as chondrocytes, and the molecular mechanisms underlying its actions remain elusive. Therefore, we examined the effects of BBR on rabbit articular chondrocytes, and the underlying molecular mechanisms, focusing on actin cytoskeletal reorganization. BBR induced dedifferentiation by inhibiting activation of phosphoinositide-3(PI3)-kinase/Akt and p38 kinase. Furthermore, inhibition of p38 kinase and PI3-kinase/Akt with SB203580 and LY294002, respectively, accelerated the BBR-induced dedifferentiation. BBR also caused actin cytoskeletal architecture reorganization and, therefore, we investigated if these effects were involved in the dedifferentiation. Disruption of the actin cytoskeleton by cytochalasin D reversed the BBR-induced dedifferentiation by activating PI3-kinase/Akt and p38 kinase. In contrast, the induction of actin filament aggregation by jasplakinolide accelerated the BBR-induced dedifferentiation via PI3-kinase/Akt inhibition and p38 kinase activation. Taken together, these data suggest that BBR strongly induces dedifferentiation, and actin cytoskeletal reorganization is a crucial requirement for this effect. Furthermore, the dedifferentiation activity of BBR appears to be mediated via PI3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes. PMID:26851252

  5. 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

  6. The inducible G protein-coupled receptor edg-1 signals via the G(i)/mitogen-activated protein kinase pathway.

    PubMed

    Lee, M J; Evans, M; Hla, T

    1996-05-10

    The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study was to investigate the G protein-coupling and -signaling properties of the edg-1 polypeptide. The third cytosolic loop (i3) of edg-1 associates with G(i) alpha and G(o) alpha polypeptides in a guanosine 5'-O-(thiotriphosphate)-sensitive manner. Immunoprecipitation of the edg-1 polypeptide in transfected cells results in the co-precipitation of G(i) alpha 1 and G(i) alpha 3 polypeptides. These data strongly suggest that edg-1 is capable of coupling to the Gi pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the sustained activation of the MAP kinase activity that is blocked by pertussis toxin treatment. Moreover, NIH3T3 cells permanently transfected with edg-1 exhibit enhanced MAP kinase and phospholipase A2 activities. These data suggest that the G(i)/mitogen-activated protein kinase pathway is a major signaling pathway regulated by the orphan receptor edg-1. PMID:8626678

  7. 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

  8. The NFKB Inducing Kinase Modulates Hematopoiesis During Stress.

    PubMed

    González-Murillo, África; Fernández, Lucía; Baena, Sara; Melen, Gustavo J; Sánchez, Rebeca; Sánchez-Valdepeñas, Carmen; Segovia, José C; Liou, Hsiou-Chi; Schmid, Roland; Madero, Luís; Fresno, Manuel; Ramírez, Manuel

    2015-09-01

    The genetic programs that maintain hematopoiesis during steady state in physiologic conditions are different from those activated during stress. Here, we show that hematopoietic stem cells (HSCs) with deficiencies in components of the alternative NFκB pathway (the NFκB inducing kinase, NIK, and the downstream molecule NFκB2) had a defect in response to stressors such as supraphysiological doses of cytokines, chemotherapy, and hematopoietic transplantation. NIK-deficient mice had peripheral blood and bone marrow leukocyte numbers within normal ranges (except for the already reported defects in B-cell maturation); however, HSCs showed significantly slower expansion capacity in in vitro cultures compared to wild-type HSCs. This was due to a delayed cell cycle and increased apoptosis. In vivo experiments showed that NIK-deficient HSCs did not recover at the same pace as controls when challenged with myeloablative chemotherapy. Finally, NIK-deficient HSCs showed a significantly decreased competitive repopulation capacity in vivo. Using HSCs from mice deficient in one of two downstream targets of NIK, that is, either NFκB2 or c-Rel, only NFκB2 deficiency recapitulated the defects detected with NIK-deficient HSCs. Our results underscore the role of NIK and the alternative NFκB pathway for the recovery of normal levels of hematopoiesis after stress.

  9. 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

  10. Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

    PubMed

    Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

    2016-04-01

    Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension

  11. Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

    PubMed

    Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

    2016-04-01

    Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension

  12. 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

  13. 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.

  14. [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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Open Stomata 1 Kinase is Essential for Yeast Elicitor-Induced Stomatal Closure in Arabidopsis.

    PubMed

    Ye, Wenxiu; Adachi, Yuji; Munemasa, Shintaro; Nakamura, Yoshimasa; Mori, Izumi C; Murata, Yoshiyuki

    2015-06-01

    We recently demonstrated that yeast elicitor (YEL)-induced stomatal closure requires a Ca(2+)-dependent kinase, CPK6. A Ca(2+)-independent kinase, Open Stomata 1 (OST1), is involved in stomatal closure induced by various stimuli including ABA. In the present study, we investigated the role of OST1 in YEL-induced stomatal closure in Arabidopsis using a knock-out mutant, ost1-3, and a kinase-deficient mutant, ost1-2. YEL did not induce stomatal closure or activation of guard cell S-type anion channels in the ost1 mutants unlike in wild-type plants. However, YEL did not increase OST1 kinase activity in wild-type guard cells. The YEL-induced stomatal closure and activation of S-type anion channels were also impaired in a gain-of-function mutant of a clade A type 2C protein phosphatase (ABA INSENSITIVE 1), abi1-1C. In the ost1 mutants like in the wild type, YEL induced H2O2 accumulation, activation of non-selective Ca(2+)-permeable cation (ICa) channels and transient elevations in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in guard cells. These results suggest that OST1 kinase is essential for stomatal closure and activation of S-type anion channels induced by YEL and that OST1 is not involved in H2O2 accumulation, ICa channel activation or [Ca(2+)]cyt elevations in guard cells induced by YEL.

  4. 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

  5. Phosphatidylinositol-3 kinase activation induced upon Fc gamma RIIIA- ligand interaction

    PubMed Central

    1994-01-01

    Induced activation of protein tyrosine kinase(s) is a central event in signal transduction mediated via the low affinity receptor for IgG (Fc gamma RIIIA, CD16) in natural killer (NK) cells. Tyrosine phosphorylation may affect the function of several protein directly, or indirectly by inducing their association with other tyrosine phosphorylated proteins. Here, we report that Fc gamma RIII stimulation induces activation of phosphatidylinositol (PI)-3 kinase in NK cells. Phosphotyrosine immunoprecipitates from Fc gamma RIII-stimulated NK cells contain PI-kinase activity and PI-3 kinase can be directly precipitated from them. Conversely, a series of tyrosine-phosphorylated proteins is coprecipitated with PI-3 kinase from the stimulated, but not from control cells. Analogous results obtained using Jurkat T cells expressing transfected Fc gamma RIIIA alpha ligand binding chain in association with gamma 2 or zeta 2 homodimers indicate that both complexes transduce this effect, although the Fc gamma RIIIA-zeta 2 complexes do so with greater efficiency. Accumulation of phosphoinositide D3 phosphorylated products in stimulated cells confirms PI-3 kinase activation, indicating the participation of this enzyme in Fc gamma RIIIA-mediated signal transduction. PMID:8294866

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. Rho-kinase-dependent F-actin rearrangement is involved in the inhibition of PI3-kinase/Akt during ischemia–reperfusion-induced endothelial cell apoptosis

    PubMed Central

    Versteilen, Amanda M. G.; Sipkema, Pieter; van Nieuw Amerongen, Geerten P.; Musters, Rene J. P.; Groeneveld, A. B. Johan

    2007-01-01

    Activation of cytoskeleton regulator Rho-kinase during ischemia–reperfusion (I/R) plays a major role in I/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I/R. Y-27632 treatment reduced I/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R2 = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity. PMID:18165899

  14. 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.

  15. Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation

    PubMed Central

    Lobbestael, E.; Civiero, L.; De Wit, T.; Taymans, J.-M.; Greggio, E.; Baekelandt, V.

    2016-01-01

    Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and is known to play a role in Parkinson’s disease (PD) pathobiology. This has stimulated the development of potent, selective LRRK2 kinase inhibitors as one of the most prevailing disease-modifying therapeutic PD strategies. Although several lines of evidence support beneficial effects of LRRK2 kinase inhibitors, many questions need to be answered before clinical applications can be envisaged. Using six different LRRK2 kinase inhibitors, we show that LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain, lung and kidney. The inhibitor-induced reduction in LRRK2 levels could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. In addition, using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Increasing our insight in the molecular and cellular consequences of LRRK2 kinase inhibition will be crucial in the further development of LRRK2-based PD therapies. PMID:27658356

  16. 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

  17. Targeting Apoptosis Signalling Kinase-1 (ASK-1) Does Not Prevent the Development of Neuropathy in Streptozotocin-Induced Diabetic Mice

    PubMed Central

    Newton, Victoria L.; Ali, Sumia; Duddy, Graham; Whitmarsh, Alan J.; Gardiner, Natalie J.

    2014-01-01

    Apoptosis signal-regulating kinase-1 (ASK1) is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K) which lies upstream of the stress-activated MAPKs, JNK and p38. ASK1 may be activated by a variety of extracellular and intracellular stimuli. MAP kinase activation in the sensory nervous system as a result of diabetes has been shown in numerous preclinical and clinical studies. As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy. We therefore wanted to characterize the expression of ASK1 in sensory neurons, and determine whether the absence of functional ASK1 would protect against the development of neuropathy in a mouse model of experimental diabetes. ASK1 mRNA and protein is constitutively expressed by multiple populations of sensory neurons of the adult mouse lumbar DRG. Diabetes was induced in male C57BL/6 and transgenic ASK1 kinase-inactive (ASK1n) mice using streptozotocin. Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes. However, levels of ASK2 mRNA increase in the spinal cord at 4 weeks of diabetes, which could represent a future target for this field. Neither motor nerve conduction velocity deficits, nor thermal or mechanical hypoalgesia were prevented or ameliorated in diabetic ASK1n mice. These results suggest that activation of ASK1 is not responsible for the nerve deficits observed in this mouse model of diabetic neuropathy. PMID:25329046

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

    SciTech Connect

    Dey, Nandini . E-mail: Don_Durden@oz.ped.emory.edu

    2005-07-01

    Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation.

  3. The involvement of an LPS inducible I kappa B kinase in endotoxin tolerance.

    PubMed

    Kohler, N G; Joly, A

    1997-03-27

    When human ovarian carcinoma cells are challenged with endotoxin, an I kappa B kinase is transiently induced within 3 to 5 min. This enzyme activity causes the hyperphosphorylation and subsequent degradation of I kappa B which allows NF-kappa B to translocate to the nucleus where it activates transcription. When endotoxin treated cells are rechallenged with a second dose of LPS, I kappa B kinase is not detected and I kappa B remains in the cytoplasm where it sequesters NF-kappa B. We report here the absence of endotoxin inducible I kappa B kinase activity in endotoxin tolerant cells suggesting that I kappa B kinase may play an important role in endotoxin tolerance. When cells tolerant to endotoxin are treated with TNF, I kappa B kinase activity is induced. Thus cells that are endotoxin tolerant are not cross tolerant to TNF. Dexamethasone, a known inhibitor of NF-kappa B activation does not inhibit endotoxin dependent induction of I kappa B kinase suggesting that the mechanism of action of dexamethasone is different from the tolerance mechanism reported here.

  4. 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

  5. Lithium protection of phencyclidine-induced neurotoxicity in developing brain: the role of phosphatidylinositol-3 kinase/Akt and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathways.

    PubMed

    Xia, Yan; Wang, Cheng Z; Liu, Jie; Anastasio, Noelle C; Johnson, Kenneth M

    2008-09-01

    Phencyclidine (PCP) and other N-methyl-D-aspartate (NMDA) receptor antagonists have been shown to be neurotoxic to developing brains and to result in schizophrenia-like behaviors later in development. Prevention of both effects by antischizophrenic drugs suggests the validity of PCP neurodevelopmental toxicity as a heuristic model of schizophrenia. Lithium is used for the treatment of bipolar and schizoaffective disorders and has recently been shown to have neuroprotective properties. The present study used organotypic corticostriatal slices taken from postnatal day 2 rat pups to investigate the protective effect of lithium and the role of the phosphatidylinositol-3 kinase (PI-3K)/Akt and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways in PCP-induced cell death. Lithium pretreatment dose-dependently reduced PCP-induced caspase-3 activation and DNA fragmentation in layers II to IV of the cortex. PCP elicited time-dependent inhibition of the MEK/ERK and PI-3K/Akt pathways, as indicated by dephosphorylation of ERK1/2 and Akt. The proapoptotic factor glycogen synthase kinase (GSK)-3beta was also dephosphorylated at serine 9 and thus activated. Lithium prevented PCP-induced inhibition of the two pathways and activation of GSK-3beta. Furthermore, blocking either PI-3K/Akt or MEK/ERK pathway abolished the protective effect of lithium, whereas inhibiting GSK-3beta activity mimicked the protective effect of lithium. However, no cross-talk between the two pathways was found. Finally, specific GSK-3beta inhibition did not prevent PCP-induced dephosphorylation of Akt and ERK. These data strongly suggest that the protective effect of lithium against PCP-induced neuroapoptosis is mediated through independent stimulation of the PI-3K/Akt and ERK pathways and suppression of GSK-3beta activity.

  6. 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

  7. 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.

  8. Genetic variation in insulin-induced kinase signaling

    PubMed Central

    Wang, Isabel Xiaorong; Ramrattan, Girish; Cheung, Vivian G

    2015-01-01

    Individual differences in sensitivity to insulin contribute to disease susceptibility including diabetes and metabolic syndrome. Cellular responses to insulin are well studied. However, which steps in these response pathways differ across individuals remains largely unknown. Such knowledge is needed to guide more precise therapeutic interventions. Here, we studied insulin response and found extensive individual variation in the activation of key signaling factors, including ERK whose induction differs by more than 20-fold among our subjects. This variation in kinase activity is propagated to differences in downstream gene expression response to insulin. By genetic analysis, we identified cis-acting DNA variants that influence signaling response, which in turn affects downstream changes in gene expression and cellular phenotypes, such as protein translation and cell proliferation. These findings show that polymorphic differences in signal transduction contribute to individual variation in insulin response, and suggest kinase modulators as promising therapeutics for diseases characterized by insulin resistance. PMID:26202599

  9. GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases.

    PubMed Central

    Heasley, L E; Storey, B; Fanger, G R; Butterfield, L; Zamarripa, J; Blumberg, D; Maue, R A

    1996-01-01

    Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G

  10. Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase.

    PubMed

    Joo, Jung Hee; Yoo, Ho Jung; Hwang, Inhwan; Lee, June Seung; Nam, Kyoung Hee; Bae, Yun Soo

    2005-02-14

    We recently reported that production of reactive oxygen species (ROS) is essential for auxin-induced gravitropic signaling. Here, we investigated the role of phosphatidylinositol 3-kinase and its product, PtdIns(3)P, in auxin-mediated ROS production and the root gravitropic response. Pretreatment with LY294002, an inhibitor of PtdIns 3-kinase activity, blocked auxin-mediated ROS generation, and reduced the sensitivity of root tissue to gravistimulation. The amount of PtdIns(3)P increased in response to auxin, and this effect was abolished by pretreatment with LY294002. In addition, sequestration of PtdIns(3)P by transient expression of the endosome binding domain in protoplasts abrogated IAA-induced ROS accumulation. These results indicate that activation of PtdIns 3-kinase and its product PtdIns(3)P are required for auxin-induced production of ROS and root gravitropism. PMID:15710420

  11. Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors.

    PubMed

    Riehl, Brandon D; Lee, Jeong Soon; Ha, Ligyeom; Lim, Jung Yul

    2015-03-01

    The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear.

  12. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    SciTech Connect

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  13. 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

  14. 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.

  15. 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

  16. Participation of protein kinases in staurosporine-induced interleukin-6 production by rat peritoneal macrophages

    PubMed Central

    Yamaki, Kouya; Ohuchi, Kazuo

    1999-01-01

    The incubation of rat peritoneal macrophages in the presence of staurosporine, a non-specific protein kinase inhibitor, induced interleukin-6 (IL-6) production in a time- and concentration-dependent manner at 6.3–63 nM, but at 210 nM, the stimulant effect on IL-6 production was reduced.The levels of IL-6 mRNA as determined by a reverse transcription-polymerase chain reaction were also increased by staurosporine in parallel with the ability to induce IL-6 production.Compounds structurally related to staurosporine including K-252a (non-specific protein kinase inhibitor) and KT-5720 (inhibitor of cyclic AMP-dependent protein kinase, PKA), did not increase IL-6 production by peritoneal macrophages.Staurosporine-induced increases in IL-6 production and expression of IL-6 mRNA were decreased by the PKC inhibitors, H-7 (2.7–27 μM), Ro 31-8425 (1–10 μM) and calphostin C (0.3–3 μM) and by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 (30–100 μM), but were further increased by the protein tyrosine kinase (PTK) inhibitor, genistein (12–37 μM).The staurosporine-induced increase in IL-6 production was not affected by the PKA inhibitor, H-89 (0.1–3 μM).These findings suggest that the induction of IL-6 production by staurosporine is secondary to elevation of IL-6 mRNA level, which, in turn, is positively regulated by the activation of PKC and PI 3-kinase and negatively regulated by the activation of PTK. PKA does not appear to play a significant role. PMID:10455280

  17. 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

  18. 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

  19. 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

  20. 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.

  1. Sphingosine induces phospholipase D and mitogen activated protein kinase in vascular smooth muscle cells.

    PubMed

    Taher, M M; Abd-Elfattah, A S; Sholley, M M

    1998-12-01

    The enzymes phospholipase D and diacylglycerol kinase generate phosphatidic acid which is considered to be a mitogen. Here we report that sphingosine produced a significant amount of phosphatidic acid in vascular smooth muscle cells from the rat aorta. The diacylglycerol kinase inhibitor R59 949 partially depressed sphingosine induced phosphatidic acid formation, suggesting that activation of phospholipase C and diacylglycerol kinase can not account for the bulk of phosphatidic acid produced and that additional pathways such as phospholipase D may contribute to this. Further, we have shown that phosphatidylethanol was produced by sphingosine when vascular smooth muscle cells were stimulated in the presence of ethanol. Finally, as previously shown for other cell types, sphingosine stimulated mitogen-activated protein kinase in vascular smooth muscle cells.

