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Sample records for induces creb phosphorylation

  1. Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells

    PubMed Central

    Barlow, Christy A.; Kitiphongspattana, Kajorn; Siddiqui, Nazli; Roe, Michael W.; Mossman, Brooke T.

    2008-01-01

    Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H2O2) initiates an increase in Ca2+/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 (ERK1/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1/2 and CREB in C10 cells. Application of H2O2 increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H2O2 increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H2O2-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1/2 and CREB that mediates cell survival during oxidant stress. PMID:18392938

  2. Chronic morphine administration induces over-expression of aldolase C with reduction of CREB phosphorylation in the mouse hippocampus.

    PubMed

    Yang, Hai-Yu; Pu, Xiao-Ping

    2009-05-01

    In recent studies, alterations in the activity and expression of metabolic enzymes, such as those involved in glycolysis, have been detected in morphine-dependent patients and animals. Increasing evidence demonstrates that the hippocampus is an important brain region associated with morphine dependence, but the molecular events occurring in the hippocampus following chronic exposure to morphine are poorly understood. Aldolase C is the brain-specific isoform of fructose-1, 6-bisphosphate aldolase which is a glycolytic enzyme catalyzing reactions in the glycolytic, gluconeogenic, and fructose metabolic pathways. Using Western blot and immunofluorescence assays, we found the expression of aldolase C was markedly increased in the mouse hippocampus following chronic morphine treatment. Naloxone pretreatment before morphine administration suppressed withdrawal jumping, weight loss, and overexpression of aldolase C. CREB is a transcription factor regulated through phosphorylation on Ser133, which is known to play a key role in the mechanism of morphine dependence. When detecting the expression of phosphorylated CREB (p-CREB) in the mouse hippocampus using Western blot and immunohistochemistry, we found CREB phosphorylation was clearly decreased following chronic morphine treatment. Interestingly, laser-confocal microscopy showed that overexpression of aldolase C in mouse hippocampal neurons was concomitant with the decreased immunoreactivity of p-CREB. The results suggest potential links between the morphine-induced alteration of aldolase C and the regulation of CREB phosphorylation, a possible mechanism of morphine dependence.

  3. Nucleotide P2Y13-stimulated phosphorylation of CREB is required for ADP-induced proliferation of late developing retinal glial progenitors in culture.

    PubMed

    Jacques, Flavia Jesus; Silva, Thayane Martins; da Silva, Flavia Emenegilda; Ornelas, Isis Moraes; Ventura, Ana Lucia Marques

    2017-03-24

    Nucleotides stimulate phosphorylation of CREB to induce cell proliferation and survival in diverse cell types. We report here that ADP induces the phosphorylation of CREB in a time- and concentration-dependent manner in chick embryo retinal progenitors in culture. ADP-induced increase in phospho-CREB is mediated by P2 receptors as it is blocked by PPADS but not by the adenosine antagonists DPCPX or ZM241385. Incubation of the cultures with the CREB inhibitor KG-501 prevents ADP-induced incorporation of [(3)H]-thymidine, indicating that CREB is involved in retinal cell proliferation. No effect of this compound is observed on the viability of retinal progenitors. While no significant increase in CREB phosphorylation is observed with the P2Y1 receptor agonist MRS2365, ADP-induced phosphorylation of CREB is blocked by the P2Y13 receptor selective antagonist MRS2211, but not by MRS2179 or PSB0739, two antagonists of the P2Y1 and P2Y12 receptors, respectively, suggesting that ADP-induced CREB phosphorylation is mediated by P2Y13 receptors. ADP-induced increase in phospho-CREB is attenuated by the PI3K inhibitor LY241385 and completely prevented by the MEK inhibitor U0126, suggesting that at least ERK is involved in ADP-induced CREB phosphorylation. A pharmacological profile similar to the activation and inhibition of CREB phosphorylation is observed in the phosphorylation of ERK, suggesting that P2Y13 receptors mediate ADP induced ERK/CREB pathway in the cultures. While no increase in [(3)H]-thymidine incorporation is observed with the P2Y1 receptor agonist MRS2365, both MRS2179 and MRS2211 prevent ADP-mediated increase in [(3)H]-thymidine incorporation, but not progenitor's survival, suggesting that both P2Y1 and P2Y13 receptor subtypes are involved in ADP-induced cell proliferation. P2Y1 receptor-mediated increase in [Ca(2+)]i is observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca

  4. Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

    PubMed Central

    Nichols, M; Weih, F; Schmid, W; DeVack, C; Kowenz-Leutz, E; Luckow, B; Boshart, M; Schütz, G

    1992-01-01

    Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation. Images PMID:1354612

  5. GHRP-6 induces CREB phosphorylation and growth hormone secretion via a protein kinase Csigma-dependent pathway in GH3 cells.

    PubMed

    Tian, Chunlei; Ye, Fei; Xu, Tongjiang; Wang, Sheng; Wang, Xiaodan; Wang, Heping; Wan, Feng; Lei, Ting

    2010-04-01

    This study examined the effect of GHRP-6, a known GHSs receptor agonist, on the phosphorylation of cAMP-responsive element-binding protein (CREB) and the underly mechanism. GH3 cells were cultured and subjected to different treatments as follows: GHRP-6, GHRP-6 plus GHRH, phorbol ester (PMA), an activator of PKC, alone or in combination with GHRP-6, Gö6983, a general inhibitor of PKCs, in the presence or absence of GHRP-6, rottlerin, an inhibitor of PKCs, alone or plus GHRP-6. The cells were transiently transfected with PKCsigma-specific siRNA and then treated with GHRP-6. GH level was measured by enzyme-linked immunosorbent assay (ELISA). The expression of phosphor-CREB, PKCsigma, PKCtheta and phosphor-PKCsigma was determined by Western blotting. The results showed that GHRP-6 stimulated GH secretion in both time- and dose-dependent manners and enhanced the effect of GHRH on GH secretion. GHRP-6 was also found to induce CREB phosphorylation. Moreover, GH secretion was enhanced by the PKC activator PMA and reduced by the PKC inhibitors (Gö6983, rottlerin) and knockdown of PKCsigma. PKCsigma could be activated by GHRP-6. It is concluded that PKC, especially PKCsigma, mediates CREB phosphorylation and GHRP-6-induced GH secretion.

  6. CP-154,526 Modifies CREB Phosphorylation and Thioredoxin-1 Expression in the Dentate Gyrus following Morphine-Induced Conditioned Place Preference.

    PubMed

    García-Carmona, Juan-Antonio; Camejo, Daymi M; Almela, Pilar; Jiménez, Ana; Milanés, María-Victoria; Sevilla, Francisca; Laorden, María-Luisa

    2015-01-01

    Corticotropin-releasing factor (CRF) acts as neuro-regulator of the behavioral and emotional integration of environmental and endogenous stimuli associated with drug dependence. Thioredoxin-1 (Trx-1) is a functional protein controlling the redox status of several proteins, which is involved in addictive processes. In the present study, we have evaluated the role of CRF1 receptor (CRF1R) in the rewarding properties of morphine by using the conditioned place preference (CPP) paradigm. We also investigate the effects of the CRF1R antagonist, CP-154,526, on the morphine CPP-induced activation of CRF neurons, CREB phosphorylation and Trx expression in paraventricular nucleus (PVN) and dentate gyrus (DG) of the mice brain. CP-154,526 abolished the acquisition of morphine CPP and the increase of CRF/pCREB positive neurons in PVN. Moreover, this CRF1R antagonist prevented morphine-induced CRF-immunoreactive fibers in DG, as well as the increase in pCREB expression in both the PVN and DG. In addition, morphine exposure induced an increase in Trx-1 expression in DG without any alterations in PVN. We also observed that the majority of pCREB positive neurons in DG co-expressed Trx-1, suggesting that Trx-1 could activate CREB in the DG, a brain region involved in memory consolidation. Altogether, these results support the idea that CRF1R antagonist blocked Trx-1 expression and pCREB/Trx-1 co-localization, indicating a critical role of CRF, through CRF1R, in molecular changes involved in morphine associated behaviors.

  7. Serine 133 Phosphorylation Is Not Required for Hippocampal CREB-Mediated Transcription and Behavior

    ERIC Educational Resources Information Center

    Brian, Lisa A.; Lee, Bridgin G.; Lelay, John; Kaestner, Klaus H.; Blendy, Julie A.

    2015-01-01

    The cAMP response element (CRE)-binding protein, CREB, is a transcription factor whose activity in the brain is critical for long-term memory formation. Phosphorylation of Ser133 in the kinase-inducible domain (KID), that in turn leads to the recruitment of the transcriptional coactivator CREB-binding protein (CBP), is thought to mediate the…

  8. CaMKII-Mediated CREB Phosphorylation Is Involved in Ca2+-Induced BDNF mRNA Transcription and Neurite Outgrowth Promoted by Electrical Stimulation

    PubMed Central

    Ye, Zhengxu; Huang, Jinghui; He, Fei; Xiao, Wei; Hu, Xueyu; Luo, Zhuojing

    2016-01-01

    Electrical stimulation (ES)-triggered up-regulation of brain-derived neurotrophic factor (BDNF) and neurite outgrowth in cultured rat postnatal dorsal root ganglion neurons (DRGNs) is calcium (Ca2+)-dependent. The effects of increased Ca2+ on BDNF up-regulation and neurite outgrowth remain unclear. We showed here that ES increased phosphorylation of the cAMP-response element binding protein (CREB). Blockade of Ca2+ suppressed CREB phosphorylation and neurite outgrowth. Down-regulation of phosphorylated (p)-CREB reduced BDNF transcription and neurite outgrowth triggered by ES. Furthermore, blockade of calmodulin-dependent protein kinase II (CaMKII) using the inhibitors KN93 or KN62 reduced p-CREB, and specific knockdown of the CaMKIIα or CaMKIIβ subunit was sufficient to suppress p-CREB. Recombinant BDNF or hyperforin reversed the effects of Ca2+ blockade and CaMKII knockdown. Taken together, these data establish a potential signaling pathway of Ca2+-CaMKII-CREB in neuronal activation. To our knowledge, this is the first report of the mechanisms of Ca2+-dependent BDNF transcription and neurite outgrowth triggered by ES. These findings might help further investigation of complex molecular signaling networks in ES-triggered nerve regeneration in vivo. PMID:27611779

  9. Mevastatin ameliorates sphingosine 1‐phosphate‐induced COX‐2/PGE2‐dependent cell migration via FoxO1 and CREB phosphorylation and translocation

    PubMed Central

    Hsu, Chih‐Kai; Lin, Chih‐Chung; Hsiao, Li‐Der

    2015-01-01

    Background and Purpose Sphingosine 1‐phosphate (S1P), an important inflammatory mediator, has been shown to regulate COX‐2 production and promote various cellular responses such as cell migration. Mevastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMG‐CoA), effectively inhibits inflammatory responses. However, the mechanisms underlying S1P‐evoked COX‐2‐dependent cell migration, which is modulated by mevastatin in human tracheal smooth muscle cells (HTSMCs) remain unclear. Experimental Approach The expression of COX‐2 was determined by Western blotting, real time‐PCR and promoter analyses. The signalling molecules were investigated by pretreatment with respective pharmacological inhibitors or transfection with siRNAs. The interaction between COX‐2 promoter and transcription factors was determined by chromatin immunoprecipitation assay. Finally, the effect of mevastatin on HTSMC migration and leukocyte counts in BAL fluid and COX‐2 expression induced by S1P was determined by a cell migration assay, cell counting and Western blot. Key Results S1P stimulated mTOR activation through the Nox2/ROS and PI3K/Akt pathways, which can further stimulate FoxO1 phosphorylation and translocation to the cytosol. We also found that S1P induced CREB activation and translocation via an mTOR‐independent signalling pathway. Finally, we showed that pretreatment with mevastatin markedly reduced S1P‐induced cell migration and COX‐2/PGE2 production via a PPARγ‐dependent signalling pathway. Conclusions and Implications Mevastatin attenuates the S1P‐induced increased expression of COX‐2 and cell migration via the regulation of FoxO1 and CREB phosphorylation and translocation by PPARγ in HTSMCs. Mevastatin could be beneficial for prevention of airway inflammation in the future. PMID:26359950

  10. Ketamine plus imipramine treatment induces antidepressant-like behavior and increases CREB and BDNF protein levels and PKA and PKC phosphorylation in rat brain.

    PubMed

    Réus, Gislaine Z; Stringari, Roberto B; Ribeiro, Karine F; Ferraro, Ana K; Vitto, Marcelo F; Cesconetto, Patrícia; Souza, Claúdio T; Quevedo, João

    2011-08-01

    A growing body of evidence has pointed to the N-methyl-d-aspartate (NMDA) receptor antagonists as a potential therapeutic target for the treatment of major depression. The present study investigated the possibility of synergistic interactions between antidepressant imipramine with the uncompetitive NMDA receptor antagonist ketamine. Wistar rats were acutely treated with ketamine (5 and 10mg/kg) and imipramine (10 and 20mg/kg) and then subjected to forced swimming tests. The cAMP response element bindig (CREB) and brain-derived neurotrophic factor (BDNF) protein levels and protein kinase C (PKC) and protein kinase A (PKA) phosphorylation were assessed in the prefrontal cortex, hippocampus and amygdala by imunoblot. Imipramine at the dose of 10mg/kg and ketamine at the dose of 5mg/kg did not have effect on the immobility time; however, the effect of imipramine (10 and 20mg/kg) was enhanced by both doses of ketamine. Ketamine and imipramine alone or in combination at all doses tested did not modify locomotor activity. Combined treatment with ketamine and imipramine produced stronger increases of CREB and BDNF protein levels in the prefrontal cortex, hippocampus and amygdala, and PKA phosphorylation in the hippocampus and amygdala and PKC phosphorylation in prefrontal cortex. The results described indicate that co-administration of antidepressant imipramine with ketamine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone. This finding may be of particular importance in the case of drug-resistant patients and could suggest a method of obtaining significant antidepressant actions whilst limiting side effects.

  11. Impairment of Memory Consolidation by Galanin Correlates with In-Vivo Inhibition of Both LTP and CREB Phosphorylation

    PubMed Central

    Kinney, Jefferson W.; Sanchez-Alavez, Manuel; Barr, Alasdair M.; Criado, Jose R.; Crawley, Jacqueline N.; Behrens, M. Margarita; Henriksen, Steven J.; Bartfai, Tamas

    2009-01-01

    Changes in the state of CREB phosphorylation and in LTP in the hippocampus have been associated with learning and memory. Here we show that galanin, the neuropeptide released in the hippocampal formation from cholinergic and noradrenergic fibers, that has been shown to produce impairments in memory consolidation in the Morris water maze task inhibits both LTP and CREB phosphorylation in the rat hippocampus in-vivo. While there are many transmitters regulating CREB phosphorylation none has been shown to suppress behaviorally-induced hippocampal CREB phosphorylation as potently as galanin. The in-vivo inhibition of dentate gyrus-LTP and of CREB phosphorylation by the agonist occupancy of GalR1 and GALR2-type galanin receptors provides strong in-vivo cellular and molecular correlates to galanin-induced learning deficits and designates galanin as a major regulator of the memory consolidation process. PMID:19531380

  12. Proliferation of hepatic stellate cells is inhibited by phosphorylation of CREB on serine 133.

    PubMed Central

    Houglum, K; Lee, K S; Chojkier, M

    1997-01-01

    Proliferating, activated, hepatic stellate cells have a high level of collagen type I expression. Therefore, stellate cell proliferation is a critical step in hepatic fibrosis. Here we show that proliferation of activated primary rat stellate cells was blocked by elevation of cAMP with 8 Br-cAMP or isomethylbutyl xanthine, a phosphodiesterase inhibitor, and by stimulation of Ca2+ fluxes with the Ca2+ ionophore A-23187. Because phosphorylation of CREB on Ser133 is an important mediator of cAMP-protein kinase (PKA) and Ca2+-calmodulin kinase II (CAMK-II) activation, we tested whether CREB-PSer133 was essential for stellate cell quiescence. Nuclear extracts from quiescent, but not from activated, stellate cells contained CREB-PSer133. Moreover, the phosphorylation of CREB on Ser133 was stimulated in activated cells by inducing the activity of PKA or CAMK-II. In addition, coexpression of CREB and either a constitutively active PKA or a constitutively active CAMK-II inhibited the proliferation of activated stellate cells. In contrast, expression of CREB alone, PKA or CAMK-II alone, CREB-Ala 133 (which lacks the Ser133 phosphoacceptor) with PKA or CAMK-II, or CREB with inactive PKA or CAMK-II mutants did not affect stellate cell proliferation, suggesting that CREB-PSer133 is necessary for blocking the stellate cell cycle. Conversely, expression of a trans-dominant negative CREB-Ala 133 mutant (which competes with CREB/CREB-PSer133 for cognate DNA binding sites and presumably for protein interactions) induced a greater than fivefold entry into S-phase of quiescent stellate cells, compared with control cells expressing either beta-galactosidase or wt CREB, indicating that CREB-PSer133 may be indispensable for the quiescent stellate cell phenotype. This study suggests that PKA and CAMK-II play an essential role on stellate cell activation through the induction of CREB phosphorylation on Ser133, and provides potential approaches for the treatment of hepatic fibrogenesis in

  13. Involvement of Phosphorylated "Apis Mellifera" CREB in Gating a Honeybee's Behavioral Response to an External Stimulus

    ERIC Educational Resources Information Center

    Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene; Eisenhardt, Dorothea

    2016-01-01

    The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees ("Apis mellifera") we recently demonstrated a particular high…

  14. Blockade of Cannabinoid CB1 receptor attenuates the acquisition of morphine-induced conditioned place preference along with a downregulation of ERK, CREB phosphorylation, and BDNF expression in the nucleus accumbens and hippocampus.

    PubMed

    Zhang, Jianbo; Wang, Na; Chen, Bo; Wang, Yi'nan; He, Jing; Cai, Xintong; Zhang, Hongbo; Wei, Shuguang; Li, Shengbin

    2016-09-06

    Cannabinoid CB1 receptor (CB1R) is highly expressed in the mesocorticolimbic system and associated with drug craving and relapse. Clinical trials suggest that CB1R antagonists may represent new therapies for drug addiction. However, the downstream signaling of CB1R is not fully elucidated. In the present study, we investigated the relationship between CB1R and the extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF) signaling in the nucleus accumbens (NAc) and hippocampus in morphine-induced conditioned place preference (CPP), which is used to assess the morphine-induced reward memory. The protein level of CB1R, ERK, CREB, and BDNF were detected by western blotting. Additionally, a CB1R antagonist, AM251, was used to study whether blockade of CB1R altered the CPP and above-mentioned molecules. We found an increase of CB1R expression in the NAc and hippocampus of the mice following morphine CPP, but not those after repeated morphine in home cage without context exposure (NO-CPP). Both morphine CPP and NO-CPP induced an upregulation of ERK, CREB phosphorylation and BDNF expression. Furthermore, pretreatment with AM251 before morphine attenuated the CPP acquisition and CB1R expression as well as the activation of ERK-CREB-BDNF cascade. Collectively, these findings demonstrate that (1) Repeated morphine with context exposures but not merely the pharmacological effects of morphine increased CB1R expression both in the NAc and hippocampus. (2) CB1R antagonist mediated blockade of ERK-CREB-BDNF signaling activation in the NAc and hippocampus may be an important mechanism underlying the attenuation of morphine CPP.

  15. CREB phosphorylation and melatonin biosynthesis in the rat pineal gland: involvement of cyclic AMP dependent protein kinase type II.

    PubMed

    Maronde, E; Wicht, H; Taskén, K; Genieser, H G; Dehghani, F; Olcese, J; Korf, H W

    1999-10-01

    Phosphorylation of cyclic AMP response element binding protein (CREB) at amino acid serine 133 appears as an important link between the norepinephrine (NE)-induced activation of second messenger systems and the stimulation of melatonin biosynthesis. Here we investigated in the rat pineal gland: 1) the type of protein kinase that mediates CREB phosphorylation: and 2) its impact on melatonin biosynthesis. Immunochemical or immunocytochemical demonstration of serine133-phosphorylated cyclic AMP regulated element binding protein (pCREB) and radioimmunological detection of melatonin revealed that only cyclic AMP-dependent protein kinase (PKA) inhibitors suppressed NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis, whereas inhibitors of cyclic GMP-dependent protein kinase (PKG), mitogen-activated protein kinase kinase, protein kinase C, or calcium-calmodulin-dependent protein kinase (CaMK) were ineffective. Investigations with cyclic AMP-agonist pairs that selectively activate either PKA type I or II link NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis to the activation of PKA type II. Our data suggest that PKA type II plays an important role in the transcriptional control of melatonin biosynthesis in the rat pineal organ.

  16. Oxidative stress-induced CREB upregulation promotes DNA damage repair prior to neuronal cell death protection.

    PubMed

    Pregi, Nicolás; Belluscio, Laura María; Berardino, Bruno Gabriel; Castillo, Daniela Susana; Cánepa, Eduardo Tomás

    2017-01-01

    cAMP response element-binding (CREB) protein is a cellular transcription factor that mediates responses to different physiological and pathological signals. Using a model of human neuronal cells we demonstrate herein, that CREB is phosphorylated after oxidative stress induced by hydrogen peroxide. This phosphorylation is largely independent of PKA and of the canonical phosphoacceptor site at ser-133, and is accompanied by an upregulation of CREB expression at both mRNA and protein levels. In accordance with previous data, we show that CREB upregulation promotes cell survival and that its silencing results in an increment of apoptosis after oxidative stress. Interestingly, we also found that CREB promotes DNA repair after treatment with hydrogen peroxide. Using a cDNA microarray we found that CREB is responsible for the regulation of many genes involved in DNA repair and cell survival after oxidative injury. In summary, the neuroprotective effect mediated by CREB appears to follow three essential steps following oxidative injury. First, the upregulation of CREB expression that allows sufficient level of activated and phosphorylated protein is the primordial event that promotes the induction of genes of the DNA Damage Response. Then and when the DNA repair is effective, CREB induces detoxification and survival genes. This kinetics seems to be important to completely resolve oxidative-induced neuronal damages.

  17. Effect of delta9-tetrahydrocannabinol on phosphorylated CREB in rat cerebellum: an immunohistochemical study.

    PubMed

    Casu, Maria Antonietta; Pisu, Carla; Sanna, Angela; Tambaro, Simone; Spada, Gabriele Pinna; Mongeau, Raymond; Pani, Luca

    2005-06-28

    Several converging lines of evidence indicate that drugs of abuse may exert their long-term effects on the central nervous system by modulating signaling pathways controlling gene expression. Cannabinoids produce, beside locomotor effects, cognitive impairment through central CB1 cannabinoid receptors. Data clearly indicate that the cerebellum, an area enriched with CB1 receptors, has a role not only in motor function but also in cognition. This immunohistochemical study examines the effect of delta9-tetrahydrocannabinol (delta9-THC), the principal psychoactive component of marijuana, on the levels of phosphorylated CREB (p-CREB) in the rat cerebellum. Acute treatments with delta9-THC at doses of 5 or 10 mg/kg induced a significant increase of p-CREB in the granule cell layer of the cerebellum, an effect blocked by the CB1 receptor antagonist SR 141716A. Following chronic delta9-THC administration (10 mg/kg/day for 4 weeks), the density of p-CREB was markedly attenuated compared to controls, and this attenuation persisted 3 weeks after withdrawal from delta9-THC. These data provide evidence for the involvement of cerebellar granule cells in the adaptive changes occurring during acute and chronic delta9-THC exposure. This might be a mechanism by which delta9-THC interferes with motor and cognitive functions.

  18. Selective phosphorylation of nuclear CREB by fluoxetine is linked to activation of CaM kinase IV and MAP kinase cascades.

    PubMed

    Tiraboschi, Ettore; Tardito, Daniela; Kasahara, Jiro; Moraschi, Stefania; Pruneri, Paolo; Gennarelli, Massimo; Racagni, Giorgio; Popoli, Maurizio

    2004-10-01

    Regulation of gene expression is purported as a major component in the long-term action of antidepressants. The transcription factor cAMP-response element-binding protein (CREB) is activated by chronic antidepressant treatments, although a number of studies reported different effects on CREB, depending on drug types used and brain areas investigated. Furthermore, little is known as to what signaling cascades are responsible for CREB activation, although cAMP-protein kinase A (PKA) cascade was suggested to be a central player. We investigated how different drugs (fluoxetine (FLX), desipramine (DMI), reboxetine (RBX)) affect CREB expression and phosphorylation of Ser(133) in the hippocampus and prefrontal/frontal cortex (PFCX). Acute treatments did not induce changes in these mechanisms. Chronic FLX increased nuclear phospho-CREB (pCREB) far more markedly than pronoradrenergic drugs, particularly in PFCX. We investigated the function of the main signaling cascades that were shown to phosphorylate and regulate CREB. PKA did not seem to account for the selective increase of pCREB induced by FLX. All drug treatments markedly increased the enzymatic activity of nuclear Ca2+/calmodulin (CaM) kinase IV (CaMKIV), a major neuronal CREB kinase, in PFCX. Activation of this kinase was due to increased phosphorylation of the activatory residue Thr196, with no major changes in the expression levels of alpha- and beta-CaM kinase kinase, enzymes that phosphorylate CaMKIV. Again in PFCX, FLX selectively increased the expression level of MAP kinases Erk1/2, without affecting their phosphorylation. Our results show that FLX exerts a more marked effect on CREB phosphorylation and suggest that CaMKIV and MAP kinase cascades are involved in this effect.

  19. Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain

    PubMed Central

    Böer, Ulrike; Eglins, Julia; Krause, Doris; Schnell, Susanne; Schöfl, Christof; Knepel, Willhart

    2007-01-01

    The molecular mechanism of the action of lithium salts in the treatment of bipolar disorder is not well understood. As their therapeutic action requires chronic treatment, adaptive neuronal processes are suggested to be involved. The molecular basis of this are changes in gene expression regulated by transcription factors such as CREB (cAMP-response-element-binding protein). CREB contains a transactivation domain, in which Ser119 is phosphorylated upon activation, and a bZip (basic leucine zipper domain). The bZip is involved in CREB dimerization and DNA-binding, but also contributes to CREB transactivation by recruiting the coactivator TORC (transducer of regulated CREB). In the present study, the effect of lithium on CRE (cAMP response element)/CREB-directed gene transcription was investigated. Electrically excitable cells were transfected with CRE/CREB-driven luciferase reporter genes. LiCl (6 mM or higher) induced an up to 4.7-fold increase in 8-bromo-cAMP-stimulated CRE/CREB-directed transcription. This increase was not due to enhanced Ser119 phosphorylation or DNA-binding of CREB. Also, the known targets inositol monophosphatase and GSK3β (glycogen-synthase-kinase 3β) were not involved as specific GSK3β inhibitors and inositol replenishment did not mimic and abolish respectively the effect of lithium. However, lithium no longer enhanced CREB activity when the CREB-bZip was deleted or the TORC-binding site inside the CREB-bZip was specifically mutated (CREB-R300A). Otherwise, TORC overexpression conferred lithium responsiveness on CREB-bZip or the CRE-containing truncated rat somatostatin promoter. This indicates that lithium enhances cAMP-induced CRE/CREB-directed transcription, conferred by TORC on the CREB-bZip. We thus support the hypothesis that lithium salts modulate CRE/CREB-dependent gene transcription and suggest the CREB coactivator TORC as a new molecular target of lithium. PMID:17696880

  20. Phosphorylation of CREB and mechanical hyperalgesia is reversed by blockade of the cAMP pathway in a time-dependent manner after repeated intramuscular acid injections.

    PubMed

    Hoeger-Bement, Marie K; Sluka, Kathleen A

    2003-07-02

    Spinal activation of the cAMP pathway produces mechanical hyperalgesia, sensitizes nociceptive spinal neurons, and phosphorylates the transcription factor cAMP-responsive element binding protein (CREB), which initiates gene transcription. This study examined the role of the cAMP pathway in a model of chronic muscle pain by assessing associated behavioral changes and phosphorylation of CREB. Bilateral mechanical hyperalgesia of the paw was induced by administering two injections of acidic saline, 5 d apart, into the gastrocnemius muscle of male Sprague Dawley rats. Interestingly, the increases in immunoreactivity for CREB and phosphorylated CREB (p-CREB) in the spinal dorsal horn occur 24 hr, but not 1 week, after the second injection of acidic saline compared with pH 7.2 intramuscular injections. Spinal blockade of adenylate cyclase prevents the expected increase in p-CREB that occurs after intramuscular acid injection. The reversal of mechanical hyperalgesia by adenylate cyclase or protein kinase A inhibitors spinally follows a similar pattern with reversal at 24 hr, but not 1 week, compared with the vehicle controls. The p-CREB immunoreactivity in the superficial dorsal horn correlates with the mechanical withdrawal threshold such that increases in p-CREB are associated with decreases in threshold. Therefore, activation of the cAMP pathway in the spinal cord phosphorylates CREB and produces mechanical hyperalgesia associated with intramuscular acid injections. The mechanical hyperalgesia and phosphorylation of CREB depend on early activation of the cAMP pathway during the first 24 hr but are independent of the cAMP pathway by 1 week after intramuscular injection of acid.

  1. Enhanced CREB phosphorylation in immature dentate gyrus granule cells precedes neurotrophin expression and indicates a specific role of CREB in granule cell differentiation

    PubMed Central

    Bender, R. A.; Lauterborn, J. C.; Gall, C. M.; Cariaga, W.; Baram, T. Z.

    2011-01-01

    Differentiation and maturation of dentate gyrus granule cells requires coordinated interactions of numerous processes. These must be regulated by protein factors capable of integrating signals mediated through diverse signalling pathways. Such integrators of inter and intracellular physiological stimuli include the cAMP-response element binding protein (CREB), a leucine-zipper class transcription factor that is activated through phosphorylation. Neuronal activity and neurotrophic factors, known to be involved in granule cell differentiation, are major physiologic regulators of CREB function. To examine whether CREB may play a role in governing coordinated gene transcription during granule cell differentiation, we determined the spatial and temporal profiles of phosphorylated (activated) CREB throughout postnatal development in immature rat hippocampus. We demonstrate that CREB activation is confined to discrete, early stages of granule cell differentiation. In addition, CREB phosphorylation occurs prior to expression of the neurotrophins BDNF and NT-3. These data indicate that in a signal transduction cascade connecting CREB and neurotrophins in the process of granule cell maturation, CREB is located upstream of neurotrophins. Importantly, CREB may be a critical component of the machinery regulating the coordinated transcription of genes contributing to the differentiation of granule cells and their integration into the dentate gyrus network. PMID:11207803

  2. Cadmium up-regulates transcription of the steroidogenic acute regulatory protein (StAR) gene through phosphorylated CREB rather than SF-1 in K28 cells.

    PubMed

    Park, Soo-Yun; Gomes, Cynthia; Oh, Sung-Dug; Soh, Jaemog

    2015-04-01

    Cadmium is a widely used heavy metal in industry and affects the male reproductive system of animals, including humans, as a result of occupational and environmental exposures. However, the molecular mechanism underlying its effect on steroidogenesis in gonads remains unclear. In this study, we demonstrated that exposure of K28 mouse testicular Leydig tumor cells to cadmium led to a significant increase in the mRNA level, promoter activity and protein level of the steroidogenic acute regulatory protein (StAR), an essential factor for steroid biosynthesis. It has been well documented that StAR gene transcription is regulated by multiple transcription factors, including cAMP-responsive element binding protein (CREB) family members and SF-1. Cadmium treatment caused an increase in CREB phosphorylation but did not alter the CREB protein level in the nucleus. EMSA studies revealed that cadmium-induced phosphorylated CREB formed specific complexes with the proximal region of the StAR gene promoter. Furthermore, co-transfection with a CREB expression plasmid significantly increased cadmium-induced StAR promoter activity. However, the nuclear level and the affinity of SF-1 protein for the StAR proximal promoter were dramatically decreased upon exposure to cadmium. Taken together, these results suggest that cadmium up-regulates StAR gene expression through phosphorylated CREB rather than through SF-1 in mouse testicular Leydig cells.

  3. Abundance of phosphorylated Apis mellifera CREB in the honeybee's mushroom body inner compact cells varies with age.

    PubMed

    Gehring, Katrin B; Heufelder, Karin; Kersting, Isabella; Eisenhardt, Dorothea

    2016-04-15

    Hymenopteran eusociality has been proposed to be associated with the activity of the transcription factor CREB (cAMP-response element binding protein). The honeybee (Apis mellifera) is a eusocial insect displaying a pronounced age-dependent division of labor. In honeybee brains, CREB-dependent genes are regulated in an age-dependent manner, indicating that there might be a role for neuronal honeybee CREB (Apis mellifera CREB, or AmCREB) in the bee's division of labor. In this study, we further explore this hypothesis by asking where in the honeybee brain AmCREB-dependent processes might take place and whether they vary with age in these brain regions. CREB is activated following phosphorylation at a conserved serine residue. An increase of phosphorylated CREB is therefore regarded as an indicator of CREB-dependent transcriptional activation. Thus, we here examine the localization of phosphorylated AmCREB (pAmCREB) in the brain and its age-dependent variability. We report prominent pAmCREB staining in a subpopulation of intrinsic neurons of the mushroom bodies. In these neurons, the inner compact cells (IC), pAmCREB is located in the nuclei, axons, and dendrites. In the central bee brain, the IC somata and their dendritic region, we observed an age-dependent increase of pAmCREB. Our results demonstrate the IC to be candidate neurons involved in age-dependent division of labor. We hypothesize that the IC display a high level of CREB-dependent transcription that might be related to neuronal and behavioral plasticity underlying a bee's foraging behavior.

  4. Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

    PubMed Central

    Barbosa, Sónia; Carreira, Suzanne; Bailey, Daniel; Abaitua, Fernando; O'Hare, Peter

    2015-01-01

    CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1a E3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV. PMID:26108621

  5. Activation of NR2A receptors induces ischemic tolerance through CREB signaling.

    PubMed

    Terasaki, Yasukazu; Sasaki, Tsutomu; Yagita, Yoshiki; Okazaki, Shuhei; Sugiyama, Yukio; Oyama, Naoki; Omura-Matsuoka, Emi; Sakoda, Saburo; Kitagawa, Kazuo

    2010-08-01

    Previous exposure to a nonlethal ischemic insult protects the brain against subsequent harmful ischemia. N-methyl-D-aspartate (NMDA) receptors are a highly studied target of neuroprotection after ischemia. Recently, NMDA receptor subtypes were implicated in neuronal survival and death. We focused on the contribution of NR2A and cyclic-AMP response element (CRE)-binding protein (CREB) signaling to ischemic tolerance using primary cortical neurons. Ischemia in vitro was modeled by oxygen-glucose deprivation (OGD). Ischemic tolerance was induced by applying 45-mins OGD 24 h before 180-mins OGD. Sublethal OGD also induced cross-tolerance against lethal glutamate and hydrogen peroxide. After sublethal OGD, expression of phosphorylated CREB and CRE transcriptional activity were significantly increased. When CRE activity was inhibited by CREB-S133A, a mutant CREB, ischemic tolerance was abolished. Inhibiting NR2A using NVP-AAM077 attenuated preconditioning-induced neuroprotection and correlated with decreased CRE activity levels. Activating NR2A using bicuculline and 4-aminopiridine induced resistance to lethal ischemia accompanied by elevated CRE activity levels, and this effect was abolished by NVP-AAM077. Elevated brain-derived neurotrophic factor (BDNF) transcriptional activities were observed after sublethal OGD and administration of bicuculline and 4-aminopiridine. NR2A-containing NMDA receptors and CREB signaling have important functions in the induction of ischemic tolerance. This may provide potential novel therapeutic strategies to treat ischemic stroke.

  6. CREB-Induced Inflammation Is Important for Malignant Mesothelioma Growth

    PubMed Central

    Westbom, Catherine M.; Shukla, Anurag; MacPherson, Maximilian B.; Yasewicz, Elizabeth C.; Miller, Jill M.; Beuschel, Stacie L.; Steele, Chad; Pass, Harvey I.; Vacek, Pamela M.; Shukla, Arti

    2015-01-01

    Malignant mesothelioma (MM) is an aggressive tumor with no treatment regimen. Previously we have demonstrated that cyclic AMP response element binding protein (CREB) is constitutively activated in MM tumor cells and tissues and plays an important role in MM pathogenesis. To understand the role of CREB in MM tumor growth, we generated CREB-inhibited MM cell lines and performed in vitro and in vivo experiments. In vitro experiments demonstrated that CREB inhibition results in significant attenuation of proliferation and drug resistance of MM cells. CREB-silenced MM cells were then injected into severe combined immunodeficiency mice, and tumor growth in s.c. and i.p. models of MM was followed. We observed significant inhibition in MM tumor growth in both s.c. and i.p. models and the presence of a chemotherapeutic drug, doxorubicin, further inhibited MM tumor growth in the i.p. model. Peritoneal lavage fluids from CREB-inhibited tumor-bearing mice showed a significantly reduced total cell number, differential cell counts, and pro-inflammatory cytokines and chemokines (IL-6, IL-8, regulated on activation normal T cell expressed and secreted, monocyte chemotactic protein-1, and vascular endothelial growth factor). In vitro studies showed that asbestos-induced inflammasome/inflammation activation in mesothelial cells was CREB dependent, further supporting the role of CREB in inflammation-induced MM pathogenesis. In conclusion, our data demonstrate the involvement of CREB in the regulation of MM pathogenesis by regulation of inflammation. PMID:25111229

  7. CREB phosphorylation regulates striatal transcriptional responses in the self-administration model of methamphetamine addiction in the rat.

    PubMed

    Krasnova, Irina N; Chiflikyan, Margarit; Justinova, Zuzana; McCoy, Michael T; Ladenheim, Bruce; Jayanthi, Subramaniam; Quintero, Cynthia; Brannock, Christie; Barnes, Chanel; Adair, Jordan E; Lehrmann, Elin; Kobeissy, Firas H; Gold, Mark S; Becker, Kevin G; Goldberg, Steven R; Cadet, Jean Lud

    2013-10-01

    Neuroplastic changes in the dorsal striatum participate in the transition from casual to habitual drug use and might play a critical role in the development of methamphetamine (METH) addiction. We examined the influence of METH self-administration on gene and protein expression that may form substrates for METH-induced neuronal plasticity in the dorsal striatum. Male Sprague-Dawley rats self-administered METH (0.1mg/kg/injection, i.v.) or received yoked saline infusions during eight 15-h sessions and were euthanized 2h, 24h, or 1month after cessation of METH exposure. Changes in gene and protein expression were assessed using microarray analysis, RT-PCR and Western blots. Chromatin immunoprecipitation (ChIP) followed by PCR was used to examine epigenetic regulation of METH-induced transcription. METH self-administration caused increases in mRNA expression of the transcription factors, c-fos and fosb, the neurotrophic factor, Bdnf, and the synaptic protein, synaptophysin (Syp) in the dorsal striatum. METH also caused changes in ΔFosB, BDNF and TrkB protein levels, with increases after 2 and 24h, but decreases after 1month of drug abstinence. Importantly, ChIP-PCR showed that METH self-administration caused enrichment of phosphorylated CREB (pCREB), but not of histone H3 trimethylated at lysine 4 (H3K4me3), on promoters of c-fos, fosb, Bdnf and Syp at 2h after cessation of drug intake. These findings show that METH-induced changes in gene expression are mediated, in part, by pCREB-dependent epigenetic phenomena. Thus, METH self-administration might trigger epigenetic changes that mediate alterations in expression of genes and proteins serving as substrates for addiction-related synaptic plasticity.

  8. DPPC regulates COX-2 expression in monocytes via phosphorylation of CREB

    SciTech Connect

    Morris, R.H.K. Tonks, A.J.; Jones, K.P.; Ahluwalia, M.K.; Thomas, A.W.; Tonks, A.; Jackson, S.K.

    2008-05-23

    The major phospholipid in pulmonary surfactant dipalmitoyl phosphatidylcholine (DPPC) has been shown to modulate inflammatory responses. Using human monocytes, this study demonstrates that DPPC significantly increased PGE{sub 2} (P < 0.05) production by 2.5-fold when compared to untreated monocyte controls. Mechanistically, this effect was concomitant with an increase in COX-2 expression which was abrogated in the presence of a COX-2 inhibitor. The regulation of COX-2 expression was independent of NF-{kappa}B activity. Further, DPPC increased the phosphorylation of the cyclic AMP response element binding protein (CREB; an important nuclear transcription factor important in regulating COX-2 expression). In addition, we also show that changing the fatty acid groups of PC (e.g. using L-{alpha}-phosphatidylcholine {beta}-arachidonoyl-{gamma}-palmitoyl (PAPC)) has a profound effect on the regulation of COX-2 expression and CREB activation. This study provides new evidence for the anti-inflammatory activity of DPPC and that this activity is at least in part mediated via CREB activation of COX-2.

  9. CYCLIC AMP-DEPENDENT PROTEIN KINASE INDUCTION BY POLYCHLORINATED BIPHENYLS (PCBS) STIMULATES CREB PHOSPHORYLATION VIA A CALCIUM-DEPENDENT, PKC-INDEPENDENT PATHWAY IN CORTICAL NEURONS.

    EPA Science Inventory

    We have previously demonstrated that the PCB mixture, Aroclor 1254 (A1254), increases the phosphorylated form of CREB (pCREB), the cAMP-responsive element binding protein. This transcription factor is important in nervous system development and plasticity. Phosphorylation
    of C...

  10. Changes in CREB activation in the prefrontal cortex and hippocampus blunt ethanol-induced behavioral sensitization in adolescent mice

    PubMed Central

    Soares-Simi, Sabrina L.; Pastrello, Daniel M.; Ferreira, Zulma S.; Yonamine, Mauricio; Marcourakis, Tania; Scavone, Cristoforo; Camarini, Rosana

    2013-01-01

    Drug dependence is a major health problem in adults and has been recognized as a significant problem in adolescents. We previously demonstrated that repeated treatment with a behaviorally sensitizing dose of ethanol in adult mice induced tolerance or no sensitization in adolescents and that repeated ethanol-treated adolescents expressed lower Fos and Egr-1 expression than adult mice in the prefrontal cortex (PFC). In the present work, we investigated the effects of acute and repeated ethanol administration on cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) DNA-binding activity using the electrophoretic mobility shift assay (EMSA) and the phosphorylated CREB (pCREB)/CREB ratio using immunoblotting in both the PFC and hippocampus in adolescent and adult mice. Adult mice exhibited typical locomotor sensitization after 15 days of daily treatment with 2.0 g/kg ethanol, whereas adolescent mice did not exhibit sensitization. Overall, adolescent mice displayed lower CREB binding activity in the PFC compared with adult mice, whereas opposite effects were observed in the hippocampus. The present results indicate that ethanol exposure induces significant and differential neuroadaptive changes in CREB DNA-binding activity in the PFC and hippocampus in adolescent mice compared with adult mice. These differential molecular changes may contribute to the blunted ethanol-induced behavioral sensitization observed in adolescent mice. PMID:24379765

  11. CREB Selectively Controls Learning-Induced Structural Remodeling of Neurons

    ERIC Educational Resources Information Center

    Middei, Silvia; Spalloni, Alida; Longone, Patrizia; Pittenger, Christopher; O'Mara, Shane M.; Marie, Helene; Ammassari-Teule, Martine

    2012-01-01

    The modulation of synaptic strength associated with learning is post-synaptically regulated by changes in density and shape of dendritic spines. The transcription factor CREB (cAMP response element binding protein) is required for memory formation and in vitro dendritic spine rearrangements, but its role in learning-induced remodeling of neurons…

  12. GSK-3 mediated phosphorylation couples ER-Golgi transport and nuclear stabilisation of the CREB-H transcription factor to mediate Apolipoprotein secretion.

    PubMed

    Barbosa, Sónia; Carreira, Suzanne; O'Hare, Peter

    2017-04-05

    CREB-H, an ER-anchored transcription factor plays a key role in regulating secretion in metabolic pathways, particularly triglyceride homeostasis. It controls the production both of secretory pathway components and cargoes including apolipoproteins ApoA-IV and ApoC-II, contributing to VLDL/HDL distribution and lipolysis. The key mechanism controlling CREB-H activity involves its ER retention and forward transport to the Golgi, where it is cleaved by Golgi-resident proteases releasing the N-terminal product which traffics to the nucleus to effect transcriptional responses. Here we show that a serine-rich motif, termed the P-motif located in the N-terminus between serines 73 to 90, controls release of the precursor transmembrane form from the ER and its forward transport to the Golgi. This motif is subject to GSK-3 phosphorylation promoting ER-retention while mutation of target serines or drug inhibition of GSK-3 activity, co-ordinately induces both forward transport of the precursor and cleavage, resulting in nuclear import. We previously showed that for the nuclear product, the P-motif is subject to multiple phosphorylations which regulate stability by targeting the protein to the SCF(Fbw1a) E3 ubiquitin ligase. Thus phosphorylation at the P-motif provides integrated control of CREB-H function, coupling intercompartmental transport in the cytoplasm with stabilisation of the active form in the nucleus.

  13. Song-induced phosphorylation of cAMP response element-binding protein in the songbird brain.

    PubMed

    Sakaguchi, H; Wada, K; Maekawa, M; Watsuji, T; Hagiwara, M

    1999-05-15

    We have investigated the participation of cAMP response element-binding protein (CREB) in the response of the songbird brain to a natural auditory stimulus, a conspecific song. The cells in the two song control nuclei, the higher vocal center (HVC) and area X of zebra finches (Taeniopygia guttata), were intensely stained with an anti-CREB monoclonal antibody. Double-labeling studies showed that CREB immunoreactivity was detected only in area X-projecting neurons in the HVC. The cloned CREB cDNA from zebra finches (zCREB) is highly homologous to mammalian delta CREB. Phosphorylation of zCREB at Ser119 in area X-projecting HVC neurons was induced by hearing tape-recorded conspecific songs of zebra finches, but not by birdsongs of another species or white noise. These results raise the possibility that zCREB plays a crucial role in the sensory process of song learning.

  14. CREB Phosphorylation Coincides with Transient Synapse Formation in the Rat Hippocampal Dentate Gyrus Following Avoidance Learning

    PubMed Central

    O'Connell, Cormac; Gallagher, Helen C.; O'Malley, Aoibheinn; Bourke, Mary; Regan, Ciaran M.

    2000-01-01

    Spine density change in the hippocampal dentate gyrus accompanies memory consolidation and coincides with the increased expression of ribosome-rich, hyperchromatic granule cells. Although this suggests increased protein synthesis to be required for synaptic growth in the 5 to 7 h post-training period, little temporal mapping of the associated molecular mechanisms has been done. Here, we demonstrate a similar frequency of hyperchromatic cells in naïve animals and in those sacrificed 6 h post-training, suggesting a transient repression of protein synthesis in the early post-training period. Immunoblot analysis of CREB phosphorylation in the dentate gyrus supported this view, with downregulation from basal levels observed at 2 to 3 h and at 12 h posttraining. Protein synthesis reactivation appears to be specific for de novo spine production as no change in spine frequency accompanies the immediate post-training period of depressed protein synthesis. These findings support the view that CREB-mediated gene transcription is a requirement for long-term memory consolidation and may be directly implicated in the process of synaptic growth. PMID:11486487

  15. A high-fat diet or galanin in the PVN decreases phosphorylation of CREB in the nucleus accumbens

    PubMed Central

    Bocarsly, Miriam E.; Avena, Nicole M.

    2013-01-01

    A high-fat diet (HFD) can increase hypothalamic galanin (GAL). GAL has recently been shown to inhibit opiate reward, which in turn, decreases cAMP response element-binding protein (CREB) in the nucleus accumbens (NAc). We hypothesized that injection of GAL into the PVN, or consumption of a HFD, would be associated with a decrease in NAc CREB. In Exp. 1, GAL in the paraventricular nucleus (PVN) of naïve rats decreased phosphorylated-CREB (pCREB) in the NAc compared to saline injected controls. In Exp. 2, rats fed ad libitum HFD for 4 wks had reduced NAc pCREB levels compared to rats with sporadic tastes of the HFD. Body weight, serum triglyceride and leptin levels were also raised in the chronic HFD-fed rats. These data suggest that PVN GAL or chronic intake of a HFD can decrease NAc pCREB. The implications of these findings may help to explain the lack of opiate-like withdrawal that has been reported in response to overeating a high fat diet, thereby providing a potential mechanism underlying behavioral differences seen with addiction-like overconsumption of different types of palatable foods. PMID:23747305

  16. YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

    SciTech Connect

    Hwang, Tsong-Long; Tang, Ming-Chi; Kuo, Liang-Mou; Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching

    2012-04-15

    Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation.

  17. Dorsal hippocampal NMDA receptor blockade impairs extinction of naloxone-precipitated conditioned place aversion in acute morphine-treated rats by suppressing ERK and CREB phosphorylation in the basolateral amygdala

    PubMed Central

    Wang, Wei-Sheng; Chen, Zhong-Guo; Liu, Wen-Tao; Chi, Zhi-Qiang; He, Ling; Liu, Jing-Gen

    2015-01-01

    BACKGROUND AND PURPOSE Substantial evidence shows that negative reinforcement resulting from the aversive affective consequences of opiate withdrawal may play a crucial role in drug relapse. Understanding the mechanisms underlying the loss (extinction) of conditioned aversion of drug withdrawal could facilitate the treatment of drug addiction. EXPERIMENTAL APPROACH Naloxone-induced conditioned place aversion (CPA) of Sprague-Dawley rats was used to measure conditioned aversion. An NMDA receptor antagonist and MAPK kinase inhibitor were applied through intracranial injections. The phosphorylation of ERK and cAMP response element-binding protein (CREB) was detected using Western blot. KEY RESULTS The extinction of CPA behaviour increased the phosphorylation of ERK and CREB in the dorsal hippocampus (DH) and basolateral amygdala (BLA), but not in the central amygdala (CeA). Intra-DH injection of AP5 or intra-BLA injection of AP-5 or U0126 before extinction training significantly attenuated ERK and CREB phosphorylation in the BLA and impaired the extinction of CPA behaviour. Although intra-DH injections of AP-5 attenuated extinction training-induced activation of the ERK-CREB pathway in the BLA, intra-BLA injection of AP5 had no effect on extinction training-induced activation of the ERK-CREB pathway in the DH. CONCLUSIONS AND IMPLICATIONS These results suggest that activation of ERK and CREB in the BLA and DH is involved in the extinction of CPA behaviour and that the DH, via a direct or indirect pathway, modulates the activity of ERK and CREB in the BLA through activation of NMDA receptors after extinction training. Understanding the mechanisms underlying the extinction of conditioned aversion could facilitate the treatment of drug addiction. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24597568

  18. Exercise Prevents Memory Impairment Induced by Arsenic Exposure in Mice: Implication of Hippocampal BDNF and CREB.

    PubMed

    Sun, Bao-Fei; Wang, Qing-Qing; Yu, Zi-Jiang; Yu, Yan; Xiao, Chao-Lun; Kang, Chao-Sheng; Ge, Guo; Linghu, Yan; Zhu, Jun-De; Li, Yu-Mei; Li, Qiang-Ming; Luo, Shi-Peng; Yang, Dang; Li, Lin; Zhang, Wen-Yan; Tian, Guang

    2015-01-01

    High concentrations of arsenic, which can be occasionally found in drinking water, have been recognized as a global health problem. Exposure to arsenic can disrupt spatial memory; however, the underlying mechanism remains unclear. In the present study, we tested whether exercise could interfere with the effect of arsenic exposure on the long-term memory (LTM) of object recognition in mice. Arsenic (0, 1, 3, and 10 mg/ kg, i.g.) was administered daily for 12 weeks. We found that arsenic at dosages of 1, 3, and 10 mg/kg decreased body weight and increased the arsenic content in the brain. The object recognition LTM (tested 24 h after training) was disrupted by 3 mg/ kg and 10 mg/ kg, but not 1 mg/ kg arsenic exposure. Swimming exercise also prevented LTM impairment induced by 3 mg/ kg, but not with 10 mg/ kg, of arsenic exposure. The expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP-response element binding protein (pCREB) in the CA1 and dentate gyrus areas (DG) of the dorsal hippocampus were decreased by 3 mg/ kg and 10 mg/ kg, but not by 1 mg/ kg, of arsenic exposure. The decrease in BDNF and pCREB in the CA1 and DG induced by 3 mg/ kg, but not 10 mg/ kg, of arsenic exposure were prevented by swimming exercise. Arsenic exposure did not affect the total CREB expression in the CA1 or DG. Taken together, these results indicated that swimming exercise prevented the impairment of object recognition LTM induced by arsenic exposure, which may be mediated by BDNF and CREB in the dorsal hippocampus.

  19. Exercise Prevents Memory Impairment Induced by Arsenic Exposure in Mice: Implication of Hippocampal BDNF and CREB

    PubMed Central

    Yu, Zi-Jiang; Yu, Yan; Xiao, Chao-Lun; Kang, Chao-Sheng; Ge, Guo; Linghu, Yan; Zhu, Jun-De; Li, Yu-Mei; Li, Qiang-Ming; Luo, Shi-Peng; Yang, Dang; Li, Lin; Zhang, Wen-Yan; Tian, Guang

    2015-01-01

    High concentrations of arsenic, which can be occasionally found in drinking water, have been recognized as a global health problem. Exposure to arsenic can disrupt spatial memory; however, the underlying mechanism remains unclear. In the present study, we tested whether exercise could interfere with the effect of arsenic exposure on the long-term memory (LTM) of object recognition in mice. Arsenic (0, 1, 3, and 10 mg/ kg, i.g.) was administered daily for 12 weeks. We found that arsenic at dosages of 1, 3, and 10 mg/kg decreased body weight and increased the arsenic content in the brain. The object recognition LTM (tested 24 h after training) was disrupted by 3 mg/ kg and 10 mg/ kg, but not 1 mg/ kg arsenic exposure. Swimming exercise also prevented LTM impairment induced by 3 mg/ kg, but not with 10 mg/ kg, of arsenic exposure. The expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP-response element binding protein (pCREB) in the CA1 and dentate gyrus areas (DG) of the dorsal hippocampus were decreased by 3 mg/ kg and 10 mg/ kg, but not by 1 mg/ kg, of arsenic exposure. The decrease in BDNF and pCREB in the CA1 and DG induced by 3 mg/ kg, but not 10 mg/ kg, of arsenic exposure were prevented by swimming exercise. Arsenic exposure did not affect the total CREB expression in the CA1 or DG. Taken together, these results indicated that swimming exercise prevented the impairment of object recognition LTM induced by arsenic exposure, which may be mediated by BDNF and CREB in the dorsal hippocampus. PMID:26368803

  20. Running exercise-induced up-regulation of hippocampal brain-derived neurotrophic factor is CREB-dependent

    PubMed Central

    Chen, Michael J.; Russo-Neustadt, Amelia A.

    2009-01-01

    The past decade has witnessed burgeoning evidence that antidepressant medications and physical exercise increase the expression of hippocampal brain-derived neurotrophic factor (BDNF). This phenomenon has gained widespread appeal because BDNF is one of the first macromolecules observed to play a central role not only in the treatment of mood disorders, but also in neuronal survival-, growth-, and plasticity-related signaling cascades. Thus, it has become critical to understand how BDNF synthesis is regulated. Much evidence exists that changes in BDNF expression result from the activation/phosphorylation of the transcription factor, cAMP-response-element binding protein (CREB) following the administration of antidepressant medications. Utilizing a mouse model genetically engineered with an inducible CREB repressor, our current study provides evidence that increases in BDNF expression and cellular survival signaling resulting from physical exercise are also dependent upon activation of this central transcription factor. The transcription and expression of hippocampal BDNF, as well as the activation of Akt, a key survival signaling molecule, were measured following acute exercise, and also following short-term treatment with the norepinephrine re-uptake inhibitor, reboxetine. We found that both interventions led to a marked increase in hippocampal BDNF mRNA, BDNF protein and Akt phosphorylation (as well as CREB phosphorylation) in wild-type mice. As expected, activation of the CREB repressor in mutant mice sharply decreased CREB phosphorylation. In addition, all measures noted above remained at baseline levels when mutant mice exercised or received reboxetine. Increases in BDNF and phospho-Akt were also prevented when mutant mice received a combination of exercise and antidepressant treatment. The results are discussed in the context of what is currently known about BDNF signaling. PMID:19294650

  1. EPINEPHRINE AND GLUCOSE MODULATE TRAINING-RELATED CREB PHOSPHORYLATION IN OLD RATS: RELATIONSHIPS TO AGE-RELATED MEMORY IMPAIRMENTS

    PubMed Central

    Morris, Ken A.; Gold, Paul E.

    2012-01-01

    Epinephrine enhances memory in young adult rats, in part, by increasing blood glucose levels needed to modulate memory. In old rats, epinephrine is deficient at raising blood glucose levels and thus is only moderately effective at enhancing memory. In contrast, systemic glucose injections improve memory in old rats, with resulting memory performance equal to that of young rats. The diminished response of glucose to training in old rats may blunt downstream neurochemical and molecular mechanisms needed to upregulate memory processes. In the first experiment, young adult and old rats were trained on an inhibitory avoidance task with immediate post-training injections of aCSF or glucose into the dorsal hippocampus. Old rats had significant memory impairments compared to young rats 7 days after training. Intrahippocampal injections of glucose reversed age-related deficits, improving memory scores in old rats to values seen in young rats. A second experiment examined age-related changes in activation of the transcription factor CREB, which is widely implicated in memory formation and may act downstream of hormonal and metabolic signals. Activation was assessed in response to training with systemic injections of epinephrine and glucose at doses known to enhance memory. Young adult and old rats were trained on inhibitory avoidance with immediate post-training systemic injections of saline, epinephrine, or glucose. After training, old rats had significant impairments in CREB phosphorylation in area CA1 and the dentate gyrus region of the hippocampus, and in the basolateral and lateral amygdala. Epinephrine and glucose attenuated age-related deficits in CREB phosphorylation, but were more effective in the amygdala and hippocampus, respectively. Together, these results support the view that age-related changes in blood glucose responses to epinephrine contribute to memory impairments, which may be related to alterations in regional patterns of CREB phosphorylation. PMID

  2. Epinephrine and glucose modulate training-related CREB phosphorylation in old rats: relationships to age-related memory impairments.

    PubMed

    Morris, Ken A; Gold, Paul E

    2013-02-01

    Epinephrine enhances memory in young adult rats, in part, by increasing blood glucose levels needed to modulate memory. In old rats, epinephrine is deficient at raising blood glucose levels and thus is only moderately effective at enhancing memory. In contrast, systemic glucose injections improve memory in old rats, with resulting memory performance equal to that of young rats. The diminished response of glucose to training in old rats may blunt downstream neurochemical and molecular mechanisms needed to upregulate memory processes. In the first experiment, young adult and old rats were trained on an inhibitory avoidance task with immediate post-training injections of aCSF or glucose into the dorsal hippocampus. Old rats had significant memory impairments compared to young rats 7 days after training. Intrahippocampal injections of glucose reversed age-related deficits, improving memory scores in old rats to values seen in young rats. A second experiment examined age-related changes in activation of the transcription factor CREB, which is widely implicated in memory formation and may act downstream of hormonal and metabolic signals. Activation was assessed in response to training with systemic injections of epinephrine and glucose at doses known to enhance memory. Young adult and old rats were trained on inhibitory avoidance with immediate post-training systemic injections of saline, epinephrine, or glucose. After training, old rats had significant impairments in CREB phosphorylation in area CA1 and the dentate gyrus region of the hippocampus, and in the basolateral and lateral amygdala. Epinephrine and glucose attenuated age-related deficits in CREB phosphorylation, but were more effective in the amygdala and hippocampus, respectively. Together, these results support the view that age-related changes in blood glucose responses to epinephrine contribute to memory impairments, which may be related to alterations in regional patterns of CREB phosphorylation.

  3. Increased phosphorylation of Ser473-Akt, Ser9-GSK-3beta and Ser133-CREB in the rat frontal cortex after MK-801 intraperitoneal injection.

    PubMed

    Ahn, Yong Min; Seo, Myoung Suk; Kim, Se Hyun; Kim, Yeni; Yoon, Se Chang; Juhnn, Yong-Sung; Kim, Yong Sik

    2005-12-01

    GSK-3beta is regarded as playing an important part in the pathogenesis of schizophrenia and the action of psychotomimetic agents. We observed phosphorylation of molecules associated with the GSK-3beta signalling pathway in the rat brain after MK-801 injection, which induces a schizophrenia-like state in humans. Ser9-GSK-3beta phosphorylation was increased after injection of 1 mg/kg MK-801 in the rat frontal cortex but not in the hippocampus or cerebellum. This increase peaked at 30 min and was maintained until 90 min after injection. The phosphorylation showed a dose-dependent increase up to 1 mg/kg MK-801, followed by a decrease at higher dosage. Furthermore, phosphorylation of Ser473-Akt and Ser133-CREB showed similar temporal, dose-dependent and regionally specific patterns with those of Ser9-GSK-3beta. However, phosphorylation of Dvl and Ser33-beta-catenin was not affected by MK-801. These results suggest that GSK-3beta phosphorylation by MK-801 may be associated with the Akt-GSK-3beta pathway rather than with the Wnt-Dvl-GSK3beta pathway.

  4. PERK mediates eIF2α phosphorylation responsible for BACE1 elevation, CREB dysfunction and neurodegeneration in a mouse model of Alzheimer's disease.

    PubMed

    Devi, Latha; Ohno, Masuo

    2014-10-01

    Emerging evidence suggests that aberrant phosphorylation of eukaryotic initiation factor-2α (eIF2α) may induce synaptic failure and neurodegeneration through persistent translational inhibition of global protein synthesis. However, elevated phospho-eIF2α also paradoxically causes translational activation of a subset of messenger RNAs such as the β-secretase enzyme, β-site APP-cleaving enzyme 1 (BACE1) and cAMP response element binding protein (CREB) repressor, activating transcription factor 4 (ATF4). Therefore, we tested whether genetic reduction of the eIF2α kinase PERK may prevent these deleterious events and mitigate Alzheimer's disease (AD)-like neuropathology and cognitive impairments in the 5XFAD mouse model. PERK haploinsufficiency blocked overactivation of the PERK-eIF2α pathway, as evidenced by significant reductions in phosphorylation of PERK and eIF2α, in 5XFAD mice. PERK haploinsufficiency was sufficient to rescue memory deficits and cholinergic neurodegeneration in this AD model. Notably, PERK haploinsufficiency also prevented BACE1 elevations, resulting in reduced levels of amyloid-β peptides and plaque burden in 5XFAD mice. Moreover, CREB dysfunction was restored in PERK(+/-)·5XFAD mice concomitant with reversal of ATF4 upregulation. Together, these findings suggest that PERK may be a disease-modifying therapeutic target to prevent multiple memory-disrupting mechanisms associated with AD.

  5. Peptide IDR-1002 Inhibits NF-κB Nuclear Translocation by Inhibition of IκBα Degradation and Activates p38/ERK1/2–MSK1-Dependent CREB Phosphorylation in Macrophages Stimulated with Lipopolysaccharide

    PubMed Central

    Huante-Mendoza, Alejandro; Silva-García, Octavio; Oviedo-Boyso, Javier; Hancock, Robert E. W.; Baizabal-Aguirre, Víctor M.

    2016-01-01

    not lead to expression of IL-4, IL-10, and IL-13, even though it induced an increase in phospho-CREB relative abundance. In conclusion, our results indicated that IDR-1002 has a dual effect. On one hand, it inhibited NF-κB nuclear translocation through a mechanism that involved inhibition of IκBα phosphorylation, and on the other, it activated a protein kinase signaling cascade that phosphorylated CREB to selectively influence cytokine gene expression. Based on these results, we think IDR-1002 could be a potential good biopharmaceutical candidate to control inflammation. PMID:27933067

  6. Differential effects of chronic amphetamine and baclofen administration on cAMP levels and phosphorylation of CREB in distinct brain regions of wild type and monoamine oxidase B-deficient mice.

    PubMed

    Yin, Hsiang-Shu; Chen, Kevin; Kalpana, Sriram; Shih, Jean C

    2006-12-15

    Roles of GABA(B) transmission were explored in the action of amphetamine (Amph) on the brain. Adult male wild type (WT) and monoamine oxidase B-knocked out (MAOBKO) mice received i.p. injections of saline, d-Amph (5 mg/kg), plus baclofen (GABA(B) receptor agonist, 10 mg/kg), or baclofen and Amph, twice daily for 3 days and single treatments on day 4, followed by immuno-cyclic-AMP (cAMP) and immunoblotting assays on the brain tissue. The WT mice responded with higher levels of behavioral responses than the KO to the daily Amph injection; however, baclofen blocked the Amph-induced behavioral hyperactivity of both WT and KO mice. After the last treatment, levels of cAMP and phosphorylated (p) cyclic-AMP response element binding protein (CREB) were up-regulated in the striatum and somatosensory cortex of Amph-treated WT mice, while similar to the saline-controls in the baclofen+Amph-treated group, indicating the blockade by baclofen to Amph. Baclofen similarly suppressed the Amph-induced increases in pCREB levels of WT hippocampus and amygdala, and decreases of olfactory bulb and thalamus. For MAOBKO mice, baclofen hindered the Amph-generated increases in motor cortical cAMP and pCREB, and amygdaloid pCREB, and the decrease in olfactory bulb pCREB, whereas did not affect the Amph-raised hippocampal pCREB. Furthermore, the levels of CREB were variably modified in distinct regions by the drug exposures. The data reveal that the GABA(B)-mediated intracellular signaling differentially participates in mechanisms underlying Amph perturbation to various regions, and may thereby contribute explanations to the behavioral consequences. Moreover, MAOB is region-dependently involved in responses of the brain to Amph and baclofen, supporting interactions between GABA and monoamines.