  2. Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632.

    PubMed

    Yamaguchi, Hiroto; Miwa, Yukiko; Kasa, Miyuki; Kitano, Ken; Amano, Mutsuki; Kaibuchi, Kozo; Hakoshima, Toshio

    2006-09-01

    Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability. PMID:16891330

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer

    PubMed Central

    Guo, Chengcheng; Hao, Chuncheng; Shao, RuPing; Fang, Bingliang; Correa, Arlene M.; Hofstetter, Wayne L.; Roth, Jack A.; Behrens, Carmen; Kalhor, Neda; Wistuba, Ignacio I.; Swisher, Stephen G.; Pataer, Apar

    2015-01-01

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expresson of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinaseT172 [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  12. Serine-threonine kinase 38 is regulated by glycogen synthase kinase-3 and modulates oxidative stress-induced cell death.

    PubMed

    Enomoto, Atsushi; Kido, Naoki; Ito, Michihiko; Takamatsu, Nobuhiko; Miyagawa, Kiyoshi

    2012-01-15

    Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-like family. In the present study, we investigated the regulatory mechanism of STK38 and assessed its role in the cellular stress response. Among various environmental stresses, STK38 was specifically activated by H(2)O(2), and the phosphatidylinositol 3-kinase inhibitor wortmannin or AKT inhibitor IV suppressed this activation. STK38 was also activated by a constitutively active AKT1 or by GSK-3β inhibitor VII. The phosphorylation level of GSK-3β was correlated with the STK38 activity, in response to various stimuli and in different cell lines. Co-immunoprecipitation analysis revealed that GSK-3β physically interacted with STK38 in cells. GSK-3β overexpression inhibited the H(2)O(2)-stimulated STK38 activity. GSK-3β phosphorylated STK38 on residues S6 and T7 in vitro, depending largely on a PKA-mediated priming phosphorylation of STK38 on residues S10 and S11, respectively. STK38's H(2)O(2)-stimulated activity was enhanced by alanine substitution at its priming sites and/or at S6 and T7, and it was partially reduced by a phosphomimetic mutation at S6 or T7. STK38 knockdown enhanced the H(2)O(2)-induced JNK phosphorylation and cell death. Our results indicate that that GSK-3β inhibits STK38's full activation, and suggest that STK38 activation is required to prevent cell death in response to oxidative stress.

  13. Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    PubMed Central

    Zhou, Ling; Yan, Chunli; Gieling, Roben G; Kida, Yujiro; Garner, Warren; Li, Wei; Han, Yuan-Ping

    2009-01-01

    Background Expressed in embryonic development, matrix metalloprotein-9 (MMP-9) is absent in most of developed adult tissues, but recurs in inflammation during tissue injury, wound healing, tumor formation and metastasis. Expression of MMP-9 is tightly controlled by extracellular cues including pro-inflammatory cytokines and extracellular matrix (ECM). While the pathologic functions of MMP-9 are evident, the intracellular signaling pathways to control its expression are not fully understood. In this study we investigated mechanism of cytokine induced MMP-9 with particular emphasis on the role of p21-activated-kinase-1 (PAK1) and the down stream signaling. Results In response to TNF-alpha or IL-1alpha, PAK1 was promptly activated, as characterized by a sequential phosphorylation, initiated at threonine-212 followed by at threonine-423 in the activation loop of the kinase, in human skin keratinocytes, dermal fibroblasts, and rat hepatic stellate cells. Ectopic expression of PAK1 variants, but not p38 MAP kinase, impaired the TNF-alpha-induced MMP-9 expression, while other MMPs such as MMP-2, -3 and -14 were not affected. Activation of Jun N-terminal kinase (JNK) and NF-kappaB has been demonstrated to be essential for MMP-9 expression. Expression of inactive PAK1 variants impaired JNK but not NF-kappaB activation, which consequently suppressed the 5'-promoter activities of the MMP-9 gene. After the cytokine-induced phosphorylation, both ectopically expressed and endogenous PAK1 proteins were promptly accumulated even in the condition of suppressing protein synthesis, suggesting the PAK1 protein is stabilized upon TNF-alpha stimulation. Stabilization of PAK1 protein by TNF-alpha treatment is independent of the kinase catalytic activity and p21 GTPase binding capacities. In contrast to epithelial cells, mesenchymal cells require 3-dimensional type-I collagen in response to TNF-alpha to massively express MMP-9. The collagen effect is mediated, in part, by boost JNK

  14. 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

  15. Antibody targeting of anaplastic lymphoma kinase induces cytotoxicity of human neuroblastoma.

    PubMed

    Carpenter, E L; Haglund, E A; Mace, E M; Deng, D; Martinez, D; Wood, A C; Chow, A K; Weiser, D A; Belcastro, L T; Winter, C; Bresler, S C; Vigny, M; Mazot, P; Asgharzadeh, S; Seeger, R C; Zhao, H; Guo, R; Christensen, J G; Orange, J S; Pawel, B R; Lemmon, M A; Mossé, Y P

    2012-11-15

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies-as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.

  16. Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

    ERIC Educational Resources Information Center

    Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

    2016-01-01

    In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

  17. Hippocampal Src kinase is required for novelty-induced enhancement of contextual fear extinction.

    PubMed

    Wang, Bing; Liang, Ri-Chu; Liu, Zheng-Shan; Luo, Bing; Ding, Ya; Chen, Zi-Xiang; Liao, Yong-Shi; Wang, Xiao-Guang

    2016-04-15

    Exposure to a novel environment enhances the extinction of contextual fear through the "tagging-and-capture" process. However, the underlying molecular mechanisms of novelty-induced enhancement of fear extinction are still unclear. NMDA receptor activity was recently revealed to be required for the enhancement of fear extinction caused by exposure to novelty. Src family kinases (SFKs) act as a molecular hub for regulation of NMDA receptors. We hypothesized that SFKs might be involved in novelty-induced enhancement of fear extinction. We found that the enhancement of fear extinction induced by novelty exposure is accompanied by Src kinase phosphorylation and activation in a restricted time window. Furthermore, intrahippocampal infusion of SFKs inhibitor PP2 inhibits Src kinase phosphorylation and activation, attenuates the activation of NR2B-containing NMDA receptors, and thereby reverses the enhancement of fear extinction induced by novelty exposure. These results suggested that Src kinase may serve as a behavioral tag in the procedural enhancement of fear extinction by novelty exposure.

  18. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    SciTech Connect

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  19. PIM2 kinase is induced by cisplatin in ovarian cancer cells and limits drug efficacy.

    PubMed

    Musiani, Daniele; Hammond, Dean E; Cirillo, Luca; Erriquez, Jessica; Olivero, Martina; Clague, Michael J; Di Renzo, Maria Flavia

    2014-11-01

    Platinum-based chemotherapy is widely used to treat various cancers, but many patients ultimately relapse due to drug resistance. We employed phosphoproteomic analysis and functional assays of the response of SK-OV-3 ovarian cancer cells to cisplatin as a strategy to identify kinases as candidate druggable targets to sensitize cells to platinum. A SILAC-based approach combined with TiO2-based phosphopeptide enrichment allowed the direct identification of ERK1/2, p90RSK, and ERBB2 as kinases whose phosphorylation is regulated by cisplatin. Bioinformatic analysis revealed enrichment in linear phosphorylation motifs predicted to be targets of p38MAPK, CDK2, and PIM2. All three PIM kinases were found expressed in a panel of 10 ovarian cancer cell lines, with the oncogenic PIM2 being the most commonly induced by cisplatin. Targeting PIM2 kinase by either biochemical inhibitors or RNA interference impaired cell growth, decreased cisplatin-triggered BAD phosphorylation, and sensitized ovarian cancer cells to drug-induced apoptosis. Overexpression of PIM2 triggered anchorage-independent growth and resulted in increased BAD phosphorylation and cell resistance to DNA damaging agents. The data show that the PIM2 kinase plays a role in the response of ovarian cancer cells to platinum drugs and suggest that PIM inhibitors may find clinical application as an adjunct to platinum-based therapies. PMID:25099161

  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.

  1. 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

  2. Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

    SciTech Connect

    Suzuki, Toshihide; Miyazaki, Koichi; Kita, Kayoko; Ochi, Takafumi

    2009-12-15

    Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvement of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.

  3. 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

  4. 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

  5. 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

  6. Degradation of the interferon-induced 68,000-M(r) protein kinase by poliovirus requires RNA.

    PubMed Central

    Black, T L; Barber, G N; Katze, M G

    1993-01-01

    Control of the interferon-induced double-stranded RNA (dsRNA) activated protein kinase (referred to as P68 because of its M(r) of 68,000 in human cells) by animal viruses is essential to avoid decreases in protein synthetic rates during infection. We have previously demonstrated that poliovirus establishes a unique way of regulating the protein kinase, namely by inducing the specific degradation of P68 during infection (T. L. Black, B. Safer, A. Hovanessian, and M. G. Katze, J. Virol. 63:2244-2251, 1989). In the present study we investigated the mechanisms by which P68 degradation occurred. To do this we used an in vitro degradation assay which faithfully reproduced the in vivo events. Although viral gene expression was required for P68 degradation, the major poliovirus proteases, 2A and 3C, were found not to be directly involved with P68 proteolysis. However, the protease responsible for P68 degradation required divalent cations for maximal activity and probably has both an RNA and a protein component since trypsin and ribonuclease abrogated the activity. Despite this requirement for divalent cations and RNA, activation of the kinase was not required for proteolysis since a catalytically inactive P68 was still degraded. Mapping of P68 protease-sensitive sites by using in vitro translated truncation and deletion mutants revealed that sites required for degradation resided in the amino terminus and colocalized to dsRNA-binding domains. Finally, we found that preincubation of cell extracts with the synthetic dsRNA poly(I-C) largely prevented P68 proteolysis, providing additional evidence for the critical role of RNA. On the basis of these data, we present a hypothetical model depicting possible mechanisms of P68 degradation in poliovirus-infected cells. Images PMID:7678306

  7. Reactive oxygen species induce tyrosine phosphorylation of and Src kinase recruitment to NO-sensitive guanylyl cyclase.

    PubMed

    Meurer, Sabine; Pioch, Sylke; Gross, Steffen; Müller-Esterl, Werner

    2005-09-30

    Soluble guanylyl cyclase (sGC) is the major cytosolic receptor for nitric oxide (NO) that converts GTP into the second messenger cGMP in a NO-dependent manner. Other factors controlling this key enzyme are intracellular proteins such as Hsp90 and PSD95, which bind to sGC and modulate its activity, stability, and localization. To date little is known about the effects of posttranslational modifications of sGC, although circumstantial evidence suggests that reversible phosphorylation may contribute to sGC regulation. Here we demonstrate that inhibitors of protein-tyrosine phosphatases such as pervanadate and bisperoxo(1,10-phenanthroline)oxovanadate(V) as well as reactive oxygen species such as H2O2 induce specific tyrosine phosphorylation of the beta1 but not of the alpha1 subunit of sGC. Tyrosine phosphorylation of sGCbeta1 is also inducible by pervanadate and H2O2 in intact PC12 cells, rat aortic smooth muscle cells, and in rat aortic tissues, indicating that tyrosine phosphorylation of sGC may also occur in vivo. We have mapped the major tyrosine phosphorylation site to position 192 of beta1, where it forms part of a highly acidic phospho-acceptor site for Src-like kinases. In the phosphorylated state Tyr(P)-192 exposes a docking site for SH2 domains and efficiently recruits Src and Fyn to sGCbeta1, thereby promoting multiple phosphorylation of the enzyme. Our results demonstrate that sGC is subject to tyrosine phosphorylation and interaction with Src-like kinases, revealing an unexpected cross-talk between the NO/cGMP and tyrosine kinase signaling pathways at the level of sGC.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Salt-Inducible Kinase 1 (SIK1) Is Induced by Gastrin and Inhibits Migration of Gastric Adenocarcinoma Cells

    PubMed Central

    Selvik, Linn-Karina M.; Rao, Shalini; Steigedal, Tonje S.; Haltbakk, Ildri; Misund, Kristine; Bruland, Torunn; Prestvik, Wenche S.; Lægreid, Astrid; Thommesen, Liv

    2014-01-01

    Salt-inducible kinase 1 (SIK1/Snf1lk) belongs to the AMP-activated protein kinase (AMPK) family of kinases, all of which play major roles in regulating metabolism and cell growth. Recent studies have shown that reduced levels of SIK1 are associated with poor outcome in cancers, and that this involves an invasive cellular phenotype with increased metastatic potential. However, the molecular mechanism(s) regulated by SIK1 in cancer cells is not well explored. The peptide hormone gastrin regulates cellular processes involved in oncogenesis, including proliferation, apoptosis, migration and invasion. The aim of this study was to examine the role of SIK1 in gastrin responsive adenocarcinoma cell lines AR42J, AGS-GR and MKN45. We show that gastrin, known to signal through the Gq/G11-coupled CCK2 receptor, induces SIK1 expression in adenocarcinoma cells, and that transcriptional activation of SIK1 is negatively regulated by the Inducible cAMP early repressor (ICER). We demonstrate that gastrin-mediated signalling induces phosphorylation of Liver Kinase 1B (LKB1) Ser-428 and SIK1 Thr-182. Ectopic expression of SIK1 increases gastrin-induced phosphorylation of histone deacetylase 4 (HDAC4) and enhances gastrin-induced transcription of c-fos and CRE-, SRE-, AP1- and NF-κB-driven luciferase reporter plasmids. We also show that gastrin induces phosphorylation and nuclear export of HDACs. Next we find that siRNA mediated knockdown of SIK1 increases migration of the gastric adenocarcinoma cell line AGS-GR. Evidence provided here demonstrates that SIK1 is regulated by gastrin and influences gastrin elicited signalling in gastric adenocarcinoma cells. The results from the present study are relevant for the understanding of molecular mechanisms involved in gastric adenocarcinomas. PMID:25384047

  13. Salt-inducible kinase 1 (SIK1) is induced by gastrin and inhibits migration of gastric adenocarcinoma cells.

    PubMed

    Selvik, Linn-Karina M; Rao, Shalini; Steigedal, Tonje S; Haltbakk, Ildri; Misund, Kristine; Bruland, Torunn; Prestvik, Wenche S; Lægreid, Astrid; Thommesen, Liv

    2014-01-01

    Salt-inducible kinase 1 (SIK1/Snf1lk) belongs to the AMP-activated protein kinase (AMPK) family of kinases, all of which play major roles in regulating metabolism and cell growth. Recent studies have shown that reduced levels of SIK1 are associated with poor outcome in cancers, and that this involves an invasive cellular phenotype with increased metastatic potential. However, the molecular mechanism(s) regulated by SIK1 in cancer cells is not well explored. The peptide hormone gastrin regulates cellular processes involved in oncogenesis, including proliferation, apoptosis, migration and invasion. The aim of this study was to examine the role of SIK1 in gastrin responsive adenocarcinoma cell lines AR42J, AGS-GR and MKN45. We show that gastrin, known to signal through the Gq/G11-coupled CCK2 receptor, induces SIK1 expression in adenocarcinoma cells, and that transcriptional activation of SIK1 is negatively regulated by the Inducible cAMP early repressor (ICER). We demonstrate that gastrin-mediated signalling induces phosphorylation of Liver Kinase 1B (LKB1) Ser-428 and SIK1 Thr-182. Ectopic expression of SIK1 increases gastrin-induced phosphorylation of histone deacetylase 4 (HDAC4) and enhances gastrin-induced transcription of c-fos and CRE-, SRE-, AP1- and NF-κB-driven luciferase reporter plasmids. We also show that gastrin induces phosphorylation and nuclear export of HDACs. Next we find that siRNA mediated knockdown of SIK1 increases migration of the gastric adenocarcinoma cell line AGS-GR. Evidence provided here demonstrates that SIK1 is regulated by gastrin and influences gastrin elicited signalling in gastric adenocarcinoma cells. The results from the present study are relevant for the understanding of molecular mechanisms involved in gastric adenocarcinomas. PMID:25384047

  14. The clinically approved drugs dasatinib and bosutinib induce anti-inflammatory macrophages by inhibiting the salt-inducible kinases

    PubMed Central

    Ozanne, James; Prescott, Alan R.; Clark, Kristopher

    2014-01-01

    Macrophages switch to an anti-inflammatory, ‘regulatory’-like phenotype characterized by the production of high levels of interleukin (IL)-10 and low levels of pro-inflammatory cytokines to promote the resolution of inflammation. A potential therapeutic strategy for the treatment of chronic inflammatory diseases would be to administer drugs that could induce the formation of ‘regulatory’-like macrophages at sites of inflammation. In the present study, we demonstrate that the clinically approved cancer drugs bosutinib and dasatinib induce several hallmark features of ‘regulatory’-like macrophages. Treatment of macrophages with bosutinib or dasatinib elevates the production of IL-10 while suppressing the production of IL-6, IL-12p40 and tumour necrosis factor α (TNFα) in response to Toll-like receptor (TLR) stimulation. Moreover, macrophages treated with bosutinib or dasatinib express higher levels of markers of ‘regulatory’-like macrophages including LIGHT, SPHK1 and arginase 1. Bosutinib and dasatinib were originally developed as inhibitors of the protein tyrosine kinases Bcr-Abl and Src but we show that, surprisingly, the effects of bosutinib and dasatinib on macrophage polarization are the result of the inhibition of the salt-inducible kinases. Consistent with the present finding, bosutinib and dasatinib induce the dephosphorylation of CREB-regulated transcription co-activator 3 (CRTC3) and its nuclear translocation where it induces a cAMP-response-element-binding protein (CREB)-dependent gene transcription programme including that of IL-10. Importantly, these effects of bosutinib and dasatinib on IL-10 gene expression are lost in macrophages expressing a drug-resistant mutant of salt-inducible kinase 2 (SIK2). In conclusion, our study identifies the salt-inducible kinases as major targets of bosutinib and dasatinib that mediate the effects of these drugs on the innate immune system and provides novel mechanistic insights into the anti

  15. Downregulation of the Ras–Mitogen-Activated Protein Kinase Pathway by the EphB2 Receptor Tyrosine Kinase Is Required for Ephrin-Induced Neurite Retraction

    PubMed Central

    Elowe, Sabine; Holland, Sacha J.; Kulkarni, Sarang; Pawson, Tony

    2001-01-01

    Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones. PMID:11585923

  16. 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.