  7. Association of MMP7 -181A→G Promoter Polymorphism with Gastric Cancer Risk: INFLUENCE OF NICOTINE IN DIFFERENTIAL ALLELE-SPECIFIC TRANSCRIPTION VIA INCREASED PHOSPHORYLATION OF cAMP-RESPONSE ELEMENT-BINDING PROTEIN (CREB).

    PubMed

    Kesh, Kousik; Subramanian, Lakshmi; Ghosh, Nillu; Gupta, Vinayak; Gupta, Arnab; Bhattacharya, Samir; Mahapatra, Nitish R; Swarnakar, Snehasikta

    2015-06-05

    Elevated expression of matrix metalloproteinase7 (MMP7) has been demonstrated to play a pivotal role in cancer invasion. The -181A→G (rs11568818) polymorphism in the MMP7 promoter modulates gene expression and possibly affects cancer progression. Here, we evaluated the impact of -181A→G polymorphism on MMP7 promoter activity and its association with gastric cancer risk in eastern Indian case-control cohorts (n = 520). The GG genotype as compared with the AA genotype was predisposed (p = 0.02; odds ratio = 1.9, 95% confidence interval = 1.1-3.3) to gastric cancer risk. Stratification analysis showed that tobacco addiction enhanced gastric cancer risk in GG subjects when compared with AA subjects (p = 0.03, odds ratio = 2.46, and 95% confidence interval = 1.07-5.68). Meta-analysis revealed that tobacco enhanced the risk for cancer more markedly in AG and GG carriers. Activity and expression of MMP7 were significantly higher in GG than in AA carriers. In support, MMP7 promoter-reporter assays showed greater transcriptional activity toward A to G transition under basal/nicotine-induced/cAMP-response element-binding protein (CREB) overexpressed conditions in gastric adenocarcinoma cells. Moreover, nicotine (a major component of tobacco) treatment significantly up-regulated MMP7 expression due to enhanced CREB phosphorylation followed by its nuclear translocation in gastric adenocarcinoma cells. Furthermore, chromatin immunoprecipitation experiments revealed higher binding of phosphorylated CREB with the -181G than the -181A allele. Altogether, specific binding of phosphorylated CREB to the G allele-carrying promoter enhances MMP7 gene expression that is further augmented by nicotine due to increased CREB phosphorylation and thereby increases the risk for gastric cancer.

  8. Leptin Induces Hippocampal Synaptogenesis via CREB-Regulated MicroRNA-132 Suppression of p250GAP

    PubMed Central

    Dhar, Matasha; Zhu, Mingyan; Impey, Soren; Lambert, Talley J.; Bland, Tyler; Karatsoreos, Ilia N.; Nakazawa, Takanobu

    2014-01-01

    Leptin acts in the hippocampus to enhance cognition and reduce depression and anxiety. Cognitive and emotional disorders are associated with abnormal hippocampal dendritic spine formation and synaptogenesis. Although leptin has been shown to induce synaptogenesis in the hypothalamus, its effects on hippocampal synaptogenesis and the mechanism(s) involved are not well understood. Here we show that leptin receptors (LepRs) are critical for hippocampal dendritic spine formation in vivo because db/db mice lacking the long form of the leptin receptor (LepRb) have reduced spine density on CA1 and CA3 neurons. Leptin promotes the formation of mature spines and functional glutamate synapses on hippocampal pyramidal neurons in both dissociated and slice cultures. These effects are blocked by short hairpin RNAs specifically targeting the LepRb and are absent in cultures from db/db mice. Activation of the LepR leads to cAMP response element–binding protein (CREB) phosphorylation and initiation of CREB-dependent transcription via the MAPK kinase/Erk pathway. Furthermore, both Mek/Erk and CREB activation are required for leptin-induced synaptogenesis. Leptin also increases expression of microRNA-132 (miR132), a well-known CREB target, which is also required for leptin-induced synaptogenesis. Last, leptin suppresses the expression of p250GAP, a miR132 target, and this suppression is obligatory for leptin's effects as is the downstream target of p250GAP, Rac1. LepRs appear to be critical in vivo as db/db mice have lowered hippocampal miR132 levels and elevated p250GAP expression. In conclusion, we identify a novel signaling pathway by which leptin increases synaptogenesis through inducing CREB transcription and increasing microRNA-mediated suppression of p250GAP activity, thus removing a known inhibitor of Rac1-stimulated synaptogenesis. PMID:24877561

  9. Heat induces interleukin-6 in skeletal muscle cells via TRPV1/PKC/CREB pathways.

    PubMed

    Obi, Syotaro; Nakajima, Toshiaki; Hasegawa, Takaaki; Kikuchi, Hironobu; Oguri, Gaku; Takahashi, Masao; Nakamura, Fumitaka; Yamasoba, Tatsuya; Sakuma, Masashi; Toyoda, Shigeru; Tei, Chuwa; Inoue, Teruo

    2017-03-01

    Interleukin-6 (IL-6) is released from skeletal muscle cells and induced by exercise, heat, catecholamine, glucose, lipopolysaccharide, reactive oxygen species, and inflammation. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-4 play vital roles in cellular functions. In this study we hypothesized that TRPV1 senses heat, transmits a signal into the nucleus, and produces IL-6. The purpose of the present study is to investigate the underlying mechanisms whereby skeletal muscle cells sense and respond to heat. When mouse myoblast cells were exposed to 37-42°C for 2 h, mRNA expression of IL-6 increased in a temperature-dependent manner. Heat also increased IL-6 secretion in myoblast cells. A fura 2 fluorescence dual-wavelength excitation method showed that heat increased intracellular calcium flux in a temperature-dependent manner. Intracellular calcium flux and IL-6 mRNA expression were increased by the TRPV1 agonists capsaicin and N-arachidonoyldopamine and decreased by the TRPV1 antagonists AMG9810 and SB366791 and siRNA-mediated knockdown of TRPV1. TRPV2, 3, and 4 agonists did not change intracellular calcium flux. Western blotting with inhibitors demonstrated that heat increased phosphorylation levels of TRPV1, followed by PKC and cAMP response element-binding protein (CREB). PKC inhibitors, Gö6983 and staurosporine, CREB inhibitors, curcumin and naphthol AS-E, and knockdown of CREB suppressed the heat-induced increases in IL-6. These results indicate that heat increases IL-6 in skeletal muscle cells through the TRPV1, PKC, and CREB signal transduction pathway.NEW & NOTEWORTHY Heat increases the release of interleukin-6 (IL-6) from skeletal muscle cells. IL-6 has been shown to serve immune responses and metabolic functions in muscle. It can be anti-inflammatory as well as proinflammatory. However, the mechanism that induces release of IL-6

  10. Reduced CREB phosphorylation after chronic lithium treatment is associated with down-regulation of CaM kinase IV in rat hippocampus.

    PubMed

    Tardito, Daniela; Tiraboschi, Ettore; Kasahara, Jiro; Racagni, Giorgio; Popoli, Maurizio

    2007-08-01

    Lithium is widely used in the treatment of bipolar disorder, although its mechanism of action is not fully clear. This study was undertaken to assess the effects of prolonged lithium administration on cAMP responsive element-binding protein (CREB) phosphorylation and CaM kinase IV (CaMKIV), one of the main kinases phosphorylating CREB in neurons following synaptic activation. CREB total protein expression and phosphorylation (Ser133), as well as CaMKIV enzymatic activity, phosphorylation of Thr196 (the activator residue) and kinase expression level were assessed in total homogenates and nuclei from the hippocampus and prefrontal/frontal cortex following 5 wk lithium treatment. Whereas no significant effects were found in prefrontal/frontal cortex, lithium administration reduced CREB phosphorylation and at the same time down-regulated CaMKIV (enzymatic activity, phospho-Thr196 and protein expression level) in cell nuclei from the hippocampus. These data suggest for the first time the involvement of CaMKIV in the mechanism of action of lithium.

  11. CREB modulates calcium signaling in cAMP-induced bone marrow stromal cells (BMSCs).

    PubMed

    Zhang, Linxia; Liu, Li; Thompson, Ryan; Chan, Christina

    2014-10-01

    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response.

  12. Prevention of vascular smooth muscle cell proliferation and injury-induced neointimal hyperplasia by CREB-mediated p21 induction: An insight from a plant polyphenol.

    PubMed

    Sun, Lan; Zhao, Rui; Zhang, Li; Zhang, Weiku; He, GuoRong; Yang, Shengqian; Song, Junke; Du, Guanhua

    2016-03-01

    Cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element (CRE)-binding protein (CREB) signaling cascade negatively regulates platelet-derived growth factor BB (PDGF-BB)-induced smooth muscle cell (SMC) proliferation, which is a critical event in the initiation and development of restenosis and atherosclerotic lesions. Salvianolic acid A (SAA) is one of the most abundant polyphenols extracted from salvia. The aim of this study is to investigate whether SAA exerts an action on PDGF-BB-induced proliferation via cAMP/PKA/CREB mechanism. SAA blunts PDGF-BB-induced human umbilical artery smooth muscle cell (hUASMC) proliferation via p21 induction, as evidenced by its increased mRNA and protein expression levels. The SAA-induced upregulation of p21 involves the cAMP/PKA signaling pathway; a cAMP analog mimicked the effects of SAA and a specific cAMP/PKA inhibitor opposed these effects. SAA also activated CREB, including phosphorylation at Ser133, and induced its nuclear translocation. Deletion and mutational analysis of p21 promoters, co-immunoprecipitation, and western blot analysis showed that CRE is essential for SAA-induced p21 protein expression. Transfection of dominant-negative CREB (mutated Ser133) plasmids into hUASMCs attenuated SAA-stimulated p21 expression. SAA upregulated p21 expression and activated CREB in the neointima of balloon-injured arteries in vivo. Our results indicate that SAA promotes p21 expression in SMCs through the cAMP/PKA/CREB signaling cascade in vitro and prevents injury-induced neointimal hyperplasia.

  13. MicroRNA-34b promoter hypermethylation induces CREB overexpression and contributes to myeloid transformation

    PubMed Central

    Pigazzi, Martina; Manara, Elena; Bresolin, Silvia; Tregnago, Claudia; Beghin, Alessandra; Baron, Emma; Giarin, Emanuela; Cho, Er-Chieh; Masetti, Riccardo; Rao, Dinesh S.; Sakamoto, Kathleen M.; Basso, Giuseppe

    2013-01-01

    MicroRNA-34b down-regulation in acute myeloid leukemia was previously shown to induce CREB overexpression, thereby causing leukemia proliferation in vitro and in vivo. The role of microRNA-34b and CREB in patients with myeloid malignancies has never been evaluated. We examined microRNA-34b expression and the methylation status of its promoter in cells from patients diagnosed with myeloid malignancies. We used gene expression profiling to identify signatures of myeloid transformation. We established that microRNA-34b has suppressor ability and that CREB has oncogenic potential in primary bone marrow cell cultures and in vivo. MicroRNA-34b was found to be up-regulated in pediatric patients with juvenile myelomonocytic leukemia (n=17) and myelodysplastic syndromes (n=28), but was down-regulated in acute myeloid leukemia patients at diagnosis (n=112). Our results showed that hypermethylation of the microRNA-34b promoter occurred in 66% of cases of acute myeloid leukemia explaining the low microRNA-34b levels and CREB overexpression, whereas preleukemic myelodysplastic syndromes and juvenile myelomonocytic leukemia were not associated with hypermethylation or CREB overexpression. In paired samples taken from the same patients when they had myelodysplastic syndrome and again during the subsequent acute myeloid leukemia, we confirmed microRNA-34b promoter hypermethylation at leukemia onset, with 103 CREB target genes differentially expressed between the two disease stages. This subset of CREB targets was confirmed to associate with high-risk myelodysplastic syndromes in a separate cohort of patients (n=20). Seventy-eight of these 103 CREB targets were also differentially expressed between healthy samples (n=11) and de novo acute myeloid leukemia (n=72). Further, low microRNA-34b and high CREB expression levels induced aberrant myelopoiesis through CREB-dependent pathways in vitro and in vivo. In conclusion, we suggest that microRNA-34b controls CREB expression and

  14. Chronic nicotine treatment reverses hypothyroidism-induced impairment of L-LTP induction phase: critical role of CREB.

    PubMed

    Alzoubi, K H; Alkadhi, K A

    2014-06-01

    We have previously shown that adult onset hypothyroidism impairs late-phase long-term potentiation (L-LTP) and reduces basal protein levels of cyclic-AMP response element binding protein (CREB), mutagen-activated protein kinase (MAPKp42/44), and calcium calmodulin kinase IV (CaMKIV) in area Cornu Ammonis 1 (CA1) of the hippocampus. These changes were reversed by chronic nicotine treatment. In the present study, levels of signaling molecules important for L-LTP were determined in CA1 area of the hippocampus during the induction phase. Standard multiple high-frequency stimulation (MHFS) was used to evoke L-LTP in the CA1 area of the hippocampus of hypothyroid, nicotine-treated hypothyroid, nicotine, and sham control anaesthetized adult rats. Chronic nicotine treatment reversed hypothyroidism-induced impairment of L-LTP at the induction phase. Five minutes after MHFS, Western blotting showed an increase in the levels of P-CREB, and P-MAPKp42/44 in sham-operated control, nicotine, and nicotine-treated hypothyroid animals, but not in hypothyroid animals. The protein levels of total CREB, total MAPK p42/44, BDNF, and CaMKIV were not altered in all groups 5 min after MHFS. Therefore, normalized phosphorylation of essential kinases such as P-CREB and P-MAPK p42/44 in the CA1 area of nicotine-treated hypothyroid animals plays a crucial role in nicotine-induced rescue of L-LTP induction during hypothyroidism.

  15. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

    PubMed

    Olala, Lawrence O; Choudhary, Vivek; Johnson, Maribeth H; Bollag, Wendy B

    2014-07-01

    Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

  16. Early induction of CREB activation and CREB-regulating signalling by antidepressants.

    PubMed

    Tardito, Daniela; Musazzi, Laura; Tiraboschi, Ettore; Mallei, Alessandra; Racagni, Giorgio; Popoli, Maurizio

    2009-11-01

    Converging evidence points to adaptive changes in neuroplasticity and gene expression as mediators of therapeutic action of antidepressants. Activation of cAMP response-element binding protein (CREB) and CREB-regulating signalling are considered main effectors in these mechanisms. We analysed the temporal profile of intracellular changes induced by antidepressants, by measuring activation of major CREB-regulating signalling cascades and activation (Ser133 phosphorylation) of CREB. The main aims of the study were to investigate how these different variables are modulated with time, whether stronger activation of signalling cascades corresponds to stronger activation of CREB, and whether these changes are different in distinct brain areas. Rat groups were treated for 1, 2 or 3 wk with the antidepressants fluoxetine or reboxetine; in additional groups drug treatment was followed by a washout week (3+1). Activation of CREB and major effectors in signalling cascades were analysed by Western blot analysis with phospho-antibodies, in nuclear and cytosolic fractions from hippocampus and prefrontal/frontal cortex (P/FC). Surprisingly, CREB activation was already maximal after 1-wk treatment. In hippocampus early and stronger CREB activation was consistent with early and stronger activation of signalling. For both drugs, the profile of activation in P/FC was different from that observed in hippocampus. The results also showed that, contrary to the activatory role of MAP-ERKs and CaM kinase IV, nuclear alphaCaM kinase II was inactivated in parallel with activation of CREB.

  17. Hypoxic adaptation engages the CBP/CREST-induced coactivator complex of Creb-HIF-1α in transactivating murine neuroblastic glucose transporter

    PubMed Central

    Thamotharan, Shanthie; Raychaudhuri, Nupur; Tomi, Masatoshi; Shin, Bo-Chul

    2013-01-01

    We have shown in vitro a hypoxia-induced time-dependent increase in facilitative glucose transporter isoform 3 (GLUT3) expression in N2A murine neuroblasts. This increase in GLUT3 expression is partially reliant on a transcriptional increase noted in actinomycin D and cycloheximide pretreatment experiments. Transient transfection assays in N2A neuroblasts using murine glut3-luciferase reporter constructs mapped the hypoxia-induced enhancer activities to −857- to −573-bp and −203- to −177-bp regions. Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1α and p-Creb binding to HRE (−828 to −824 bp) and AP-1 (−187 to −180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays. In addition, the interaction of CBP with Creb and HIF-1α and CREST with CBP in hypoxia was detected by coimmunoprecipitation. Furthermore, small interference (si)RNA targeting Creb in these cells decreased endogenous Creb concentrations that reduced by twofold hypoxia-induced glut3 gene transcription. Thus, in N2A neuroblasts, phosphorylated HIF-1α and Creb mediated the hypoxia-induced increase in glut3 transcription. Coactivation by the Ca++-dependent CREST and CBP proteins may enhance cross-talk between p-Creb-AP-1 and HIF-1α/HRE of the glut3 gene. Collectively, these processes can facilitate an adaptive response to hypoxic energy depletion targeted at enhancing glucose transport and minimizing injury while fueling the proliferative potential of neuroblasts. PMID:23321477

  18. Viral vector induction of CREB expression in the periaqueductal gray induces a predator stress-like pattern of changes in pCREB expression, neuroplasticity, and anxiety in rodents.

    PubMed

    Adamec, Robert; Berton, Olivier; Abdul Razek, Waleed

    2009-01-01

    Predator stress is lastingly anxiogenic. Phosphorylation of CREB to pCREB (phosphorylated cyclic AMP response element binding protein) is increased after predator stress in fear circuitry, including in the right lateral column of the PAG (periaqueductal gray). Predator stress also potentiates right but not left CeA-PAG (central amygdala-PAG) transmission up to 12 days after stress. The present study explored the functional significance of pCREB changes by increasing CREB expression in non-predator stressed rats through viral vectoring, and assessing the behavioral, electrophysiological and pCREB expression changes in comparison with handled and predator stressed controls. Increasing CREB expression in right PAG was anxiogenic in the elevated plus maze, had no effect on risk assessment, and increased acoustic startle response while delaying startle habituation. Potentiation of the right but not left CeA-PAG pathway was also observed. pCREB expression was slightly elevated in the right lateral column of the PAG, while the dorsal and ventral columns were not affected. The findings of this study suggest that by increasing CREB and pCREB in the right lateral PAG, it is possible to produce rats that exhibit behavioral, brain, and molecular changes that closely resemble those seen in predator stressed rats.

  19. Spatial Memory in the Morris Water Maze and Activation of Cyclic AMP Response Element-Binding (CREB) Protein within the Mouse Hippocampus

    ERIC Educational Resources Information Center

    Porte, Yves; Buhot, Marie Christine; Mons, Nicole E.

    2008-01-01

    We investigated the spatio-temporal dynamics of learning-induced cAMP response element-binding protein activation/phosphorylation (pCREB) in mice trained in a spatial reference memory task in the water maze. Using immunohistochemistry, we examined pCREB immunoreactivity (pCREB-ir) in hippocampal CA1 and CA3 and related brain structures. During the…

  20. Resveratrol abrogates lipopolysaccharide-induced depressive-like behavior, neuroinflammatory response, and CREB/BDNF signaling in mice.

    PubMed

    Ge, Li; Liu, Liwei; Liu, Hansen; Liu, Song; Xue, Hao; Wang, Xueer; Yuan, Lin; Wang, Zhen; Liu, Dexiang

    2015-12-05

    Current evidence supports that depression is accompanied by the activation of the inflammatory-response system, and overproduction of pro-inflammatory cytokines may play a role in the pathophysiology of depressive disorders. Resveratrol has anti-inflammatory, antioxidant and anti-depressant-like properties. Using an animal model of depression induced by a single administration of lipopolysaccharide (LPS), the present study investigated the effects of resveratrol on LPS-induced depressive-like behavior and inflammatory-response in adult mice. Our results showed that pretreatment with resveratrol (80mg/kg, i.p.) for 7 consecutive days reversed LPS-increased the immobility time in the forced swimming test and tail suspension test, and LPS-reduced sucrose preference test. Moreover, the antidepressant action of resveratrol was paralleled by significantly reducing the expression levels of pro-inflammatory cytokines, and up-regulating phosphorylated cAMP response-element-binding protein (pCREB)/brain-derived neurotrophic factor (BDNF) expression in prefrontal cortex (PFC) and hippocampus. In addition, resveratrol ameliorated LPS-induced NF-κB activation in the PFC and hippocampus. The results demonstrate that resveratrol may be an effective therapeutic agent for LPS-induced depressive-like behavior, partially due to its anti-inflammatory aptitude and by modulating pCREB and BDNF expression in the brain region of mice.

  1. Ginger improves cognitive function via NGF-induced ERK/CREB activation in the hippocampus of the mouse.

    PubMed

    Lim, Soonmin; Moon, Minho; Oh, Hyein; Kim, Hyo Geun; Kim, Sun Yeou; Oh, Myung Sook

    2014-10-01

    Ginger (the rhizome of Zingiber officinale Roscoe) has been used worldwide for many centuries in cooking and for treatment of several diseases. The main pharmacological properties of ginger include anti-inflammatory, antihyperglycemic, antiarthritic, antiemetic and neuroprotective actions. Recent studies demonstrated that ginger significantly enhances cognitive function in various cognitive disorders as well as in healthy brain. However, the biochemical mechanisms underlying the ginger-mediated enhancement of cognition have not yet been studied in normal or diseased brain. In the present study, we assessed the memory-enhancing effects of dried ginger extract (GE) in a model of scopolamine-induced memory deficits and in normal animals by performing a novel object recognition test. We found that GE administration significantly improved the ability of mice to recognize novel objects, indicating improvements in learning and memory. Furthermore, to elucidate the mechanisms of GE-mediated cognitive enhancement, we focused on nerve growth factor (NGF)-induced signaling pathways. NGF enzyme-linked immunosorbent assay analysis revealed that GE administration led to elevated NGF levels in both the mouse hippocampus and rat glioma C6 cells. GE administration also resulted in phosphorylation of extracellular-signal-regulated kinase (ERK) and cyclic AMP response element-binding protein (CREB), as revealed by Western blotting analysis. Neutralization of NGF with a specific NGF antibody inhibited GE-triggered activation of ERK and CREB in the hippocampus. Also, GE treatment significantly increased pre- and postsynaptic markers, synaptophysin and PSD-95, which are related to synapse formation in the brain. These data suggest that GE has a synaptogenic effect via NGF-induced ERK/CREB activation, resulting in memory enhancement.

  2. Dynorphin up-regulation in the dentate granule cell mossy fiber pathway following chronic inhibition of GluN2B-containing NMDAR is associated with increased CREB (Ser 133) phosphorylation, but is independent of BDNF/TrkB signaling pathways

    PubMed Central

    Rittase, W. Bradley; Dong, Yu; Barksdale, DaRel; Galdzicki, Zygmunt; Bausch, Suzanne B.

    2014-01-01

    Emerging evidence suggests that neuronal responses to N-methyl-d-aspartate (NMDAR) activation/inactivation are influenced by subunit composition. For example, activation of synaptic NMDAR (comprised of GluN2A > GluN2B) phosphorylates cAMP-response-element-binding protein (CREB) at Ser 133, induces BDNF expression and promotes neuronal survival. Activation of extrasynaptic NMDAR (comprised of GluN2B>GluN2), dephosphorylates CREB (Ser 133), reduces BDNF expression and triggers neuronal death. These results led us to hypothesize that chronic inhibition of GluN2B-containing NMDAR would increase CREB (Ser 133) phosphorylation, increase BDNF levels and subsequently alter downstream dynorphin (DYN) and neuropeptide Y (NPY) expression. We focused on DYN and NPY because these neuropeptides can decrease excitatory neurotransmission and seizure occurrence and we reported previously that seizure-like events are reduced following chronic treatment with GluN2B antagonists. Consistent with our hypothesis, chronic treatment (17-21 days) of hippocampal slice cultures with the GluN2B-selective antagonists ifenprodil or Ro25,6981 increased both CREB (Ser 133) phosphorylation and granule cell mossy fiber pathway DYN expression. Similar treatment with the non-subtype-selective NMDAR antagonists D-APV or memantine had no significant effect on either CREB (Ser 133) phosphorylation or DYN expression. In contrast to our hypothesis, BDNF levels were decreased following chronic treatment with Ro25,6981, but not ifenprodil, D-APV or memantine. Blockade of BDNF actions and TrkB activation did not significantly augment hilar DYN expression in vehicle-treated cultures and had no effect in Ro25,6981 treated cultures. These finding suggest that chronic exposure to GluN2B-selective NMDAR antagonists increased DYN expression through a putatively pCREB-dependent, but BDNF/TrkB-independent mechanism. PMID:24769103

  3. Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects

    SciTech Connect

    Corona, Giulia; Deiana, Monica; Incani, Alessandra; Vauzour, David; Assunta Dessi, M.; Spencer, Jeremy P.E.

    2007-10-26

    We investigated the anti-proliferative effects of an olive oil polyphenolic extract on human colon adenocarcinoma cells. Analysis indicated that the extract contained hydroxytyrosol, tyrosol and the various secoiridoid derivatives, including oleuropein. This extract exerted a strong inhibitory effect on cancer cell proliferation, which was linked to the induction of a G2/M phase cell cycle block. Following treatment with the extract (50 {mu}g/ml) the number of cells in the G2/M phase increased to 51.82 {+-} 2.69% relative to control cells (15.1 {+-} 2.5%). This G2/M block was mediated by the ability of olive oil polyphenols (50 {mu}g/ml) to exert rapid inhibition of p38 (38.7 {+-} 4.7%) and CREB (28.6 {+-} 5.5%) phosphorylation which led to a downstream reduction in COX-2 expression (56.9 {+-} 9.3%). Our data suggest that olive oil polyphenols may exert chemopreventative effects in the large intestine by interacting with signalling pathways responsible for colorectal cancer development.

  4. Ferulic acid prevents LPS-induced up-regulation of PDE4B and stimulates the cAMP/CREB signaling pathway in PC12 cells

    PubMed Central

    Huang, Hao; Hong, Qian; Tan, Hong-ling; Xiao, Cheng-rong; Gao, Yue

    2016-01-01

    Aim: Phosphodiesterase 4 (PDE4) isozymes are involved in different functions, depending on their patterns of distribution in the brain. The PDE4 subtypes are distributed in different inflammatory cells, and appear to be important regulators of inflammatory processes. In this study we examined the effects of ferulic acid (FA), a plant component with strong anti-oxidant and anti-inflammatory activities, on lipopolysaccharide (LPS)-induced up-regulation of phosphodiesterase 4B (PDE4B) in PC12 cells, which in turn regulated cellular cAMP levels and the cAMP/cAMP response element binding protein (CREB) pathway in the cells. Methods: PC12 cells were treated with LPS (1 μg/mL) for 8 h, and the changes of F-actin were detected using laser scanning confocal microscopy. The levels of pro-inflammatory cytokines were measured suing ELISA kits, and PDE4B-specific enzymatic activity was assessed with a PDE4B assay kit. The mRNA levels of PDE4B were analyzed with Q-PCR, and the protein levels of CREB and phosphorylated CREB (pCREB) were determined using immunoblotting. Furthermore, molecular docking was used to identify the interaction between PDE4B2 and FA. Results: Treatment of PC12 cells with LPS induced thick bundles of actin filaments appearing in the F-actin cytoskeleton, which were ameliorated by pretreatment with FA (10–40 μmol/L) or with a PDE4B inhibitor rolipram (30 μmol/L). Pretreatment with FA dose-dependently inhibited the LPS-induced production of TNF-α and IL-1β in PC12 cells. Furthermore, pretreatment with FA dose-dependently attenuated the LPS-induced up-regulation of PDE4 activity in PC12 cells. Moreover, pretreatment with FA decreased LPS-induced up-regulation of the PDE4B mRNA, and reversed LPS-induced down-regulation of CREB and pCREB in PC12 cells. The molecular docking results revealed electrostatic and hydrophobic interactions between FA and PDE4B2. Conclusion: The beneficial effects of FA in PC12 cells might be conferred through inhibition of LPS-induced

  5. TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration.

    PubMed

    Lee, Guan-Lin; Wu, Jing-Yiing; Yeh, Chang-Ching; Kuo, Cheng-Chin

    2016-05-13

    Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis.

  6. Dioscin Derived from Solanum melongena L. "Usukawamarunasu" Attenuates α-MSH-Induced Melanogenesis in B16 Murine Melanoma Cells via Downregulation of Phospho-CREB and MITF.

    PubMed

    Nishina, Atsuyoshi; Ebina, Kodai; Ukiya, Motohiko; Fukatsu, Makoto; Koketsu, Mamoru; Ninomiya, Masayuki; Sato, Daisuke; Kimura, Hirokazu

    2015-10-01

    This study aimed to chemically isolate and explore an antimelanogenesis inducer in extracts of Solanum melongena L. "Usukawamarunasu" eggplant. We successfully identified dioscin ([25R]-Spirost-5-en-3β-yl) 2-O-(6-deoxy-α-L-mannopyranosyl) - 4-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranoside] in the plant, and examined the effects of α-melanocyte-stimulating hormone (MSH)-induced melanogenesis in B16 murine melanoma cells by this plant-derived dioscin. Immunoblot analysis suggested that dioscin reduced the expression of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2, resulting in inhibition of intracellular production of melanin. In addition, dioscin caused reduction of phosphorylated cAMP-responsive element binding protein 1 transcription factors (CREB), which led to a reduction of microphthalmia-related transcription factor (MITF) in α-MSH-stimulated cells, but did not affect phosphorylation of extracellular signal-regulated kinase. Furthermore, dioscin significantly downregulated the expression of tyrosinase, TRP-1, and TRP-2, which led to the reduction of α-MSH-induced melanogenesis in B16 cells. These results suggest that dioscin may decrease the level of MITF via inhibition of phosphorylation of CREB in α-MSH-induced melanogenesis in B16 cells.

  7. Differences in the Flexibility of Switching Learning Strategies and CREB Phosphorylation Levels in Prefrontal Cortex, Dorsal Striatum and Hippocampus in Two Inbred Strains of Mice

    PubMed Central

    Cho, Woo-Hyun; Han, Jung-Soo

    2016-01-01

    Flexibility in using different learning strategies was assessed in two different inbred strains of mice, the C57BL/6 and DBA/2 strains. Mice were trained sequentially in two different Morris water maze protocols that tested their ability to switch their learning strategy to complete a new task after first being trained in a different task. Training consisted either of visible platform trials (cued training) followed by subsequent hidden platform trials (place training) or the reverse sequence (place training followed by cued training). Both strains of mice showed equivalent performance in the type of training (cued or place) that they received first. However, C57BL/6 mice showed significantly better performances than DBA/2 mice following the switch in training protocols, irrespective of the order of training. After completion of the switched training session, levels of cAMP response element-binding protein (CREB) and phosphorylated CREB (pCREB) were measured in the hippocampus, striatum and prefrontal cortex of the mice. Prefrontal cortical and hippocampal pCREB levels differed by strain, with higher levels found in C57BL/6 mice than in DBA/2 mice. No strain differences were observed in the medial or lateral region of the dorsal striatum. These findings indicate that the engagement (i.e., CREB signaling) of relevant neural structures may vary by the specific demands of the learning strategy, and this is closely tied to differences in the flexibility of C57BL/6 and DBA/2 mice to switch their learning strategies when given a new task. PMID:27695401

  8. Differences in the Flexibility of Switching Learning Strategies and CREB Phosphorylation Levels in Prefrontal Cortex, Dorsal Striatum and Hippocampus in Two Inbred Strains of Mice.

    PubMed

    Cho, Woo-Hyun; Han, Jung-Soo

    2016-01-01

    Flexibility in using different learning strategies was assessed in two different inbred strains of mice, the C57BL/6 and DBA/2 strains. Mice were trained sequentially in two different Morris water maze protocols that tested their ability to switch their learning strategy to complete a new task after first being trained in a different task. Training consisted either of visible platform trials (cued training) followed by subsequent hidden platform trials (place training) or the reverse sequence (place training followed by cued training). Both strains of mice showed equivalent performance in the type of training (cued or place) that they received first. However, C57BL/6 mice showed significantly better performances than DBA/2 mice following the switch in training protocols, irrespective of the order of training. After completion of the switched training session, levels of cAMP response element-binding protein (CREB) and phosphorylated CREB (pCREB) were measured in the hippocampus, striatum and prefrontal cortex of the mice. Prefrontal cortical and hippocampal pCREB levels differed by strain, with higher levels found in C57BL/6 mice than in DBA/2 mice. No strain differences were observed in the medial or lateral region of the dorsal striatum. These findings indicate that the engagement (i.e., CREB signaling) of relevant neural structures may vary by the specific demands of the learning strategy, and this is closely tied to differences in the flexibility of C57BL/6 and DBA/2 mice to switch their learning strategies when given a new task.

  9. Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB

    PubMed Central

    Mair, William; Morantte, Ianessa; Rodrigues, Ana P. C.; Manning, Gerard; Montminy, Marc; Shaw, Reuben J.; Dillin, Andrew

    2011-01-01

    Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans1,2 and both have been implicated as therapeutic targets for age-related pathology in mammals3–5. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs)6 are a family of cofactors involved in diverse physiological processes including energy homeostasis7–9, cancer10 and endoplasmic reticulum stress11. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity. PMID:21331044

  10. CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

    PubMed Central

    Bilbao, Ainhoa; Rieker, Claus; Cannella, Nazzareno; Parlato, Rosanna; Golda, Slawomir; Piechota, Marcin; Korostynski, Michal; Engblom, David; Przewlocki, Ryszard; Schütz, Günther; Spanagel, Rainer; Parkitna, Jan R.