  17. 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

  18. Characterization of a tomato protein kinase gene induced by infection by Potato spindle tuber viroid.

    PubMed

    Hammond, R W; Zhao, Y

    2000-09-01

    Viroids--covalently closed, circular RNA molecules in the size range of 250 to 450 nucleotides-are the smallest known infectious agents and cause a number of diseases of crop plants. Viroids do not encode proteins and replicate within the nucleus without a helper virus. In many cases, viroid infection results in symptoms of stunting, epinasty, and vein clearing. In our study of the molecular basis of the response of tomato cv. Rutgers to infection by Potato spindle tuber viroid (PSTVd), we have identified a specific protein kinase gene, pkv, that is transcriptionally activated in plants infected with either the intermediate or severe strain of PSTVd, at a lower level in plants inoculated with a mild strain, and not detectable in mock-inoculated plants. A full-length copy of the gene encoding the 55-kDa PKV (protein kinase viroid)-induced protein has been isolated and sequence analysis revealed significant homologies to cyclic nucleotide-dependent protein kinases. Although the sequence motifs in the catalytic domain suggest that it is a serine/threonine protein kinase, the recombinant PKV protein autophosphorylates in vitro on serine and tyrosine residues, suggesting that it is a putative member of the class of dual-specificity protein kinases. PMID:10975647

  19. p53 deficiency enhances mitotic arrest and slippage induced by pharmacological inhibition of Aurora kinases.

    PubMed

    Marxer, M; Ma, H T; Man, W Y; Poon, R Y C

    2014-07-01

    A number of small-molecule inhibitors of Aurora kinases have been developed and are undergoing clinical trials for anti-cancer therapies. Different Aurora kinases, however, behave as very different targets: while inhibition of Aurora A (AURKA) induces a delay in mitotic exit, inhibition of Aurora B (AURKB) triggers mitotic slippage. Furthermore, while it is evident that p53 is regulated by Aurora kinase-dependent phosphorylation, how p53 may in turn regulate Aurora kinases remains mysterious. To address these issues, isogenic p53-containing and -negative cells were exposed to classic inhibitors that target both AURKA and AURKB (Alisertib and ZM447439), as well as to new generation of inhibitors that target AURKA (MK-5108), AURKB (Barasertib) individually. The fate of individual cells was then tracked with time-lapse microscopy. Remarkably, loss of p53, either by gene disruption or small interfering RNA-mediated depletion, sensitized cells to inhibition of both AURKA and AURKB, promoting mitotic arrest and slippage respectively. As the p53-dependent post-mitotic checkpoint is also important for preventing genome reduplication after mitotic slippage, these studies indicate that the loss of p53 in cancer cells represents a major opportunity for anti-cancer drugs targeting the Aurora kinases.

  20. Cbl participates in shikonin-induced apoptosis by negatively regulating phosphoinositide 3-kinase/protein kinase B signaling.

    PubMed

    Qu, Dan; Xu, Xiao-Man; Zhang, Meng; Jiang, Ting-Shu; Zhang, Yi; Li, Sheng-Qi

    2015-07-01

    Shikonin, a naturally occurring naphthoquinone, exhibits anti-tumorigenic activity. However, its precise mechanisms of action have remained elusive. In the present study, the involvement in the action of shikonin of the ubiquitin ligases Cbl-b and c-Cbl, which are negative regulators of phosphoinositide 3-kinase (PI3K) activation, was investigated. Shikonin was observed to reduce cell viability and induce apoptosis and G2/M phase arrest in lung cancer cells. In addition, shikonin increased the protein levels of B-cell lymphoma 2 (Bcl-2)-associated X and p53 and reduced those of Bcl-2. Additionally, shikonin inhibited PI3k/Akt activity and upregulated Cbl protein expression. In addition, a specific inhibitor of PI3K, LY294002, was observed to have a synergistic effect on the proliferation inhibition and apoptotic induction of A549 cells with shikonin. In conclusion, the results of the present study suggested that Cbl proteins promote shikonin-induced apoptosis by negatively regulating PI3K/Akt signaling in lung cancer cells.

  1. Serum- and glucocorticoid-inducible kinase 1 in doxorubicin-induced nephrotic syndrome.

    PubMed

    Artunc, Ferruh; Nasir, Omaima; Amann, Kerstin; Boini, Krishna M; Häring, Hans-Ulrich; Risler, Teut; Lang, Florian

    2008-12-01

    Doxorubicin-induced nephropathy leads to epithelial sodium channel (ENaC)-dependent volume retention and renal fibrosis. The aldosterone-sensitive serum- and glucocorticoid-inducible kinase SGK1 has been shown to participate in the stimulation of ENaC and to mediate renal fibrosis following mineralocorticoid and salt excess. The present study was performed to elucidate the role of SGK1 in the volume retention and fibrosis during nephrotic syndrome. To this end, doxorubicin (15 mug/g body wt) was injected intravenously into gene-targeted mice lacking SGK1 (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)). Doxorubicin treatment resulted in heavy proteinuria (>100 mg protein/mg crea) in 15/44 of sgk1(+/+) and 15/44 of sgk1(-/-) mice leading to severe nephrotic syndrome with ascites, lipidemia, and hypoalbuminemia in both genotypes. Plasma aldosterone levels increased in nephrotic mice of both genotypes and was followed by increased SGK1 protein expression in sgk1(+/+) mice. Urinary sodium excretion reached signficantly lower values in sgk1(+/+) mice (15 +/- 5 mumol/mg crea) than in sgk1(-/-) mice (35 +/- 5 mumol/mg crea) and was associated with a significantly higher body weight gain in sgk1(+/+) compared with sgk1(-/-) mice (+6.6 +/- 0.7 vs. +4.1 +/- 0.8 g). During the course of nephrotic syndrome, serum urea concentrations increased significantly faster in sgk1(-/-) mice than in sgk1(+/+) mice leading to uremia and a reduced median survival in sgk1(-/-) mice (29 vs. 40 days in sgk1(+/+) mice). In conclusion, gene-targeted mice lacking SGK1 showed blunted volume retention, yet were not protected against renal fibrosis during experimental nephrotic syndrome. PMID:18768591

  2. 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

  3. 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.

  4. Exogenous Mg-ATP induces a large inhibition of pyruvate kinase in intact rat hepatocytes.

    PubMed

    Ichai, C; El-Mir, M Y; Nogueira, V; Piquet, M A; Chauvin, C; Fontaine, E; Leverve, X M

    2001-03-01

    Mg-ATP infusion in vivo has been reported to be beneficial both to organ function and survival rate in various models of shock. Moreover, a large variety of metabolic effects has been shown to occur in several tissues due to purinergic receptor activation. In the present work we studied the effects of exogenous Mg-ATP in rat liver cells perifused with dihydroxyacetone to investigate simultaneously gluconeogenetic and glycolytic pathways. We found a significant effect on oxidative phosphorylation as characterized by a decrease in oxygen consumption rate and in the cellular ATP-to-ADP ratio associated with an increase in lactate-to-pyruvate ratio. In addition, exogenous Mg-ATP induced rapid and reversible inhibition of both gluconeogenesis and glycolysis. The main effect on gluconeogenesis was located at the level of the fructose cycle, whereas the decrease in glycolysis was due to a strong inhibition of pyruvate kinase. Although pyruvate kinase inhibition induced by exogenous Mg-ATP was allosteric when assessed in vitro after enzyme extraction, we found a large decrease in the apparent maximal velocity when kinetics were assessed in vivo in intact perifused hepatocytes. This newly described short-term regulation of pyruvate kinase occurs only in the intact cell and may open new potentials for the pharmacological regulation of pyruvate kinase in vivo. PMID:11104754

  5. LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission.

    PubMed

    Kim, J-E; Ryu, H J; Kim, M J; Kang, T-C

    2014-07-01

    Although the aberrant activation of cell cycle proteins has a critical role in neuronal death, effectors or mediators of cyclin D1/cyclin-dependent kinase 4 (CDK4)-mediated death signal are still unknown. Here, we describe a previously unsuspected role of LIM kinase 2 (LIMK2) in programmed necrotic neuronal death. Downregulation of p27(Kip1) expression by Rho kinase (ROCK) activation induced cyclin D1/CDK4 expression levels in neurons vulnerable to status epilepticus (SE). Cyclin D1/CDK4 complex subsequently increased LIMK2 expression independent of caspase-3 and receptor interacting protein kinase 1 activity. In turn, upregulated LIMK2 impaired dynamic-related protein-1 (DRP1)-mediated mitochondrial fission without alterations in cofilin phosphorylation/expression and finally resulted in necrotic neuronal death. Inhibition of LIMK2 expression and rescue of DRP1 function attenuated this programmed necrotic neuronal death induced by SE. Therefore, we suggest that the ROCK-p27(Kip1)-cyclin D1/CDK4-LIMK2-DRP1-mediated programmed necrosis may be new therapeutic targets for neuronal death.

  6. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  7. Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

    SciTech Connect

    Chakraborty, Goutam; Saito, Mitsuo; Mao, Rui-Fen; Wang, Ray; Vadasz, Csaba; Saito, Mariko

    2008-03-14

    Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3{beta} (GSK-3{beta}), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3{beta}, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3{beta}, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.

  8. Pro-nerve growth factor induces autocrine stimulation of breast cancer cell invasion through tropomyosin-related kinase A (TrkA) and sortilin protein.

    PubMed

    Demont, Yohann; Corbet, Cyril; Page, Adeline; Ataman-Önal, Yasemin; Choquet-Kastylevsky, Genevieve; Fliniaux, Ingrid; Le Bourhis, Xuefen; Toillon, Robert-Alain; Bradshaw, Ralph A; Hondermarck, Hubert

    2012-01-13

    The precursor of nerve growth factor (proNGF) has been described as a biologically active polypeptide able to induce apoptosis in neuronal cells, via the neurotrophin receptor p75(NTR) and the sortilin receptor. Herein, it is shown that proNGF is produced and secreted by breast cancer cells, stimulating their invasion. Using Western blotting and mass spectrometry, proNGF was detected in a panel of breast cancer cells as well as in their conditioned media. Immunohistochemical analysis indicated an overproduction of proNGF in breast tumors, when compared with benign and normal breast biopsies, and a relationship to lymph node invasion in ductal carcinomas. Interestingly, siRNA against proNGF induced a decrease of breast cancer cell invasion that was restored by the addition of non-cleavable proNGF. The activation of TrkA, Akt, and Src, but not the MAP kinases, was observed. In addition, the proNGF invasive effect was inhibited by the Trk pharmacological inhibitor K252a, a kinase-dead TrkA, and siRNA against TrkA sortilin, neurotensin, whereas siRNA against p75(NTR) and the MAP kinase inhibitor PD98059 had no impact. These data reveal the existence of an autocrine loop stimulated by proNGF and mediated by TrkA and sortilin, with the activation of Akt and Src, for the stimulation of breast cancer cell invasion.

  9. The Secretome of Endothelial Progenitor Cells Promotes Brain Endothelial Cell Activity through PI3-Kinase and MAP-Kinase

    PubMed Central

    Di Santo, Stefano; Seiler, Stefanie; Fuchs, Anna-Lena; Staudigl, Jennifer; Widmer, Hans Rudolf

    2014-01-01

    Background Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. Methods Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. Results Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. Conclusion The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects. PMID:24755675

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. Hepatitis B virus x protein induces autophagy via activating death-associated protein kinase.

    PubMed

    Zhang, H-T; Chen, G G; Hu, B-G; Zhang, Z-Y; Yun, J-P; He, M-L; Lai, P B S

    2014-01-01

    Hepatitis B virus x protein (HBX), a product of hepatitis B virus (HBV), is a multifunctional protein that regulates viral replication and various cellular functions. Recently, HBX has been shown to induce autophagy; however, the responsible mechanism is not fully known. In this study, we established stable HBX-expressing epithelial Chang cells as the platform to study how HBX induced autophagy. The results showed that the overexpression of HBX resulted in starvation-induced autophagy. HBX-induced autophagy was related to its ability to dephosphorylate/activate death-associated protein kinase (DAPK). The block of DAPK by its siRNA significantly counteracted HBX-mediated autophagy, confirming the positive role of DAPK in this process. HBX also induced Beclin 1, which functions at the downstream of the DAPK-mediated autophagy pathway. Although HBX could activate JNK, a kinase known to participate in autophagy in certain conditions, the change in JNK failed to influence HBX-induced autophagy. In conclusion, HBX induces autophagy via activating DAPK in a pathway related to Beclin 1, but not JNK. This new finding should help us to understand the role of autophagy in HBX-mediated pathogenesis and thus may provide targets for intervening HBX-related disorders.

  15. TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

    SciTech Connect

    Li, Feifei; Jiang, Yinan; Zheng, Qiping; Yang, Xiaoming; Wang, Siying

    2011-01-07

    Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.

  16. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis

    PubMed Central

    Weigel, Christoph; Veldwijk, Marlon R.; Oakes, Christopher C.; Seibold, Petra; Slynko, Alla; Liesenfeld, David B.; Rabionet, Mariona; Hanke, Sabrina A.; Wenz, Frederik; Sperk, Elena; Benner, Axel; Rösli, Christoph; Sandhoff, Roger; Assenov, Yassen; Plass, Christoph; Herskind, Carsten; Chang-Claude, Jenny; Schmezer, Peter; Popanda, Odilia

    2016-01-01

    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy. PMID:26964756

  17. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis.

    PubMed

    Weigel, Christoph; Veldwijk, Marlon R; Oakes, Christopher C; Seibold, Petra; Slynko, Alla; Liesenfeld, David B; Rabionet, Mariona; Hanke, Sabrina A; Wenz, Frederik; Sperk, Elena; Benner, Axel; Rösli, Christoph; Sandhoff, Roger; Assenov, Yassen; Plass, Christoph; Herskind, Carsten; Chang-Claude, Jenny; Schmezer, Peter; Popanda, Odilia

    2016-03-11

    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy.

  18. 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

  19. Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes.

    PubMed

    Brutman-Barazani, Tamar; Horovitz-Fried, Miriam; Aga-Mizrachi, Shlomit; Brand, Chagit; Brodie, Chaya; Rosa, Jagoda; Sampson, Sanford R

    2012-06-01

    The liver is a major insulin-responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin-induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin-induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin-induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML-12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin-induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin-induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin-induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin-induced glucose uptake and glycogenesis in hepatocytes.

  20. 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.

  1. Glutamate signalling via a MEKK1 kinase-dependent pathway induces changes in Arabidopsis root architecture.

    PubMed

    Forde, Brian G; Cutler, Sean R; Zaman, Najia; Krysan, Patrick J

    2013-07-01

    A chemical genetic approach has been used to investigate the mechanism by which external glutamate (l-Glu) is able to trigger major changes in root architecture in Arabidopsis thaliana L. An initial screen of 80 agonists and antagonists of mammalian glutamate and GABA receptors, using a specially developed 96-well microphenotyping system, found none that replicated the response of the root to l-Glu or antagonized it. However, a larger screen using >1500 molecules bioactive in Saccharomyces cerevisiae (yeast) identified two groups that interfered with the l-Glu response. One of the antagonists, 2-(4-chloro-3-methylphenyl)-2-oxoethyl thiocyanate (CMOT), has been reported to target Ste11, an evolutionarily conserved MAP kinase kinase kinase (MAP3K) in yeast. This led to the discovery that root growth in a triple mekk1 mekk2 mekk3 mutant (mekk1/2/3), defective in a set of three tandemly arranged MAP3Ks, was almost insensitive to l-Glu. However, the sensitivity of mekk1/2/3 roots to inhibition by other amino acids reported to act as agonists of glutamate receptor-like (GLR) channels in Arabidopsis roots (Asn, Cys, Gly and Ser) was unaffected. The l-Glu sensitivity of the mekk1/2/3 mutant was restored by transformation with a construct carrying the intact MEKK1 gene. These results demonstrate that MEKK1 plays a key role in transducing the l-Glu signal that elicits large-scale changes in root architecture, and provide genetic evidence for the existence in plants of an l-Glu signalling pathway analogous to that found in animals.

  2. Salinomycin causes migration and invasion of human fibrosarcoma cells by inducing MMP-2 expression via PI3-kinase, ERK-1/2 and p38 kinase pathways.