    2014-01-01

    It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB. PMID:24966820

  11. Hydrocortisone-induced parkin prevents dopaminergic cell death via CREB pathway in Parkinson's disease model.

    PubMed

    Ham, Sangwoo; Lee, Yun-Il; Jo, Minkyung; Kim, Hyojung; Kang, Hojin; Jo, Areum; Lee, Gum Hwa; Mo, Yun Jeong; Park, Sang Chul; Lee, Yun Song; Shin, Joo-Ho; Lee, Yunjong

    2017-04-03

    Dysfunctional parkin due to mutations or post-translational modifications contributes to dopaminergic neurodegeneration in Parkinson's disease (PD). Overexpression of parkin provides protection against cellular stresses and prevents dopamine cell loss in several PD animal models. Here we performed an unbiased high-throughput luciferase screening to identify chemicals that can increase parkin expression. Among promising parkin inducers, hydrocortisone possessed the most favorable profiles including parkin induction ability, cell protection ability, and physicochemical property of absorption, distribution, metabolism, and excretion (ADME) without inducing endoplasmic reticulum stress. We found that hydrocortisone-induced parkin expression was accountable for cell protection against oxidative stress. Hydrocortisone-activated parkin expression was mediated by CREB pathway since gRNA to CREB abolished hydrocortisone's ability to induce parkin. Finally, hydrocortisone treatment in mice increased brain parkin levels and prevented 6-hydroxy dopamine induced dopamine cell loss when assessed at 4 days after the toxin's injection. Our results showed that hydrocortisone could stimulate parkin expression via CREB pathway and the induced parkin expression was accountable for its neuroprotective effect. Since glucocorticoid is a physiological hormone, maintaining optimal levels of glucocorticoid might be a potential therapeutic or preventive strategy for Parkinson's disease.

  12. Topiramate Confers Neuroprotection Against Methylphenidate-Induced Neurodegeneration in Dentate Gyrus and CA1 Regions of Hippocampus via CREB/BDNF Pathway in Rats.

    PubMed

    Motaghinejad, Majid; Motevalian, Manijeh; Abdollahi, Mohammad; Heidari, Mansour; Madjd, Zahra

    2017-04-01

    Methylphenidate (MPH) abuse can cause serious neurological damages. The neuroprotective effects of topiramate (TPM) have been reported already, but its mechanism of action still remains unclear. The current study evaluates in vivo role of CREB/BDNF in TPM protection of the rat hippocampal cells from methylphenidate-induced apoptosis, oxidative stress, and inflammation. A total of 60 adult male rats were divided into six groups. Groups 1 and 2 received normal saline (0.7 ml/rat) and MPH (10 mg/kg) respectively for 14 days. Groups 3 and 4 were concurrently treated with MPH (10 mg/kg) and TPM 50 and 100 mg/kg respectively for 14 days. Groups 5 and 6 were treated with 50 and 100 mg/kg TPM only respectively. After drug administration, open field test (OFT) was used to investigate motor activity. The hippocampus was then isolated and the apoptotic, antiapoptotic, oxidative, antioxidant, and inflammatory factors were measured. Expression of the total and phosphorylated CREB and BDNF in gene and protein levels, and gene expression of Ak1, CaMK4, MAPK3, PKA, and c-Fos levels were also measured. MPH significantly decreased motor activity in OFT. TPM (50 and 100 mg/kg) decreased MPH-induced motor activity disturbance. Additionally, MPH significantly increased Bax protein level, CaMK4 gene expression, lipid peroxidation, catalase activity, mitochondrial GSH, IL-1β, and TNF-α levels in isolated hippocampal cells. Also CREB, in total and phosphorylated forms, BDNF and Bcl-2 protein levels, Ak1, MAPK3, PKA and c-Fos gene expression, superoxide dismutase, glutathione peroxidase, and glutathione reductase activities decreased significantly by MPH. TPM (50 and 100 mg/kg), both in the presence and absence of MPH, attenuated the effects of MPH. Immunohistochemistry data showed that TPM increased localization of the total and phosphorylated forms of CREB in dentate gyrus (DG) and CA1 areas of the hippocampus. It seems that TPM can be used as a neuroprotective agent against

  13. Scopolamine induced amnesia is reversed by Bacopa monniera through participation of kinase-CREB pathway.

    PubMed

    Saraf, Manish Kumar; Anand, Akshay; Prabhakar, Sudesh

    2010-02-01

    Scopolamine, an anticholinergic drug, is reported to produce amnesia by interference of long term potentiation and has been used for discerning the efficacy of various antiamnesic drugs. The intoxication with anticholinergics and benzodiazepines tend to produce neurodegeneration which cause memory deficits. Our earlier reports have shown the antiamnesic drug, B. monniera to be capable of alleviating diazepam induced memory deficits. We have now tested how scopolamine affects downstream signaling molecules of long term potentiation and if B. monniera can also modulate the scopolamine induced amnesia. We used Morris water maze scale to test the amnesic effect of scopolamine and its reversal by B. monniera. Rota-rod test was used to screen muscle coordination activity of mice before water maze investigations were carried out. The results showed that scopolamine downregulated protein kinase C and iNOS without affecting cAMP, protein kinase A, calmodulin, MAP kinase, nitrite, CREB and pCREB. B. monniera reversed the scopolamine induced amnesia by significantly improving calmodulin and by partially attenuating protein kinase C and pCREB. These observations suggest involvement of calmodulin in evoking antiamnesic effects of B. monniera.

  14. Puerarin protects mouse liver against nickel-induced oxidative stress and inflammation associated with the TLR4/p38/CREB pathway.

    PubMed

    Liu, Chan-Min; Ma, Jie-Qiong; Liu, Si-Si; Feng, Zhao-Jun; Wang, Ai-Min

    2016-01-05

    Nickel (Ni), one of hazardous environmental chemicals, is known to cause liver injury. Accumulating evidence showed that puerarin (PU) possessed comprehensive biological effects. The purpose of the current study was to test the hypothesis that the puerarin protects against enhanced liver injury caused by Ni in mice. ICR mice received intraperitoneally nickel sulfate (20 mg/kg/body weight, daily) for 20 days, and puerarin (200 and 400 mg/kg/body weight) was applied before Ni exposure. The results indicated that puerarin markedly inhibited Ni-induced liver injury, which was characterized by decreased aminotransferase activities and inflammation. Puerarin also inhibited the oxidative stress and decreased the metallothionein (MT) levels. Puerarin decreased the level of pro-inflammatory cytokines TNF-α and IL-6 in livers. Puerarin significantly inhibited the TLR4 activation and p38 MAPK phosphorylation, which in turn inhibited NF-κB activity. Likewise, Ni-induced inflammatory responses were diminished by puerarin as observed by a remarkable reduction in the levels of phosphorylated CREB. Furthermore, puerarin also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) levels in livers. Data from this study suggested that the inhibition of Ni-induced oxidative stress and inflammatory responses by puerarin is due to its ability to modulate the TLR4/p38/CREB signaling pathway.

  15. N-palmitoyl serotonin alleviates scopolamine-induced memory impairment via regulation of cholinergic and antioxidant systems, and expression of BDNF and p-CREB in mice.

    PubMed

    Min, A Young; Doo, Choon Nan; Son, Eun Jung; Sung, Nak Yun; Lee, Kun Jong; Sok, Dai-Eun; Kim, Mee Ree

    2015-12-05

    N-Palmitoyl-5-hydroxytryptamines (Pal-5HT), a cannabinoid, has recently been reported to express anti-allergic and anti-inflammatory actions in RBL-2H3 cells, and ameliorate glutamate-induced cytotoxicity in HT-22 cells. In this study, we examined the effect of Pal-5HT on deficits of learning and memory induced by scopolamine in mice. Memory performance was evaluated using Morris water maze test and passive avoidance test. Activities of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT), level of oxidative stress markers, and expression of brain-derived neurotrophic factor (BDNF), phosphorylation of cAMP response element-binding protein (p-CREB) were determined. Loss of neuronal cells in hippocampus was evaluated by histological examinations. Pal-5HT significantly improved the amnesia in the behavioral assessment. Pal-5HT regulated cholinergic function by inhibiting scopolamine-induced elevation of AChE activity and decline of ChAT activity. Pal-5HT suppressed oxidative stress by increasing activities of glutathione peroxidase (GPx), glutathione reductase (GR) or NAD(P)H quinine oxidoreductase-1 (NQO-1) and lowering MDA level. Additionally, it prevented against scopolamine-induced expression of iNOS and COX-2. Moreover, Pal-5HT suppressed the death of neuronal cells in CA1 and CA3 regions, while it restored expression of p-CREB and BDNF in hippocampus. Taken together, Pal-5HT is suggested to ameliorate deficits of memory and learning through regulation of cholinergic function, activation of antioxidant systems as well as restoration of BDNF and p-CREB expression. From these, Pal-5HT may be a potential candidate to prevent against neurodegeneration related to the memory deficit.

  16. IDENTIFICATION OF CHOLINERGIC AND NONCHOLINERGIC NEURONS IN THE PONS EXPRESSING PHOSPHORYLATED cAMP RESPONSE ELEMENT-BINDING PROTEIN (pCREB) AS A FUNCTION OF RAPID EYE MOVEMENT (REM) SLEEP

    PubMed Central

    DATTA, S.; SIWEK, D. F.; STACK, E. C.

    2009-01-01

    Recent studies have shown that in the pedunculopontine tegmental nucleus (PPT), increased neuronal activity and kainate receptor-mediated activation of intracellular protein kinase A (PKA) are important physiological and molecular steps for the generation of REM sleep. In the present study performed on rats, phosphorylated cAMP response element-binding protein (pCREB) immunostaining was used as a marker for increased intracellular PKA activation and as a reflection of increased neuronal activity. To identify whether activated cells were either cholinergic or noncholinergic, the PPT and laterodorsal tegmental nucleus (LDT) cells were immunostained for choline acetyltransferase (ChAT) in combination with pCREB or c-Fos. The results demonstrated that during high REM sleep (HR, ~27%), significantly higher numbers of cells expressed pCREB and c-Fos in the PPT, of which 95% of pCREB-expressing cells were ChAT-positive. With high REM sleep, the numbers of pCREB-positive cells were also significantly higher in the medial pontine reticular formation (mPRF), pontine reticular nucleus oral (PnO), and dorsal subcoeruleus nucleus (SubCD) but very few in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). Conversely, with low REM sleep (LR, ~2%), the numbers of pCREB expressing cells were very few in the PPT, mPRF, PnO, and SubCD but significantly higher in the LC and DRN. The results of regression analyses revealed significant positive relationships between the total percentages of REM sleep and numbers of ChAT+/pCREB+ (Rsqr = 0.98) cells in the PPT and pCREB+ cells in the mPRF (Rsqr = 0.88), PnO (Rsqr = 0.87), and SubCD (Rsqr = 0.84); whereas significantly negative relationships were associated with the pCREB+ cells in the LC (Rsqr = 0.70) and DRN (Rsqr = 0.60). These results provide evidence supporting the hypothesis that during REM sleep, the PPT cholinergic neurons are active, whereas the LC and DRN neurons are inactive. More importantly, the regression analysis

  17. miR-134 regulates ischemia/reperfusion injury-induced neuronal cell death by regulating CREB signaling.

    PubMed

    Huang, Weidong; Liu, Xiaobin; Cao, Jie; Meng, Facai; Li, Min; Chen, Bo; Zhang, Jie

    2015-04-01

    microRNA-134 (miR-134) has been reported to be a brain-specific miRNA and is differently expressed in brain tissues subjected to ischemic injury. However, the underlying mechanism of miR-134 in regulating cerebral ischemic injury remains poorly understood. The current study was designed to delineate the molecular basis of miR-134 in regulating cerebral ischemic injury. Using the oxygen-glucose deprivation (OGD) model of hippocampal neuron ischemia in vitro, we found that the overexpression of miR-134 mediated by recombinant adeno-associated virus (AAV) vector infection significantly promoted neuron death induced by OGD/reoxygenation, whereas the inhibition of miR-134 provided protective effects against OGD/reoxygenation-induced cell death. Moreover, cyclic AMP (cAMP) response element-binding protein (CREB) as a putative target of miR-134 was downregulated and upregulated by miR-134 overexpression or inhibition, respectively. The direct interaction between miR-134 and the 3'-untranslated region (UTR) of CREB mRNA was further confirmed by dual-luciferase reporter assay. Overexpression of miR-134 also inhibited the expression of the downstream gene of CREB, including brain-derived neurotrophic factor (BDNF) and the anti-apoptotic gene Bcl-2, whereas the inhibition of miR-134 upregulated the expression of BDNF and Bcl-2 in neurons after OGD/reoxygenation. Notably, the knockdown of CREB by CREB siRNA apparently abrogated the protective effect of anti-miR-134 on OGD/reoxygenation-induced cell death. Taken together, our study suggests that downregulation of miR-134 alleviates ischemic injury through enhancing CREB expression and downstream genes, providing a promising and potential therapeutic target for cerebral ischemic injury.

  18. Reversion of BDNF, Akt and CREB in Hippocampus of Chronic Unpredictable Stress Induced Rats: Effects of Phytochemical, Bacopa Monnieri

    PubMed Central

    Hazra, Somoday; Kumar, Sourav; Saha, Goutam Kumar

    2017-01-01

    Objective The aims of the present study were to explore the behavioural effects and to understand the possible mode of action of Bacopa monnieri extract (BME) on chronic unpredictable stress (CUS) induced depressive model and the biochemical alterations such as brain derived neurotrophic factor (BDNF), Akt, cyclic-AMP response element binding (CREB) protein level in the hippocampus of rats. Methods We examined the effects of chronic administration of BME on CUS exposed rats for 28 days. Behavioural changes were assessed by sucrose consumption and open field test to assess the effect of BME on CUS-induced depression. The mechanisms underlying antidepressant like action of BME was further evaluated by measuring levels of BDNF, Akt, and CREB in the hippocampus of rat brain and compared with the standard tricyclic antidepressant drug imipramine (20 mg/kg body weight). Results Exposure to CUS for 28 days produced depression-like behavior in rats, as indicated by significant decreases in sucrose consumption, locomotor activity including decreased BDNF, Akt and CREB levels in the hippocampus. Daily administration of BME at a dose of (80 mg/kg body weight) significantly reverses the behavioral alteration and restored the normal level of BDNF, total and phospho-Akt, total and phospho CREB in the hippocampus of CUS induced rats as compared to vehicle treated control rats. Conclusion These findings suggest that BME ameliorates CUS induced behavioural depression in rats and that can be used as a potent therapeutic agent in treating depressive like behavior. PMID:28096878

  19. RGS17, an Overexpressed Gene in Human Lung and Prostate Cancer, Induces Tumor Cell Proliferation Through the Cyclic AMP-PKA-CREB Pathway

    PubMed Central

    James, Michael A.; Lu, Yan; Liu, Yan; Vikis, Haris G.; You, Ming

    2009-01-01

    We have identified RGS17 as a commonly induced gene in lung and prostate tumors. Through microarray and gene expression analysis, we show that expression of RGS17 is up-regulated in 80% of lung tumors, and also up-regulated in prostate tumors. Through knockdown and overexpression of RGS17 in tumor cells, we show that RGS17 confers a proliferative phenotype and is required for the maintenance of the proliferative potential of tumor cells. We show through exon microarray, transcript analysis, and functional assays that RGS17 promotes cyclic AMP (cAMP)-responsive element binding protein (CREB)-responsive gene expression, increases cAMP levels, and enhances forskolin-mediated cAMP production. Furthermore, inhibition of cAMP-dependent kinase prevents tumor cell proliferation, and proliferation is partially rescued by RGS17 overexpression. In the present study, we show a role for RGS17 in the maintenance of tumor cell proliferation through induction of cAMP signaling and CREB phosphorylation. The prevalence of the induction of RGS17 in tumor tissues of various types further implicates its importance in the maintenance of tumor growth. PMID:19244110

  20. Differential regulation of c-jun and CREB by acrolein and 4-hydroxynonenal.

    PubMed

    Pugazhenthi, Subbiah; Phansalkar, Ketaki; Audesirk, Gerald; West, Anne; Cabell, Leigh

    2006-01-01

    In Alzheimer's disease (AD), oxidative stress-induced lipid peroxidation leads to accumulation of unsaturated aldehydes including acrolein and 4-hydroxynonenal (4HNE) in brain. In this study, we examined the effects of these lipid peroxidation products on apoptotic pathways in cultured neurons. Acrolein and 4HNE increased the levels of active phosphorylated forms of c-jun and CREB, the transcription factors that promote apoptosis and cell survival, respectively. However, they decreased the activity of CREB-dependent BDNF promoter while they increased the activity of promoters responsive to c-jun. We hypothesized that this differential regulation could be due to competition between proapoptotic c-jun and cytoprotective CREB for CBP (CREB-binding protein), a coactivator shared by several transcription factors. In support of this hypothesis, we demonstrate that the decrease of BDNF promoter activity by acrolein and 4HNE could be restored (i) by cotransfection with CBP, (ii) by cotransfection with VP 16-CREB, a constitutively active form of CREB that does not depend on CBP for its activation, or (iii) by inhibiting JNK-mediated c-jun activation. Finally, adenoviral transduction of hippocampal neurons with VP 16-CREB resulted in significant reduction in caspase-3 activation by acrolein and 4HNE. These observations suggest that lipid peroxidation-induced differential regulation of CREB and c-jun might play a role in neurodegeneration in AD.

  1. Essential role of CREB family proteins during Xenopus embryogenesis.

    PubMed

    Lutz, B; Schmid, W; Niehrs, C; Schütz, G

    1999-10-01

    The leucine zipper transcription factors cAMP response element binding protein (CREB), cAMP response element modulatory protein (CREM) and activating transcription factor 1 (ATF1) bind to the cAMP response element (CRE) with the palindromic consensus sequence TGACGTCA. Their transcriptional activities are dependent on serine phosphorylation induced by various extracellular signals such as hormones, growth factors and neurotransmitters. Here we show that CREB is the predominant CRE-binding protein in Xenopus embryos and that it plays an essential role during early development. The importance of CREB for morphogenetic processes was assessed by injection of RNA encoding a dominant-negative form of CREB that is fused to a truncated progesterone receptor ligand binding domain. In this fusion protein, a dominant-negative function can be induced by application of the synthetic steroid RU486 at given developmental stages. The inhibition of CREB at blastula and early gastrula stages leads to severe posterior defects of the embryos reflected by strong spina bifida, whereas the inhibition of CREB at the beginning of neurulation resulted in stunted embryos with microcephaly. In these embryos, initial induction of neural and mesodermal tissues is not dependent on CREB function, as genes such as Otx2, Krox20, Shh and MyoD are still expressed in injected embryos. But the expression domains of Otx2 and MyoD were found to be distorted reflecting the abnormal development in both neural and somitic derivatives. In summary, our data show that CREB is essential during several developmental stages of Xenopus embryogenesis.

  2. Hippocampal cAMP/PKA/CREB is required for neuroprotective effect of acupuncture.

    PubMed

    Li, Qian-Qian; Shi, Guang-Xia; Yang, Jing-Wen; Li, Zhao-Xin; Zhang, Zhen-Hua; He, Tian; Wang, Jing; Liu, Li-Ying; Liu, Cun-Zhi

    2015-02-01

    Acupuncture has beneficial effects in vascular dementia (VaD) patients. The underlying mechanism, however, remains unknown. The present study was designed to investigate whether the cAMP/PKA/CREB cascade is involved in the mechanism of acupuncture in cerebral multi-infarction rats. In this study, cerebral multi-infarction was modeled in adult Wistar rats by homologous blood clot emboli. After a two-week acupuncture treatment at Zusanli (ST36), hippocampal-dependent memory was tested by employing a radial arm maze test. The hippocampus was isolated for analyses of cAMP concentration, phosphodiesterase (PDE) activity and CREB/pCREB and ERK/pERK expressions. The Morris water maze (MWM) task and CREB phosphorylation were evaluated in the presence of PKA-selective peptide inhibitor (H89). The radial arm maze test results demonstrated that acupuncture treatment at ST36 reversed hippocampal-dependent memory in impaired animals. Compared to those of the impaired group, cAMP concentration, PKA activity and pCREB and pERK expressions were increased following acupuncture therapy. Finally, the blockade of PKA reversed the increase in CREB phosphorylation and the improvement in recognitive function induced by acupuncture treatment. These results suggest that acupuncture could improve hippocampus function by modulating the cAMP/PKA/CREB signaling pathway, which represents a molecular mechanism of acupuncture for recognitive function in cerebral multi-infarction rats.

  3. Involvement of PI3K/Akt/FoxO3a and PKA/CREB Signaling Pathways in the Protective Effect of Fluoxetine Against Corticosterone-Induced Cytotoxicity in PC12 Cells.

    PubMed

    Zeng, Bingqing; Li, Yiwen; Niu, Bo; Wang, Xinyi; Cheng, Yufang; Zhou, Zhongzhen; You, Tingting; Liu, Yonggang; Wang, Haitao; Xu, Jiangping

    2016-08-01

    The selective serotonin reuptake inhibitor fluoxetine is neuroprotective in several brain injury models. It is commonly used to treat major depressive disorder and related conditions, but its mechanism of action remains incompletely understood. Activation of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FoxO3a) and protein kinase A/cAMP-response element binding protein (PKA/CREB) signaling pathways has been strongly implicated in the pathogenesis of depression and might be the downstream target of fluoxetine. Here, we used PC12 cells exposed to corticosterone (CORT) to study the neuroprotective effects of fluoxetine and the involvement of the PI3K/Akt/FoxO3a and PKA/CREB signaling pathways. Our results show that CORT reduced PC12 cells viability by 70 %, and that fluoxetine showed a concentration-dependent neuroprotective effect. Neuroprotective effects of fluoxetine were abolished by inhibition of PI3K, Akt, and PKA using LY294002, KRX-0401, and H89, respectively. Treatment of PC12 cells with fluoxetine resulted in increased phosphorylation of Akt, FoxO3a, and CREB. Fluoxetine also dose-dependently rescued the phosphorylation levels of Akt, FoxO3a, and CREB, following administration of CORT (from 99 to 110, 56 to 170, 80 to 170 %, respectively). In addition, inhibition of PKA and PI3K/Akt resulted in decreased levels of p-CREB, p-Akt, and p-FoxO3a in the presence of fluoxetine. Furthermore, fluoxetine reversed CORT-induced upregulation of p53-upregulated modulator of apoptosis (Puma) and Bcl-2-interacting mediator of cell death (Bim) via the PI3K/Akt/FoxO3a signaling pathway. H89 treatment reversed the effect of fluoxetine on the mRNA level of brain-derived neurotrophic factor, which was decreased in the presence of CORT. Our data indicate that fluoxetine elicited neuroprotection toward CORT-induced cell death that involves dual regulation from PI3K/Akt/FoxO3a and PKA/CREB pathways.

  4. MiR-582-5p/miR-590-5p targeted CREB1/CREB5–NF-κB signaling and caused opioid-induced immunosuppression in human monocytes

    PubMed Central

    Long, X; Li, Y; Qiu, S; Liu, J; He, L; Peng, Y

    2016-01-01

    Chronic opioid abusers are more susceptible to bacterial and viral infections, but the molecular mechanism underlying opioid-induced immunosuppression is unknown. MicroRNAs (miRNAs) are emerging as key players in the control of biological processes, and may participate in immune regulation. In this study, we investigated the molecular mechanisms in opioid-induced and miRNA-mediated immunosuppression, in the context of miRNA dysregulation in opioid abusers. Blood samples of heroin abusers were collected and analyzed using miRNA microarray analysis and quantitative PCR validation. The purified primary human monocytes were cultured in vitro to explore the underlying mechanism. We found that morphine and its derivative heroin significantly decreased the expression levels of miR-582-5p and miR-590-5p in monocytes. cAMP response element-binding protein 1 (CREB1) and CREB5 were detected as direct target genes of miR-582-5p and miR-590-5p, respectively, by using dual-luciferase assay and western bolt. Functional studies showed that knockdown of CREB1/CREB5 increased tumor necrosis factor alpha (TNF-α) level and enhanced expression of phospho–NF-κB p65 and NF-κB p65. Our results demonstrated that miR-582-5p and miR-590-5p play important roles in opioid-induced immunosuppression in monocytes by targeting CREB1/CREB5–NF-κB signaling pathway. PMID:26978739

  5. Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity.

    PubMed

    Zhang, Juan; Tang, Hongju; Deng, Ruyuan; Wang, Ning; Zhang, Yuqing; Wang, Yao; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

    2015-01-01

    Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been demonstrated to lower blood glucose, blood lipid, and body weight in patients with type 2 diabetes mellitus. The anti-obesity effect of berberine has been attributed to its anti-adipogenic activity. However, the underlying molecular mechanism remains largely unknown. In the present study, we found that berberine significantly suppressed the expressions of CCAAT/enhancer-binding protein (C/EBP)α, peroxisome proliferators-activated receptor γ2 (PPARγ2), and other adipogenic genes in the process of adipogenesis. Berberine decreased cAMP-response element-binding protein (CREB) phosphorylation and C/EBPβ expression at the early stage of 3T3-L1 preadipocyte differentiation. In addition, CREB phosphorylation and C/EBPβ expression induced by 3-isobutyl-1-methylxanthine (IBMX) and forskolin were also attenuated by berberine. The binding activities of cAMP responsive element (CRE) stimulated by IBMX and forskolin were inhibited by berberine. The binding of phosphorylated CREB to the promoter of C/EBPβ was abrogated by berberine after the induction of preadipocyte differentiation. These results suggest that berberine blocks adipogenesis mainly via suppressing CREB activity, which leads to a decrease in C/EBPβ-triggered transcriptional cascades.

  6. Tetramethylpyrazine protects against scopolamine-induced memory impairments in rats by reversing the cAMP/PKA/CREB pathway.

    PubMed

    Wu, Wei; Yu, Xiao; Luo, Xiao-Ping; Yang, Shu-Hua; Zheng, Dong

    2013-09-15

    Tetramethylpyrazine is used in the treatment of many neurological diseases because of its neuroprotective effect. Here, we demonstrate that administration of tetramethylpyrazine effectively reverses memory deficits induced by scopolamine. Moreover, tetramethylpyrazine preserves postsynaptic protein synthesis and restores cAMP/PKA/CREB pathway signaling deficits. Our study not only explores the actions of tetramethylpyrazine on synapses, but also provides novel evidence for the possible therapeutic use of tetramethylpyrazine in dementia.

  7. β-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H

    PubMed Central

    Cheng, Yun; Gao, Wei-Wei; Tang, Hei-Man Vincent; Deng, Jian-Jun; Wong, Chi-Ming; Chan, Chi-Ping; Jin, Dong-Yan

    2016-01-01

    CREB-H is an endoplasmic reticulum-resident bZIP transcription factor which critically regulates lipid homeostasis and gluconeogenesis in the liver. CREB-H is proteolytically activated by regulated intramembrane proteolysis to generate a C-terminally truncated form known as CREB-H-ΔTC, which translocates to the nucleus to activate target gene expression. CREB-H-ΔTC is a fast turnover protein but the mechanism governing its destruction was not well understood. In this study, we report on β-TrCP-dependent ubiquitination and proteasomal degradation of CREB-H-ΔTC. The degradation of CREB-H-ΔTC was mediated by lysine 48-linked polyubiquitination and could be inhibited by proteasome inhibitor. CREB-H-ΔTC physically interacted with β-TrCP, a substrate recognition subunit of the SCFβ-TrCP E3 ubiquitin ligase. Forced expression of β-TrCP increased the polyubiquitination and decreased the stability of CREB-H-ΔTC, whereas knockdown of β-TrCP had the opposite effect. An evolutionarily conserved sequence, SDSGIS, was identified in CREB-H-ΔTC, which functioned as the β-TrCP-binding motif. CREB-H-ΔTC lacking this motif was stabilized and resistant to β-TrCP-induced polyubiquitination. This motif was a phosphodegron and its phosphorylation was required for β-TrCP recognition. Furthermore, two inhibitory phosphorylation sites close to the phosphodegron were identified. Taken together, our work revealed a new intracellular signaling pathway that controls ubiquitination and degradation of the active form of CREB-H transcription factor. PMID:27029215

  8. Reduced phosphoCREB in Müller glia during retinal degeneration in rd10 mice

    PubMed Central

    Bachleda, Amelia; Xiong, Yubin; Osawa, Shoji; Weiss, Ellen R.

    2017-01-01

    Purpose The mechanisms that trigger retinal degeneration are not well understood, despite the availability of several animal models with different mutations. In the present report, the rd10 mouse, a model for retinitis pigmentosa (RP) that contains a mutation in the gene for PDE6β (Pde6b), is used to evaluate gliosis, as a marker for retinal stress, and cyclic AMP response element binding protein (CREB) phosphorylation, which may be important early in retinal degeneration. Methods Wild-type C57Bl6J and rd10 mice raised under cyclic light were examined for changes in gliosis and CREB phosphorylation for approximately 3 weeks beginning at P14 to P17 using immunocytochemistry. Mice raised under normal cyclic light and in complete darkness were also compared for changes in CREB phosphorylation. Results Gliosis in rd10 mice raised under cyclic light was apparent at P17, before extensive degeneration of the photoreceptor layer is visible, and increased over time. Phosphorylation of CREB at Ser133 (pCREB) was detected in Müller glia (MG) in the wild-type and rd10 mice. However, at all phases of photoreceptor degeneration, the pCREB levels were lower in the rd10 mice. We also observed extensive migration of MG cell bodies to the outer nuclear layer (ONL) during degeneration. In contrast to the mice raised under cyclic light, the rd10 mice raised in the dark exhibited slower rates of degeneration. When the dark-reared mice were exposed to cyclic light, the photoreceptor layer degenerated within 4 days to approximately one to two rows of nuclei. Interestingly, the pCREB levels in the MG also decreased during this 4-day cyclic light exposure compared to the levels in the rd10 mice raised continuously in the dark. Conclusions The results of these studies suggest that photoreceptors communicate directly or indirectly with MG at early stages, inducing gliosis before extensive retinal degeneration is apparent in rd10 mice. Surprisingly, phosphorylation of CREB is downregulated

  9. Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

    PubMed Central

    Almeida, Luis E. F.; Murray, Peter D.; Zielke, H. Ronald; Roby, Clinton D.; Kingsbury, Tami J.; Krueger, Bruce K.

    2009-01-01

    Cyclic AMP can stimulate the transcription of many activity-dependent genes via activation of the transcription factor, CREB. However, in mouse cortical neuron cultures, prior to synaptogenesis, neither cAMP nor dopamine, which acts via cAMP, stimulated CREB-dependent gene transcription when NR2B-containing NMDA receptors (NMDARs) were blocked. Stimulation of transcription by cAMP was potentiated by inhibitors of excitatory amino acid uptake, suggesting a role for extracellular glutamate or aspartate in cAMP-induced transcription. Aspartate was identified as the extracellular messenger: enzymatic scavenging of L-aspartate, but not glutamate, blocked stimulation of CREB-dependent gene transcription by cAMP; moreover, cAMP induced aspartate but not glutamate release. Taken together, these results suggest that cAMP acts via an autocrine or paracrine pathway to release aspartate, which activates NR2B-containing NMDARs, leading to Ca2+ entry and activation of transcription. This cAMP/aspartate/NMDAR signaling pathway may mediate the effects of transmitters such as dopamine on axon growth and synaptogenesis in developing neurons or on synaptic plasticity in mature neural networks. PMID:19812345

  10. Renal Proximal Tubule Na,K-ATPase is Controlled by CREB Regulated Transcriptional CoActivators as well as Salt Inducible Kinase 1

    PubMed Central

    Taub, Mary; Garamella, Sudha; Kim, Dongwook; Rajkhowa, Trivikram; Cutuli, Facundo

    2015-01-01

    Sodium reabsorption by the kidney is regulated by locally produced natriuretic and anti-natriuretic factors, including dopamine and norepinephrine, respectively. Previous studies indicated that signaling events initiated by these natriuretic and anti-natriuretic factors achieve their effects by altering the phosphorylation of Na,K-ATPase in the renal proximal tubule, and that Protein Kinase A (PKA) and Calcium mediated signaling pathways are involved. The same signaling pathways also control the transcription of the Na,K-ATPase β subunit gene atp1b1 in renal proximal tubule cells. In this report, evidence is presented that 1) both the recently discovered cAMP-Regulated Transcriptional Coactivators (CRTCs), and Salt Inducible Kinase 1 (SIK1) contribute to the transcriptional regulation of atp1b1 in renal proximal tubule (RPT) cells, and 2) that renal effectors including norepinephrine, dopamine, prostaglandins and sodium play a role. Exogenously expressed CRTCs stimulate atp1b1 transcription. Evidence for a role of endogenous CRTCs includes the loss of transcriptional regulation of atp1b1 by a dominant negative CRTC, as well as by a CREB mutant, with an altered CRTC binding site. In a number of experimental systems, SIK phosphorylates CRTCs, which are then sequestered in the cytoplasm, preventing their nuclear effects. Consistent with such a role of SIK in primary RPT cells, atp1b1 transcription increased in the presence of a dominant negative SIK1, and in addition, regulation by dopamine, norepinephrine and monensin was disrupted by a dominant negative SIK1. These latter observations can be explained, if SIK1 is phosphorylated and inactivated in the presence of these renal effectors. Our results support the hypothesis that Na,K-ATPase in the renal proximal tubule is regulated at the transcriptional level via SIK1 and CRTCs by renal effectors, in addition to the previously reported control of the phosphorylation of Na,K-ATPase. PMID:26432356

  11. Measuring CREB activation using bioluminescent probes that detect KID-KIX interaction in living cells.

    PubMed

    Ishimoto, Tetsuya; Mano, Hiroki; Ozawa, Takeaki; Mori, Hisashi

    2012-05-16

    The cyclic adenosine monophosphate response element-binding protein (CREB) is a transcription factor that contributes to memory formation. The transcriptional activity of CREB is induced by its phosphorylation at Ser-133 and subsequent interaction with the CREB-binding protein (CBP)/p300. We designed and optimized firefly split luciferase probe proteins that detect the interaction of the kinase-inducible domain (KID) of CREB and the KIX domain of CBP/p300. The increase in the light intensity of the probe proteins results from the phosphorylation of the responsible serine corresponding to Ser-133 of CREB. Because these proteins have a high signal-to-noise ratio and are nontoxic, it has become possible for the first time to carry out long-term measurement of KID-KIX interaction in living cells. Furthermore, we examined the usefulness of the probe proteins for future high-throughput cell-based drug screening and found several herbal extracts that activated CREB.