    PubMed

    Yu, Seon-Mi; Kim, Song Ja

    2016-06-01

    Salinomycin (SAL) is a polyether ionophore antibiotic that has recently been shown to regulate a variety of cellular responses in various human cancer cells. However, the effects of SAL on metastatic capacity of HT1080 human fibrosarcoma cells have not been elucidated. We investigated the effect of SAL on migration and invasion, with emphasis on the expression and activation of matrix metalloproteinase (MMP)-2 in HT1080 human fibrosarcoma cells. Treatment of SAL promoted the expression and activation of MMP-2 in a dose- and time-dependent manner, as detected by western blot analysis, gelatin zymography, and real-time polymerase chain reaction. SAL also increased metastatic capacities, as determined by an increase in the migration and invasion of cells using the wound healing assay and the invasion assay, respectively. To confirm the detailed molecular mechanisms of these effects, we measured the activation of phosphoinositide 3 kinase (PI3-kinase) and mitogen-activated protein kinase (MAPK)s (ERK-1/2 and p38 kinase), as detected by the phosphorylated proteins through western blot analysis. SAL treatment increased the phosphorylation of Akt and MAPKs. Inhibition of PI3-kinase, ERK-1/2, and p38 kinase with LY294002, PD98059, and SB203580, respectively, in the presence of SAL suppressed the metastatic capacity by reducing MMP-2 expression, as determined by gelatin zymography. Our results indicate that the PI3-kinase and MAPK signaling pathways are involved in migration and invasion of HT1080 through induction of MMP-2 expression and activation. In conclusion, SAL significantly increases the metastatic capacity of HT1080 cells by inducing MMP-2 expression via PI3-kinase and MAPK pathways. Our results suggest that SAL may be a potential agent for the study of cancer metastatic capacities.

  3. Protein Tyrosine Kinase 6 Regulates UVB-Induced Signaling and Tumorigenesis in Mouse Skin.

    PubMed

    Chastkofsky, Michael I; Bie, Wenjun; Ball-Kell, Susan M; He, Yu-Ying; Tyner, Angela L

    2015-10-01

    Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the epithelial linings of the gastrointestinal tract and the skin, where it is expressed in nondividing differentiated cells. We found that PTK6 expression increases in the epidermis following UVB treatment. To evaluate the roles of PTK6 in the skin following UVB-induced damage, we exposed back skin of Ptk6 +/+ and Ptk6 -/- SENCAR mice to incremental doses of UVB for 30 weeks. Wild-type mice were more sensitive to UVB and exhibited increased inflammation and greater activation of signal transducer and activator of transcription-3 (STAT3) than Ptk6-/- mice. Disruption of Ptk6 did not have an impact on proliferation, although PTK6 was expressed and activated in basal epithelial cells in wild-type mice following UVB treatment. However, wild-type mice exhibited shortened tumor latency and increased tumor load compared with Ptk6-/- mice, and STAT3 activation was increased in these tumors. PTK6 activation was detected in UVB-induced tumors, and this correlated with increased activating phosphorylation of focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1). Activation of PTK6 was also detected in human squamous cell carcinomas of the skin. Although PTK6 has roles in normal differentiation, it also contributes to UVB-induced injury and tumorigenesis in vivo. PMID:25938342

  4. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

    PubMed Central

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-01-01

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  5. 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

  6. Two Dictyostelium tyrosine kinase–like kinases function in parallel, stress-induced STAT activation pathways

    PubMed Central

    Araki, Tsuyoshi; Vu, Linh Hai; Sasaki, Norimitsu; Kawata, Takefumi; Eichinger, Ludwig; Williams, Jeffrey G.

    2014-01-01

    When Dictyostelium cells are hyperosmotically stressed, STATc is activated by tyrosine phosphorylation. Unusually, activation is regulated by serine phosphorylation and consequent inhibition of a tyrosine phosphatase: PTP3. The identity of the cognate tyrosine kinase is unknown, and we show that two tyrosine kinase–like (TKL) enzymes, Pyk2 and Pyk3, share this function; thus, for stress-induced STATc activation, single null mutants are only marginally impaired, but the double mutant is nonactivatable. When cells are stressed, Pyk2 and Pyk3 undergo increased autocatalytic tyrosine phosphorylation. The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action. The signaling pathways that activate Pyk2 and Pyk3 are only partially overlapping, and there may be a structural basis for this difference because Pyk3 contains both a TKL domain and a pseudokinase domain. The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain. The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3. PMID:25143406

  7. N-Terminus of the Protein Kinase CLK1 Induces SR Protein Hyper-Phosphorylation

    PubMed Central

    Aubol, Brandon E.; Plocinik, Ryan M.; Keshwani, Malik M.; McGlone, Maria L.; Hagopian, Jonathan C.; Ghosh, Gourisankar; Fu, Xiang-Dong; Adams, Joseph A.

    2016-01-01

    SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine-serine-rich) domains by two major families of protein kinases. The SRPKs efficiently phosphorylate the arginine-serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs lack a docking groove and phosphorylate both arginine-serine and serine-proline dipeptides, modifications that generate a hyper-phosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long, flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1. This interaction not only is essential for facilitating hyper-phosphorylation but also induces cooperative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 by 2–3-fold. These findings suggest that CLK1-dependent hyper-phosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein. PMID:24869919

  8. Ribosomal protein mutations induce autophagy through S6 kinase inhibition of the insulin pathway.

    PubMed

    Heijnen, Harry F; van Wijk, Richard; Pereboom, Tamara C; Goos, Yvonne J; Seinen, Cor W; van Oirschot, Brigitte A; van Dooren, Rowie; Gastou, Marc; Giles, Rachel H; van Solinge, Wouter; Kuijpers, Taco W; Gazda, Hanna T; Bierings, Marc B; Da Costa, Lydie; MacInnes, Alyson W

    2014-01-01

    Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies. PMID:24875531

  9. RIP3 induces apoptosis independent of pro-necrotic kinase activity

    PubMed Central

    Mandal, Pratyusha; Berger, Scott B.; Pillay, Sirika; Moriwaki, Kenta; Huang, Chunzi; Guo, Hongyan; Lich, John D.; Finger, Joshua; Kasparcova, Viera; Votta, Bart; Ouellette, Michael; King, Bryan W.; Wisnoski, David; Lakdawala, Ami S.; DeMartino, Michael P.; Casillas, Linda N.; Haile, Pamela A.; Sehon, Clark A.; Marquis, Robert W.; Upton, Jason; Daley-Bauer, Lisa P.; Roback, Linda; Ramia, Nancy; Dovey, Cole M.; Carette, Jan E.; Chan, Francis; Bertin, John; Gough, Peter J.; Mocarski, Edward S.; Kaiser, William J.

    2015-01-01

    Summary Receptor interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pro-necrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound; whereas, D161G, D143N, and K51A mutants only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3K51A/K51A) are viable and fertile, in stark contrast to the perinatal lethality of Rip3D161N/D161N mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3. PMID:25459880

  10. 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...

  11. 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.

  12. 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

  13. Rutin inhibits B[a]PDE-induced cyclooxygenase-2 expression by targeting EGFR kinase activity.

    PubMed

    Choi, Seunghwan; Lim, Tae-Gyu; Hwang, Mun Kyung; Kim, Yoon-A; Kim, Jiyoung; Kang, Nam Joo; Jang, Tae Su; Park, Jun-Seong; Yeom, Myeong Hun; Lee, Ki Won

    2013-11-15

    Rutin is a well-known flavonoid that exists in various natural sources. Accumulative studies have represented the biological effects of rutin, such as anti-oxidative and anti-inflammatory effects. However, the underlying mechanisms of rutin and its direct targets are not understood. We investigated whether rutin reduced B[a]PDE-induced-COX-2 expression. The transactivation of AP-1 and NF-κB were inhibited by rutin. Rutin also attenuated B[a]PDE-induced Raf/MEK/ERK and Akt activation, but had no effect on the phosphorylation of EGFR. An in vitro kinase assay revealed rutin suppressed EGFR kinase activity. We also confirmed direct binding between rutin and EGFR, and found that the binding was regressed by ATP. The EGFR inhibitor also inhibited the B[a]PDE-induced MEK/ERK and Akt signaling pathways and subsequently, suppressed COX-2 expression and promoter activity, in addition to suppressing the transactivation of AP-1 and NF-κB. In EGFR(-/-)mouse embryonic fibroblast cells, B[a]PDE-induced COX-2 expression was also diminished. Collectively, rutin inhibits B[a]PDE-induced COX-2 expression by suppressing the Raf/MEK/ERK and Akt signaling pathways. EGFR appeared to be the direct target of rutin.

  14. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    PubMed Central

    Kuo, Chen-Tzu; Chen, Bing-Chang; Yu, Chung-Chi; Weng, Chih-Ming; Hsu, Ming-Jen; Chen, Chien-Chih; Chen, Mei-Chieh; Teng, Che-Ming; Pan, Shiow-Lin; Bien, Mauo-Ying; Shih, Chung-Hung; Lin, Chien-Huang

    2009-01-01

    In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis. PMID:19405983

  15. Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway.

    PubMed Central

    Rouyez, M C; Boucheron, C; Gisselbrecht, S; Dusanter-Fourt, I; Porteu, F

    1997-01-01

    Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation. PMID:9271377

  16. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    SciTech Connect

    Souza, C.F.; Carneiro, A.B.; Silveira, A.B.; Laranja, G.A.T.; Silva-Neto, M.A.C.; Costa, S.C. Goncalves da; Paes, M.C.

    2009-12-18

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  17. Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

    SciTech Connect

    Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

    2009-04-15

    Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G{sub 1} phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

  18. Mitochondrial APE1/Ref-1 suppressed protein kinase C-induced mitochondrial dysfunction in mouse endothelial cells.

    PubMed

    Joo, Hee Kyoung; Lee, Yu Ran; Park, Myoung Soo; Choi, Sunga; Park, Kyoungsook; Lee, Sang Ki; Kim, Cuk-Seong; Park, Jin Bong; Jeon, Byeong Hwa

    2014-07-01

    Protein kinase C (PKC) induces mitochondrial dysfunction, which is an important pathological factor in cardiovascular diseases. The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) on PKC-induced mitochondrial dysfunction has not been variously investigated. In this study, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, induced mitochondrial hyperpolarization and reactive oxygen species generation and also increased mitochondrial translocation of APE1/Ref-1. APE1/Ref-1 overexpression suppressed PMA-induced mitochondrial dysfunction. In contrast, gene silencing of APE1/Ref-1 increased the sensitivity of mitochondrial dysfunction. Moreover, mitochondrial targeting sequence (MTS)-fused APE1/Ref-1 more effectively suppressed PMA-induced mitochondrial dysfunctions. These results suggest that mitochondrial APE1/Ref-1 is contributed to the protective role to protein kinase C-induced mitochondrial dysfunction in endothelial cells.

  19. Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

    PubMed Central

    Zhong, Yiming; Dong, Shuai; Strattan, Ethan; Ren, Li; Butchar, Jonathan P.; Thornton, Kelsey; Mishra, Anjali; Porcu, Pierluigi; Bradshaw, J. Michael; Bisconte, Angelina; Owens, Timothy D.; Verner, Erik; Brameld, Ken A.; Funk, Jens Oliver; Hill, Ronald J.; Johnson, Amy J.; Dubovsky, Jason A.

    2015-01-01

    Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases. PMID:25593320

  20. Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases.

    PubMed Central

    Benn, J; Su, F; Doria, M; Schneider, R J

    1996-01-01

    The HBx protein of hepatitis B virus is a dual-specificity activator of transcription, stimulating signal transduction pathways in the cytoplasm and transcription factors in the nucleus, when expressed in cell lines in culture. In the cytoplasm, HBx was shown to stimulate the Ras-Raf-mitogen-activated protein kinase (MAP kinase) cascade, which is essential for activation of transcription factor AP-1. Here we show that HBx protein stimulates two independently regulated members of the MAP kinase family when expressed transiently in cells. HBx protein stimulates the extracellular signal-regulated kinases (ERKs) and the c-Jun N-terminal kinases (JNKs). HBx activation of ERKs and JNKs leads to induction and activation of AP-1 DNA binding activity involving transient de novo synthesis of c-Fos protein and prolonged synthesis of c-Jun, mediated by N-terminal phosphorylation of c-Jun carried out by HBx-activated JNK. New c-Jun synthesis was blocked by coexpression with a dominant-negative MAP kinase kinase (MEK kinase, MEKK-1), confirming that HBx stimulates the prolonged synthesis of c-Jun by activating JNK signalling pathways. Activation of the c-fos gene was blocked by coexpression with a Raf-C4 catalytic mutant, confirming that HBx induces c-Fos by acting on Ras-Raf linked pathways. HBx activation of ERK and JNK pathways resulted in prolonged accumulation of AP-1-c-Jun dimer complexes. HBx activation of JNK and sustained activation of c-jun, should they occur in the context of hepatitis B virus infection, might play a role in viral transformation and pathogenesis. PMID:8764004

  1. 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.

  2. 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.

  3. Resveratrol inhibits phorbol ester-induced expression of COX-2 and activation of NF-kappaB in mouse skin by blocking IkappaB kinase activity.

    PubMed

    Kundu, Joydeb Kumar; Shin, Young Kee; Kim, Sung Hoon; Surh, Young-Joon

    2006-07-01

    Aberrant expression of cyclooxygenase-2 (COX-2) has been implicated in tumor promotion. Resveratrol, a phytoalexin present in grapes, was reported to inhibit multistage mouse skin carcinogenesis. In the present study, we found that topically applied resveratrol significantly inhibited COX-2 expression induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Resveratrol-suppressed phosphorylation and subsequent degradation of IkappaBalpha, thereby inhibiting activation of nuclear factor-kappaB (NF-kappaB) in TPA-stimulated mouse skin. Pretreatment with resveratrol also suppressed TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein (MAP) kinase. Resveratrol blunted TPA-induced phosphorylation of p65 and its interaction with CBP/p300, rendering NF-kappaB transcriptionally inactive. To get further insights into the molecular basis of NF-kappaB inactivation by resveratrol, we examined the role of IkappaB kinase (IKK) in mediating TPA-induced activation of NF-kappaB and COX-2 expression. TPA treatment led to rapid induction of IKK activity in mouse skin, which was abolished either by resveratrol or an IKK inhibitor Bay 11-7082. Topical application of Bay 11-7082 also abrogated TPA-induced NF-kappaB activation and COX-2 expression, supporting the involvement of IKK in TPA-induced COX-2 expression. Taken together, the above findings suggest that resveratrol targets IKK in blocking TPA-induced NF-kappaB activation and COX-2 expression in mouse skin in vivo.

  4. 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.

  5. Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.

    PubMed

    Chibalin, Alexander V; Leng, Ying; Vieira, Elaine; Krook, Anna; Björnholm, Marie; Long, Yun Chau; Kotova, Olga; Zhong, Zhihui; Sakane, Fumio; Steiler, Tatiana; Nylén, Carolina; Wang, Jianjun; Laakso, Markku; Topham, Matthew K; Gilbert, Marc; Wallberg-Henriksson, Harriet; Zierath, Juleen R

    2008-02-01

    Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life. PMID:18267070

  6. 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

  7. Probabilistic, Seismically-Induced Landslide Hazard Mapping of Western Oregon

    NASA Astrophysics Data System (ADS)

    Olsen, M. J.; Sharifi Mood, M.; Gillins, D. T.; Mahalingam, R.

    2015-12-01

    Earthquake-induced landslides can generate significant damage within urban communities by damaging structures, obstructing lifeline connection routes and utilities, generating various environmental impacts, and possibly resulting in loss of life. Reliable hazard and risk maps are important to assist agencies in efficiently allocating and managing limited resources to prepare for such events. This research presents a new methodology in order to communicate site-specific landslide hazard assessments in a large-scale, regional map. Implementation of the proposed methodology results in seismic-induced landslide hazard maps that depict the probabilities of exceeding landslide displacement thresholds (e.g. 0.1, 0.3, 1.0 and 10 meters). These maps integrate a variety of data sources including: recent landslide inventories, LIDAR and photogrammetric topographic data, geology map, mapped NEHRP site classifications based on available shear wave velocity data in each geologic unit, and USGS probabilistic seismic hazard curves. Soil strength estimates were obtained by evaluating slopes present along landslide scarps and deposits for major geologic units. Code was then developed to integrate these layers to perform a rigid, sliding block analysis to determine the amount and associated probabilities of displacement based on each bin of peak ground acceleration in the seismic hazard curve at each pixel. The methodology was applied to western Oregon, which contains weak, weathered, and often wet soils at steep slopes. Such conditions have a high landslide hazard even without seismic events. A series of landslide hazard maps highlighting the probabilities of exceeding the aforementioned thresholds were generated for the study area. These output maps were then utilized in a performance based design framework enabling them to be analyzed in conjunction with other hazards for fully probabilistic-based hazard evaluation and risk assessment. a) School of Civil and Construction

  8. 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

  9. Salmonella induces SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling pathways in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on d...

  10. Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production

    PubMed Central

    Hashimoto, Shin; Yamamoto, Soh; Ogasawara, Noriko; Sato, Toyotaka; Yamamoto, Keisuke; Katoh, Hiroshi; Kubota, Toru; Shiraishi, Tsukasa; Kojima, Takashi; Himi, Tetsuo; Tsutsumi, Hiroyuki; Yokota, Shin-ichi

    2016-01-01

    Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection. PMID:27560627

  11. Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production.

    PubMed

    Hashimoto, Shin; Yamamoto, Soh; Ogasawara, Noriko; Sato, Toyotaka; Yamamoto, Keisuke; Katoh, Hiroshi; Kubota, Toru; Shiraishi, Tsukasa; Kojima, Takashi; Himi, Tetsuo; Tsutsumi, Hiroyuki; Yokota, Shin-Ichi

    2016-01-01

    Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection. PMID:27560627

  12. Protein kinase Cα inhibits myocardin-induced cardiomyocyte hypertrophy through the promotion of myocardin phosphorylation.

    PubMed

    Li, Weizong; Wang, Nan; Li, Man; Gong, Huiqin; Liao, Xinghua; Yang, Xiaolong; Zhang, Tongcun

    2015-09-01

    Myocardin plays a key role in the development of cardiac hypertrophy. However, the upstream signals that control the stability and transactivity of myocardin remain to be fully understood. The expression of protein kinase Cα (PKCα) also induces cardiac hypertrophy. An essential downstream molecule of PKCα, extracellular signal-regulated kinase 1/2, was reported to negatively regulate the activities of myocardin. But, the effect of cooperation between PKCα and myocardin and the potential molecular mechanism by which PKCα regulates myocardin-mediated cardiac hypertrophy are unclear. In this study, a luciferase assay was performed using H9C2 cells transfected with expression plasmids for PKCα and myocardin. Surprisingly, the results showed that PKCα inhibited the transcriptional activity of myocardin. PKCα inhibited myocardin-induced cardiomyocyte hypertrophy, demonstrated by the decrease in cell surface area and fetal gene expression, in cardiomyocyte cells overexpressing PKCα and myocardin. The potential mechanism underlying the inhibition effect of PKCα on the function of myocardin is further explored. PKCα directly promoted the basal phosphorylation of endogenous myocardin at serine and threonine residues. In myocardin-overexpressing cardiomyocyte cells, PKCα induced the excessive phosphorylation of myocardin, resulting in the degradation of myocardin and a transcriptional suppression of hypertrophic genes. These results demonstrated that PKCα inhibits myocardin-induced cardiomyocyte hypertrophy through the promotion of myocardin phosphorylation. PMID:26206583

  13. Norathyriol Suppresses Skin Cancers Induced by Solar Ultraviolet Radiation by Targeting ERK Kinases

    SciTech Connect

    Li, Jixia; Malakhova, Margarita; Mottamal, Madhusoodanan; Reddy, Kanamata; Kurinov, Igor; Carper, Andria; Langfald, Alyssa; Oi, Naomi; Kim, Myoung Ok; Zhu, Feng; Sosa, Carlos P.; Zhou, Keyuan; Bode, Ann M.; Dong, Zigang

    2012-06-27

    Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, prompting great interest in chemopreventive agents for this disease. In this study, we report the discovery of norathyriol, a plant-derived chemopreventive compound identified through an in silico virtual screening of the Chinese Medicine Library. Norathyriol is a metabolite of mangiferin found in mango, Hypericum elegans, and Tripterospermum lanceolatum and is known to have anticancer activity. Mechanistic investigations determined that norathyriol acted as an inhibitor of extracellular signal-regulated kinase (ERK)1/2 activity to attenuate UVB-induced phosphorylation in mitogen-activated protein kinases signaling cascades. We confirmed the direct and specific binding of norathyriol with ERK2 through a cocrystal structural analysis. The xanthone moiety in norathyriol acted as an adenine mimetic to anchor the compound by hydrogen bonds to the hinge region of the protein ATP-binding site on ERK2. Norathyriol inhibited in vitro cell growth in mouse skin epidermal JB6 P+ cells at the level of G{sub 2}-M phase arrest. In mouse skin tumorigenesis assays, norathyriol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis indicated that norathyriol mediates its chemopreventive activity by inhibiting the ERK-dependent activity of transcriptional factors AP-1 and NF-{kappa}B during UV-induced skin carcinogenesis. Taken together, our results identify norathyriol as a safe new chemopreventive agent that is highly effective against development of UV-induced skin cancer.

  14. Mitogen-activated protein kinase activation in UV-induced signal transduction.

    PubMed

    Bode, Ann M; Dong, Zigang

    2003-01-28

    Experimental evidence supported by epidemiological findings suggests that solar ultraviolet (UV) irradiation is the most important environmental carcinogen leading to the development of skin cancers. Because the ozone layer blocks UVC (wavelength, 180 to 280 nm) exposure, UVA (UVA I, 340 to 400 nm; UVA II, 320 to 340 nm) and UVB (280 to 320 nm) are probably the chief carcinogenic components of sunlight with relevance for human skin cancer. Substantial contributions to the elucidation of the specific signal transduction pathways involved in UV-induced skin carcinogenesis have been made over the past few years, and most evidence suggests that the cellular signaling response is UV wavelength-dependent. The mitogen-activated protein kinase (MAPK) signaling cascades are targets for UV and are important in the regulation of the multitude of UV-induced cellular responses. Experimental studies have used a range of UVA, UVB, UVC, and various combinations in multiple doses, and the observed effects on activation and phosphorylation of MAPKs are varied. This review focuses on the mechanistic data supporting a role for MAPKs in UV-induced skin carcinogenesis. Progress in understanding the mechanisms of UV-induced signal transduction could lead to the use of these protein kinases as specific targets for the prevention and control of skin cancer.

  15. The role of protein kinase C alpha translocation in radiation-induced bystander effect.

    PubMed

    Fang, Zihui; Xu, An; Wu, Lijun; Hei, Tom K; Hong, Mei

    2016-01-01

    Ionizing radiation is a well known human carcinogen. Evidence accumulated over the past decade suggested that extranuclear/extracellular targets and events may also play a critical role in modulating biological responses to ionizing radiation. However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear. In the current study, AL cells were irradiated with alpha particles and responses of bystander cells were investigated. We found out that in bystander AL cells, protein kinase C alpha (PKCα) translocated from cytosol to membrane fraction. Pre-treatment of cells with PKC translocation inhibitor chelerythrine chloride suppressed the induced extracellular signal-regulated kinases (ERK) activity and the increased cyclooxygenase 2 (COX-2) expression as well as the mutagenic effect in bystander cells. Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells. Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1. Our data suggested that PKCα translocation may occur as an early event in radiation-induced bystander responses and mediate TNFα-induced signaling pathways that lead to the activation of ERK and up-regulation of COX-2. PMID:27165942

  16. Cyclic stretch-induced stress fiber dynamics - Dependence on strain rate, Rho-kinase and MLCK

    SciTech Connect

    Lee, Chin-Fu; Haase, Candice; Deguchi, Shinji; Kaunas, Roland

    2010-10-22

    Research highlights: {yields} Cyclic stretch induces stress fiber disassembly, reassembly and fusion perpendicular to the direction of stretch. {yields} Stress fiber disassembly and reorientation were not induced at low stretch frequency. {yields} Stretch caused actin fiber formation parallel to stretch in distinct locations in cells treated with Rho-kinase and MLCK inhibitors. -- Abstract: Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor (Y27632) reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.

  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. UV irradiation-induced apoptosis leads to activation of a 36-kDa myelin basic protein kinase in HL-60 cells

    SciTech Connect

    Lu, M.L.; Sato, Mitsuhiro; Cao, Boliang; Richie, J.P.

    1996-08-20

    UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirements for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-{alpha}-and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis. 40 refs., 5 figs.

  19. Inactivation of MARK4, an AMP-activated protein kinase (AMPK)-related kinase, leads to insulin hypersensitivity and resistance to diet-induced obesity.

    PubMed

    Sun, Chao; Tian, Liang; Nie, Jia; Zhang, Hai; Han, Xiao; Shi, Yuguang

    2012-11-01

    MARK4, also known as Par-1d/MarkL1, is a member of the AMP-activated protein kinase (AMPK)-related family of kinases, which are implicated in the regulation of dynamic biological functions, including glucose and energy homeostasis. However, the physiological function of MARK4 in mammals remains elusive. Here, we investigated a role for MARK4 in regulating energy homeostasis by generating mice with targeted inactivation of the mark4 gene. We show that MARK4 deficiency in mice caused hyperphagia, hyperactivity, and hypermetabolism, leading to protection from diet-induced obesity and its related metabolic complications through up-regulation of brown fat activity. Consequently, MARK4 deficiency mitigated insulin resistance associated with diet-induced obesity by dramatically enhancing insulin-stimulated AKT phosphorylation in major metabolic tissues. Ablation of MARK4 also significantly improved glucose homeostasis by up-regulating the activity and expression of AMPK kinase in key metabolic tissues. Taken together, these data identify a key role of MARK4 in energy metabolism, implicating the kinase as a novel drug target for the treatment of obesity and type 2 diabetes. PMID:22992738

  20. Activation of AMP kinase α1 subunit induces aortic vasorelaxation in mice

    PubMed Central

    Goirand, Françoise; Solar, Myriam; Athea, Yoni; Viollet, Benoit; Mateo, Philippe; Fortin, Dominique; Leclerc, Jocelyne; Hoerter, Jacqueline; Ventura-Clapier, Renée; Garnier, Anne

    2007-01-01

    Vasodilatation is a vital mechanism of systemic blood flow regulation that occurs during periods of increased energy demand. The AMP-dependent protein kinase (AMPK) is a serine/threonine kinase that is activated by conditions that increase the AMP-to-ATP ratio, such as exercise and metabolic stress. We hypothesized that AMPK could trigger vasodilatation and participate in blood flow regulation. Rings of thoracic aorta were isolated from C57Bl6 mice and mice deficient in the AMPK catalytic α1 (AMPKα1−/−) or α2 (AMPKα2−/−) subunit and their littermate controls, and mounted in an organ bath. Aortas were preconstricted with phenylephrine (1 μm) and activation of AMPK was induced by addition of increasing concentrations of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). AICAR (0.1–3 mm) dose-dependently induced relaxation of precontracted C57BL6, AMPKα1+/+ and α2+/+ aorta (P < 0.001, n = 5–7 per group). This AICAR induced vasorelaxation was not inhibited by the addition of adenosine receptor antagonists. Moreover, when aortic rings were freed of endothelium by gentle rubbing, AICAR still induced aortic ring relaxation, suggesting a direct effect of AICAR on smooth muscle cells. When aortic rings were pretreated with l-NMMA (30 μm) to inhibit nitric oxide synthase activity, AICAR still induced relaxation. Western blot analysis of C57Bl6 mice denuded aorta showed that AMPK was phosphorylated after incubation with AICAR and that AMPKα1 was the main catalytic subunit expressed. Finally, AICAR-induced relaxation of aortic rings was completely abolished in AMPKα1−/− but not AMPKα2−/− mice. Taken together, the results show that activation of AMPKα1 but not AMPKα2 is able to induce aortic relaxation in mice, in an endothelium- and eNOS-independent manner. PMID:17446219

  1. Development of regional liquefaction-induced deformation hazard maps

    USGS Publications Warehouse

    Rosinski, A.; Knudsen, K.-L.; Wu, J.; Seed, R.B.; Real, C.R.; ,

    2004-01-01

    This paper describes part of a project to assess the feasibility of producing regional (1:24,000-scale) liquefaction hazard maps that are based-on potential liquefaction-induced deformation. The study area is the central Santa Clara Valley, at the south end of San Francisco Bay in Central California. The information collected and used includes: a) detailed Quaternary geological mapping, b) over 650 geotechnical borings, c) probabilistic earthquake shaking information, and d) ground-water levels. Predictions of strain can be made using either empirical formulations or numerical simulations. In this project lateral spread displacements are estimated and new empirical relations to estimate future volumetric and shear strain are used. Geotechnical boring data to are used to: (a) develop isopach maps showing the thickness of sediment thatis likely to liquefy and deform under earthquake shaking; and (b) assess the variability in engineering properties within and between geologic map units. Preliminary results reveal that late Holocene deposits are likely to experience the greatest liquefaction-induced strains, while Holocene and late Pleistocene deposits are likely to experience significantly less horizontal and vertical strain in future earthquakes. Development of maps based on these analyses is feasible.

  2. Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase Cδ.

    PubMed

    Shin, Eun-Joo; Duong, Chu Xuan; Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F; Kanthasamy, Anumantha G; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2012-06-15

    This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (-/-) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (-/-) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (-/-) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity.

  3. Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid.

    PubMed

    Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA. PMID:27057269

  4. Protein kinase G inhibits flow-induced Ca2+ entry into collecting duct cells.

    PubMed

    Du, Juan; Wong, Wei-Yan; Sun, Lei; Huang, Yu; Yao, Xiaoqiang

    2012-07-01

    The renal cortical collecting duct (CCD) contributes to the maintenance of K(+) homeostasis by modulating renal K(+) secretion. Cytosolic Ca(2+) ([Ca(2+)](i)) mediates flow-induced K(+) secretion in the CCD, but the mechanisms regulating flow-induced Ca(2+) entry into renal epithelial cells are not well understood. Here, we found that atrial natriuretic peptide, nitric oxide, and cyclic guanosine monophosphate (cGMP) act through protein kinase G (PKG) to inhibit flow-induced increases in [Ca(2+)](i) in M1-CCD cells. Coimmunoprecipitation, double immunostaining, and functional studies identified heteromeric TRPV4-P2 channels as the mediators of flow-induced Ca(2+) entry into M1-CCD cells and HEK293 cells that were coexpressed with both TRPV4 and TRPP2. In these HEK293 cells, introducing point mutations at two putative PKG phosphorylation sites on TRPP2 abolished the ability of cGMP to inhibit flow-induced Ca(2+) entry. In addition, treating M1-CCD cells with fusion peptides that compete with the endogenous PKG phosphorylation sites on TRPP2 also abolished the cGMP-mediated inhibition of the flow-induced Ca(2+) entry. Taken together, these data suggest that heteromeric TRPV4-P2 channels mediate the flow-induced entry of Ca(2+) into collecting duct cells. Furthermore, substances such as atrial natriuretic peptide and nitric oxide, which increase cGMP, abrogate flow-induced Ca(2+) entry through PKG-mediated inhibition of these channels. PMID:22518003

  5. Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase

    PubMed Central

    Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F.; Kanthasamy, Anumantha G.; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2014-01-01

    This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (–/–) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (–/–) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (–/–) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity. PMID:22512859

  6. Vesnarinone suppresses TNF-induced activation of NF-kappa B, c-Jun kinase, and apoptosis.

    PubMed

    Manna, S K; Aggarwal, B B

    2000-06-01

    Vesnarinone, a synthetic quinolinone derivative used in the treatment of cardiac failure, exhibits immunomodulatory, anti-inflammatory, and cell growth regulatory properties. The mechanisms underlying these properties are not understood, but due to the critical role of nuclear transcription factor NF-kappa B in these responses, we hypothesized that vesnarinone must modulate NF-kappa B activation. We investigated the effect of vesnarinone on NF-kappa B activation induced by inflammatory agents. Vesnarinone blocked TNF-induced activation of NF-kappa B in a concentration- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of I kappa B alpha, an inhibitor of NF-kappa B. The effects of vesnarinone were not cell type specific, as it blocked TNF-induced NF-kappa B activation in a variety of cells. NF-kappa B-dependent reporter gene transcription activated by TNF was also suppressed by vesnarinone. The TNF-induced NF-kappa B activation cascade involving TNF receptor 1-TNF receptor associated death domain-TNF receptor associated factor 2 NF-kappa B-inducing kinase-IKK was interrupted at the TNF receptor associated factor 2 and NF-kappa B-inducing kinase sites by vesnarinone, thus suppressing NF-kappa B reporter gene expression. Vesnarinone also blocked NF-kappa B activation induced by several other inflammatory agents, inhibited the TNF-induced activation of transcription factor AP-1, and suppressed the TNF-induced activation of c-Jun N-terminal kinase and mitogen-activated protein kinase kinase. TNF-induced cytotoxicity, caspase activation, and lipid peroxidation were also abolished by vesnarinone. Overall, our results indicate that vesnarinone inhibits activation of NF-kappa B and AP-1 and their associated kinases. This may provide a molecular basis for vesnarinone's ability to suppress inflammation, immunomodulation, and growth regulation.

  7. 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

  8. Trace metal mapping by laser-induced breakdown spectroscopy

    SciTech Connect

    Kaiser, Jozef; Novotny, Dr. Karel; Hrdlicka, A; Malina, R; Hartl, M; Kizek, R; Adam, V

    2012-01-01

    Abstract: Laser-Induced Breakdown Spectroscopy (LIBS) is a sensitive optical technique capable of fast multi-elemental analysis of solid, gaseous and liquid samples. The potential applications of lasers for spectrochemical analysis were developed shortly after its invention; however the massive development of LIBS is connected with the availability of powerful pulsed laser sources. Since the late 80s of 20th century LIBS dominated the analytical atomic spectroscopy scene and its application are developed continuously. Here we review the utilization of LIBS for trace elements mapping in different matrices. The main emphasis is on trace metal mapping in biological samples.

  9. Bub1 overexpression induces aneuploidy and tumor formation through Aurora B kinase hyperactivation

    PubMed Central

    Ricke, Robin M.; Jeganathan, Karthik B.

    2011-01-01

    High expression of the protein kinase Bub1 has been observed in a variety of human tumors and often correlates with poor clinical prognosis, but its molecular and cellular consequences and role in tumorigenesis are unknown. Here, we demonstrate that overexpression of Bub1 in mice leads to near-diploid aneuploidies and tumor formation. We found that chromosome misalignment and lagging are the primary mitotic errors responsible for the observed aneuploidization. High Bub1 levels resulted in aberrant Bub1 kinase activity and hyperactivation of Aurora B kinase. When Aurora B activity is suppressed, pharmacologically or via BubR1 overexpression, chromosome segregation errors caused by Bub1 overexpression are largely corrected. Importantly, Bub1 transgenic mice overexpressing Bub1 developed various kinds of spontaneous tumors and showed accelerated Myc-induced lymphomagenesis. Our results establish that Bub1 has oncogenic properties and suggest that Aurora B is a critical target through which overexpressed Bub1 drives aneuploidization and tumorigenesis. PMID:21646403

  10. Oncogenic kinase NPM/ALK induces expression of HIF1α mRNA.

    PubMed

    Marzec, M; Liu, X; Wong, W; Yang, Y; Pasha, T; Kantekure, K; Zhang, P; Woetmann, A; Cheng, M; Odum, N; Wasik, M A

    2011-03-17

    The mechanisms of malignant cell transformation mediated by the oncogenic anaplastic lymphoma kinase (ALK) tyrosine kinase remain only partially understood. In this study, we report that T-cell lymphoma (TCL) cells carrying the nucleophosmin (NPM)/ALK fusion protein (ALK+ TCL) strongly express hypoxia-induced factor 1α (HIF1α) mRNA, even under normoxic conditions, and markedly upregulate HIF1α protein expression under hypoxia. HIF1α expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as shown in BaF3 cells transfected with wild-type NPM/ALK and kinase-inactive NPM/ALK K210R mutant and by the inhibition of the NPM/ALK function in ALK+ TCL cells by a small-molecule ALK inhibitor. NPM/ALK induces HIF1α expression by upregulating its gene transcription through its key signal transmitter signal transducer and activator of transcription 3 (STAT3), which binds to the HIF1α gene promoter as shown by the chromatin immunoprecipitation assay and is required for HIF1α gene expression as demonstrated by its small interfering RNA-mediated depletion. In turn, depletion of HIF1α increases mammalian target of rapamycin complex 1 activation, cell growth and proliferation and decreases vascular endothelial growth factor synthesis. These results identify a novel cell-transforming property of NPM/ALK, namely its ability to induce the expression of HIF1α, a protein with an important role in carcinogenesis. These results also provide another rationale to therapeutically target NPM/ALK and STAT3 in ALK+ TCL.