  12. Stochasticity and bifurcations in a reduced model with interlinked positive and negative feedback loops of CREB1 and CREB2 stimulated by 5-HT.

    PubMed

    Hao, Lijie; Yang, Zhuoqin; Bi, Yuanhong

    2016-04-01

    The cyclic AMP (cAMP)-response element-binding protein (CREB) family of transcription factors is crucial in regulating gene expression required for long-term memory (LTM) formation. Upon exposure of sensory neurons to the neurotransmitter serotonin (5-HT), CREB1 is activated via activation of the protein kinase A (PKA) intracellular signaling pathways, and CREB2 as a transcriptional repressor is relieved possibly via phosphorylation of CREB2 by mitogen-activated protein kinase (MAPK). Song et al. [18] proposed a minimal model with only interlinked positive and negative feedback loops of transcriptional regulation by the activator CREB1 and the repressor CREB2. Without considering feedbacks between the CREB proteins, Pettigrew et al. [8] developed a computational model characterizing complex dynamics of biochemical pathways downstream of 5-HT receptors. In this work, to describe more simply the biochemical pathways and gene regulation underlying 5-HT-induced LTM, we add the important extracellular sensitizing stimulus 5-HT as well as the product Ap-uch into the Song's minimal model. We also strive to examine dynamical properties of the gene regulatory network under the changing concentration of the stimulus, [5-HT], cooperating with the varying positive feedback strength in inducing a high state of CREB1 for the establishment of long-term memory. Different dynamics including monostability, bistability and multistability due to coexistence of stable steady states and oscillations is investigated by means of codimension-2 bifurcation analysis. At the different positive feedback strengths, comparative analysis of deterministic and stochastic dynamics reveals that codimension-1 bifurcation with respect to [5-HT] as the parameter can predict diverse stochastic behaviors resulted from the finite number of molecules, and the number of CREB1 molecules more and more preferentially resides near the high steady state with increasing [5-HT], which contributes to long

  13. Region-dependent dynamics of cAMP response element-binding protein phosphorylation in the basal ganglia

    PubMed Central

    Liu, Fu-Chin; Graybiel, Ann M.

    1998-01-01

    The cAMP response element-binding protein (CREB) is an activity-dependent transcription factor that is involved in neural plasticity. The kinetics of CREB phosphorylation have been suggested to be important for gene activation, with sustained phosphorylation being associated with downstream gene expression. If so, the duration of CREB phosphorylation might serve as an indicator for time-sensitive plastic changes in neurons. To screen for regions potentially involved in dopamine-mediated plasticity in the basal ganglia, we used organotypic slice cultures to study the patterns of dopamine- and calcium-mediated CREB phosphorylation in the major subdivisions of the striatum. Different durations of CREB phosphorylation were evoked in the dorsal and ventral striatum by activation of dopamine D1-class receptors. The same D1 stimulus elicited (i) transient phosphorylation (≤15 min) in the matrix of the dorsal striatum; (ii) sustained phosphorylation (≤2 hr) in limbic-related structures including striosomes, the nucleus accumbens, the fundus striati, and the bed nucleus of the stria terminalis; and (iii) prolonged phosphorylation (up to 4 hr or more) in cellular islands in the olfactory tubercle. Elevation of Ca2+ influx by stimulation of L-type Ca2+ channels, NMDA, or KCl induced strong CREB phosphorylation in the dorsal striatum but not in the olfactory tubercle. These findings differentiate the response of CREB to dopamine and calcium signals in different striatal regions and suggest that dopamine-mediated CREB phosphorylation is persistent in limbic-related regions of the neonatal basal ganglia. The downstream effects activated by persistent CREB phosphorylation may include time-sensitive neuroplasticity modulated by dopamine. PMID:9539803

  14. cAMP Response Element-binding Protein (CREB) and Nuclear Factor κB Mediate the Tamoxifen-induced Up-regulation of Glutamate Transporter 1 (GLT-1) in Rat Astrocytes*

    PubMed Central

    Karki, Pratap; Webb, Anton; Smith, Keisha; Lee, Kyuwon; Son, Deok-Soo; Aschner, Michael; Lee, Eunsook

    2013-01-01

    Tamoxifen (TX), a selective estrogen receptor modulator, exerts antagonistic effects on breast tissue and is used to treat breast cancer. Recent evidence also suggests that it may act as an agonist in brain tissue. We reported previously that TX enhanced the expression and function of glutamate transporter 1 (GLT-1) in rat astrocytes, an effect that was mediated by TGF-α. To gain further insight into the mechanisms that mediate TX-induced up-regulation of GLT-1 (EAAT2 in humans), we investigated its effect on GLT-1 at the transcriptional level. TX phosphorylated the cAMP response element-binding protein (CREB) and recruited CREB to the GLT-1 promoter consensus site. The effect of TX on astrocytic GLT-1 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway. In addition, the effect of TX on GLT-1 promoter activity was abolished by the inhibition of the NF-κB pathway. Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1 promoter. Mutation of NF-κB (triple, −583/-282/-251) or CRE (-308) sites on the GLT-1 promoter led to significant repression of the promoter activity, but neither mutant completely abolished the TX-induced GLT-1 promoter activity. Mutation of both the NF-κB (-583/-282/-251) and CRE (-308) sites led to a complete abrogation of the effect of TX on GLT-1 promoter activity. Taken together, our findings establish that TX regulates GLT-1 via the CREB and NF-κB pathways. PMID:23955341

  15. The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells

    PubMed Central

    Menga, Alessio; Iacobazzi, Vito; Infantino, Vittoria; Avantaggiati, Maria Laura; Palmieri, Ferdinando

    2015-01-01

    The aspartate/glutamate carrier isoform 1 is an essential mitochondrial transporter that exchanges intramitochondrial aspartate and cytosolic glutamate across the inner mitochondrial membrane. It is expressed in brain, heart and muscle and is involved in important biological processes, including myelination. However, the signals that regulate the expression of this transporter are still largely unknown. In this study we first identify a CREB binding site within the aspartate/glutamate carrier gene promoter that acts as a strong enhancer element in neuronal SH-SY5Y cells. This element is regulated by active, phosphorylated CREB protein and by signal pathways that modify the activity of CREB itself and, most noticeably, by intracellular Ca2+ levels. Specifically, aspartate/glutamate carrier gene expression is induced via CREB by forskolin while it is inhibited by the PKA inhibitor, H89. Furthermore, the CREB-induced activation of gene expression is increased by thapsigargin, which enhances cytosolic Ca2+, while it is inhibited by BAPTA-AM that reduces cytosolic Ca2+ or by STO-609, which inhibits CaMK-IV phosphorylation. We further show that CREB-dependent regulation of aspartate/glutamate carrier gene expression occurs in neuronal cells in response to pathological (inflammation) and physiological (differentiation) conditions. Since this carrier is necessary for neuronal functions and is involved in myelinogenesis, our results highlight that targeting of CREB activity and Ca2+ might be therapeutically exploited to increase aspartate/glutamate carrier gene expression in neurodegenerative diseases. PMID:25597433

  16. Hemorrhage induces rapid in vivo activation of CREB and NF-kappaB in murine intraparenchymal lung mononuclear cells.

    PubMed

    Shenkar, R; Abraham, E

    1997-02-01

    Increased expression of proinflammatory cytokines appears to be an important factor contributing to the development of acute lung injury. In murine models, mRNA levels of proinflammatory and immunoregulatory cytokines, including IL-1alpha, IL-1beta, TGF-beta1, and TNF-alpha, are increased in intraparenchymal lung mononuclear cells 1 h after hemorrhage. Binding elements for the nuclear transcriptional regulatory factors, nuclear factor kappaB (NF-kappaB), CCAAT/enhancer binding protein beta (C/EBPbeta), serum protein 1 (Sp1), activator protein 1 (AP-1), and the cyclic AMP response-element binding protein (CREB) are present in the promoter regions of numerous cytokine genes, including those whose expression is increased after blood loss. To investigate early transcriptional mechanisms which may be involved in regulating pulmonary cytokine expression after hemorrhage, we examined in vivo activation of these five nuclear transcriptional factors among intraparenchymal lung mononuclear cells obtained in the immediate post-hemorrhage period. Activation of NF-kappaB and CREB, but not C/EBPbeta, Sp1, or AP-1, was present in lung mononuclear cells isolated from mice 15 min after hemorrhage. Inhibition of xanthine oxidase by prior feeding with either an allopurinol-supplemented or a tungsten-enriched diet prevented hemorrhage-induced activation of CREB, but not NF-kappaB. These results demonstrate that hemorrhage leads to rapid in vivo activation in the lung of CREB through a xanthine oxidase-dependent mechanism and of NF-kappaB through other pathways, and suggest that the activation of these transcriptional factors may have an important role in regulating pulmonary cytokine expression and the development of acute lung injury after blood loss.

  17. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    PubMed

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants.

  18. CREB-mediated synaptogenesis and neurogenesis is crucial for the role of 5-HT1a receptors in modulating anxiety behaviors

    PubMed Central

    Zhang, Jing; Cai, Cheng-Yun; Wu, Hai-Yin; Zhu, Li-Juan; Luo, Chun-Xia; Zhu, Dong-Ya

    2016-01-01

    Serotonin 1a-receptor (5-HT1aR) has been specifically implicated in the pathogenesis of anxiety. However, the mechanism underlying the role of 5-HT1aR in anxiety remains poorly understood. Here we show in mice that the transcription factor cAMP response element binding protein (CREB) in the hippocampus functions as an effector of 5-HT1aR in modulating anxiety-related behaviors. We generated recombinant lentivirus LV-CREB133-GFP expressing a dominant negative CREB which could not be phosphorylated at Ser133 to specifically reduce CREB activity, and LV-VP16-CREB-GFP expressing a constitutively active fusion protein VP16-CREB which could be phosphorylated by itself to specifically enhance CREB activity. LV-CREB133-GFP neutralized 5-HT1aR agonist-induced up-regulation of synapse density, spine density, dendrite complexity, neurogenesis, and the expression of synapsin and spinophilin, two well-characterized synaptic proteins, and abolished the anxiolytic effect of 5-HT1aR agonist; whereas LV-VP16-CREB-GFP rescued the 5-HT1aR antagonist-induced down-regulation of synapse density, spine density, dendrite complexity, neurogenesis and synapsin and spinophilin expression, and reversed the anxiogenic effect of 5-HT1aR antagonist. The deletion of neurogenesis by irradiation or the diminution of synaptogenesis by knockdown of synapsin expression abolished the anxiolytic effects of both CREB and 5-HT1aR activation. These findings suggest that CREB-mediated hippoacampus structural plasticity is crucial for the role of 5-HT1aR in modulating anxiety-related behaviors. PMID:27404655

  19. Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

    PubMed

    Wang, Guannan; Matsuura, Isao; He, Dongming; Liu, Fang

    2009-04-10

    Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.

  20. Cyclophilin D deficiency rescues Aβ-impaired PKA/CREB signaling and alleviates synaptic degeneration.

    PubMed

    Du, Heng; Guo, Lan; Wu, Xiaoping; Sosunov, Alexander A; McKhann, Guy M; Chen, John Xi; Yan, Shirley ShiDu

    2014-12-01

    The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Cyclophilin D (CypD), an integral part of mitochondrial permeability transition pore (mPTP), is involved in amyloid beta (Aβ)-instigated mitochondrial dysfunction. Blockade of CypD prevents Aβ-induced mitochondrial malfunction and the consequent cognitive impairments. Here, we showed the elimination of reactive oxygen species (ROS) by antioxidants probucol or superoxide dismutase (SOD)/catalase blocks Aβ-mediated inactivation of protein kinase A (PKA)/cAMP regulatory-element-binding (CREB) signal transduction pathway and loss of synapse, suggesting the detrimental effects of oxidative stress on neuronal PKA/CREB activity. Notably, neurons lacking CypD significantly attenuate Aβ-induced ROS. Consequently, CypD-deficient neurons are resistant to Aβ-disrupted PKA/CREB signaling by increased PKA activity, phosphorylation of PKA catalytic subunit (PKA C), and CREB. In parallel, lack of CypD protects neurons from Aβ-induced loss of synapses and synaptic dysfunction. Furthermore, compared to the mAPP mice, CypD-deficient mAPP mice reveal less inactivation of PKA-CREB activity and increased synaptic density, attenuate abnormalities in dendritic spine maturation, and improve spontaneous synaptic activity. These findings provide new insights into a mechanism in the crosstalk between the CypD-dependent mitochondrial oxidative stress and signaling cascade, leading to synaptic injury, functioning through the PKA/CREB signal transduction pathway.

  1. CREB is activated by UVC through a p38/HOG-1-dependent protein kinase.

    PubMed Central

    Iordanov, M; Bender, K; Ade, T; Schmid, W; Sachsenmaier, C; Engel, K; Gaestel, M; Rahmsdorf, H J; Herrlich, P

    1997-01-01

    Changes in environmental conditions such as the addition of growth factors or irradiation of cells in culture first affect immediate response genes. We have shown previously that short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor-dependent pathways. At the level of the immediate response gene c-fos, these pathways activate the transcription factor complex serum response factor (SRF)-p62TCF which mediates part of the UV-induced transcriptional response. These studies have, however, suggested that more that one pathway is required for full UV responsiveness of c-fos. Using appropriate promoter mutations and dominant-negative cAMP response element (CRE)-binding protein (CREB), we now find that UVC-induced transcriptional activation depends also on the CRE at position -60 of the c-fos promoter and on the functionality of a CREB. Upon UV irradiation, CREB and ATF-1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG-1 and of a p38/RK/HOG-1-dependent p108 CREB kinase. Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not, at least not decisively, participate in this signalling pathway to CREB and ATF-1 as it is not p38/RK/HOG-1 dependent, and CREB is a poor substrate for MAPKAP kinase 2 in vitro. On the basis of resistance to the growth factor receptor inhibitor suramin and of several types of cross-refractoriness experiments, the UVC-induced CREB/ATF-1 phosphorylation represents an as yet unrecognized route of UVC-induced signal transduction, independent of suramin-inhibitable growth factor receptors and different from the Erk 1,2-p62TCF pathway. PMID:9118940

  2. Enriched environment prevents hypobaric hypoxia induced memory impairment and neurodegeneration: role of BDNF/PI3K/GSK3β pathway coupled with CREB activation.

    PubMed

    Jain, Vishal; Baitharu, Iswar; Prasad, Dipti; Ilavazhagan, Govindasamy

    2013-01-01

    Adverse environmental conditions such as hypobaric hypoxia (HH) cause memory impairment by affecting cellular machinery leading to neurodegeneration. Providing enriched environment (EE) is found to be beneficial for curing several neurodegenerative disorders. The protective role of EE in preventing HH induced neuronal death has been reported previously but the involved mechanism is still not clearly understood. The present study is an attempt to verify the impact of EE on spatial memory during HH and also to explore the possible role of neurotrophin in EE mediated neuroprotection. Signaling mechanism involved in neuroprotection was also explored. Male Sprague Dawley rats were simulated to HH condition in an Animal Decompression Chamber at an altitude of 25000 feet in standard and enriched cages for 7 days. Spatial memory was assessed through Morris Water Maze. Role of different neurotrophins was explored by gene silencing and inhibitors for their respective receptors. Further, using different blockers signaling pathway was also explored. Finding of the present study suggested that EE prevents HH mediated memory impairment and neurodegeneration. Also brain-derived neurotrophic factor (BDNF) plays a major role in EE mediated neuroprotection and it effectively prevented neurodegeneration by activating PI3K/AKT pathway resulting in GSK3β inactivation which further inhibits apoptosis. Moreover GSK3β phosphorylation and hence its inactivation upregulates CREB phosphorylation which may also accounts for activation of survival machinery in cells and provides neuroprotection. From these observations it can be postulated that EE has a therapeutic potential in amelioration of HH induced memory impairment and neurodegeneration. Hence it may be used as a non invasive and non pharmacological intervention against various neurological disorders.

  3. Enhanced phosphorylation of cyclic AMP response element binding protein in Brain of mice following repetitive hypoxic exposure

    SciTech Connect

    Gao Yanan; Gao Ge; Long Caixia; Han Song; Zu Pengyu; Fang Li . E-mail: lfang@utmb.edu; Li Junfa . E-mail: junfali@cpums.edu.cn

    2006-02-10

    Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders Brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in Brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly (p < 0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2-H4, n = 6 for each group), when compared to that of the normoxic (H0, n = 6) or hypoxic exposure once group (H1, n = 6). In addition, a significant enhancement (p < 0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2-H4, n = 6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning.

  4. Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization

    PubMed Central

    Steven, André; Leisz, Sandra; Sychra, Katharina; Hiebl, Bernhard; Wickenhauser, Claudia; Mougiakakos, Dimitrios; Kiessling, Rolf; Denkert, Carsten; Seliger, Barbara

    2016-01-01

    The cAMP-responsive element-binding protein (CREB) is involved in the tumorigenicity of HER-2/neu-overexpressing murine and human tumor cells, but a link between the HER-2/neu-mediated CREB activation, its posttranslational modification and localization and changes in the cellular metabolism, due to an altered (tumor) microenvironment remains to be established. The present study demonstrated that shRNA-mediated silencing of CREB in HER-2/neu-transformed cells resulted in decreased tumor formation, which was associated with reduced angiogenesis, but increased necrotic and hypoxic areas in the tumor. Hypoxia induced pCREBSer133, but not pCREBSer121 expression in HER-2/neu-transformed cells. This was accompanied by upregulation of the hypoxia-inducible genes GLUT1 and VEGF, increased cell migration and matrix metalloproteinase-mediated invasion. Treatment of HER-2/neu+ cells with signal transduction inhibitors targeting in particular HER-2/neu was able to revert hypoxia-controlled CREB activation. In addition to changes in the phosphorylation, hypoxic response of HER-2/neu+ cells caused a transient ubiquitination and SUMOylation as well as a co-localization of nuclear CREB to the mitochondrial matrix. A mitochondrial localization of CREB was also demonstrated in hypoxic areas of HER-2/neu+ mammary carcinoma lesions. This was accompanied by an altered gene expression pattern, activity and metabolism of mitochondria leading to an increased respiratory rate, oxidative phosphorylation and mitochondrial membrane potential and consequently to an enhanced apoptosis and reduced cell viability. These data suggest that the HER-2/neu-mediated CREB activation caused by a hypoxic tumor microenvironment contributes to the neoplastic phenotype of HER-2/neu+ cells at various levels. PMID:27409833

  5. Estrogen receptor α L429 and A430 regulate 17β-estradiol-induced cell proliferation via CREB1.

    PubMed

    Pesiri, Valeria; Totta, Pierangela; Segatto, Marco; Bianchi, Fabrizio; Pallottini, Valentina; Marino, Maria; Acconcia, Filippo

    2015-12-01

    17β-Estradiol (E2)-dependent cell proliferation requires both estrogen receptor α (ERα)-based integrated control of gene transcription and kinase pathways activation. Such coordination of intracellular E2:ERα-dependent signaling mechanisms is finely tuned by receptor association with specific partner proteins. Recently, we identified the leucine (L) 429 and alanine (A) 430 within the ERα ligand binding domain as important residues for receptor non-covalent interaction to ubiquitinated species [i.e., ERα ubiquitin-binding surface (ERα UBS)] and for E2-induced ERα activation. To date, if these two ERα amino acids are involved in the control of E2-dependent pathways required for cell proliferation is unknown. Here, by using stably expressing ERα mutated in L429 and A430 (i.e., L429A,A430G-LAAG) cell lines, we show that L429 and A430 are critical for E2-induced cell proliferation, PI3K/AKT pathway activation, and ERα-mediated transcriptional changes. Moreover, we demonstrate that these two receptor structural determinants direct the E2-induced PI3K/AKT/CREB1 pathway activation and CREB1-mediated transcriptional activity that in turn control the hormone-induced cell proliferation. As a whole, our data demonstrate for the first time that the ERα UBS contributes to the modulation of E2-induced ERα-mediated cell proliferation and provide a novel connection between the receptor structure and the functional molecular mechanisms by which E2:ERα complex can regulate cell processes.

  6. CREB1/ATF1 Activation in Photoreceptor Degeneration and Protection

    PubMed Central

    Beltran, William A.; Allore, Heather G.; Johnson, Elizabeth; Towle, Virginia; Tao, Weng; Acland, Gregory M.; Aguirre, Gustavo D.

    2009-01-01

    Purpose. The cAMP response element binding protein 1 (CREB1) and activating transcription factor 1 (ATF1) are closely related members of the bZIP superfamily of transcription factors. Both are activated in response to a wide array of stimuli, including cellular stress. This study was conducted to assess the CREB1/ATF1 pathway in photoreceptor disease and protection. Methods. The expression levels of p-CREB1, CREB1, and ATF1 were examined by immunoblot and immunohistochemistry in normal canine retina and retinas of several canine models of retinal degeneration (rcd1, rcd2, erd, prcd, XLPRA1, XLPRA2, T4R RHO). Humans retinas affected with age-related macular degeneration (AMD) were also examined. p-CREB1/ATF1 immunolabeling was assessed in normal and rcd1 dogs treated with ciliary neurotrophic factor (CNTF), to examine the effect of a neuroprotective stimulus on activation of CREB1/ATF1. Results. Native CREB1 and ATF1 as well as phosphorylated CREB1/ATF1 was examined in normal canine retina by immunoblot. The p-CREB1 antibody identified phosphorylated CREB1 and ATF1 and labeled the inner retina only in normal dogs. In degenerate canine and human retinas, strong immunolabeling appeared in rod and cone photoreceptors, indicating increased expression of native CREB1 and ATF1, as well as increased phosphorylation of these proteins. Retinal protection by CNTF in rcd1 dogs was accompanied by a significant increase in the number of p-CREB1/ATF1-labeled photoreceptor nuclei. Conclusions. Positive association of CREB1/ATF1 phosphorylation with photoreceptor protection suggests that it may contribute to an innate protective response. These data identify a signaling mechanism in rods and cones of potential importance for therapies of RP and AMD. PMID:19643965

  7. Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

    PubMed

    Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2014-09-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway.

  8. Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling

    NASA Technical Reports Server (NTRS)

    Degani, CH.; Kawatsuji, M.; Halmann, M.

    1975-01-01

    It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

  9. Oxytocin is involved in the proconvulsant effects of Sildenafil: Possible role of CREB.

    PubMed

    Khoshneviszadeh, Mahsima; Rahimian, Reza; Fakhfouri, Gohar; Payandemehr, Borna; Khodagholi, Fariba; Ejtemaei Mehr, Shahram; Dehpour, Ahmad Reza

    2016-08-10

    Sildenafil is a phosphodiesterase type 5 inhibitor mainly used for male erectile dysfunction. One of rare yet serious adverse effects of Sildenafil is its potential to decrease seizure threshold. Ample evidence suggests that Sildenafil exerts central effects through induction of Oxytocin (OT) secretion and CREB phosphorylation. The aim of the present study is to evaluate potential roles of OT and CREB in the proconvulsant effects of Sildenafil. The Pentylenetetrazole-induced seizure was used as a standard convulsion model in this study. OT release and pCREB expression were evaluated in the hippocampus of mice using ELISA and western blot assays, respectively. Our results showed that Sildenafil at the dose of 10mgkg(-1) or higher, significantly decreased seizure threshold. Pretreatment with a non-effective dose of OT, potentiated while OT receptor antagonist, Atosiban, reversed fully the proconvulsant effects of Sildenafil (5mgkg(-1)). At biochemical inspection, Sildenafil markedly increased CREB which was attenuated by coadministration of Atosiban. The present study shows for the first time that OT release and the subsequent CREB phosphorylation are involved in the proconvulsant effects of acute Sildenafil treatment in an experimental model of seizure.

  10. Methamphetamine induces Shati/Nat8L expression in the mouse nucleus accumbens via CREB- and dopamine D1 receptor-dependent mechanism

    PubMed Central

    Uno, Kyosuke; Miyazaki, Toh; Sodeyama, Kengo; Miyamoto, Yoshiaki

    2017-01-01

    Shati/Nat8L significantly increased in the nucleus accumbens (NAc) of mice after repeated methamphetamine (METH) treatment. We reported that Shati/Nat8L overexpression in mouse NAc attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference. We recently found that Shati/Nat8L overexpression in NAc regulates the dopaminergic neuronal system via the activation of group II mGluRs by elevated N-acetylaspartylglutamate following N-acetylaspartate increase due to the overexpression. These findings suggest that Shati/Nat8L suppresses METH-induced responses. However, the mechanism by which METH increases the Shati/Nat8L mRNA expression in NAc is unclear. To investigate the regulatory mechanism of Shati/Nat8L mRNA expression, we performed a mouse Shati/Nat8L luciferase assay using PC12 cells. Next, we investigated the response of METH to Shati/Nat8L expression and CREB activity using mouse brain slices of NAc, METH administration to mice, and western blotting for CREB activity of specific dopamine receptor signals in vivo and ex vivo. We found that METH activates CREB binding to the Shati/Nat8L promoter to induce the Shati/Nat8L mRNA expression. Furthermore, the dopamine D1 receptor antagonist SCH23390, but not the dopamine D2 receptor antagonist sulpiride, inhibited the upregulation of Shati/Nat8L and CREB activities in the mouse NAc slices. Thus, the administration of the dopamine D1 receptor agonist SKF38393 increased the Shati/Nat8L mRNA expression in mouse NAc. These results showed that the Shati/Nat8L mRNA was increased by METH-induced CREB pathway via dopamine D1 receptor signaling in mouse NAc. These findings may contribute to development of a clinical tool for METH addiction. PMID:28319198

  11. Role of Phosphorylated HDAC4 in Stroke-Induced Angiogenesis

    PubMed Central

    Liu, Juan; Zhou, Xiang; Li, Qing; Zhou, Shu-Min; Hu, Bin; Hu, Guo-Wen; Niu, Xin; Guo, Shang-Chun

    2017-01-01

    Acetylation or deacetylation of chromatin proteins and transcription factors is part of a complex signaling system that is involved in the control of neurological disorders. Recent studies have demonstrated that histone deacetylases (HDACs) exert protective effects in attenuating neuronal injury after ischemic insults. Class IIa HDAC4 is highly expressed in the brain, and neuronal activity depends on the nucleocytoplasmic shuttling of HDAC4. However, little is known about HDAC4 and its roles in ischemic stroke. In this study, we report that phosphorylation of HDAC4 was remarkably upregulated after stroke and blockade of HDAC4 phosphorylation with GÖ6976 repressed stroke-induced angiogenesis. Phosphorylation of HDAC4 was also increased in endothelial cells hypoxia model and suppression of HDAC4 phosphorylation inhibited the tube formation and migration of endothelial cells in vitro. Furthermore, in addition to the inhibition of angiogenesis, blockade of HDAC4 phosphorylation suppressed the expression of genes downstream of HIF-VEGF signaling in vitro and in vivo. These data indicate that phosphorylated HDAC4 may serve as an important regulator in stroke-induced angiogenesis. The protective mechanism of phosphorylated HDAC4 is associated with HIF-VEGF signaling, implicating a novel therapeutic target in stroke. PMID:28127553

  12. The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site.

    PubMed Central

    Kvietikova, I; Wenger, R H; Marti, H H; Gassmann, M

    1995-01-01

    The hypoxia-inducible factor-1 (HIF-1) was first described as a DNA binding activity that specifically recognizes an 8 bp motif known to be essential for hypoxia-inducible erythropoietin gene transcription. Subsequently HIF-1 activity has also been found in cell lines which do not express erythropoietin, suggesting that HIF-1 is part of a widespread oxygen sensing mechanism. In electrophoretic mobility shift assays HIF-1 DNA binding activity is only detectable in nuclear extracts of cells cultivated in a low oxygen atmosphere. In addition to HIF-1, a constitutive DNA binding activity also specifically binds the HIF1 probe. Here we report that CRE and AP1 oligonucleotides efficiently competed for binding of the HIF1 probe to this constitutive factor, whereas HIF-1 activity itself remained unaffected. Monoclonal antibodies raised against the CRE binding factors ATF-1 and CREB-1 supershifted the constitutive factors ATF-1 and CREB-1 supershifted the constitutive factor, while Jun and Fos family members, which constitute the AP-1 factor, were immunologically undetectable. Recombinant ATF-1 and CREB-1 proteins bound HIF1 probes either as homodimers or as heterodimers, indicating a new binding specificity for ATF-1/CREB-1. Finally, reporter gene assays in HeLa cells treated with either a cAMP analogue or a phorbol ester suggest that the PKA, but not the PKC signalling pathway is involved in oxygen sensing. Images PMID:8524640

  13. Systemic Inhibition of CREB is Well-tolerated in vivo

    PubMed Central

    Li, Bingbing X.; Gardner, Ryan; Xue, Changhui; Qian, David Z.; Xie, Fuchun; Thomas, George; Kazmierczak, Steven C.; Habecker, Beth A.; Xiao, Xiangshu

    2016-01-01

    cAMP-response element binding protein (CREB) is a nuclear transcription factor activated by multiple extracellular signals including growth factors and hormones. These extracellular cues activate CREB through phosphorylation at Ser133 by various protein serine/threonine kinases. Once phosphorylated, it promotes its association with transcription coactivators CREB-binding protein (CBP) and its paralog p300 to activate CREB-dependent gene transcription. Tumor tissues of different origins have been shown to present overexpression and/or overactivation of CREB, indicating CREB as a potential cancer drug target. We previously identified 666-15 as a potent inhibitor of CREB with efficacious anti-cancer activity both in vitro and in vivo. Herein, we investigated the specificity of 666-15 and evaluated its potential in vivo toxicity. We found that 666-15 was fairly selective in inhibiting CREB. 666-15 was also found to be readily bioavailable to achieve pharmacologically relevant concentrations for CREB inhibition. Furthermore, the mice treated with 666-15 showed no evidence of changes in body weight, complete blood count, blood chemistry profile, cardiac contractility and tissue histologies from liver, kidney and heart. For the first time, these results demonstrate that pharmacological inhibition of CREB is well-tolerated in vivo and indicate that such inhibitors should be promising cancer therapeutics. PMID:27694829

  14. Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

    SciTech Connect

    Pistollato, Francesca; Louisse, Jochem; Scelfo, Bibiana; Mennecozzi, Milena; Accordi, Benedetta; Basso, Giuseppe; Gaspar, John Antonydas; Zagoura, Dimitra; Barilari, Manuela; Palosaari, Taina; Sachinidis, Agapios; Bremer-Hoffmann, Susanne

    2014-10-15

    According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2{sup +} neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations. - Highlights: • HESCs derived neuronal cells serve as benchmark for iPSC based neuronal toxicity test development. • Comparisons between hESCs and hiPSCs demonstrated variability of the epigenetic state • CREB pathway modulation have been explored in relation to the neurotoxicant exposure KG-501 • hiPSC might be promising tools to translate theoretical AoPs into toxicological in vitro tests.

  15. Morphine-induced conditioned place preference and the alterations of p-ERK, p-CREB and c-fos levels in hypothalamus and hippocampus: the effects of physical stress.