  11. 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

  12. 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

  13. 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.

  14. Protein tyrosine kinase 6 promotes ERBB2-induced mammary gland tumorigenesis in the mouse.

    PubMed

    Peng, M; Ball-Kell, S M; Tyner, A L

    2015-01-01

    Protein tyrosine kinase 6 (PTK6) expression, activation, and amplification of the PTK6 gene have been reported in ERBB2/HER2-positive mammary gland cancers. To explore contributions of PTK6 to mammary gland tumorigenesis promoted by activated ERBB2, we crossed Ptk6-/- mice with the mouse mammary tumor virus-ERBB2 transgenic mouse line expressing activated ERBB2 and characterized tumor development and progression. ERBB2-induced tumorigenesis was significantly delayed and diminished in mice lacking PTK6. PTK6 expression was induced in the mammary glands of ERBB2 transgenic mice before tumor development and correlated with activation of signal transducer and activator of transcription 3 (STAT3) and increased proliferation. Disruption of PTK6 impaired STAT3 activation and proliferation. Phosphorylation of the PTK6 substrates focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1; p130CAS) was decreased in Ptk6-/- mammary gland tumors. Reduced numbers of metastases were detected in the lungs of Ptk6-/- mice expressing activated ERBB2, compared with wild-type ERBB2 transgenic mice. PTK6 activation was detected at the edges of ERBB2-positive tumors. These data support roles for PTK6 in both ERBB2-induced mammary gland tumor initiation and metastasis, and identify STAT3, FAK, and BCAR1 as physiologically relevant PTK6 substrates in breast cancer. Including PTK6 inhibitors as part of a treatment regimen could have distinct benefits in ERBB2/HER2-positive breast cancers.

  15. Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

    SciTech Connect

    Nakatsu, Yusuke; Kotake, Yaichiro Hino, Atsuko; Ohta, Shigeru

    2008-08-01

    AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release.

  16. Phosphodiesterase 5 Inhibition Limits Doxorubicin-induced Heart Failure by Attenuating Protein Kinase G Iα Oxidation.

    PubMed

    Prysyazhna, Oleksandra; Burgoyne, Joseph Robert; Scotcher, Jenna; Grover, Steven; Kass, David; Eaton, Philip

    2016-08-12

    Phosphodiesterase 5 (PDE5) inhibitors limit myocardial injury caused by stresses, including doxorubicin chemotherapy. cGMP binding to PKG Iα attenuates oxidant-induced disulfide formation. Because PDE5 inhibition elevates cGMP and protects from doxorubicin-induced injury, we reasoned that this may be because it limits PKG Iα disulfide formation. To investigate the role of PKG Iα disulfide dimerization in the development of apoptosis, doxorubicin-induced cardiomyopathy was compared in male wild type (WT) or disulfide-resistant C42S PKG Iα knock-in (KI) mice. Echocardiography showed that doxorubicin treatment caused loss of myocardial tissue and depressed left ventricular function in WT mice. Doxorubicin also reduced pro-survival signaling and increased apoptosis in WT hearts. In contrast, KI mice were markedly resistant to the dysfunction induced by doxorubicin in WTs. In follow-on experiments the influence of the PDE5 inhibitor tadalafil on the development of doxorubicin-induced cardiomyopathy in WT and KI mice was investigated. In WT mice, co-administration of tadalafil with doxorubicin reduced PKG Iα oxidation caused by doxorubicin and also protected against cardiac injury and loss of function. KI mice were again innately resistant to doxorubicin-induced cardiotoxicity, and therefore tadalafil afforded no additional protection. Doxorubicin decreased phosphorylation of RhoA (Ser-188), stimulating its GTPase activity to activate Rho-associated protein kinase (ROCK) in WTs. These pro-apoptotic events were absent in KI mice and were attenuated in WTs co-administered tadalafil. PKG Iα disulfide formation triggers cardiac injury, and this initiation of maladaptive signaling can be blocked by pharmacological therapies that elevate cGMP, which binds kinase to limit its oxidation. PMID:27342776

  17. 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

  18. Apoptosis during spontaneous and prostaglandin F(2alpha)-induced luteal regression in the buffalo cow (Bubalus bubalis): involvement of mitogen-activated protein kinases.

    PubMed

    Yadav, Vijay K; Sudhagar, Ranga R; Medhamurthy, R

    2002-09-01

    The present study was conducted to evaluate whether the corpus luteum (CL) of the water buffalo (Bubalus bubalis) cow undergoes luteal regression by the process of apoptosis and to examine the involvement of mitogen-activated protein (MAP) kinases during prostaglandin (PG) F(2alpha)-induced luteolysis. Sections of CL from late in the estrous cycle, i.e., during spontaneous luteolysis, stained for 4',6'-diamidino-2-phenylindole revealed increased numbers of condensed nuclei, indicating cell death by apoptosis, which was confirmed further by the occurrence of pronounced oligonucleosome formation. For morphological and biochemical characterization during PGF(2alpha)-induced apoptosis, CL were collected at 0, 4, 12, and 18 h after injection of 750 micro g of Tiaprost, a synthetic analogue of PGF(2alpha), to midestrous buffalo cows. Serum progesterone concentrations fell within 4 h and decreased (P < 0.05) maximally by 18 h. Concomitant decreases (P < 0.05) in the levels of steroidogenic acute regulatory mRNA and protein were observed in CL during 12-18 h, with the more profound effect on mRNA levels. Quantitative analysis of the genomic DNA showed a >5-fold increase (P < 0.05) in the low molecular weight DNA fragments by 18 h postinjection. Immunoblot analysis of CL tissue lysates showed increased (P < 0.05) levels of phospho-Jun N-terminal kinase (JNK) 1 (4- to 14-fold during 4-18 h) and phospho-p38 (2- to 4-fold at 18 h). Immunohistochemical evaluation of CL sections revealed an increased nuclear localization of phospho-JNK after treatment. These findings demonstrate that the CL of the buffalo cow undergoes cell death by the process of apoptosis both during spontaneous and PGF(2alpha)-induced luteolysis and that MAP kinases are involved during PGF(2alpha)-mediated apoptosis in the CL.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. Insulin-induced decrease in protein phosphorylation in rat adipocytes not explained by decreased A-kinase activity

    SciTech Connect

    Egan, J.J.; Greenberg, A.S.; Chang, M.K.; Londos, C.

    1987-05-01

    In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase (A-kinase), from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from /sup 32/P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The insulin-induced decrease in lipolysis was associated with a decrease in the /sup 32/P content of several proteins, an effect not explained by the modest reduction in A-kinase activity by insulin. This effect of insulin on protein phosphorylation was lost as the A-kinase activity ratios exceeded 0.5. The results suggest that insulin promotes the dephosphorylation of those adipocyte proteins which are subject to phosphorylation by A-kinase.

  4. Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase*

    PubMed Central

    Zmijewski, Jaroslaw W.; Banerjee, Sami; Bae, Hongbeom; Friggeri, Arnaud; Lazarowski, Eduardo R.; Abraham, Edward

    2010-01-01

    Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H2O2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H2O2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H2O2 on kinase activity. Similar to the results obtained with H2O2-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H2O2 are increased. These results demonstrate that physiologically relevant concentrations of H2O2 can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. PMID:20729205

  5. Rosuvastatin intensifies the beneficial effects of rho-kinase inhibitor in reversal of monocrotaline-induced pulmonary hypertension

    PubMed Central

    Owczarek, Jacek; Sołtysiak, Urszula; Orszulak-Michalak, Daria

    2015-01-01

    Introduction It remains controversial whether statins have a beneficial effect on pulmonary arterial hypertension (PAH). This study is intended to evaluate whether statin, co-administered with Rho-kinase inhibitor, could enhance its efficacy. Although Rho-kinase inhibitors, including fasudil, have been reported to improve pulmonary hypertension in experimental and clinical studies, the combination of these agents has not been tested in the treatment of pulmonary hypertension (PH). Material and methods The effects of such a regimen on hemodynamics, right ventricle hypertrophy, and Rho-associated protein kinase (ROCK) activity in experimental monocrotaline (MCT)-induced pulmonary hypertension were examined. Fourteen days after monocrotaline injection (60 mg/kg), male rats were treated orally for another 14 days with fasudil (15 mg/kg per day), or with a combination of fasudil + rosuvastatin (10 mg/kg per day). Results The drug combination reversed the MCT-induced increase in right ventricle pressure (RVP) and reduced right ventricular hypertrophy (RV/LV + S ratio) more than Rho kinase inhibitor alone. The simultaneous administration of fasudil and rosuvastatin caused a further decrease of RhoA kinase activity in isolated lung tissues as compared to fasudil alone. Conclusions The results indicate that rosuvastatin intensifies the beneficial effects of Rho-kinase inhibitor on the Rho/Rho-kinase pathway and such a combination may represent an option for the treatment of pulmonary arterial hypertension. PMID:27478473

  6. Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia.

    PubMed

    Ferrelli, Francesca; Pastore, Donatella; Capuani, Barbara; Lombardo, Marco F; Blot-Chabaud, Marcel; Coppola, Andrea; Basello, Katia; Galli, Angelica; Donadel, Giulia; Romano, Maria; Caratelli, Sara; Pacifici, Francesca; Arriga, Roberto; Di Daniele, Nicola; Sbraccia, Paolo; Sconocchia, Giuseppe; Bellia, Alfonso; Tesauro, Manfredi; Federici, Massimo; Della-Morte, David; Lauro, Davide

    2015-02-01

    Diabetic hyperglycaemia causes endothelial dysfunction mainly by impairing endothelial nitric oxide (NO) production. Moreover, hyperglycaemia activates several noxious cellular pathways including apoptosis, increase in reactive oxygen species (ROS) levels and diminishing Na(+)-K(+) ATPase activity which exacerbate vascular damage. Serum glucocorticoid kinase (SGK)-1, a member of the serine/threonine kinases, plays a pivotal role in regulating NO production through inducible NO synthase activation and other cellular mechanisms. Therefore, in this study, we aimed to investigate the protective role of SGK-1 against hyperglycaemia in human umbilical endothelial cells (HUVECs). We used retrovirus to infect HUVECs with either SGK-1, SGK-1Δ60 (lacking of the N-60 amino acids-increase SGK-1 activity) or SGK-1Δ60KD (kinase-dead constructs). We tested our hypothesis in vitro after high glucose and glucosamine incubation. Increase in SGK-1 expression and activity (SGK-1Δ60) resulted in higher production of NO, inhibition of ROS synthesis and lower apoptosis in endothelial cell after either hyperglycaemia or glucosamine treatments. Moreover, in this study, we showed increased GLUT-1 membrane translocation and Na(+)-K(+) ATPase activity in cell infected with SGK-1Δ60 construct. These results suggest that as in endothelial cells, an increased SGK-1 activity and expression reduces oxidative stress, improves cell survival and restores insulin-mediated NO production after different noxae stimuli. Therefore, SGK-1 may represent a specific target to further develop novel therapeutic options against diabetic vascular disease.

  7. Properties of a novel thymidine kinase induced by an acyclovir-resistant herpes simplex virus type 1 mutant.

    PubMed Central

    Larder, B A; Darby, G

    1982-01-01

    The acyclovir-resistant mutant of herpes simplex virus type 1, SC16 S1, induced reduced levels of thymidine kinase activity (ca. 25% reduction) in infected cells. The activity appeared with kinetics similar to that in wild type-infected cells, and pulse-labeling experiments showed that the thymidine kinase polypeptide was synthesized at a similar rate. We showed that the enzyme was virus specific by inactivating it with antiserum raised against herpes simplex virus-infected cell proteins. The enzyme induced by the mutant had reduced electrophoretic mobility in nondenaturing gels, decreased thermal stability, and decreased affinity for several different substrates (assessed by measurement of Km values) compared with the enzyme induced by the wild type. From the data obtained we conclude that the thymidine kinase induced by the mutant has an altered specificity, probably resulting from an amino acid substitution which affects the primary binding site for nucleosides and nucleoside analogs. Images PMID:6283175

  8. Frutalin, a galactose-binding lectin, induces chemotaxis and rearrangement of actin cytoskeleton in human neutrophils: involvement of tyrosine kinase and phosphoinositide 3-kinase.

    PubMed

    Brando-Lima, Aline C; Saldanha-Gama, Roberta F; Henriques, Maria das Graças M O; Monteiro-Moreira, Ana C O; Moreira, Renato A; Barja-Fidalgo, Christina

    2005-10-15

    Several lectin-like molecules have been shown as potent activators of leukocytes. Galactose-binding lectins are of special interest since they could interact with several endogenous molecules involved in the innate and specific immune responses. The effects of Frutalin (FTL), an alpha-D-galactose (Gal)-binding plant lectin, on the modulation of neutrophil (PMN) functions were investigated. FTL induced a dose-dependent PMN migration in mice pleural cavity. Moreover, FTL was also a potent direct chemotactic for human PMN, in vitro, and triggered oxidative burst in these cells. These effects were accompanied by a rearrangement of the actin cytoskeleton dynamic, activation of tyrosine kinase (TK) pathways, increase in focal adhesion kinase (FAK) phosphorylation, and its subsequent association to phosphoinositide3-kinase (PI3K). All those effects were inhibited in the presence of Gal, suggesting specific carbohydrate recognition for FTL effects. The activations of TK and PI3K pathways are essential events for FTL-induced chemotaxis, since inhibitors of these pathways, genistein and LY294002, inhibited neutrophil migration in vitro. The data indicate that sugar-protein interactions between a soluble lectin and galacto-components on neutrophil surface trigger the TK pathway, inducing FAK and PI3K activation, interfering with cell motility and oxidative response.

  9. 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

  10. Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

    PubMed Central

    Liu, Bing-Chen; Yang, Li-Li; Lu, Xiao-Yu; Song, Xiang; Li, Xue-Chen; Chen, Guangping; Li, Yichao; Yao, Xincheng; Humphrey, Donald R.; Eaton, Douglas C.

    2015-01-01

    We recently showed that lovastatin attenuates cyclosporin A (CsA)-induced damage of cortical collecting duct (CCD) principal cells by reducing intracellular cholesterol. Previous studies showed that, in cell expression models or artificial membranes, exogenous cholesterol directly inhibits inward rectifier potassium channels, including Kir1.1 (Kcnj1; the gene locus for renal outer medullary K+ [ROMK1] channels). Therefore, we hypothesized that lovastatin might stimulate ROMK1 by reducing cholesterol in CCD cells. Western blots showed that mpkCCDc14 cells express ROMK1 channels with molecular masses that approximate the molecular masses of ROMK1 in renal tubules detected before and after treatment with DTT. Confocal microscopy showed that ROMK1 channels were not in the microvilli, where cholesterol-rich lipid rafts are located, but rather, the planar regions of the apical membrane of mpkCCDc14 cells. Furthermore, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], an activator of ROMK channels, was detected mainly in the microvilli under resting conditions along with the kinase responsible for PI(4,5)P2 synthesis, phosphatidylinositol-4-phosphate 5-kinase, type I γ [PI(4)P5K I γ], which may explain the low basal open probability and increased sensitivity to tetraethylammonium observed here for this channel. Notably, lovastatin induced PI(4)P5K I γ diffusion into planar regions and elevated PI(4,5)P2 and ROMK1 open probability in these regions through a cholesterol-associated mechanism. However, exogenous cholesterol alone did not induce these effects. These results suggest that lovastatin stimulates ROMK1 channels, at least in part, by inducing PI(4,5)P2 synthesis in planar regions of the renal CCD cell apical membrane, suggesting that lovastatin could reduce cyclosporin-induced nephropathy and associated hyperkalemia. PMID:25349201

  11. Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels.