    PubMed

    Pahlevani, P; Fatahi, Z; Moradi, M; Haghparast, A

    2014-12-08

    The hypothalamus and hippocampus are important areas involved in stress responses and reward processing. In addition, ERK/CREB pathway plays a critical role in the control of cellular responses to stress and reward. In the current study, effects of acute and subchronic stress on the alteration of p-ERK, p-CREB and c-fos levels in the hypothalamus and hippocampus of saline- or morphine-treated animals during morphine-induced conditioned place preference (CPP) procedure were investigated. Male Wistar rats were divided into two saline- and morphine-treated supergroups. Each supergroup includes of control, acute stress and subchronic stress groups. In all of groups, the CPP procedure was done, afterward the alternation of p-ERK/ERK ratio, p-CREB/CREB ratio and c-fos level in the hypothalamus and hippocampus were estimated by Western blot analysis. The results indicated that in saline- or morphine-treated animals, p-ERK/ERK ratio, p-CREB/CREB ratio and c-fos level increased after application of acute and subchronic stress (except for p-ERK/ERK ratio in morphine-control group). Our findings revealed that in saline- or morphine-treated animals, acute and subcronic stress increased the p-ERK/ERK ratio, p-CREB/CREB ratio and c-fos level in the hypothalamus and hippocampus and this enhancement in morphine-treated animals, was more considerable than that in saline-treated animals.

  16. Neuroprotective action of N-acetyl serotonin in oxidative stress-induced apoptosis through the activation of both TrkB/CREB/BDNF pathway and Akt/Nrf2/Antioxidant enzyme in neuronal cells.

    PubMed

    Yoo, Jae-Myung; Lee, Bo Dam; Sok, Dai-Eun; Ma, Jin Yuel; Kim, Mee Ree

    2017-04-01

    N-acetyl serotonin (NAS) as a melatonin precursor has neuroprotective actions. Nonetheless, it is not clarified how NAS protects neuronal cells against oxidative stress. Recently, we have reported that N-palmitoyl serotonins possessed properties of antioxidants and neuroprotection. Based on those, we hypothesized that NAS, a N-acyl serotonin, may have similar actions in oxidative stress-induced neuronal cells, and examined the effects of NAS based on in vitro and in vivo tests. NAS dose-dependently inhibited oxidative stress-induced cell death in HT-22 cells. Moreover, NAS suppressed glutamate-induced apoptosis by suppressing expression of AIF, Bax, calpain, cytochrome c and cleaved caspase-3, whereas it enhanced expression of Bcl-2. Additionally, NAS improved phosphorylation of tropomyosin-related kinase receptor B (TrkB) and cAMP response element-binding protein (CREB) as well as expression of brain-derived neurotrophic factor (BDNF), whereas the inclusion of each inhibitor of JNK, p38 or Akt neutralized the neuroprotective effect of NAS, but not that of ERK. Meanwhile, NAS dose-dependently reduced the level of reactive oxygen species, and enhanced the level of glutathione in glutamate-treated HT-22 cells. Moreover, NAS significantly increased expression of heme oxygenase-1, NAD(P)H quinine oxidoreductase-1 and glutamate-cysteine ligase catalytic subunit as well as nuclear translocation of NF-E2-related factor-2. Separately, NAS at 30mg/kg suppressed scopolamine-induced memory impairment and cell death in CA1 and CA3 regions in mice. In conclusion, NAS shows actions of antioxidant and anti-apoptosis by activating TrkB/CREB/BDNF pathway and expression of antioxidant enzymes in oxidative stress-induced neurotoxicity. Therefore, such effects of NAS may provide the information for the application of NAS against neurodegenerative diseases.

  17. Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation.

    PubMed

    Li, Jiaxin; Dou, Xiaobing; Li, Songtao; Zhang, Ximei; Zeng, Yong; Song, Zhenyuan

    2015-11-01

    Nicotinamide (NAM) is the amide of nicotinic acid and a predominant precursor for NAD(+) biosynthesis via the salvage pathway. Sirt1 is a NAD(+)-dependent deacetylase, playing an important role in regulating cellular functions. Although hepatoprotective effect of NAM has been reported, the underlying mechanism remains elusive. ER stress, induced by saturated fatty acids, in specific palmitate, plays a pathological role in the development of nonalcoholic fatty liver disease. This study aims to determine the effect of NAM on palmitate-induced ER stress in hepatocytes and to elucidate molecular mechanisms behind. Both HepG2 cells and primary mouse hepatocytes were exposed to palmitate (conjugated to BSA at a 2:1 M ratio), NAM, or their combination for different durations. Cellular NAD(+) level, Sirt1 expression/activity, ER stress, as well as cAMP/PKA/CREB pathway activation were determined. NAM increased Sirt1 expression and enzymatic activity, which contributes to the ameliorative effect of NAM on palmitate-triggered ER stress. NAM increased intracellular NAD(+) level in hepatocytes, however, blocking the salvage pathway, a pathway for NAD(+) synthesis from NAM, only partially prevented NAM-induced Sirt1 upregulation while completely prevented NAD+ increase in response to NAM. Further mechanistic investigations revealed that NAM elevated intracellular cAMP level via suppressing PDE activity, leading to downstream PKA and CREB activation. Importantly, cAMP/PKA/CREB pathway blockade abolished not only NAM-induced Sirt1 upregulation, but also its protective effect against ER stress. Our results demonstrate that NAM protects hepatocytes against palmitate-induced ER stress in hepatocytes via upregulating Sirt1. Activation of the cAMP/PKA/CREB pathway plays a key role in NAM-induced Sirt1 upregulation.

  18. Chronic light deprivation inhibits appetitive associative learning induced by ethanol and its respective c-Fos and pCREB expression.

    PubMed

    Varela, Patrícia; Escosteguy-Neto, João Carlos; Coelho, Carolina Tesone; Mello, Luiz Eugênio; da Silveira, Dartiu Xavier; Santos-Junior, Jair Guilherme

    2014-11-01

    To address the role of mixed anxiety/mood disorder on appetitive associative learning, we verify whether previous chronic light deprivation changes ethanol-induced conditioned place preference and its respective expression of c-Fos and pCREB, markers of neuronal activity and plasticity. The experimental group was maintained in light deprivation for 24 h for a period of 4 wk. Subsequently, it was adapted to a standard light-dark cycle for 1 wk. As a control, some mice were maintained in standard cycle for a period of 4 wk (Naïve group). Then, all animals were submitted to behavioral tests to assess emotionality: elevated plus maze; open field; and forced swim. After that, they were submitted to ethanol-induced conditioned place preference. Ninety minutes after the place preference test, they were perfused, and their brains processed for c-Fos and pCREB immunohistochemistry. Light deprivation induced anxiety-like trait (elevated plus maze), despair (forced swim), and hyperlocomotion (open field), common features seen in other animal models of depression. Ethanol-induced conditioned place preference was accompanied by increases on c-Fos and pCREB in the hippocampus, prefrontal cortex and striatum. Interestingly, mice previously submitted to light deprivation did not develop either acquisition and/or expression of ethanol-induced conditioned place preference or increases in c-Fos and pCREB. Therefore, chronic light deprivation mimics several behavioral aspects of other animal models of depression. Furthermore, it could be useful to study the neurochemical mechanisms involved in the dual diagnosis. However, given its likely deleterious effects on appetitive associative memory, it should be used with caution to investigate the cognitive aspects related to the dual diagnosis.

  19. Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy.

    PubMed

    Qian, Xu; Li, Xinjian; Cai, Qingsong; Zhang, Chuanbao; Yu, Qiujing; Jiang, Yuhui; Lee, Jong-Ho; Hawke, David; Wang, Yugang; Xia, Yan; Zheng, Yanhua; Jiang, Bing-Hua; Liu, David X; Jiang, Tao; Lu, Zhimin

    2017-03-02

    Autophagy is crucial for maintaining cell homeostasis. However, the precise mechanism underlying autophagy initiation remains to be defined. Here, we demonstrate that glutamine deprivation and hypoxia result in inhibition of mTOR-mediated acetyl-transferase ARD1 S228 phosphorylation, leading to ARD1-dependent phosphoglycerate kinase 1 (PGK1) K388 acetylation and subsequent PGK1-mediated Beclin1 S30 phosphorylation. This phosphorylation enhances ATG14L-associated class III phosphatidylinositol 3-kinase VPS34 activity by increasing the binding of phosphatidylinositol to VPS34. ARD1-dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumorigenesis. Furthermore, PGK1 K388 acetylation levels correlate with Beclin1 S30 phosphorylation levels and poor prognosis in glioblastoma patients. Our study unearths an important mechanism underlying cellular-stress-induced autophagy initiation in which the protein kinase activity of the metabolic enzyme PGK1 plays an instrumental role and reveals the significance of the mutual regulation of autophagy and cell metabolism in maintaining cell homeostasis.

  20. Activation of CREB by St. John's wort may diminish deletorious effects of aging on spatial memory.

    PubMed

    Trofimiuk, Emil; Holownia, Adam; Braszko, Jan J

    2010-03-01

    St. John's wort (Hypericum perforatum) is one of the leading psychotherapeutic phytomedicines. Beneficial effects of this herb in the treatment of mild to moderate depression are well known. In this study we tested a hypothesis that St. John's wort alleviates age-related memory impairments by increasing the levels of cyclic adenosine 3', 5'-monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) in hippocampus. Middleaged rats (18 month-old) displayed a decline in the acquisition of spatial working memory (p < 0.001) in the Morris water maze (MWM). Chronic administration of Hypericum perforatum (HP) (350 mg/kg for 21 days), potently and significantly improved the processing of spatial information in the aged rats (p < 0.001). Also the herb increased the levels of pCREB in the aged rat's hippocampus (p < 0.01) as measured by western immunoblotting. Aging caused significant locomotor impairments as tested in the open field (p < 0.001) but not in the MWM test. However, these were unaffected by treatment with HP. Thus, this study indicates that St. John's wort effectively prevents aging-induced deterioration of spatial memory in 18 month-old rats, possibly by the activation of CREB regulated genes associated with memory formation. It appears that mechanism is probably inactive in young rats.

  1. Involvement of BDNF/TrkB and ERK/CREB axes in nitroglycerin-induced rat migraine and effects of estrogen on these signals in the migraine

    PubMed Central

    Guo, Jiu-Qing; Deng, Hui-Hui; Bo, Xiao

    2017-01-01

    ABSTRACT Migraine is a highly prevalent headache disorder, especially in women. Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinases (TrkB), as well as extracellular signal-regulated kinase (ERK) and its downstream target c-AMP-responsive element binding protein (CREB) are strongly associated with the transmission of nociceptive information. However, the involvement of these substances in migraine has rarely been examined. In the present study, intraperitoneal injection of nitroglycerin (NTC) successfully induced rat migraine attack, as evidenced by behavioral testing. The location and abundance of these substances in the migraine model were determined by immunohistochemistry, real-time polymerase chain reaction (RT-PCR), western blot and enzyme-linked immunosorbant assays (ELISA). Results showed that BDNF, TrkB, phosphor(p)-ERK and p-CREB were up-regulated in the brain neurons of both male and female rats with NTG-induced migraine compared to non-migraine control, whereas their expression levels were decreased in headache-free intervals of the migraine compared to migraine attacks. Estrogen is an important contributor to migraine. Female ovariectomized rats showed significant reduction in the expression of BDNF, TrkB, p-CREB and p-ERK in both attacks and intervals of NTG-induced migraine, relative to rats that have their ovaries. But, intraperitoneal administration of exogenous estrogen recovered their expression in ovariectomized rats. Collectively, this study unveiled a positive correlation of BDNF/TrkB and ERK/CREB axes in NTG-induced migraine and promoting effects of estrogen on their signals in the migraine. These findings contribute to further understanding the pathogenesis of migraine in the molecular basis. PMID:27875242

  2. The cyclic AMP response element-binding protein antisense oligonucleotide induced anti-nociception and decreased the expression of KIF17 in spinal cord after peripheral nerve injury in mice

    PubMed Central

    Bo, Jinhua; Zhang, Wei; Sun, Xiaofeng; Yang, Yan; Liu, Xiaojie; Jiang, Ming; Ma, Zhengliang; Gu, Xiaoping

    2014-01-01

    Backgrounds: The cyclic AMP response element-binding protein (CREB) plays an important role in neuropathic pain. Kinesin superfamily motor protein 17 (KIF17) is involved in long-term memory formation. CREB could increase the level of KIF17 when activated by synaptic input. This study is to investigate the role and mechanism of CREB antisense oligonucleotide (ODN) in neuropathic pain induced by chronic constriction injury (CCI) in mice. Results: CCI surgery decreased thresholds of mechanical allodynia and thermal hyperalgesia whereas CREB antisense oligonucleotide ODN significantly attenuated these pain behaviors (P < 0.05). CCI significantly induced the protein expression of phosphorylated CREB (pCREB) and KIF17, but not KIF5B, in the spinal cord of CCI mice (P < 0.05). Additionally, the mRNA expression of CREB and KIF17 was significantly increased by CCI (P < 0.05). However, CREB antisense ODN significantly decreased the protein expression of pCREB and KIF17 (but not KIF5B), and the mRNA expression of CREB and KIF17 (P < 0.05). Conclusions: CREB antisense oligonucleotide ODN may reduce neuropathic pain through targeting CREB and decreasing the expression of pCREB and KIF17. PMID:25664020

  3. CREB SUMOylation by the E3 ligase PIAS1 enhances spatial memory.

    PubMed

    Chen, Yan-Chu; Hsu, Wei-Lun; Ma, Yun-Li; Tai, Derek J C; Lee, Eminy H Y

    2014-07-16

    cAMP-responsive element binding protein (CREB) phosphorylation and signaling plays an important role in long-term memory formation, but other posttranslational modifications of CREB are less known. Here, we found that CREB1Δ, the short isoform of CREB, could be sumoylated by the small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) at Lys271 and Lys290 and PIAS1 SUMOylation of CREB1Δ increased the expression level of CREB1Δ. CREB1Δ could also be sumoylated by other PIAS family proteins, but not by the E3 ligases RanBP2 and Pc2 or by the E2 ligase Ubc9. Furthermore, water maze training increased the level of endogenous CREB SUMOylation in rat CA1 neurons determined by in vitro SUMOylation assay, but this effect was not observed in other brain areas. Moreover, transduction of Lenti-CREBWT to rat CA1 area facilitated, whereas transduction of Lenti-CREB double sumo-mutant (CREBK271RK290R) impaired, spatial learning and memory performance. Transduction of Lenti-CREBWT-SUMO1 fusion vector to rat CA1 area showed a more significant effect in enhancing spatial learning and memory and CREB SUMOylation. Lenti-CREBWT transduction increased, whereas Lenti-CREBK271RK290R transduction decreased, CREB DNA binding to the brain-derived neurotrophic factor (bdnf) promoter and decreased bdnf mRNA expression. Knock-down of PIAS1 expression in CA1 area by PIAS1 siRNA transfection impaired spatial learning and memory and decreased endogenous CREB SUMOylation. In addition, CREB SUMOylation was CREB phosphorylation dependent and lasted longer. Therefore, CREB phosphorylation may be responsible for signal transduction during the early phase of long-term memory formation, whereas CREB SUMOylation sustains long-term memory.

  4. Astaxanthin Inhibits Acetaldehyde-Induced Cytotoxicity in SH-SY5Y Cells by Modulating Akt/CREB and p38MAPK/ERK Signaling Pathways

    PubMed Central

    Yan, Tingting; Zhao, Yan; Zhang, Xia; Lin, Xiaotong

    2016-01-01

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. Acetaldehyde, the most toxic metabolite of ethanol, mediates the brain tissue damage and cognitive dysfunction induced by chronic excessive alcohol consumption. In this study, the effect of astaxanthin, a marine bioactive compound, on acetaldehyde-induced cytotoxicity was investigated in SH-SY5Y cells. It was found that astaxanthin protected cells from apoptosis by ameliorating the effect of acetaldehyde on the expression of Bcl-2 family proteins, preventing the reduction of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bak induced by acetaldehyde. Further analyses showed that astaxanthin treatment inhibited acetaldehyde-induced reduction of the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). Astaxanthin treatment also prevented acetaldehyde-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK) and decrease of the level of activated extracellular signal-regulated kinases (ERKs). Activation of Akt/CREB pathway promotes cell survival and is involved in the upregulation of Bcl-2 gene. P38MAPK plays a critical role in apoptotic events while ERKs mediates the inhibition of apoptosis. Thus, astaxanthin may inhibit acetaldehyde-induced apoptosis through promoting the activation of Akt/CREB and ERKs and blocking the activation of p38MAPK. In addition, astaxanthin treatment suppressed the oxidative stress induced by acetaldehyde and restored the antioxidative capacity of SH-SY5Y cells. Therefore, astaxanthin may protect cells against acetaldehyde-induced cytotoxicity through maintaining redox balance and modulating apoptotic and survival signals. The results suggest that astaxanthin treatment may be beneficial for preventing neurotoxicity associated with acetaldehyde and excessive alcohol consumption. PMID:26978376

  5. Astaxanthin Inhibits Acetaldehyde-Induced Cytotoxicity in SH-SY5Y Cells by Modulating Akt/CREB and p38MAPK/ERK Signaling Pathways.

    PubMed

    Yan, Tingting; Zhao, Yan; Zhang, Xia; Lin, Xiaotong

    2016-03-10

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. Acetaldehyde, the most toxic metabolite of ethanol, mediates the brain tissue damage and cognitive dysfunction induced by chronic excessive alcohol consumption. In this study, the effect of astaxanthin, a marine bioactive compound, on acetaldehyde-induced cytotoxicity was investigated in SH-SY5Y cells. It was found that astaxanthin protected cells from apoptosis by ameliorating the effect of acetaldehyde on the expression of Bcl-2 family proteins, preventing the reduction of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bak induced by acetaldehyde. Further analyses showed that astaxanthin treatment inhibited acetaldehyde-induced reduction of the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). Astaxanthin treatment also prevented acetaldehyde-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK) and decrease of the level of activated extracellular signal-regulated kinases (ERKs). Activation of Akt/CREB pathway promotes cell survival and is involved in the upregulation of Bcl-2 gene. P38MAPK plays a critical role in apoptotic events while ERKs mediates the inhibition of apoptosis. Thus, astaxanthin may inhibit acetaldehyde-induced apoptosis through promoting the activation of Akt/CREB and ERKs and blocking the activation of p38MAPK. In addition, astaxanthin treatment suppressed the oxidative stress induced by acetaldehyde and restored the antioxidative capacity of SH-SY5Y cells. Therefore, astaxanthin may protect cells against acetaldehyde-induced cytotoxicity through maintaining redox balance and modulating apoptotic and survival signals. The results suggest that astaxanthin treatment may be beneficial for preventing neurotoxicity associated with acetaldehyde and excessive alcohol consumption.

  6. Cranial irradiation regulates CREB-BDNF signaling and variant BDNF transcript levels in the mouse hippocampus.

    PubMed

    Son, Yeonghoon; Yang, Miyoung; Kang, Sohi; Lee, Sueun; Kim, Jinwook; Kim, Juhwan; Park, Seri; Kim, Joong-Sun; Jo, Sung-Kee; Jung, Uhee; Shin, Taekyun; Kim, Sung-Ho; Wang, Hongbing; Moon, Changjong

    2015-05-01

    The brain can be exposed to ionizing radiation in various ways, and such irradiation can trigger adverse effects, particularly on learning and memory. However, the precise mechanisms of cognitive impairments induced by cranial irradiation remain unknown. In the hippocampus, brain-derived neurotrophic factor (BDNF) plays roles in neurogenesis, neuronal survival, neuronal differentiation, and synaptic plasticity. The significance of BDNF transcript variants in these contexts is becoming clearer. In the present study, both object recognition memory and contextual fear conditioning task performance in adult C57BL/6 mice were assessed 1 month after a single exposure to cranial irradiation (10 Gy) to evaluate hippocampus-related behavioral dysfunction following such irradiation. Furthermore, changes in the levels of BDNF, the cAMP-response element binding protein (CREB) phosphorylation, and BDNF transcript variants were measured in the hippocampus 1 month after cranial irradiation. On object recognition memory and contextual fear conditioning tasks, mice evaluated 1 month after irradiation exhibited significant memory deficits compared to sham-irradiated controls, but no apparent change was evident in locomotor activity. Both phosphorylated CREB and BDNF protein levels were significantly downregulated after irradiation of the hippocampus. Moreover, the levels of mRNAs encoding common BDNF transcripts, and exons IIC, III, IV, VII, VIII, and IXA, were significantly downregulated after irradiation. The reductions in CREB phosphorylation and BDNF expression induced by differential regulation of BDNF hippocampal exon transcripts may be associated with the memory deficits evident in mice after cranial irradiation.

  7. Stress induces altered CRE/CREB pathway activity and BDNF expression in the hippocampus of glucocorticoid receptor-impaired mice.

    PubMed

    Alboni, Silvia; Tascedda, Fabio; Corsini, Daniela; Benatti, Cristina; Caggia, Federica; Capone, Giacomo; Barden, Nicholas; Blom, Joan M C; Brunello, Nicoletta

    2011-06-01

    The gene coding for the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) is a stress-responsive gene. Changes in its expression may underlie some of the pathological effects of stress-related disorders like depression. Data on the stress-induced regulation of the expression of BDNF in pathological conditions are rare because often research is conducted using healthy animals. In our experiments, we used transgenic mice with glucocorticoid receptor impaired (GR-i) expression in the hypothalamus created as a tool to study the neuroendocrine changes occurring in stress-related disorders. First, under basal condition, GR-i mice displayed lower levels of BDNF exons IX and IV and decreased CRE(BDNF) binding activity with respect to wild-type (WT) mice in the hippocampus. Then, we exposed GR-i and WT mice to an acute restraint stress (ARS) to test the hypothesis that GR-i mice display: 1] different ARS induced expression of BDNF, and 2] altered activation of signaling pathways implicated in regulating BDNF gene expression in the hippocampus with respect to WT mice. Results indicate that ARS enhanced BDNF mRNA expression mainly in the CA3 hippocampal sub-region of GR-i mice in the presence of enhanced levels of pro-BDNF protein, while no effect was observed in WT mice. Moreover, ARS reduced CREB signaling and binding to the BDNF promoter in GR-i mice but enhanced signaling and binding, possibly through ERK1/2 activation, in WT mice. Thus, life-long central GR dysfunction resulted in an altered sensitivity at the transcriptional level that may underlie an impaired response to an acute psycho-physical stress. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

  8. Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

    PubMed

    Moraz, Marie-Laurence; Pythoud, Christelle; Turk, Rolf; Rothenberger, Sylvia; Pasquato, Antonella; Campbell, Kevin P; Kunz, Stefan

    2013-05-01

    The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.

  9. Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan

    PubMed Central

    Moraz, Marie-Laurence; Pythoud, Christelle; Turk, Rolf; Rothenberger, Sylvia; Pasquato, Antonella; Campbell, Kevin P.; Kunz, Stefan

    2013-01-01

    The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a hemorrhagic fever with high mortality in man. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process. PMID:23279385

  10. Interplay of CREB and ATF2 in Ionizing Radiation-Induced Neuroendocrine Differentiation of Prostate Cancer Cells

    DTIC Science & Technology

    2011-06-01

    and NSE expression was barely detectable by immunoblotting even for wild-type LNCaP cells. Cell viability and growth inhibition assay. Wild-type or...by IR treatment (Fig. 2C). Interestingly, pCREB was also detected in the cytoplasm in proliferating LNCaP cells, and IR treatment did not seem to...cell lysates were prepared 3 d after the induction, and Flag-nATF2 and HA-CREB-S133A were detected with anti-ATF2 and anti-HA antibodies, respectively

  11. Endogenous prostaglandin E2 potentiates anti-inflammatory phenotype of macrophage through the CREB-C/EBP-β cascade.

    PubMed

    Na, Yi Rang; Jung, Daun; Yoon, Bo Ruem; Lee, Won Woo; Seok, Seung Hyeok

    2015-09-01

    Macrophages have important functions in tissue homeostasis, but the exact mechanisms regarding wide spectrum of macrophage phenotype remain unresolved. In this study, we report that mouse bone marrow derived naïve macrophages produce prostaglandin E2 (PGE2 ) endogenously, resulting in anti-inflammatory gene expression upon differentiation induced by macrophage colony stimulating factor (M-CSF). Cyclooxygenase (COX) inhibition by indomethacin reduced endogenous PGE2 production of macrophages and subsequently reduced arg1, IL10 and Mrc1, YmI and FizzI gene expressions. Of note, PGE2 phosphorylates CREB via EP2 and EP4 receptor ligation, thereby transcriptionally increasing C/EBP-β expression in BALB/c bone marrow derived macrophages. Activated CREB directly binds to the CREB-responsive element of the C/EBP-β promoter, such that PGE2 ultimately reinforces arg1, IL10 and Mrc1 gene expression. Cyclic AMP activator forskolin also phosphorylated CREB and induced the C/EBP-β cascade, but this was completely blocked by the PKA inhibitor, H89. Consequently, M-CSF grown macrophages inhibited T-cell proliferation but the inhibition ability was reduced when the COX is inhibited by indomethacin or macrophage C/EBP-β expression was decreased by siRNA transduction. Our results collectively describe the molecular basis for homeostatic macrophage differentiation by endogenous PGE2 .

  12. DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

    PubMed

    Won, Jong Hoon; Ahn, Kyong Hoon; Back, Moon Jung; Ha, Hae Chan; Jang, Ji Min; Kim, Ha Hyung; Choi, Sang-Zin; Son, Miwon; Kim, Dae Kyong

    2015-01-01

    In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

  13. GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis.

    PubMed

    Zibrova, Darya; Vandermoere, Franck; Göransson, Olga; Peggie, Mark; Mariño, Karina V; Knierim, Anne; Spengler, Katrin; Weigert, Cora; Viollet, Benoit; Morrice, Nicholas A; Sakamoto, Kei; Heller, Regine

    2017-03-07

    Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

  14. Neuroprotective effects of various doses of topiramate against methylphenidate-induced oxidative stress and inflammation in isolated rat amygdala: the possible role of CREB/BDNF signaling pathway.

    PubMed

    Motaghinejad, Majid; Motevalian, Manijeh; Falak, Reza; Heidari, Mansour; Sharzad, Mahshid; Kalantari, Elham

    2016-12-01

    Methylphenidate (MPH) abuse damages brain cells. The neuroprotective effects of topiramate (TPM) have been reported previously, but its exact mechanism of action still remains unclear. This study investigated the in vivo role of various doses of TPM in the protection of rat amygdala cells against methylphenidate-induced oxidative stress and inflammation. Seventy adult male rats were divided into seven groups. Groups 1 and 2 received normal saline (0.7 ml/rat) and MPH (10 mg/kg), respectively, for 21 days. Groups 3, 4, 5, 6, and 7 were concurrently treated with MPH (10 mg/kg) and TPM (10, 30, 50, 70, and 100 mg/kg), respectively, for 21 days. elevated plus maze (EPM) was used to assess motor activity disturbances. In addition, oxidative, antioxidantand inflammatory factors and CREB, Ak1, CAMK4, MAPK3, PKA, BDNF, and c FOS gene levels were measured by RT-PCR, and also, CREB and BDNF protein levels were measured by WB in isolated amygdalae. MPH significantly disturbed motor activity and TPM (70 and 100 mg/kg) neutralized its effects. MPH significantly increased lipid peroxidation, mitochondrial GSSG levels and IL-1β and TNF-α level and CAMK4 gene expression in isolated amygdala cells. In contrast, superoxide dismutase, glutathione peroxidase, and glutathione reductase activities and CREB, BDNF Ak1, MAPK3, PKA, BDNF, and c FOS expression significantly decreased. The various doses of TPM attenuated these effects of MPH. It seems that TPM can be used as a neuroprotective agent and is a good candidate against MPH-induced neurodegeneration.

  15. cAMP/PKA/CREB/GLT1 signaling involved in the antidepressant-like effects of phosphodiesterase 4D inhibitor (GEBR-7b) in rats

    PubMed Central

    Liu, Xu; Guo, Haibiao; Sayed, Mohammad Daud SOM; Lu, Yang; Yang, Ting; Zhou, Dongsheng; Chen, Zhongming; Wang, Haitao; Wang, Chuang; Xu, Jiangping

    2016-01-01

    Objectives GEBR-7b, a potential phosphodiesterase 4D inhibitor, has been shown to have memory-enhancing effects in rodents. However, it is still unknown whether GEBR-7b also has the antidepressant-like effects in rats. Herein, we examined the potential of GEBR-7b to attenuate depression-like behaviors in the rat model of depression induced by chronic unpredictable stress (CUS). Next, we also investigated the alterations of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) catalytic subunit (PKAca), cAMP response element-binding (CREB), and glutamate transporter 1 (GLT1) levels produced by GEBR-7b in the rats model of depression. Methods Effects of GEBR-7b on CUS (35 days)-induced depression-like behaviors were examined by measuring immobility time in the forced swimming test (FST). Hippocampal cAMP levels were examined by enzyme-linked immunosorbent assay, whereas PKAca, phosphorylation of CREB (pCREB), CREB, and GLT1 in the hippocampus of rats were subjected to Western blot analysis. Results CUS exposure caused a depression-like behavior evidenced by the increased immobility time in FST. Depression-like behavior induced by CUS was accompanied by a significant increased GLT, decreased cAMP, PKAca, pCREB activities in hippocampus. However, repeated GEBR-7b administration significantly reversed CUS-induced depression-like behavior and changes of cAMP/PKA/CREB/GLT1 signaling. No alteration was observed in locomotor activity in open field test. Conclusion These findings indicate that GEBR-7b reversed the depression-like behaviors induced by CUS in rats, which is at least in part mediated by modulating cAMP, PKAca, pCREB, and GLT1 levels in the hippocampus of rats, supporting its neuroprotective potential against behavioral and biochemical dysfunctions induced by CUS. PMID:26855578

  16. Cigarette sidestream smoke induces phosphorylated histone H2AX.

    PubMed

    Toyooka, Tatsushi; Ibuki, Yuko

    2009-05-31

    Cigarette sidestream smoke (CSS) is a widespread environmental pollutant having highly genotoxic potency. In spite of the overwhelming evidence that CSS induces a wide range of DNA damage such as oxidative base damage and DNA adducts, evidence that CSS can result in DNA double strand breaks (DSBs) is little. In this study, we showed that CSS generated phosphorylated histone H2AX (gamma-H2AX), recently considered as a sensitive marker of the generation of DSBs, in a human pulmonary epithelial cell model, A549. Treatment with CSS drastically induced discrete foci of gamma-H2AX within the nucleus in a dose-dependent manner. CSS increased intracellular oxidation, and N-acetylcysteine (NAC), an antioxidant, significantly attenuated the formation of gamma-H2AX, suggesting that reactive oxygen species produced from CSS partially contributed to the phosphorylation. The generation of gamma-H2AX is considered to be accompanied the induction of DSBs. CSS in fact induced DSBs, which was also inhibited by NAC. DSBs are the worst type of DNA damage, related to genomic instability and carcinogenesis. Our results would increase the evidence of the strong genotoxicity of passive smoking.

  17. Rapid changes in protein phosphorylation associated with light-induced gravity perception in corn roots

    NASA Technical Reports Server (NTRS)

    McFadden, J. J.; Poovaiah, B. W.

    1988-01-01

    The effect of light and calcium depletion on in vivo protein phosphorylation was tested using dark-grown roots of Merit corn. Light caused rapid and specific promotion of phosphorylation of three polypeptides. Pretreatment of roots with ethylene glycol bis N,N,N',N' tetraacetic acid and A23187 prevented light-induced changes in protein phosphorylation. We postulate that these changes in protein phosphorylation are involved in the light-induced gravity response.

  18. The Insulin-Regulated CREB Coactivator TORC Promotes Stress Resistance in Drosophila

    PubMed Central

    Wang, Biao; Goode, Jason; Best, Jennifer; Meltzer, Jodi; Schilman, Pablo E.; Chen, Jian; Garza, Dan; Thomas, John B.; Montminy, Marc

    2013-01-01

    SUMMARY In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB) coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1. Here we show that starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augmented TORC activity in adult flies, our results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance. PMID:18460334

  19. Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB.

    PubMed

    Ahluwalia, Amrita; Baatar, Dolgor; Jones, Michael K; Tarnawski, Andrzej S

    2014-09-15

    Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.

  20. AGE-RELATED IMPAIRMENTS IN MEMORY AND IN CREB AND pCREB EXPRESSION IN HIPPOCAMPUS AND AMYGDALA FOLLOWING INHIBITORY AVOIDANCE TRAINING

    PubMed Central

    Morris, Ken A.; Gold, Paul E.