    PubMed

    Liu, Bing-Chen; Yang, Li-Li; Lu, Xiao-Yu; Song, Xiang; Li, Xue-Chen; Chen, Guangping; Li, Yichao; Yao, Xincheng; Humphrey, Donald R; Eaton, Douglas C; Shen, Bao-Zhong; Ma, He-Ping

    2015-07-01

    We recently showed that lovastatin attenuates cyclosporin A (CsA)-induced damage of cortical collecting duct (CCD) principal cells by reducing intracellular cholesterol. Previous studies showed that, in cell expression models or artificial membranes, exogenous cholesterol directly inhibits inward rectifier potassium channels, including Kir1.1 (Kcnj1; the gene locus for renal outer medullary K(+) [ROMK1] channels). Therefore, we hypothesized that lovastatin might stimulate ROMK1 by reducing cholesterol in CCD cells. Western blots showed that mpkCCDc14 cells express ROMK1 channels with molecular masses that approximate the molecular masses of ROMK1 in renal tubules detected before and after treatment with DTT. Confocal microscopy showed that ROMK1 channels were not in the microvilli, where cholesterol-rich lipid rafts are located, but rather, the planar regions of the apical membrane of mpkCCDc14 cells. Furthermore, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], an activator of ROMK channels, was detected mainly in the microvilli under resting conditions along with the kinase responsible for PI(4,5)P2 synthesis, phosphatidylinositol-4-phosphate 5-kinase, type I γ [PI(4)P5K I γ], which may explain the low basal open probability and increased sensitivity to tetraethylammonium observed here for this channel. Notably, lovastatin induced PI(4)P5K I γ diffusion into planar regions and elevated PI(4,5)P2 and ROMK1 open probability in these regions through a cholesterol-associated mechanism. However, exogenous cholesterol alone did not induce these effects. These results suggest that lovastatin stimulates ROMK1 channels, at least in part, by inducing PI(4,5)P2 synthesis in planar regions of the renal CCD cell apical membrane, suggesting that lovastatin could reduce cyclosporin-induced nephropathy and associated hyperkalemia. PMID:25349201

  12. Critical role of c‐jun (NH2) terminal kinase in paracetamol‐ induced acute liver failure

    PubMed Central

    Henderson, Neil C; Pollock, Katharine J; Frew, John; Mackinnon, Alison C; Flavell, Richard A; Davis, Roger J; Sethi, Tariq; Simpson, Kenneth J

    2007-01-01

    Background Acute hepatic failure secondary to paracetamol poisoning is associated with high mortality. C‐jun (NH2) terminal kinase (JNK) is a member of the mitogen‐activated protein kinase family and is a key intracellular signalling molecule involved in controlling the fate of cells. Aim To examine the role of JNK in paracetamol‐induced acute liver failure (ALF). Methods A previously developed mouse model of paracetamol poisoning was used to examine the role of JNK in paracetamol‐induced ALF. Results Paracetamol‐induced hepatic JNK activation both in human and murine paracetamol hepatotoxicity and in our murine model preceded the onset of hepatocyte death. JNK inhibition in vivo (using two JNK inhibitors with different mechanisms of action) markedly reduced mortality in murine paracetamol hepatotoxicity, with a significant reduction in hepatic necrosis and apoptosis. In addition, delayed administration of the JNK inhibitor was more effective than N‐acetylcysteine after paracetamol poisoning in mice. JNK inhibition was not protective in acute carbon tetrachloride‐mediated or anti‐Fas antibody‐mediated hepatic injury, suggesting specificity for the role of JNK in paracetamol hepatotoxicity. Furthermore, disruption of the JNK1 or JNK2 genes did not protect against paracetamol‐induced hepatic damage. Pharmacological JNK inhibition had no effect on paracetamol metabolism, but markedly inhibited hepatic tumour necrosis foctor α (TNF α) production after paracetamol poisoning. Conclusions These data demonstrated a central role for JNK in the pathogenesis of paracetamol‐induced liver failure, thereby identifying JNK as an important therapeutic target in the treatment of paracetamol hepatotoxicity. PMID:17185352

  13. Pim1 kinase protects airway epithelial cells from cigarette smoke-induced damage and airway inflammation.

    PubMed

    de Vries, M; Heijink, I H; Gras, R; den Boef, L E; Reinders-Luinge, M; Pouwels, S D; Hylkema, M N; van der Toorn, M; Brouwer, U; van Oosterhout, A J M; Nawijn, M C

    2014-08-01

    Exposure to cigarette smoke (CS) is the main risk factor for developing chronic obstructive pulmonary disease and can induce airway epithelial cell damage, innate immune responses, and airway inflammation. We hypothesized that cell survival factors might decrease the sensitivity of airway epithelial cells to CS-induced damage, thereby protecting the airways against inflammation upon CS exposure. Here, we tested whether Pim survival kinases could protect from CS-induced inflammation. We determined expression of Pim kinases in lung tissue, airway inflammation, and levels of keratinocyte-derived cytokine (KC) and several damage-associated molecular patterns in bronchoalveolar lavage in mice exposed to CS or air. Human bronchial epithelial BEAS-2B cells were treated with CS extract (CSE) in the presence or absence of Pim1 inhibitor and assessed for loss of mitochondrial membrane potential, induction of cell death, and release of heat shock protein 70 (HSP70). We observed increased expression of Pim1, but not of Pim2 and Pim3, in lung tissue after exposure to CS. Pim1-deficient mice displayed a strongly enhanced neutrophilic airway inflammation upon CS exposure compared with wild-type controls. Inhibition of Pim1 activity in BEAS-2B cells increased the loss of mitochondrial membrane potential and reduced cell viability upon CSE treatment, whereas release of HSP70 was enhanced. Interestingly, we observed release of S100A8 but not of double-strand DNA or HSP70 in Pim1-deficient mice compared with wild-type controls upon CS exposure. In conclusion, we show that expression of Pim1 protects against CS-induced cell death in vitro and neutrophilic airway inflammation in vivo. Our data suggest that the underlying mechanism involves CS-induced release of S100A8 and KC. PMID:24816488

  14. Sphingosine Kinase 1 Deficiency Confers Protection against Hyperoxia-Induced Bronchopulmonary Dysplasia in a Murine Model

    PubMed Central

    Harijith, Anantha; Pendyala, Srikanth; Reddy, Narsa M.; Bai, Tao; Usatyuk, Peter V.; Berdyshev, Evgeny; Gorshkova, Irina; Huang, Long Shuang; Mohan, Vijay; Garzon, Steve; Kanteti, Prasad; Reddy, Sekhar P.; Raj, J. Usha; Natarajan, Viswanathan

    2014-01-01

    Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1−/− (Sphk1−/−), sphingosine kinase 2−/− (Sphk2−/−), and S1P lyase+/− (Sgpl1+/−) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1−/−, but not Sphk2−/− or Sgpl1+/−, mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling–regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia. PMID:23933064

  15. MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

    PubMed Central

    Kobayashi, Miho; Nishita, Michiru; Mishima, Toshiaki; Ohashi, Kazumasa; Mizuno, Kensaku

    2006-01-01

    Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells. PMID:16456544

  16. 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

  17. 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

  18. 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...

  19. 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

  20. 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.

  1. 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

  2. Investigating combinatorial approaches in virtual screening on human inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3): a case study for small molecule kinases.

    PubMed

    Crochet, Robert B; Cavalier, Michael C; Seo, Minsuh; Kim, Jeong Do; Yim, Young-Sun; Park, Seung-Jong; Lee, Yong-Hwan

    2011-11-01

    Efforts toward improving the predictiveness in tier-based approaches to virtual screening (VS) have mainly focused on protein kinases. Despite their significance as drug targets, small molecule kinases have been rarely tested with these approaches. In this paper, we investigate the efficacy of a pharmacophore screening-combined structure-based docking approach on the human inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, an emerging target for cancer chemotherapy. Six out of a total 1364 compounds from NCI's Diversity Set II were selected as true actives via throughput screening. Using a database constructed from these compounds, five programs were tested for structure-based docking (SBD) performance, the MOE of which showed the highest enrichments and second highest screening rates. Separately, using the same database, pharmacophore screening was performed, reducing 1364 compounds to 287 with no loss in true actives, yielding an enrichment of 4.75. When SBD was retested with the pharmacophore filtered database, 4 of the 5 SBD programs showed significant improvements to enrichment rates at only 2.5% of the database, with a 7-fold decrease in an average VS time. Our results altogether suggest that combinatorial approaches of VS technologies are easily applicable to small molecule kinases and, moreover, that such methods can decrease the variability associated with single-method SBD approaches.

  3. 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.

  4. Protein kinase C-mediated sodium glucose transporter 1 activation in precondition-induced cardioprotection

    PubMed Central

    Kanwal, Abhinav; Kasetti, Sujatha; Putcha, Uday Kumar; Asthana, Shailendra; Banerjee, Sanjay K

    2016-01-01

    The concept of cardioprotection through preconditioning against ischemia–reperfusion (I/R) injury is well known and established. However, among different proposed mechanisms regarding the concept of ischemic preconditioning, protein kinase C (PKC)-mediated cardioprotection through ischemic preconditioning plays a key role in myocardial I/R injury. Thus, this study was designed to find the relationship between PKC and sodium glucose transporter 1 (SGLT1) in preconditioning-induced cardioprotection, which is ill reported till now. By applying a multifaceted approach, we demonstrated that PKC activates SGLT1, which curbed oxidative stress and apoptosis against I/R injury. PKC activation enhances cardiac glucose uptake through SGLT1 and seems essential in preventing I/R-induced cardiac injury, indicating a possible cross-talk between PKC and SGLT1.

  5. Protein kinase C-mediated sodium glucose transporter 1 activation in precondition-induced cardioprotection

    PubMed Central

    Kanwal, Abhinav; Kasetti, Sujatha; Putcha, Uday Kumar; Asthana, Shailendra; Banerjee, Sanjay K

    2016-01-01

    The concept of cardioprotection through preconditioning against ischemia–reperfusion (I/R) injury is well known and established. However, among different proposed mechanisms regarding the concept of ischemic preconditioning, protein kinase C (PKC)-mediated cardioprotection through ischemic preconditioning plays a key role in myocardial I/R injury. Thus, this study was designed to find the relationship between PKC and sodium glucose transporter 1 (SGLT1) in preconditioning-induced cardioprotection, which is ill reported till now. By applying a multifaceted approach, we demonstrated that PKC activates SGLT1, which curbed oxidative stress and apoptosis against I/R injury. PKC activation enhances cardiac glucose uptake through SGLT1 and seems essential in preventing I/R-induced cardiac injury, indicating a possible cross-talk between PKC and SGLT1. PMID:27695290

  6. 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

  7. Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK)*

    PubMed Central

    Chan, Kenny L.; Pillon, Nicolas J.; Sivaloganathan, Darshan M.; Costford, Sheila R.; Liu, Zhi; Théret, Marine; Chazaud, Benedicte; Klip, Amira

    2015-01-01

    A rise in tissue-embedded macrophages displaying “M1-like” proinflammatory polarization is a hallmark of metabolic inflammation during a high fat diet or obesity. Here we show that bone marrow-derived macrophages (BMDM) from high fat-fed mice retain a memory of their dietary environment in vivo (displaying the elevated proinflammatory genes Cxcl1, Il6, Tnf, Nos2) despite 7-day differentiation and proliferation ex vivo. Notably, 6-h incubation with palmitoleate (PO) reversed the proinflammatory gene expression and cytokine secretion seen in BMDM from high fat-fed mice. BMDM from low fat-fed mice exposed to palmitate (PA) for 18 h ex vivo also showed elevated expression of proinflammatory genes (Cxcl1, Il6, Tnf, Nos2, and Il12b) associated with M1 polarization. Conversely, PO treatment increased anti-inflammatory genes (Mrc1, Tgfb1, Il10, Mgl2) and oxidative metabolism, characteristic of M2 macrophages. Therefore, saturated and unsaturated fatty acids bring about opposite macrophage polarization states. Coincubation of BMDM with both fatty acids counteracted the PA-induced Nos2 expression in a PO dose-dependent fashion. PO also prevented PA-induced IκBα degradation, RelA nuclear translocation, NO production, and cytokine secretion. Mechanistically, PO exerted its anti-inflammatory function through AMP-activated protein kinase as AMP kinase knockout or inhibition by Compound C offset the PO-dependent prevention of PA-induced inflammation. These results demonstrate a nutritional memory of BMDM ex vivo, highlight the plasticity of BMDM polarization in response to saturated and unsaturated fatty acids, and identify the potential to reverse diet- and saturated fat-induced M1-like polarization by administering palmitoleate. These findings could have applicability to reverse obesity-linked inflammation in metabolically relevant tissues. PMID:25987561

  8. Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A.

    PubMed Central

    Hu, D E; Fan, T P

    1995-01-01

    1. Vascular endothelial growth factor (VEGF) is a heparin-binding angiogenic factor which specifically acts on endothelial cells via distinct membrane-spanning tyrosine kinase receptors. Here we used the rat sponge implant model to test the hypothesis that the angiogenic activity of VEGF can be suppressed by protein tyrosine kinase (PTK) inhibitors. 2. Neovascular responses in subcutaneous sponge implants were determined by measurements of relative sponge blood flow by use of a 133Xe clearance technique, and confirmed by histological studies and morphometric analysis. 3. Daily local administration of 250 ng VEGF165 accelerated the rate of 133Xe clearance from the sponges and induced an intense neovascularisation. This VEGF165-induced angiogenesis was inhibited by daily co-administration of the selective PTK inhibitor, lavendustin A (10 micrograms), but not its negative control, lavendustin B (10 micrograms). Blood flow measurements and morphometric analysis of 8-day-old sponges showed that lavendustin A reduced the 133Xe clearance of VEGF165-treated sponges from 32.9 +/- 1.5% to 20.9 +/- 1.6% and the total fibrovascular growth area from 62.4 +/- 6.1% to 21.6 +/- 6.8% (n = 12, P < 0.05). 4. Co-injection of suramin (3 mg), an inhibitor of heparin-binding growth factors, also suppressed the VEGF165-elicited neovascular response. In contrast, neither lavendustin A nor suramin produced any effect on the basal sponge-induced angiogenesis. 5. When given alone, low doses of VEGF165 (25 ng) or basic fibroblast growth factor (bFGF; 10 ng) did not modify the basal sponge-induced neovascularisation.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2 Figure 2 PMID:7533611

  9. Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK).

    PubMed

    Chan, Kenny L; Pillon, Nicolas J; Sivaloganathan, Darshan M; Costford, Sheila R; Liu, Zhi; Théret, Marine; Chazaud, Benedicte; Klip, Amira

    2015-07-01

    A rise in tissue-embedded macrophages displaying "M1-like" proinflammatory polarization is a hallmark of metabolic inflammation during a high fat diet or obesity. Here we show that bone marrow-derived macrophages (BMDM) from high fat-fed mice retain a memory of their dietary environment in vivo (displaying the elevated proinflammatory genes Cxcl1, Il6, Tnf, Nos2) despite 7-day differentiation and proliferation ex vivo. Notably, 6-h incubation with palmitoleate (PO) reversed the proinflammatory gene expression and cytokine secretion seen in BMDM from high fat-fed mice. BMDM from low fat-fed mice exposed to palmitate (PA) for 18 h ex vivo also showed elevated expression of proinflammatory genes (Cxcl1, Il6, Tnf, Nos2, and Il12b) associated with M1 polarization. Conversely, PO treatment increased anti-inflammatory genes (Mrc1, Tgfb1, Il10, Mgl2) and oxidative metabolism, characteristic of M2 macrophages. Therefore, saturated and unsaturated fatty acids bring about opposite macrophage polarization states. Coincubation of BMDM with both fatty acids counteracted the PA-induced Nos2 expression in a PO dose-dependent fashion. PO also prevented PA-induced IκBα degradation, RelA nuclear translocation, NO production, and cytokine secretion. Mechanistically, PO exerted its anti-inflammatory function through AMP-activated protein kinase as AMP kinase knockout or inhibition by Compound C offset the PO-dependent prevention of PA-induced inflammation. These results demonstrate a nutritional memory of BMDM ex vivo, highlight the plasticity of BMDM polarization in response to saturated and unsaturated fatty acids, and identify the potential to reverse diet- and saturated fat-induced M1-like polarization by administering palmitoleate. These findings could have applicability to reverse obesity-linked inflammation in metabolically relevant tissues.

  10. Involvement of Extracellular Signal Regulated Kinases in Traumatic Brain Injury-Induced Depression in Rodents

    PubMed Central

    Kuo, Jinn-Rung; Cheng, Yi-Hsuan; Chen, Yi-Shion

    2013-01-01

    Abstract Traumatic brain injury (TBI) is the most common cause of death and acquired disability among children and young adults in the developed countries. In clinical studies, the incidence of depression is high after TBI, and the mechanisms behind TBI-induced depression remain unclear. In the present study, we subjected rats to a moderate fluid percussion into the closed cranial cavity to induce TBI. After 3 days of recovery, injured rats were given a forced swim test (FST) and novelty-suppressed feeding tests. We found that TBI rats exhibited increased duration of immobility and longer latency to begin chewing food in a new environment compared with sham-operated rats. Western blot analysis showed that TBI led to a decrease in the phosphorylated levels of extracellular signal regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK). Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), significantly reduced the duration of immobility when administered once per day for 14 days. Consistent with behavioral tests, fluoxetine treatment reversed TBI-induced decrease in p-ERK1/2 and p-p38 MAPK levels. Pre-treatment with a selective tryptophan hydroxylase inhibitor para-chlorophenylalanine (PCPA) blocked the antidepressant effect of fluoxetine. PCPA also prevented the effect of fluoxetine on ERK1/2 phosphorylation without affecting p38 MAPK phosphorylation. Pre-treatment with ERK inhibitor SL327 but not p38 MAPK inhibitor SB203580 prevented the antidepressant effect of fluoxetine. These results suggest that ERK1/2 plays a critical role in TBI-induced depression. PMID:23360216

  11. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    PubMed

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

  12. A comparative study of the aneugenic and polyploidy-inducing effects of fisetin and two model Aurora kinase inhibitors.

    PubMed

    Gollapudi, P; Hasegawa, L S; Eastmond, D A

    2014-06-01

    Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements.

  13. Blueberry anthocyanins ameliorate radiation-induced lung injury through the protein kinase RNA-activated pathway.

    PubMed

    Liu, Yunen; Tan, Dehong; Tong, Changci; Zhang, Yubiao; Xu, Ying; Liu, Xinwei; Gao, Yan; Hou, Mingxiao

    2015-12-01

    The purpose of this study was to explore the effect of blueberry anthocyanins (BA) on radiation-induced lung injury and investigate the mechanism of action. Seven days after BA(20 and 80 mg/kg/d)administration, 6 weeks old male Sprague-Dawley rats rats were irradiated by LEKTA precise linear accelerator at a single dose of 20 Gy only once. and the rats were continuously treated with BA for 4 weeks. Moreover, human pulmonary alveolar epithelial cells (HPAEpiC) were transfected with either control-siRNA or siRNA targeting protein kinase R (PKR). Cells were then irradiated and treated with 75 μg/mL BA for 72 h. The results showed that BA significantly ameliorated radiation-induced lung inflammation, lung collagen deposition, apoptosis and PKR expression and activation. In vitro, BA significantly protected cells from radiation-induced cell death through modulating expression of Bcl-2, Bax and Caspase-3. Suppression of PKR by siRNA resulted in ablation of BA protection on radiation-induced cell death and modulation of anti-apoptotic and pro-apoptotic proteins, as well as Caspase-3 expression. These findings suggest that BA is effective in ameliorating radiation-induced lung injury, likely through the PKR signaling pathway. PMID:26551926

  14. Blueberry anthocyanins ameliorate radiation-induced lung injury through the protein kinase RNA-activated pathway.

    PubMed

    Liu, Yunen; Tan, Dehong; Tong, Changci; Zhang, Yubiao; Xu, Ying; Liu, Xinwei; Gao, Yan; Hou, Mingxiao

    2015-12-01

    The purpose of this study was to explore the effect of blueberry anthocyanins (BA) on radiation-induced lung injury and investigate the mechanism of action. Seven days after BA(20 and 80 mg/kg/d)administration, 6 weeks old male Sprague-Dawley rats rats were irradiated by LEKTA precise linear accelerator at a single dose of 20 Gy only once. and the rats were continuously treated with BA for 4 weeks. Moreover, human pulmonary alveolar epithelial cells (HPAEpiC) were transfected with either control-siRNA or siRNA targeting protein kinase R (PKR). Cells were then irradiated and treated with 75 μg/mL BA for 72 h. The results showed that BA significantly ameliorated radiation-induced lung inflammation, lung collagen deposition, apoptosis and PKR expression and activation. In vitro, BA significantly protected cells from radiation-induced cell death through modulating expression of Bcl-2, Bax and Caspase-3. Suppression of PKR by siRNA resulted in ablation of BA protection on radiation-induced cell death and modulation of anti-apoptotic and pro-apoptotic proteins, as well as Caspase-3 expression. These findings suggest that BA is effective in ameliorating radiation-induced lung injury, likely through the PKR signaling pathway.

  15. Pressure overload-induced cardiac hypertrophy response requires janus kinase 2-histone deacetylase 2 signaling.

    PubMed

    Ying, Huang; Xu, Mao-Chun; Tan, Jing-Hua; Shen, Jing-Hua; Wang, Hao; Zhang, Dai-Fu

    2014-01-01

    Pressure overload induces cardiac hypertrophy through activation of Janus kinase 2 (Jak2), however, the underlying mechanisms remain largely unknown. In the current study, we tested whether histone deacetylase 2 (HDAC2) was involved in the process. We found that angiotensin II (Ang-II)-induced re-expression of fetal genes (Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)) in cultured cardiomyocytes was prevented by the Jak2 inhibitor AG-490 and HDAC2 inhibitor Trichostatin-A (TSA), or by Jak2/HDAC2 siRNA knockdown. On the other hand, myocardial cells with Jak2 or HDAC2 over-expression were hyper-sensitive to Ang-II. In vivo, pressure overload by transverse aorta binding (AB) induced a significant cardiac hypertrophic response as well as re-expression of ANP and BNP in mice heart, which were markedly reduced by AG-490 and TSA. Significantly, AG-490, the Jak2 inhibitor, largely suppressed pressure overload-/Ang-II-induced HDAC2 nuclear exportation in vivo and in vitro. Meanwhile, TSA or HDAC2 siRNA knockdown reduced Ang-II-induced ANP/BNP expression in Jak2 over-expressed H9c2 cardiomyocytes. Together, these results suggest that HDAC2 might be a downstream effector of Jak2 to mediate cardiac hypertrophic response by pressure overload or Ang-II. PMID:25380525

  16. Involvement of calcium/calmodulin-dependent protein kinase II in methamphetamine-induced neural damage.

    PubMed

    Chen, Xufeng; Xing, Jingjing; Jiang, Lei; Qian, Wenyi; Wang, Yixin; Sun, Hao; Wang, Yu; Xiao, Hang; Wang, Jun; Zhang, Jinsong

    2016-11-01

    Methamphetamine (METH), an illicit drug, is widely abused in many parts of the world. Mounting evidence shows that METH exposure contributes to neurotoxicity, particularly for the monoaminergic neurons. However, to date, only a few studies have tried to unravel the mechanisms involved in METH-induced non-monoaminergic neural damage. Therefore, in the present study, we tried to explore the mechanisms for METH-induced neural damage in cortical neurons. Our results showed that METH significantly increased intracellular [Ca(2) (+) ]i in Ca(2) (+) -containing solution rather than Ca(2) (+) -free solution. Moreover, METH also upregulated calmodulin (CaM) expression and activated CaM-dependent protein kinase II (CaMKII). Significantly, METH-induced neural damage can be partially retarded by CaM antagonist W7 as well as CaMKII blocker KN93. In addition, L-type Ca(2) (+) channel was also proved to be involved in METH-induced cell damage, as nifedipine, the L-type Ca(2) (+) channel-specific inhibitor, markedly attenuated METH-induced neural damage. Collectively, our results suggest that Ca(2) (+) -CaM-CaMKII is involved in METH-mediated neurotoxicity, and it might suggest a potential target for the development of therapeutic strategies for METH abuse. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Dual leucine zipper kinase is required for excitotoxicity-induced neuronal degeneration

    PubMed Central

    Pozniak, Christine D.; Sengupta Ghosh, Arundhati; Gogineni, Alvin; Hanson, Jesse E.; Lee, Seung-Hye; Larson, Jessica L.; Solanoy, Hilda; Bustos, Daisy; Li, Hong; Ngu, Hai; Jubb, Adrian M.; Ayalon, Gai; Wu, Jiansheng; Scearce-Levie, Kimberly; Zhou, Qiang; Weimer, Robby M.; Kirkpatrick, Donald S.

    2013-01-01

    Excessive glutamate signaling is thought to underlie neurodegeneration in multiple contexts, yet the pro-degenerative signaling pathways downstream of glutamate receptor activation are not well defined. We show that dual leucine zipper kinase (DLK) is essential for excitotoxicity-induced degeneration of neurons in vivo. In mature neurons, DLK is present in the synapse and interacts with multiple known postsynaptic density proteins including the scaffolding protein PSD-95. To examine DLK function in the adult, DLK-inducible knockout mice were generated through Tamoxifen-induced activation of Cre-ERT in mice containing a floxed DLK allele, which circumvents the neonatal lethality associated with germline deletion. DLK-inducible knockouts displayed a modest increase in basal synaptic transmission but had an attenuation of the JNK/c-Jun stress response pathway activation and significantly reduced neuronal degeneration after kainic acid–induced seizures. Together, these data demonstrate that DLK is a critical upstream regulator of JNK-mediated neurodegeneration downstream of glutamate receptor hyper-activation and represents an attractive target for the treatment of indications where excitotoxicity is a primary driver of neuronal loss. PMID:24166713

  18. 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.

  19. Protein Kinase RNA-Like Endoplasmic Reticulum Kinase-Mediated Bcl-2 Protein Phosphorylation Contributes to Evodiamine-Induced Apoptosis of Human Renal Cell Carcinoma Cells

    PubMed Central

    Wu, Wen-Shin; Chien, Chih-Chiang; Chen, Yen-Chou; Chiu, Wen-Ta

    2016-01-01

    We investigated the anticancer mechanism of evodiamine (EVO) against the viability of human A498 renal cell carcinoma (RCC) cells in vitro and in vivo. The in vitro study showed that EVO decreased the viability of A498 cells with the occurrence of apoptotic characteristics such as hypodiploid cells, DNA ladders, chromatin-condensed cells, and cleaved caspase (Casp)-3/poly(ADP ribose) polymerase (PARP) proteins. Pharmacological studies using chemical inhibitors of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) indicated that phosphorylation of the c-Jun N-terminal kinase (JNK) protein participated in EVO-induced cell death of A498 cells, and application of the JNK inhibitor, SP600125 (SP), inhibited EVO-induced cleavage of the Casp-3/PARP proteins and chromatin condensation according to Giemsa staining. EVO disruption of the mitochondrial membrane potential (MMP) with increased protein levels of the phosphorylated Bcl-2 protein (p-Bcl-2) was prevented by JNK inhibitors in A498 cells. A structure-activity relationship study showed that a methyl group at position 14 in EVO was important for its apoptotic effects and increased p-Bcl-2 protein in A498 cells. Furthermore, significant increases in the phosphorylated endoplasmic reticular stress protein, protein kinase RNA-like endoplasmic reticulum kinase (p-PERK at Thr980), by EVO were detected in A498 cells, and the PERK inhibitor, GSK2606414, significantly suppressed EVO-induced apoptosis, p-JNK, p-PERK, and cleaved PARP proteins. The in vivo study showed that EVO significantly reduced RCC growth elicited by a subcutaneous injection of A498 cells, and an increased protein level of p-PERK was observed according to an immunohistochemical analysis. Apoptosis by EVO was also demonstrated in other RCC cells such as 786-O, ACHN, and Caki-1 cells. This is the first study to demonstrate the anti-RCC effect of EVO via apoptosis in vitro and in vivo, and activation of JNK and PERK to induce Bcl-2

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. Glycogen synthase kinase-3 facilitates con a-induced IFN-γ-- mediated immune hepatic injury.

    PubMed

    Tsai, Cheng-Chieh; Huang, Wei-Ching; Chen, Chia-Ling; Hsieh, Chia-Yuan; Lin, Yee-Shin; Chen, Shun-Hua; Yang, Kao-Chi; Lin, Chiou-Feng

    2011-10-01

    Immune hepatic injury induced by Con A results primarily from IFN-γ-mediated inflammation, followed by hepatic cell death. Glycogen synthase kinase (GSK)-3, which acts proapoptotically and is proinflammatory, is also important for facilitating IFN-γ signaling. We hypothesized a pathogenic role for GSK-3 in Con A hepatic injury. Con A stimulation caused GSK-3 activation in the livers of C57BL/6 mice. Inhibiting GSK-3 reduced Con A hepatic injury, including hepatic necrosis and apoptosis, inflammation, infiltration of T cells and granulocytes, and deregulated expression of adhesion molecule CD54. Con A induced hepatic injury in an IFN-γ receptor 1-dependent manner. Con A/IFN-γ induced activation and expression of STAT1 in a GSK-3-dependent manner. GSK-3 facilitated IFN-γ-induced inducible NO synthase, but had limited effects on CD95 upregulation and CD95-mediated hepatocyte apoptosis in vitro. Notably, inhibiting GSK-3 decreased Con A-induced IFN-γ production in both wild-type and IFN-γ receptor 1-deficient C57BL/6 mice. In Con A-activated NKT cells, GSK-3 was also activated and was required for nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, but it was not required for CD95 ligand expression or activation-induced cell death. These results demonstrate the dual and indispensable role of GSK-3 in Con A hepatic injury by facilitating IFN-γ-induced hepatopathy.

  8. 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.

  9. Glycogen synthase kinase-3 (GSK3) inhibition induces prosurvival autophagic signals in human pancreatic cancer cells.

    PubMed

    Marchand, Benoît; Arsenault, Dominique; Raymond-Fleury, Alexandre; Boisvert, François-Michel; Boucher, Marie-Josée

    2015-02-27

    Glycogen synthase kinase-3 (GSK3) are ubiquitously expressed serine-threonine kinases involved in a plethora of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK3 inhibition triggers JNK-cJUN-dependent apoptosis in human pancreatic cancer cells. However, the comprehensive picture of downstream GSK3-regulated pathways/functions remains elusive. Herein, counterbalancing the death signals, we show that GSK3 inhibition induces prosurvival signals through increased activity of the autophagy/lysosomal network. Our data also reveal a contribution of GSK3 in the regulation of the master transcriptional regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB) in pancreatic cancer cells. Similarly to mammalian target of rapamycin (mTOR) inhibition, GSK3 inhibitors promote TFEB nuclear localization and leads to TFEB dephosphorylation through endogenous serine/threonine phosphatase action. However, GSK3 and mTOR inhibition impinge differently and independently on TFEB phosphorylation suggesting that TFEB is regulated by a panel of kinases and/or phosphatases. Despite their differential impact on TFEB phosphorylation, both GSK3 and mTOR inhibitors promote 14-3-3 dissociation and TFEB nuclear localization. Quantitative mass spectrometry analyses further reveal an increased association of TFEB with nuclear proteins upon GSK3 and mTOR inhibition suggesting a positive impact on TFEB transcriptional function. Finally, a predominant nuclear localization of TFEB is unveiled in fully fed pancreatic cancer cells, whereas a reduction in TFEB expression significantly impairs their capacity for growth in an anchorage-independent manner. In addition, TFEB-restricted cells are more sensitive to apoptosis upon GSK3 inhibition. Altogether, our data uncover new functions under the control of GSK3 in pancreatic cancer cells in addition to providing key insight into TFEB regulation.

  10. Highly specific fluorescence detection of T4 polynucleotide kinase activity via photo-induced electron transfer.

    PubMed

    Tao, Mangjuan; Shi, Zhilu; Cheng, Rui; Zhang, Jing; Li, Baoxin; Jin, Yan

    2015-09-15

    Sensitive and reliable study of the activity of polynucleotide kinase (PNK) and its potential inhibitors is of great importance for biochemical interaction related to DNA phosphorylation as well as development of kinase-targeted drug discovery. To achieve facile and reliable detection of PNK activity, we report here a novel fluorescence method for PNK assay based on a combination of exonuclease cleavage reaction and photo-induced electron transfer (PIET) by using T4 PNK as a model target. The fluorescence of 3'-carboxyfluorescein-labeled DNA probe (FDNA) is effectively quenched by deoxyguanosines at the 5' end of its complementary DNA (cDNA) due to an effective PIET between deoxyguanosines and fluorophore. Whereas FDNA/cDNA hybrid is phosphorylated by PNK and then immediately cleaved by lambda exonuclease (λ exo), fluorescence is greatly restored due to the break of PIET. This homogeneous PNK activity assay does not require a complex design by taking advantage of the quenching ability of deoxyguanosines, making the proposed strategy facile and cost-effective. The activity of PNK can be sensitively detected in the range of 0.005 to 10 U mL(-1) with a detection limit of 2.1×10(-3) U mL(-1). Research on inhibition efficiency of different inhibitors demonstrated that it can be explored to evaluate inhibition capacity of inhibitors. The application for detection of PNK activity in complex matrix achieved satisfactory results. Therefore, this PIET strategy opens a promising avenue for studying T4 PNK activity as well as evaluating PNK inhibitors, which is of great importance for discovering kinase-targeted drugs. PMID:26050629

  11. Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1).

    PubMed

    Brickley, Deanna R; Mikosz, Christina A; Hagan, Christy R; Conzen, Suzanne D

    2002-11-01

    The serum and glucocorticoid-induced protein kinase gene (sgk-1) encodes a multifunctional kinase that can be phosphorylated and activated through a phosphatidylinositol 3-kinase-dependent signaling pathway. In many cell types, endogenous SGK-1 steady-state protein levels are very low but can be acutely up-regulated after glucocorticoid receptor-mediated transcriptional activation; in breast epithelial and cancer cell lines, this up-regulation is associated with promotion of cell survival. We and others have noted that ectopically introduced full-length SGK-1 is poorly expressed, although SGK-1 lacking the first 60 amino acids (delta60SGK-1) is expressed at much higher-fold protein levels than wild-type SGK-1 in both human embryonic kidney 293T and MCF10A mammary epithelial cells. In this report, we demonstrate for the first time that the low steady-state expression level of SGK-1 is due to polyubiquitination and subsequent degradation by the 26S proteasome. Deletion of the amino-terminal 60 amino acids of SGK-1 results in a mutant SGK-1 protein that is neither efficiently polyubiquitinated nor degraded by the 26S proteasome, accounting for the higher steady-state levels of the truncated protein. We also demonstrate that a subset of SGK-1 localizes to the plasma membrane and that the polyubiquitin-modified SGK-1 localizes to a membrane-associated fraction of the cell. Taken together, these data suggest that a significant fraction of SGK-1 is membrane-associated and ubiquitinated. These findings are consistent with the recently described role of SGK-1 in phosphorylating the membrane-associated protein Nedd4-2 and the integral membrane Na+/H+ exchanger isoform 3 (NHE3) and suggest a novel mechanism of regulation of SGK-1.

  12. Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes.

    PubMed

    Itoh, Yumi; Sanosaka, Masato; Fuchino, Hiroyuki; Yahara, Yasuhito; Kumagai, Ayako; Takemoto, Daisaku; Kagawa, Mai; Doi, Junko; Ohta, Miho; Tsumaki, Noriyuki; Kawahara, Nobuo; Takemori, Hiroshi

    2015-07-17

    Salt-inducible kinases (SIKs), members of the 5'-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.

  13. Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes*

    PubMed Central

    Itoh, Yumi; Sanosaka, Masato; Fuchino, Hiroyuki; Yahara, Yasuhito; Kumagai, Ayako; Takemoto, Daisaku; Kagawa, Mai; Doi, Junko; Ohta, Miho; Tsumaki, Noriyuki; Kawahara, Nobuo; Takemori, Hiroshi

    2015-01-01

    Salt-inducible kinases (SIKs), members of the 5′-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes. PMID:26048985

  14. Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

    SciTech Connect

    Faellman, M.; Stendahl, O.; Andersson, T. )

    1989-03-01

    Human neutrophils stimulated with a phorbol ester (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 minutes. In contrast, 4-{alpha}-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.

  15. 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

  16. Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration*

    PubMed Central

    Daher, João P. L.; Abdelmotilib, Hisham A.; Hu, Xianzhen; Volpicelli-Daley, Laura A.; Moehle, Mark S.; Fraser, Kyle B.; Needle, Elie; Chen, Yi; Steyn, Stefanus J.; Galatsis, Paul; Hirst, Warren D.; West, Andrew B.

    2015-01-01

    Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression. PMID:26078453

  17. 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 explanati