    2012-01-01

    This experiment examined whether age-related changes in CREB and pCREB contribute to the rapid forgetting seen in aged animals. Young (3-month-old) and aged (24-month-old) Fischer-344 rats received inhibitory avoidance training with a low (0.2 mA, 0.4 sec) or moderate (0.5 mA, 0.5 sec) footshock; memory was measured 7 days later. Other rats were euthanized 30 minutes after training, and CREB and pCREB expression levels were examined in the hippocampus, amygdala, and piriform cortex using immunohistochemistry. CREB levels decreased with age in the hippocampus and amygdala. After training with either shock level, young rats exhibited good memory and increases in pCREB levels in the hippocampus and amygdala. Aged rats exhibited good memory for the moderate but not the low shock but did not show increases in pCREB levels after either shock intensity. These results suggest that decreases in total CREB and in pCREB activation in the hippocampus and amygdala may contribute to rapid forgetting in aged rats. After moderate footshock, the stable memory in old rats together with absence of CREB activation suggests either that CREB was phosphorylated in a spatiotemporal pattern other than analyzed here or that the stronger training conditions engaged alternate mechanisms that promote long-lasting memory. PMID:22445851

  1. Antiproliferative effect of panaxynol on RASMCs via inhibition of ERK1/2 and CREB.

    PubMed

    Jiang, Li-Ping; Lu, Yang; Nie, Bao-Ming; Chen, Hong-Zhuan

    2008-02-15

    Panaxynol (PNN) occurs in many foods such as carrot, celery, and several reports have shown that it has neuritogenic and neuroprotective properties. In this study, we have investigated the antiproliferative effect and the mechanism of PNN on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (RASMCs). PNN significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of RASMCs in a concentration-dependent manner. Flow cytometry analysis showed that PNN blocked the cell cycle progression at the G(1)/S phase. Preincubation of RASMCs with 9 microM PNN resulted in a significant inhibition of PDGF-BB-induced extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation expression and PDGF-BB-induced CREB phosphorylation expression. The results indicated that the inhibitory effect of PNN on the PDGF-BB-induced proliferation of RASMCs might be mediated by blocking phosphorylation of ERK1/2 and that of CREB.

  2. Phosphorylation induces distinct alpha-synuclein strain formation

    PubMed Central

    Ma, Meng-Rong; Hu, Zhi-Wen; Zhao, Yu-Fen; Chen, Yong-Xiang; Li, Yan-Mei

    2016-01-01

    Synucleinopathies are a group of neurodegenerative diseases associated with alpha-synuclein (α-Syn) aggregation. Recently, increasing evidence has demonstrated the existence of different structural characteristics or ‘strains’ of α-Syn, supporting the concept that synucleinopathies share several common features with prion diseases and possibly explaining how a single protein results in different clinical phenotypes within synucleinopathies. In earlier studies, the different strains were generated through the regulation of solution conditions, temperature, or repetitive seeded fibrillization in vitro. Here, we synthesize homogeneous α-Syn phosphorylated at serine 129 (pS129 α-Syn), which is highly associated with the pathological changes, and demonstrate that phosphorylation at Ser129 induces α-Syn to form a distinct strain with different structures, propagation properties, and higher cytotoxicity compared with the wild-type α-Syn. The results are the first demonstration that post-translational modification of α-Syn can induce different strain formation, offering a new mechanism for strain formation. PMID:27853185

  3. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    SciTech Connect

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  4. Protective Effect of Curcumin by Modulating BDNF/DARPP32/CREB in Arsenic-Induced Alterations in Dopaminergic Signaling in Rat Corpus Striatum.

    PubMed

    Srivastava, Pranay; Dhuriya, Yogesh K; Gupta, Richa; Shukla, Rajendra K; Yadav, Rajesh S; Dwivedi, Hari N; Pant, Aditya B; Khanna, Vinay K

    2016-12-13

    Earlier, protective role of curcumin in arsenic-induced dopamine (DA)-D2 receptor dysfunctions in corpus striatum has been demonstrated by us. In continuation to that, the present study is focused to decipher the molecular mechanisms associated with alterations in dopaminergic signaling on arsenic exposure in corpus striatum and assess the protective efficacy of curcumin. Exposure to arsenic (20 mg/kg, body weight p.o. for 28 days) in rats resulted to decrease the expression of presynaptic proteins-tyrosine hydroxylase and VMAT2 while no effect was observed on the expression of DAT in comparison to controls. A significant decrease in the expression of DA-D2 receptors associated with alterations in the expression of PKA, pDARPP32 (Thr 34), and PP1 α was clearly evident on arsenic exposure. Expression of BDNF and pGSK3β in corpus striatum was found decreased in arsenic-exposed rats. Simultaneous treatment with curcumin (100 mg/kg, body weight p.o. for 28 days) resulted to protect arsenic-induced alterations in the expression of DA-D2 receptors, PKA, pDARPP32, pCREB, and pPP1α. Neuroprotective efficacy of curcumin can possibly be attributed to its antioxidant potential which significantly protected arsenic-induced mitochondrial dysfunctions by modulating the ROS generation and apoptosis. Modulation in the expression of BDNF and pGSK3β in corpus striatum by curcumin exhibits the importance of neuronal survival pathway in arsenic-induced dopaminergic dysfunctions. Interestingly, curcumin was also found to protect arsenic-induced ultrastructural changes in corpus striatum. The results exhibit that curcumin modulates BDNF/DARPP32/CREB in arsenic-induced alterations in dopaminergic signaling in rat corpus striatum.

  5. The selective glucocorticoid receptor antagonist ORG 34116 decreases immobility time in the forced swim test and affects cAMP-responsive element-binding protein phosphorylation in rat brain.

    PubMed

    Bachmann, Cornelius G; Bilang-Bleuel, Alicia; De Carli, Sonja; Linthorst, Astrid C E; Reul, Johannes M H M

    2005-01-01

    Glucocorticoid receptor (GR) antagonists can block the retention of the immobility response in the forced swimming test. Recently, we showed that forced swimming evokes a distinct spatiotemporal pattern of cAMP-responsive element-binding protein (CREB) phosphorylation in the dentate gyrus (DG) and neocortex. In the present study, we found that chronic treatment of rats with the selective GR antagonist ORG 34116 decreased the immobility time in the forced swim test, increased baseline levels of phosphorylated CREB (P-CREB) in the DG and neocortex and affected the forced swimming-induced changes in P-CREB levels in a time- and site-specific manner. Overall, we observed that, in control rats, forced swimming evoked increases in P-CREB levels in the DG and neocortex, whereas in ORG 34116-treated animals a major dephosphorylation of P-CREB was observed. These observations underscore an important role of GRs in the control of the phosphorylation state of CREB which seems to be of significance for the immobility response in the forced swim test and extend the molecular mechanism of action of GRs in the brain.

  6. Small molecule kaempferol modulates PDX-1 protein expression and subsequently promotes pancreatic β-cell survival and function via CREB.

    PubMed

    Zhang, Yanling; Zhen, Wei; Maechler, Pierre; Liu, Dongmin

    2013-04-01

    Chronic hyperlipidemia causes β-cell apoptosis and dysfunction, thereby contributing to the pathogenesis of type 2 diabetes (T2D). Thus, searching for agents to promote pancreatic β-cell survival and improve its function could be a promising strategy to prevent and treat T2D. We investigated the effects of kaempferol, a small molecule isolated from ginkgo biloba, on apoptosis and function of β-cells and further determined the mechanism underlying its actions. Kaempferol treatment promoted viability, inhibited apoptosis and reduced caspase-3 activity in INS-1E cells and human islets chronically exposed to palmitate. In addition, kaempferol prevented the lipotoxicity-induced down-regulation of antiapoptotic proteins Akt and Bcl-2. The cytoprotective effects of kaempferol were associated with improved insulin secretion, synthesis, and pancreatic and duodenal homeobox-1 (PDX-1) expression. Chronic hyperlipidemia significantly diminished cyclic adenosine monophosphate (cAMP) production, protein kinase A (PKA) activation, cAMP-responsive element binding protein (CREB) phosphorylation and its regulated transcriptional activity in β-cells, all of which were restored by kaempferol treatment. Disruption of CREB expression by transfection of CREB siRNA in INS-1E cells or adenoviral transfer of dominant-negative forms of CREB in human islets ablated kaempferol protection of β-cell apoptosis and dysfunction caused by palmitate. Incubation of INS-1E cells or human islets with kaempferol for 48h induced PDX-1 expression. This effect of kaempferol on PDX-1 expression was not shared by a host of structurally related flavonoid compounds. PDX-1 gene knockdown reduced kaempferol-stimulated cAMP generation and CREB activation in INS-1E cells. These findings demonstrate that kaempferol is a novel survivor factor for pancreatic β-cells via up-regulating the PDX-1/cAMP/PKA/CREB signaling cascade.

  7. Distinctive Roles for Amygdalar CREB in Reconsolidation and Extinction of Fear Memory

    ERIC Educational Resources Information Center

    Tronson, Natalie C.; Wiseman, Shari L.; Neve, Rachael L.; Nestler, Eric J.; Olausson, Peter; Taylor, Jane R.

    2012-01-01

    Cyclic AMP response element binding protein (CREB) plays a critical role in fear memory formation. Here we determined the role of CREB selectively within the amygdala in reconsolidation and extinction of auditory fear. Viral overexpression of the inducible cAMP early repressor (ICER) or the dominant-negative mCREB, specifically within the lateral…

  8. Allicin inhibits oxidative stress-induced mitochondrial dysfunction and apoptosis by promoting PI3K/AKT and CREB/ERK signaling in osteoblast cells

    PubMed Central

    DING, GUOLIANG; ZHAO, JIANQUAN; JIANG, DIANMING

    2016-01-01

    Osteoporosis is a disease of the skeleton that is characterized by the loss of bone mass and degeneration of bone microstructure, resulting in an increased risk of fracture. Oxidative stress, which is known to promote oxidative damage to mitochondrial function and also cell apoptosis, has been recently indicated to be implicated in osteoporosis. However, there are few agents that counteract oxidative stress in osteoporosis. In the present study, the protective effects of allicin against the oxidative stress-induced mitochondrial dysfunction and apoptosis were investigated in murine osteoblast-like MC3T3-E1 cells. The results demonstrated that allicin counteracted the reduction of cell viability and induction of apoptosis caused by hydrogen peroxide (H2O2) exposure. The inhibition of apoptosis by allicin was confirmed by the inhibition of H2O2-induced cytochrome c release and caspase-3 activation. Moreover, the inhibition of apoptosis by allicin was identified to be associated with the counteraction of H2O2-induced mitochondrial dysfunction. In addition, allicin was demonstrated to be able to significantly ameliorate the repressed phosphoinositide 3-kinase (PI3K)/AKT and cyclic adenosine monophosphate response element-binding protein (CREB)/extracellular-signal-regulated kinase (ERK) signaling pathways by H2O2, which may also be associated with the anti-oxidative stress effects of allicin. In conclusion, allicin protects osteoblasts from H2O2-induced oxidative stress and apoptosis in MC3T3-E1 cells by improving mitochondrial function and the activation of PI3K/AKT and CREB/ERK signaling. The present study implies a promising role of allicin in oxidative stress-associated osteoporosis. PMID:27284348

  9. Neuregulin1 signaling targets SRF and CREB and activates the muscle spindle-specific gene Egr3 through a composite SRF-CREB-binding site.

    PubMed

    Herndon, Carter A; Ankenbruck, Nick; Lester, Bridget; Bailey, Julie; Fromm, Larry

    2013-03-10

    Muscle spindles are sensory receptors embedded within muscle that detect changes in muscle length. Each spindle is composed of specialized muscle fibers, known as intrafusal muscle fibers, along with the endings of axons from sensory neurons that innervate these muscle fibers. Formation of muscle spindles requires neuregulin1 (NRG1), which is released by sensory axons, activating ErbB receptors in muscle cells that are contacted. In muscle cells, the transcription factor Egr3 is transcriptionally induced by NRG1, which in turn activates various target genes involved in forming the intrafusal fibers of muscle spindles. The signaling relay within the NRG1-ErbB pathway that acts to induce Egr3 is presumably critical for muscle spindle formation but for the most part has not been determined. In the current studies, we examined, using cultured muscle cells, transcriptional regulatory mechanisms by which Egr3 responds to NRG1. We identified a composite regulatory element for the Egr3 gene, consisting adjacent sites that bind cAMP response element binding protein (CREB) and serum response factor (SRF), with a role in NRG1 responsiveness. The SRF element also influences Egr3 basal expression in unstimulated myotubes, and in the absence of the SRF element, the CREB element influences basal expression. We show that NRG1 signaling, to target SRF, acts on the SRF coactivators myocardian-related transcription factor (MRTF)-A and MRTF-B, which are known to activate SRF-mediated transcription, by stimulating their translocation from the cytoplasm to the nucleus. CREB is phosphorylated, which is known to contribute to its activation, in response to NRG1. These results suggest that NRG1 induces expression of the muscle spindle-specific gene Egr3 by stimulating the transcriptional activity of CREB and SRF.

  10. Tunable regulation of CREB DNA binding activity couples genotoxic stress response and metabolism

    PubMed Central

    Kim, Sang Hwa; Trinh, Anthony T.; Larsen, Michele Campaigne; Mastrocola, Adam S.; Jefcoate, Colin R.; Bushel, Pierre R.; Tibbetts, Randal S.

    2016-01-01

    cAMP response element binding protein (CREB) is a key regulator of glucose metabolism and synaptic plasticity that is canonically regulated through recruitment of transcriptional coactivators. Here we show that phosphorylation of CREB on a conserved cluster of Ser residues (the ATM/CK cluster) by the DNA damage-activated protein kinase ataxia-telangiectasia-mutated (ATM) and casein kinase1 (CK1) and casein kinase2 (CK2) positively and negatively regulates CREB-mediated transcription in a signal dependent manner. In response to genotoxic stress, phosphorylation of the ATM/CK cluster inhibited CREB-mediated gene expression, DNA binding activity and chromatin occupancy proportional to the number of modified Ser residues. Paradoxically, substoichiometric, ATM-independent, phosphorylation of the ATM/CK cluster potentiated bursts in CREB-mediated transcription by promoting recruitment of the CREB coactivator, cAMP-regulated transcriptional coactivators (CRTC2). Livers from mice expressing a non-phosphorylatable CREB allele failed to attenuate gluconeogenic genes in response to DNA damage or fully activate the same genes in response to glucagon. We propose that phosphorylation-dependent regulation of DNA binding activity evolved as a tunable mechanism to control CREB transcriptional output and promote metabolic homeostasis in response to rapidly changing environmental conditions. PMID:27431323

  11. 5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB).

    PubMed Central

    Giono, L E; Varone, C L; Cánepa, E T

    2001-01-01

    The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression. PMID:11139395

  12. Effect of rhynchophylline on the expression of p-CREB and sc-Fos in triatum and hippocampal CA1 area of methamphetamine-induced conditioned place preference rats.

    PubMed

    Liu, Wei; Peng, Qiu-Xian; Lin, Xiao-Liang; Luo, Chao-Hua; Jiang, Ming-Jin; Mo, Zhi-Xian; Yung, Ken Kin-Lam

    2014-01-01

    To explore the effect of rhynchophylline (Rhy) on the expression of p-CREB and c-Fos in the striatum and hippocampal CA1 area of methamphetamine-induced conditioned place preference (CPP) rat, methamphetamine (2 mg/kg) was injected to rats and the conditioned place preference was observed in these rats treated with or without Rhy. An immunohistochemistry assay was used to determine the expression of p-CREB and c-Fos in the striatum and hippocampal CA1 area. Methamphetamine induced significant behavior alteration in CPP, while after pretreatment with rhynchophylline or ketamine, the time of staying in methamphetamine-paired compartment of rats was significantly reduced. Methamphetamine also increased the number of p-CREB positive cells in the striatum and hippocampal CA1 zone, as well as p-Fos positive cells. However, the compound Rhy could attenuate the effect. These findings show that Rhy can suppress the acquisition of CPP in rats induced by methamphetamine and the action may be related with the reduced expression of p-CREB and p-Fos in the striatum and hippocampus.

  13. Differences in basal and morphine-induced FosB/DeltaFosB and pCREB immunoreactivities in dopaminergic brain regions of alcohol-preferring AA and alcohol-avoiding ANA rats.

    PubMed

    Kaste, Kristiina; Kivinummi, Tanja; Piepponen, T Petteri; Kiianmaa, Kalervo; Ahtee, Liisa

    2009-06-01

    Besides alcohol, alcohol-preferring AA and alcohol-avoiding ANA rats differ also with respect to other abused drugs. To study the molecular basis of these differences, we examined the expression of two transcription factors implicated in addiction, DeltaFosB and pCREB, in brain dopaminergic regions of AA and ANA rats. The effects of morphine and nicotine were studied to relate the behavioral and molecular changes induced by these drugs. Baseline FosB/DeltaFosB immunoreactivity (IR) in the nucleus accumbens core and pCREB IR in the prefrontal cortex (PFC) were elevated in AA rats. Morphine increased DeltaFosB-like IR more readily in the caudate-putamen of AA rats than in ANA rats. In the PFC morphine decreased pCREB IR in AA rats, but increased it in ANA rats. In addition to enhanced locomotor response, the development of place preference to morphine was enhanced in AA rats. The enhanced nicotine-induced locomotor sensitization found in AA compared with ANA rats seems to depend in addition to dopamine and DeltaFosB on other mechanisms. These findings suggest that enhanced sensitivity of AA rats to morphine is related to augmented morphine-induced expression of FosB/DeltaFosB and morphine-induced reduction of pCREB levels. Moreover, altered innate expression of FosB/DeltaFosB and pCREB in AA rats is likely to affect the sensitivity of these rats to abused drugs.

  14. Synergistic Neuroprotective Effects of Two Herbal Ingredients via CREB-Dependent Pathway

    PubMed Central

    Liu, Xu; Wang, Dongxiao; Zhao, Runqing; Dong, Xianzhe; Hu, Yuan; Liu, Ping

    2016-01-01

    As two natural oligosaccharide esters, 3,6’-Disinapoyl sucrose (DISS) and tenuifolisideA (TFSA) are originating from the root of Polygala tenuifolia Willd, a traditional Chinese medicine used in treatment of mental disorders. Previous reports have shown that both of them possess in vitro neuroprotective effects by stimulating different upstream pathways related with cyclic AMP-responsive element-binding protein (CREB). In the present study, we investigated the additive neuroprotective effects of DISS and TFSA on Glu-induced damage of SY5Y cells and purposed the possible underlying mechanism. The interaction between DISS and TFSA showed a clear-cut synergistic effect as evidenced by combination index (CI). Additional evidence from biochemical (NOS activity) assays confirmed their additive inhibition on the Glu-induced NOS hyperactivation. Moreover, we showed that co-treatment of DISS and TFSA resulted in an additively up-regulated phosphorylation of CREB as well as increased expressions of CRTC1 and BDNF. Neuroprotective effects of DISS and TFSA on Glu-induced decrease in cell viability were blocked by MAPK/ERK1/2 inhibitor (U0126) and PI3-K inhibitor (LY290042). Nevertheless, the CRTC1 or BDNF expression induced by these two compounds was significantly reduced in the presence of either ERK or PI3-K inhibitor, indicating that the two oligosaccharide esters shared some common pathways in the regulation of CREB-BDNF pathway. Taken together, we, for the first time, showed that DISS and TFSA exerted the additive neuroprotective effects on CREB-BDNF signaling pathway through complementary mechanisms. PMID:27729863

  15. Mdivi-1 Protects Against Ischemic Brain Injury via Elevating Extracellular Adenosine in a cAMP/CREB-CD39-Dependent Manner.

    PubMed

    Cui, Mei; Ding, Hongyan; Chen, Fangzhe; Zhao, Yanxin; Yang, Qi; Dong, Qiang

    2016-01-01

    This study aimed to examine whether the neuroprotective effects of Mdivi-1 are attributable to extracellular ATP and adenosine. Mdivi-1 was administered prior to or post middle cerebral artery occlusion (MCAO). The extracellular adenosine was measured by in vivo microdialysis and high-pressure liquid chromatography (HPLC) in MCAO mouse model. Western blot was done to determine the influence of Mdivi-1 on the expression of CD39 and CREB phosphorylation both in vivo and in the cultured astrocytes. Intracellular cAMP and protein kinase A (PKA) activity were detected in primary astrocytes. Results showed that Mdivi-1 significantly reduced infarct volume and neurological scores when administered either prior to or post MCAO. Interestingly, pretreatment with Mdivi-1 resulted in marked increase of extracellular adenosine and concomitant decrease in ATP. The expression of CD39, but not CD73, was upregulated by Mdivi-1, which was associated with the elevated phosphorylated cAMP response element-binding protein (CREB), a transcription factor potentially regulating CD39 expression. In primary astrocytes, Mdivi-1 treatment induced increases in intracellular cAMP, PKA activity and CREB phosphorylation, and PKA-specific inhibitor completely reversed Mdivi-1-induced CD39 expression. Our results demonstrate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by cAMP/PKA/CREB cascade. Figure Potential mechanisms by which Mdivi-1 mediates the neuroprotection on cerebral ischemic stroke. Results from the present study indicate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by the cAMP/PKA/CREB cascades.

  16. Storage of passive motion pattern in hippocampal CA1 region depends on CaMKII/CREB signaling pathway in a motion sickness rodent model

    PubMed Central

    Wang, Junqin; Liu, Jiluo; Pan, Leilei; Qi, Ruirui; Liu, Peng; Zhou, Wei; Cai, Yiling

    2017-01-01

    Sensory mismatch between actual motion information and anticipated sensory patterns (internal model) is the etiology of motion sickness (MS). Some evidence supports that hippocampus might involve the neural storage of the “internal model”. This study established an “internal model” acquisition-retention behavioral model using a repeated habituation rotation training protocol. We tried to identify the hippocampal subregion involved in “internal model” retention using chemical lesion methods. Hippocampal kinases (CaMK, CaMKIV, CREB and ERK1/2) phosphorylation in the target subregion was assayed and the effects of kinase inhibitors (KN93 or U0126) on “internal model” retention were investigated. The activities of potential kinases (CaMKII and CREB) were also examined in otoliths deficit het/het mice. In habituated rats, CA1 lesion reproduced MS-related behavioral responses on “internal model” retention day. Habituation training increased CaMKII and CREB activity but had no effect on CaMKIV and ERK1/2 activity in the CA1, while inhibition of CaMKII but not ERK1/2 impaired “internal model” retention. In het/het mice, CaMKII and CREB were not activated in the CA1 on the retention day. These results suggested that CaMKII/CREB pathway might potentially contribute to the storage of the “internal model” in the hippocampal CA1 after motion sickness induced by vestibular stimulation. PMID:28230177

  17. 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.

  18. Essential oil of Syzygium aromaticum reverses the deficits of stress-induced behaviors and hippocampal p-ERK/p-CREB/brain-derived neurotrophic factor expression.

    PubMed

    Liu, Bin-Bin; Luo, Liu; Liu, Xiao-Long; Geng, Di; Li, Cheng-Fu; Chen, Shao-Mei; Chen, Xue-Mei; Yi, Li-Tao; Liu, Qing

    2015-02-01

    Syzygium aromaticum has been widely used in traditional medicine. Our study investigated the safety and antidepressant-like effects of the essential oil of S. aromaticum after acute or long-term treatment. Using GC-MS, a total of eight volatile constituents were identified in the essential oil of S. aromaticum. The single LD50 was approximately 4500 mg/kg based on a 24-h acute oral toxicity study. In a long-term repeated toxicity study of this essential oil (100, 200, and 400 mg/kg, p. o.), only 400 mg/kg induced a significant decrease in body weight. In addition, no significant changes in relative organ weights and histopathological analysis were observed in all doses of essential oil-treated mice compared with the control group. Furthermore, acute S. aromaticum essential oil administration by gavage exerted antidepressant-like effects in the forced swimming test (200 mg/kg, p < 0.05) and tail suspension test (100 and 200 mg/kg, p < 0.05). Long-term S. aromaticum essential oil treatment via gavage significantly increased sucrose preference (50 mg/kg, p < 0.05; 100 and 200 mg/kg, p < 0.01) as well as elevated the protein levels of hippocampal p-ERK, p-CREB, and brain-derived neurotrophic factor in mice exposed to chronic unpredictable mild stress. These results confirmed the safety of the essential oil of S. aromaticum and suggested that its potent antidepressant-like property might be attributed to the improvement in the hippocampal pERK1/2-pCREB-BDNF pathway in rats exposed to chronic unpredictable mild stress.

  19. Rit-mediated Stress Resistance Involves a p38-Mitogen- and Stress-activated Protein Kinase 1 (MSK1)-dependent cAMP Response Element-binding Protein (CREB) Activation Cascade*

    PubMed Central

    Shi, Geng-Xian; Cai, Weikang; Andres, Douglas A.

    2012-01-01

    The cAMP response element (CRE)-binding protein (CREB) is a key regulatory factor of gene transcription, and plays an essential role in development of the central nervous system and for neuroprotection. Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both ERK and p38 mitogen-activated protein (MAP) kinases cascades. Recent studies have identified the Ras-related small G-protein, Rit, as a central regulator of a p38-MK2-HSP27 signaling cascade that functions as a critical survival mechanism for cells adapting to stress. Here, we examine the contribution of Rit-p38 signaling to the control of stress-dependent gene transcription. Using a pheochromocytoma cell model, we find that a novel Rit-p38-MSK1/2 pathway plays a critical role in stress-mediated CREB activation. RNAi-mediated Rit silencing, or inhibition of p38 or MSK1/2 kinases, was found to disrupt stress-mediated CREB-dependent transcription, resulting in increased cell death. Furthermore, ectopic expression of active Rit stimulates CREB-Ser133 phosphorylation, induces expression of the anti-apoptotic Bcl-2 and BclXL proteins, and promotes cell survival. These data indicate that the Rit-p38-MSK1/2 signaling pathway may have an important role in the stress-dependent regulation of CREB-dependent gene expression. PMID:23038261

  20. In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

    PubMed Central

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

    2016-01-01

    XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells. PMID:26666690

  1. Fyn is required for oxidative- and hyperosmotic-stress-induced tyrosine phosphorylation of caveolin-1.

    PubMed Central

    Sanguinetti, Amy R; Cao, Haiming; Corley Mastick, Cynthia

    2003-01-01

    Caveolin-1 is phosphorylated on Tyr(14) in response to both oxidative and hyperosmotic stress. In the present paper, we show that this phosphorylation requires activation of the Src family kinase Fyn. Stress-induced caveolin phosphorylation was abolished by three Src kinase inhibitors, SU6656, PP2 and PD180970, and was not observed in fibroblasts derived from a Src, Yes and Fyn triple-knockout mouse (SYF-/-). Using cell lines derived from single-kinase-knockout mice (Src-/-, Yes-/- and Fyn-/-), we show that expression of Fyn, but not Src or Yes, is required for stress-induced caveolin phosphorylation. Heterologous expression of Fyn in the SYF-/- and Fyn-/- cells was sufficient to reconstitute stress-induced caveolin phosphorylation, and overexpression of Fyn in wild-type cells induced hyperphosphorylation of caveolin. Fyn was autophosphorylated following oxidative stress, verifying activation of this kinase. Interestingly, there was a concomitant increase in the phosphorylation of Fyn on its Csk (C-terminal Src kinase) site, indicating feedback inhibition. Csk binds to phosphocaveolin [Cao, Courchesne and Mastick (2002) J. Biol. Chem. 277, 8771-8774] and should phosphorylate any co-localized Src-family kinases. Oxidative-stress-induced phosphorylation of caveolin-1 also requires expression of Abl [Sanguinetti and Mastick (2003) Cell Signal. 15, 289-298]. Using inhibitors and cells derived from knockout mice, we verified a requirement for both Abl and Fyn in stress-induced caveolin phosphorylation in a single cell type. Our data suggest a novel mechanism for attenuation of Src-kinase activity by Abl: stable tyrosine phosphorylation of a scaffolding protein, caveolin, and recruitment of Csk. Paxillin, a substrate of both Abl and Src, organizes a similar regulatory complex. PMID:12921535

  2. The neuroprotective effects of α-iso-cubebene on dopaminergic cell death: involvement of CREB/Nrf2 signaling.

    PubMed

    Park, Sun Young; Son, Beung Gu; Park, Young Hoon; Kim, Cheol-Min; Park, Geuntae; Choi, Young-Whan

    2014-09-01

    As a part of ongoing studies to elucidate pharmacologically active components of Schisandra chinensis, we isolated and studied α-iso-cubebene. The neuroprotective mechanisms of α-iso-cubebene in human neuroblastoma SH-SY5Y cells were investigated. α-Iso-cubebene significantly inhibited cytotoxicity and apoptosis due to 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in dopaminergic SH-SY5Y cells. Pretreatment of cells with α-iso-cubebene reduced intracellular accumulation of ROS and calcium in response to 6-OHDA. The neuroprotective effects of α-iso-cubebene were found to result from protecting the mitochondrial membrane potential. Notably, α-iso-cubebene inhibited the release of apoptosis-inducing factor from the mitochondria into the cytosol and nucleus after 6-OHDA treatment. α-Iso-cubebene also induced the activation of PKA/PKB/CREB/Nrf2 and suppressed 6-OHDA-induced neurotoxicity. α-Iso-cubebene was found to induce phosphorylation of PKA and PKB and activate Nrf2 and CREB signaling pathways in a dose-dependent manner. Additionally, α-iso-cubebene stimulated the expression of the antioxidant response genes NQO1 and HO-1. Finally, α-iso-cubebene-mediated neuroprotective effects were found to be reversible after transfection with CREB and Nrf2 small interfering RNAs.

  3. Stress-induced inhibition of translation independently of eIF2α phosphorylation.

    PubMed

    Knutsen, Jon Halvor Jonsrud; Rødland, Gro Elise; Bøe, Cathrine Arnason; Håland, Tine Weise; Sunnerhagen, Per; Grallert, Beáta; Boye, Erik

    2015-12-01

    Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution.

  4. Stress-induced inhibition of translation independently of eIF2α phosphorylation

    PubMed Central

    Knutsen, Jon Halvor Jonsrud; Rødland, Gro Elise; Bøe, Cathrine Arnason; Håland, Tine Weise; Sunnerhagen, Per; Grallert, Beáta; Boye, Erik

    2015-01-01

    ABSTRACT Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution. PMID:26493332

  5. Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation.

    PubMed

    Röhrig, Horst; Schmidt, Jürgen; Colby, Thomas; Bräutigam, Anne; Hufnagel, Peter; Bartels, Dorothea

    2006-08-01

    Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LCMS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.

  6. CREB influences timing and entrainment of the SCN circadian clock.

    PubMed

    Lee, Boyoung; Li, Aiqing; Hansen, Katelin F; Cao, Ruifeng; Yoon, Jae Hwa; Obrietan, Karl

    2010-12-01

    The transcriptional feedback circuit, which is at the core of the suprachiasmatic nucleus (SCN) circadian (i.e., 24 h) clock, is tightly coupled to both external entrainment cues, such as light, as well as rhythmic cues that arise on a system-wide level within the SCN. One potential signaling pathway by which these cues are conveyed to the molecular clock is the CREB/CRE transcriptional cascade. In this study, we employed a tetracycline-inducible CREB repressor mouse strain, in which approximately 60% of the SCN neurons express the transgene, to test CREB functionality in the clock and its effects on overt rhythmicity. We show that attenuated CREB signaling in the SCN led to a significant reduction in light-evoked clock entrainment. An examination of circadian timing revealed that CREB repressor mice exhibited normal free-running rhythms in the absence of external lighting cues. However, under conditions of constant light, which typically leads to a lengthening of the circadian period, CREB repressor mice exhibited a dramatic arrhythmic phenotype, which could be reversed with doxycycline. At a cellular level, the repression of CREB led to a significant reduction in both the expression of the circadian clock proteins PERIOD1 and PERIOD2 and the clock output hormones AVP and VIP. Together, these data support the idea that the CRE transcriptional pathway orchestrates transcriptional events that are essential for both the maintenance of SCN timing and light entrainment of the circadian clock.

  7. FAK and Src kinases are required for netrin-induced tyrosine phosphorylation of UNC5.

    PubMed

    Li, Weiquan; Aurandt, Jennifer; Jürgensen, Claudia; Jürgense, Claudia; Rao, Yi; Guan, Kun-Liang

    2006-01-01

    During neuronal development, netrin and its receptors UNC5 and DCC (deleted in colorectal cancer) guide axonal growth cones in navigating to their targets. Netrin also plays important roles in the regulation of cell migration, tissue morphogenesis and tumor growth. Here, we show that netrin induces UNC5 tyrosine phosphorylation and that this effect of netrin is dependent on its co-receptor DCC. UNC5 tyrosine phosphorylation is known to be important for netrin to induce cell migration and axonal repulsion. Src tyrosine kinase activity is required for netrin to stimulate UNC5 tyrosine phosphorylation in neurons and transfected cells. The SH2 domain of Src kinase directly interacts with the cytosolic domain of UNC5 in a tyrosine-phosphorylation-dependent manner. Furthermore, the tyrosine kinase focal adhesion kinase (FAK) is also involved in netrin-induced UNC5 tyrosine phosphorylation. Both Src and FAK can phosphorylate UNC5. Our data suggest a model in which netrin stimulates UNC5 tyrosine phosphorylation and signaling in a manner dependent on the co-receptor DCC, through the recruitment of Src and FAK kinases.

  8. α-Melanocyte-stimulating hormone ameliorates ocular surface dysfunctions and lesions in a scopolamine-induced dry eye model via PKA-CREB and MEK-Erk pathways.

    PubMed

    Ru, Yusha; Huang, Yue; Liu, Huijuan; Du, Juan; Meng, Zhu; Dou, Zexia; Liu, Xun; Wei, Rui Hua; Zhang, Yan; Zhao, Shaozhen

    2015-12-21

    Dry eye is a highly prevalent, chronic, and multifactorial disease that compromises quality of life and generates socioeconomic burdens. The pathogenic factors of dry eye disease (DED) include tear secretion abnormalities, tear film instability, and ocular surface inflammation. An effective intervention targeting the pathogenic factors is needed to control this disease. Here we applied α-Melanocyte-stimulating hormone (α-MSH) twice a day to the ocular surface of a scopolamine-induced dry eye rat model. The results showed that α-MSH at different doses ameliorated tear secretion, tear film stability, and corneal integrity, and corrected overexpression of proinflammatory factors, TNF-α, IL-1β, and IFN-γ, in ocular surface of the dry eye rats. Moreover, α-MSH, at 10(-4) μg/μl, maintained corneal morphology, inhibited apoptosis, and restored the number and size of conjunctival goblet cells in the dry eye rats. Mechanistically, α-MSH activated both PKA-CREB and MEK-Erk pathways in the dry eye corneas and conjunctivas; pharmacological blockade of either pathway abolished α-MSH's protective effects, suggesting that both pathways are necessary for α-MSH's protection under dry eye condition. The peliotropic protective functions and explicit signaling mechanism of α-MSH warrant translation of the α-MSH-containing eye drop into a novel and effective intervention to DED.

  9. Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB.

    PubMed

    Lee, Jinwoo; Tong, Tiegang; Takemori, Hiroshi; Jefcoate, Colin

    2015-06-15

    In mouse steroidogenic cells the activation of cholesterol metabolism is mediated by steroidogenic acute regulatory protein (StAR). Here, we visualized a coordinated regulation of StAR transcription, splicing and post-transcriptional processing, which are synchronized by salt inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2). To detect primary RNA (pRNA), spliced primary RNA (Sp-RNA) and mRNA in single cells, we generated probe sets by using fluorescence in situ hybridization (FISH). These methods allowed us to address the nature of StAR gene expression and to visualize protein-nucleic acid interactions through direct detection. We show that SIK1 represses StAR expression in Y1 adrenal and MA10 testis cells through inhibition of processing mediated by CRTC2. Digital image analysis matches qPCR analyses of the total cell culture. Evidence is presented for spatially separate accumulation of StAR pRNA and Sp-RNA at the gene loci in the nucleus. These findings establish that cAMP, SIK and CRTC mediate StAR expression through activation of individual StAR gene loci.

  10. Acetylcholine-induced phosphorylation in isolated outer hair cells.

    PubMed

    Szõnyi, M; Csermely, P; Sziklai, I

    1999-03-01

    Two groups of isolated, surviving outer hair cells (OHCs) of guinea pig cochleas (n = 20, for each group) were treated with 10 microM acetylcholine or acetylcholine plus strichnine (an alpha9 nAChR antagonist), respectively, under short-term tissue culture conditions. The protein content of the cell homogenates was separated by SDS-polyacrylamide gel electrophoresis, Western blotted and labelled with an antibody against phosphoserine residues. Signals were detected using the ECL system. Acetylcholine challenge of the OHCs resulted in a difference in the pattern of phosphorylated proteins from those of strichnine pretreated cells. A 220 kDa and a 120 kDa protein expressed a more intense phosphorylated state in the ACh group compared with the ACh plus strichnine group. The 220 kDa phosphoprotein is in the range of the cytoskeletal protein beta-fodrin, whereas the 120 kDa fraction is similar to alpha-fodrin or an ankyrin isoform. Phosphorylation of proteins due to activation of the AChR by agonist can play a role in the signalling mechanism between receptor activation and increase in the electromotile capability of isolated OHCs.

  11. Impact of oncogenic K-RAS on YB-1 phosphorylation induced by ionizing radiation

    PubMed Central

    2011-01-01

    Introduction Expression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation. Methods IR-induced YB-1 phosphorylation in K-RAS wild-type (K-RASwt) and K-RAS-mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using γ-H2AX foci and standard colony formation assays, we investigated the function of YB-1 in repair of IR-induced DNA double-stranded breaks (DNA-DSB) and postirradiation survival was investigated. Results The average level of phosphorylation of YB-1 in the breast cancer cell lines SKBr3, MCF-7, HBL100 and MDA-MB-231 was significantly higher than that in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF-7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similarly to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 as a result of irradiation or K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS siRNA as well as YB-1 siRNA blocked

  12. Dexmedetomidine-Induced Contraction Involves CPI-17 Phosphorylation in Isolated Rat Aortas

    PubMed Central

    Ok, Seong-Ho; Kwon, Seong-Chun; Baik, Jiseok; Hong, Jeong-Min; Oh, Jiah; Han, Jeong Yeol; Sohn, Ju-Tae

    2016-01-01

    Dexmedetomidine, a highly selective α-2 adrenoceptor agonist, produces vasoconstriction, which leads to transiently increased blood pressure. The goal of this study was to investigate specific protein kinases and the associated cellular signal pathways responsible for the increased calcium sensitization induced by dexmedetomidine in isolated rat aortas, with a particular focus on phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17). The effect of Y-27632 and chelerythrine on the dexmedetomidine-induced intracellular calcium concentration ([Ca2+]i) and tension were assessed using fura-2-loaded aortic strips. The effects of rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride on the dexmedetomidine-induced phosphorylation of CPI-17 or of the 20-kDa regulatory light chain of myosin (MLC20) were investigated in rat aortic vascular smooth muscle cells. The effects of rauwolscine, Y-27632, and chelerythrine on the membrane translocation of Rho-kinase and protein kinase C (PKC) phosphorylation induced by dexmedetomidine were assessed. Y-27632 and chelerythrine each reduced the slopes of the [Ca2+]i-tension curves of dexmedetomidine-induced contraction, and Y-27632 more strongly reduced these slopes than did chelerythrine. Rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride attenuated the dexmedetomidine-induced phosphorylation of CPI-17 and MLC20. Taken together, these results suggest that dexmedetomidine-induced contraction involves calcium sensitization, which appears to be mediated by CPI-17 phosphorylation via Rho-kinase or PKC. PMID:27706026

  13. Neuroprotective effect of arctigenin via upregulation of P-CREB in mouse primary neurons and human SH-SY5Y neuroblastoma cells.

    PubMed

    Zhang, Nan; Wen, Qingping; Ren, Lu; Liang, Wenbo; Xia, Yang; Zhang, Xiaodan; Zhao, Dan; Sun, Dong; Hu, Yv; Hao, Haiguang; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian; Kang, Tingguo

    2013-09-10

    Arctigenin (Arc) has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ) production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1) protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN) expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB) induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB.

  14. Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

    PubMed

    Min, Jiho; Jin, Yoon-Mi; Moon, Je-Sung; Sung, Min-Sun; Jo, Sangmee Ahn; Jo, Inho

    2006-06-01

    Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.

  15. Γ-Ionizing radiation-induced activation of the EGFR-p38/ERK-STAT3/CREB-1-EMT pathway promotes the migration/invasion of non-small cell lung cancer cells and is inhibited by podophyllotoxin acetate.

    PubMed

    Cho, Jeong Hyun; Hong, Wan Gi; Jung, Yu-Jin; Lee, Jaeseok; Lee, Eunah; Hwang, Sang-Gu; Um, Hong-Duck; Park, Jong Kuk

    2016-06-01

    Here, we report a new intracellular signaling pathway involved in γ-ionizing radiation (IR)-induced migration/invasion and show that podophyllotoxin acetate (PA) inhibits the IR-induced invasion and migration of A549 cells (a non-small cell lung cancer (NSCLC) cell line). Our results revealed that IR increased the invasion/migration of A549 cells, and this effect was decreased by 10 nM PA treatment. PA also inhibited the expressions/activities of matrix metalloprotase (MMP) -2, MMP-9, and vimentin, suggesting that PA could block the IR-induced epithelial-mesenchymal transition (EMT). The IR-induced increases in invasion/migration were associated with the activation of EGFR-AKT, and PA inhibited this effect. P38 and p44/42 ERK were also involved in IR-induced invasion/migration, and combined treatments with PA plus inhibitors of each MAPK synergistically blocked this invasion/migration. In terms of transcription factors (TFs), IR-induced increases in cyclic AMP response element-binding protein-1 (CREB-1) and signal transducer and activator of transcription 3 (STAT3) increased invasion/migration and EMT. PA also inhibited these transcription factors and then blocked IR-induced invasion/migration. Collectively, these results indicate that IR induces cancer cell invasion/migration by activating the EGFR-p38/ERK-CREB-1/STAT3-EMT pathway and that PA blocks this pathway to inhibit IR-induced invasion/migration.

  16. Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified

    PubMed Central

    Hart, Katherine; Kothari, Anisha; Mackintosh, Samuel G.; Kovak, Matthew R.; Chambers, Timothy C.

    2016-01-01

    Microtubule targeting agents (MTAs) characteristically promote phosphorylation and degradation of Mcl-1, and this represents a critical pro-apoptotic signal in mitotic death. While several phosphorylation sites and kinases have been implicated in mitotic arrest-induced Mcl-1 phosphorylation, a comprehensive biochemical analysis has been lacking. Contrary to previous reports suggesting that T92 phosphorylation by Cdk1 regulates Mcl-1 degradation, a T92A Mcl-1 mutant expressed in HeLa cells was phosphorylated and degraded with the same kinetics as wild-type Mcl-1 following vinblastine treatment. Similarly, when Mcl-1 with alanine replacements of all five putative Cdk sites (S64, T92, S121, S159, T163) was expressed, it was also phosphorylated and degraded in response to vinblastine. To analyze Mcl-1 phosphorylation in more detail, two-dimensional gel electrophoresis (2D-PAGE) was performed. While untreated cells expressed mainly unphosphorylated Mcl-1 with two minor phosphorylated species, Mcl-1 from vinblastine treated cells migrated during 2D-PAGE as a train of acidic spots representing nine or more phosphorylated species. Immunopurification and mass spectrometry of phosphorylated Mcl-1 derived from mitotically arrested HeLa cells revealed nine distinct sites, including several previously unreported. Mcl-1 bearing substitutions of all nine sites had a longer half-life than wild-type Mcl-1 under basal conditions, but still underwent phosphorylation and degradation in response to vinblastine treatment, and, like wild-type Mcl-1, was unable to protect cells from MTA treatment. These results reveal an unexpected complexity in Mcl-1 phosphorylation in response to MTAs and indicate that previous work has severely underestimated the number of sites, and thus encourage major revisions to the current model. PMID:27738316

  17. Uncoupling Stress-Inducible Phosphorylation of Heat Shock Factor 1 from Its Activation

    PubMed Central

    Budzyński, Marek A.; Puustinen, Mikael C.; Joutsen, Jenny

    2015-01-01

    In mammals the stress-inducible expression of genes encoding heat shock proteins is under the control of the heat shock transcription factor 1 (HSF1). Activation of HSF1 is a multistep process, involving trimerization, acquisition of DNA-binding and transcriptional activities, which coincide with several posttranslational modifications. Stress-inducible phosphorylation of HSF1, or hyperphosphorylation, which occurs mainly within the regulatory domain (RD), has been proposed as a requirement for HSF-driven transcription and is widely used for assessing HSF1 activation. Nonetheless, the contribution of hyperphosphorylation to the activity of HSF1 remains unknown. In this study, we generated a phosphorylation-deficient HSF1 mutant (HSF1Δ∼PRD), where the 15 known phosphorylation sites within the RD were disrupted. Our results show that the phosphorylation status of the RD does not affect the subcellular localization and DNA-binding activity of HSF1. Surprisingly, under stress conditions, HSF1Δ∼PRD is a potent transactivator of both endogenous targets and a reporter gene, and HSF1Δ∼PRD has a reduced activation threshold. Our results provide the first direct evidence for uncoupling stress-inducible phosphorylation of HSF1 from its activation, and we propose that the phosphorylation signature alone is not an appropriate marker for HSF1 activity. PMID:25963659

  18. Resveratrol ameliorates spatial learning memory impairment induced by Aβ1-42 in rats.

    PubMed

    Wang, Rui; Zhang, Yu; Li, Jianguo; Zhang, Ce

    2017-03-06

    β-amyloid (Aβ) deposition is considered partially responsible for cognitive dysfunction in Alzheimer's disease (AD). Recently, resveratrol has been reported to play a potential role as a neuroprotective biofactor by modulating Aβ pathomechanisms, including through anti-neuronal apoptotic, anti-oxidative stress, and anti-neuroinflammatory effects. In addition, SIRT1 has been demonstrated to modulate learning and memory function by regulating the expression of cAMP response binding protein (CREB), which involves in modulating the expression of SIRT1. However, whether resveratrol can alleviate Aβ-induced cognitive dysfunction, whether SIRT1 expression and CREB phosphorylation in the hippocampus are affected by Aβ, and whether resveratrol influences these effects remain unknown. In the present study, we used a hippocampal injection model in rats to investigate the effects of resveratrol on Aβ1-42-induced impairment of spatial learning, memory and synaptic plasticity as well as on alterations of SIRT1 expression and CREB phosphorylation. We found that resveratrol significantly reversed the water maze behavioral impairment and the attenuation of long-term potentiation (LTP) in area CA1 that were induced by hippocampal injection of Aβ1-42. Interestingly, resveratrol also prevented the Aβ1-42-induced reductions in SIRT1 expression and CREB phosphorylation in rat hippocampus. In conclusion, in rats, resveratrol protects neurons against Aβ1-42-induced disruption of spatial learning, memory and hippocampal LTP. The mechanisms underlying the neuroprotective effects may involve rescue of SIRT1 expression and CREB phosphorylation.

  19. Phosphorylated tau potentiates Aβ-induced mitochondrial damage in mature neurons.

    PubMed

    Quintanilla, Rodrigo A; von Bernhardi, Rommy; Godoy, Juan A; Inestrosa, Nibaldo C; Johnson, Gail V W

    2014-11-01

    Tau phosphorylated at the PHF-1 epitope (S396/S404) is likely involved in the pathogenesis of Alzheimer's disease (AD). However, the molecular mechanisms by which tau phosphorylated at these sites negatively impacts neuronal functions are still under scrutiny. Previously, we showed that expression of tau truncated at D421 enhances mitochondrial dysfunction induced by Aβ in cortical neurons. To extend these findings, we expressed tau pseudo-phosphorylated at S396/404 (T42EC) in mature and young cortical neurons and evaluated different aspects of mitochondrial function in response to Aβ. Expression of T42EC did not induce significant changes in mitochondrial morphology, mitochondrial length, or mitochondrial transport, compared to GFP and full-length tau. However, T42EC expression enhanced Aβ-induced mitochondrial membrane potential loss and increased superoxide levels compared to what was observed in mature neurons expressing full-length tau. The same effect was observed in mature neurons that expressed both pseudo-phosphorylated and truncated tau when they were treated with Aβ. Interestingly, the mitochondrial failure induced by Aβ in mature neurons that expressed T42EC, was not observed in young neurons expressing T42EC. These novel findings suggest that phosphorylated tau (PHF-1 epitope) enhances Aβ-induced mitochondrial injury, which contributes to neuronal dysfunction and to the pathogenesis of AD.

  20. Rapamycin induces Bad phosphorylation in association with its resistance to human lung cancer cells.

    PubMed

    Liu, Yan; Sun, Shi-Yong; Owonikoko, Taofeek K; Sica, Gabriel L; Curran, Walter J; Khuri, Fadlo R; Deng, Xingming

    2012-01-01

    Inhibition of mTOR signaling by rapamycin has been shown to activate extracellular signal-regulated kinase 1 or 2 (ERK1/2) and Akt in various types of cancer cells, which contributes to rapamycin resistance. However, the downstream effect of rapamycin-activated ERKs and Akt on survival or death substrate(s) remains unclear. We discovered that treatment of human lung cancer cells with rapamycin results in enhanced phosphorylation of Bad at serine (S) 112 and S136 but not S155 in association with activation of ERK1/2 and Akt. A higher level of Bad phosphorylation was observed in rapamycin-resistant cells compared with parental rapamycin-sensitive cells. Thus, Bad phosphorylation may contribute to rapamycin resistance. Mechanistically, rapamycin promotes Bad accumulation in the cytosol, enhances Bad/14-3-3 interaction, and reduces Bad/Bcl-XL binding. Rapamycin-induced Bad phosphorylation promotes its ubiquitination and degradation, with a significant reduction of its half-life (i.e., from 53.3-37.5 hours). Inhibition of MEK/ERK by PD98059 or depletion of Akt by RNA interference blocks rapamycin-induced Bad phosphorylation at S112 or S136, respectively. Simultaneous blockage of S112 and S136 phosphorylation of Bad by PD98059 and silencing of Akt significantly enhances rapamycin-induced growth inhibition in vitro and synergistically increases the antitumor efficacy of rapamycin in lung cancer xenografts. Intriguingly, either suppression of Bad phosphorylation at S112 and S136 sites or expression of the nonphosphorylatable Bad mutant (S112A/S136A) can reverse rapamycin resistance. These findings uncover a novel mechanism of rapamycin resistance, which may promote the development of new strategies for overcoming rapamycin resistance by manipulating Bad phosphorylation at S112 and S136 in human lung cancer.

  1. Detection of tyrosine phosphorylated peptides via skimmer collision-induced dissociation/ion trap mass spectrometry.

    PubMed

    Zolodz, Melissa D; Wood, Karl V

    2003-03-01

    Phosphorylation of proteins is an important post-translational protein modification in cellular response to environmental change and occurs in both prokaryotes and eukaryotes. Identification of the amino acid on individual proteins that become phosphorylated in response to extracellular stimulus is essential for understanding the mechanisms involved in the intracellular signals that these modifications facilitate. Most protein kinases catalyze the phosphorylation of proteins on serine, threonine or tyrosine. Although tyrosine phosphorylation is often the least abundant of the three major phosphorylation sites, it is important owing to its role in signal pathways. Currently available methods for the identification of phosphorylation sites can often miss low levels of tyrosine phosphorylations. This paper describes a method for the identification of phosphotyrosine-containing peptides using electrospray ionization on an ion trap mass spectrometer. Skimmer-activated collision-induced dissociation (CID) was used to generate the phosphotyrosine immonium ion at m/z 216. This method is gentle enough that the protonated molecule of the intact peptide is still observed. In-trap CID was employed for the verification of the phosphotyrosine immonium ion. Using this technique, low levels of phosphotyrosine-containing peptides can be identified from peptide mixtures separated by nanoflow micro liquid chromatography/mass spectrometry.

  2. Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

    PubMed Central

    Unoki, Takamitsu; Abiko, Yumi; Toyama, Takashi; Uehara, Takashi; Tsuboi, Koji; Nishida, Motohiro; Kaji, Toshiyuki; Kumagai, Yoshito

    2016-01-01

    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death. PMID:27357941

  3. Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells.

    PubMed

    Choi, Yoon Jung; Kim, So Young; Oh, Jung Min; Juhnn, Yong Sung

    2009-08-31

    Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the alpha subunit of Gs (GalphasQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GalphasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GalphasQL-stimulated Bak reporter luciferase activity. Expression of GalphasQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Galphas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Galphas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.

  4. Downregulation of CREB expression in Alzheimer's brain and in Aβ-treated rat hippocampal neurons

    PubMed Central

    2011-01-01

    Background Oxidative stress plays an important role in neuronal dysfunction and neuron loss in Alzheimer's brain. Previous studies have reported downregulation of CREB-mediated transcription by oxidative stress and Aβ. The promoter for CREB itself contains cyclic AMP response elements. Therefore, we examined the expression of CREB in the hippocampal neurons of Tg2576 mice, AD post-mortem brain and in cultured rat hippocampal neurons exposed to Aβ aggregates. Results Laser Capture Microdissection of hippocampal neurons from Tg2576 mouse brain revealed decreases in the mRNA levels of CREB and its target, BDNF. Immunohistochemical analysis of Tg2576 mouse brain showed decreases in CREB levels in hippocampus and cortex. Markers of oxidative stress were detected in transgenic mouse brain and decreased CREB staining was observed in regions showing abundance of astrocytes. There was also an inverse correlation between SDS-extracted Aβ and CREB protein levels in Alzheimer's post-mortem hippocampal samples. The levels of CREB-regulated BDNF and BIRC3, a caspase inhibitor, decreased and the active cleaved form of caspase-9, a marker for the intrinsic pathway of apoptosis, was elevated in these samples. Exposure of rat primary hippocampal neurons to Aβ fibrils decreased CREB promoter activity. Decrease in CREB mRNA levels in Aβ-treated neurons was reversed by the antioxidant, N-acetyl cysteine. Overexpression of CREB by adenoviral transduction led to significant protection against Aβ-induced neuronal apoptosis. Conclusions Our findings suggest that chronic downregulation of CREB-mediated transcription results in decrease of CREB content in the hippocampal neurons of AD brain which may contribute to exacerbation of disease progression. PMID:21854604

  5. The Myb-p300-CREB axis modulates intestine homeostasis, radiosensitivity and tumorigenesis

    PubMed Central

    Sampurno, S; Bijenhof, A; Cheasley, D; Xu, H; Robine, S; Hilton, D; Alexander, W S; Pereira, L; Mantamadiotis, T; Malaterre, J; Ramsay, R G

    2013-01-01

    The gastrointestinal (GI) epithelium is constantly renewing, depending upon the intestinal stem cells (ISC) regulated by a spectrum of transcription factors (TFs), including Myb. We noted previously in mice with a p300 mutation (plt6) within the Myb-interaction-domain phenocopied Myb hypomorphic mutant mice with regard to thrombopoiesis, and here, changes in GI homeostasis. p300 is a transcriptional coactivator for many TFs, most prominently cyclic-AMP response element-binding protein (CREB), and also Myb. Studies have highlighted the importance of CREB in proliferation and radiosensitivity, but not in the GI. This prompted us to directly investigate the p300–Myb–CREB axis in the GI. Here, the role of CREB has been defined by generating GI-specific inducible creb knockout (KO) mice. KO mice show efficient and specific deletion of CREB, with no evident compensation by CREM and ATF1. Despite complete KO, only modest effects on proliferation, radiosensitivity and differentiation in the GI under homeostatic or stress conditions were evident, even though CREB target gene pcna (proliferating cell nuclear antigen) was downregulated. creb and p300 mutant lines show increased goblet cells, whereas a reduction in enteroendocrine cells was apparent only in the p300 line, further resembling the Myb hypomorphs. When propagated in vitro, crebKO ISC were defective in organoid formation, suggesting that the GI stroma compensates for CREB loss in vivo, unlike in MybKO studies. Thus, it appears that p300 regulates GI differentiation primarily through Myb, rather than CREB. Finally, active pCREB is elevated in colorectal cancer (CRC) cells and adenomas, and is required for the expression of drug transporter, MRP2, associated with resistance to Oxaliplatin as well as several chromatin cohesion protein that are relevant to CRC therapy. These data raise the prospect that CREB may have a role in GI malignancy as it does in other cancer types, but unlike Myb, is not critical for GI

  6. Ethacrynic Acid Inhibits Sphingosylphosphorylcholine-Induced Keratin 8 Phosphorylation and Reorganization via Transglutaminase-2 Inhibition.

    PubMed

    Byun, Hyun Jung; Kang, Kyung Jin; Park, Mi Kyung; Lee, Hye Ja; Kang, June Hee; Lee, Eun Ji; Kim, You Ri; Kim, Hyun Ji; Kim, Young Woo; Jung, Kyung Chae; Kim, Soo Youl; Lee, Chang Hoon

    2013-09-30

    Sphingosylphosphorylcholine (SPC) is significantly increased in the malicious ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments in PANC-1 cells. The reorganization contributes to the viscoelasticity of metastatic cancer cells resulting in increased migration. Recently, we reported that transglutaminase-2 (Tgase-2) is involved in SPC-induced K8 phosphorylation and reorganization. However, effects of Tgase-2 inhibitors on SPC-induced K8 phosphorylation and reorganization were not clearly studied. We found that ethacrynic acid (ECA) concentration-dependently inhibited Tgase-2. Therefore, we examined the effects of ECA on SPC-induced K8 phosphorylation and reorganization. ECA concentration-dependently suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8. ECA also suppressed the SPC-induced migration and invasion. SPC induced JNK activation through Tgase-2 expression and ECA suppressed the activation and expression of JNK in PANC-1 cells. These results suggested that ECA might be useful to control Tgase-2 dependent metastasis of cancer cells such as pancreatic cancer and lung cancers.

  7. Activation of cAMP-response-element-binding protein (CREB) after focal cerebral ischemia stimulates neurogenesis in the adult dentate gyrus

    PubMed Central

    Zhu, Dong Ya; Lau, Lorraine; Liu, Shu Hong; Wei, Jian She; Lu, You Ming

    2004-01-01

    New neurons are generated in adult mammalians and may contribute to repairing the brain after injury. Here, we show that the number of new neurons in the dentate gyrus of adult rats increased in cerebral ischemic stroke and correlated with activation of the cAMP-response-element-binding protein (CREB). Inhibition of endogenous CREB by expression of a dominant-negative mutant of CREB (CREB-S133A or CREB-R287L) blocked ischemia-induced neurogenesis in the dentate gyrus of adult rats, whereas expression of constitutively active CREB, VP16-CREB, increased the number of new neurons. Thus, our findings provide roles and regulatory mechanisms for CREB in adult neurogenesis and possibly suggest a practical strategy for replacing dead neurons in brain injury. PMID:15197280

  8. Neuroinflammation is not a Prerequisite for Diabetes-induced Tau Phosphorylation.

    PubMed

    van der Harg, Judith M; Eggels, Leslie; Ruigrok, Silvie R; Hoozemans, Jeroen J M; la Fleur, Susanne E; Scheper, Wiep

    2015-01-01

    Abnormal phosphorylation and aggregation of tau is a key hallmark of Alzheimer's disease (AD). AD is a multifactorial neurodegenerative disorder for which Diabetes Mellitus (DM) is a risk factor. In animal models for DM, the phosphorylation and aggregation of tau is induced or exacerbated, however the underlying mechanism is unknown. In addition to the metabolic dysfunction, DM is characterized by chronic low-grade inflammation. This was reported to be associated with a neuroinflammatory response in the hypothalamus of DM animal models. Neuroinflammation is also implicated in the development and progression of AD. It is unknown whether DM also induces neuroinflammation in brain areas affected in AD, the cortex and hippocampus. Here we investigated whether neuroinflammation could be the mechanistic trigger to induce tau phosphorylation in the brain of DM animals. Two distinct diabetic animal models were used; rats on free-choice high-fat high-sugar (fcHFHS) diet that are insulin resistant and streptozotocin-treated rats that are insulin deficient. The streptozotocin-treated animals demonstrated increased tau phosphorylation in the brain as expected, whereas the fcHFHS diet fed animals did not. Remarkably, neither of the diabetic animal models showed reactive microglia or increased GFAP and COX-2 levels in the cortex or hippocampus. From this, we conclude: 1. DM does not induce neuroinflammation in brain regions affected in AD, and 2. Neuroinflammation is not a prerequisite for tau phosphorylation. Neuroinflammation is therefore not the mechanism that explains the close connection between DM and AD.

  9. Localized α4 Integrin Phosphorylation Directs Shear Stress-Induced Endothelial Cell Alignment

    PubMed Central

    Goldfinger, Lawrence E.; Tzima, Eleni; Stockton, Rebecca; Kiosses, William B.; Kinbara, Kayoko; Tkachenko, Eugene; Gutierrez, Edgar; Groisman, Alex; Nguyen, Phu; Chien, Shu; Ginsberg1, Mark H.

    2009-01-01

    Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to athero-protection. Here we report that localized α4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress-induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial α4 integrins became phosphorylated on Ser988. In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced α4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a non-phosphorylatable α4(S988A) mutation failed to align in response to shear stress, thus establishing α4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent α4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress. PMID:18583710

  10. Leptin signaling plays a critical role in the geniposide-induced decrease of tau phosphorylation.

    PubMed

    Liu, Jianhui; Liu, Zixuan; Zhang, Yonglan; Yin, Fei

    2015-12-01

    We have previously demonstrated that geniposide attenuates the production of Aβ1-42 both in vitro and in vivo via enhancing leptin receptor signaling. But the role played by geniposide in the phosphorylation of tau and its underlying molecular mechanisms remain unclear. In this study, we investigated the effect of geniposide on the phosphorylation of tau and the role of leptin signaling in this process. Our data suggested that, accompanied by the up-regulation of leptin receptor expression, geniposide significantly decreased the phosphorylation of tau in rat primary cultured cortical neurons and in APP/PS1 transgenic mice, and this geniposide-induced decrease of tau phosphorylation could be prevented by leptin antagonist (LA). Furthermore, LA also prevented the phosphorylation of Akt at Ser-473 site and GSK-3β at Ser-9 site induced by geniposide. All these results indicate that geniposide may regulate tau phosphorylation through leptin signaling, and geniposide may be a promising therapeutic compound for the treatment of Alzheimer's disease in the future.

  11. Phosphodiesterases Regulate BAY 41-2272-Induced VASP Phosphorylation in Vascular Smooth Muscle Cells

    PubMed Central

    Adderley, Shaquria P.; Joshi, Chintamani N.; Martin, Danielle N.; Tulis, David Anthony

    2012-01-01

    BAY 41-2272 (BAY), a stimulator of soluble guanylyl cyclase, increases cyclic nucleotides and inhibits proliferation of vascular smooth muscle cells (VSMCs). In this study, we elucidated mechanisms of action of BAY in its regulation of vasodilator-stimulated phosphoprotein (VASP) with an emphasis on VSMC phosphodiesterases (PDEs). BAY alone increased phosphorylation of VASPSer239 and VASPSer157, respective indicators of PKG and PKA signaling. IBMX, a non-selective inhibitor of PDEs, had no effect on BAY-induced phosphorylation at VASPSer239 but inhibited phosphorylation at VASPSer157. Selective inhibitors of PDE3 or PDE4 attenuated BAY-mediated increases at VASPSer239 and VASPSer157, whereas PDE5 inhibition potentiated BAY-mediated increases only at VASPSer157. In comparison, 8Br-cGMP increased phosphorylation at VASPSer239 and VASPSer157 which were not affected by selective PDE inhibitors. In the presence of 8Br-cAMP, inhibition of either PDE4 or PDE5 decreased VASPSer239 phosphorylation and inhibition of PDE3 increased phosphorylation at VASPSer239, while inhibition of PDE3 or PDE4 increased and PDE5 inhibition had no effect on VASPSer157 phosphorylation. These findings demonstrate that BAY operates via cAMP and cGMP along with regulation by PDEs to phosphorylate VASP in VSMCs and that the mechanism of action of BAY in VSMCs is different from that of direct cyclic nucleotide analogs with respect to VASP phosphorylation and the involvement of PDEs. Given a role for VASP as a critical cytoskeletal protein, these findings provide evidence for BAY as a regulator of VSMC growth and a potential therapeutic agent against vasculoproliferative disorders. PMID:22347188

  12. Overexpression of CREB in the nucleus accumbens shell increases cocaine reinforcement in self-administering rats.

    PubMed

    Larson, Erin B; Graham, Danielle L; Arzaga, Rose R; Buzin, Nicole; Webb, Joseph; Green, Thomas A; Bass, Caroline E; Neve, Rachael L; Terwilliger, Ernest F; Nestler, Eric J; Self, David W

    2011-11-09

    Chronic exposure to addictive drugs enhances cAMP response element binding protein (CREB)-regulated gene expression in nucleus accumbens (NAc), and these effects are thought to reduce the positive hedonic effects of passive cocaine administration. Here, we used viral-mediated gene transfer to produce short- and long-term regulation of CREB activity in NAc shell of rats engaging in volitional cocaine self-administration. Increasing CREB expression in NAc shell markedly enhanced cocaine reinforcement of self-administration behavior, as indicated by leftward (long-term) and upward (short-term) shifts in fixed ratio dose-response curves. CREB also increased the effort exerted by rats to obtain cocaine on more demanding progressive ratio schedules, an effect highly correlated with viral-induced modulation of BDNF protein in the NAc shell. CREB enhanced cocaine reinforcement when expressed either throughout acquisition of self-administration or when expression was limited to postacquisition tests, indicating a direct effect of CREB independent of reinforcement-related learning. Downregulating endogenous CREB in NAc shell by expressing a short hairpin RNA reduced cocaine reinforcement in similar tests, while overexpression of a dominant-negative CREB(S133A) mutant had no significant effect on cocaine self-administration. Finally, increasing CREB expression after withdrawal from self-administration enhanced cocaine-primed relapse, while reducing CREB levels facilitated extinction of cocaine seeking, but neither altered relapse induced by cocaine cues or footshock stress. Together, these findings indicate that CREB activity in NAc shell increases the motivation for cocaine during active self-administration or after withdrawal from cocaine. Our results also highlight that volitional and passive drug administration can lead to substantially different behavioral outcomes.

  13. Interplay of CREB and ATF2 in Ionizing Radiation-Induced Neuroendocrine Differentiation of Prostate Cancer Cells

    DTIC Science & Technology

    2012-06-01

    cross-talk between Hh and androgen signaling in prostate cancer. Source of Funding: NCI RO1-CA111618; DOD W81XWH-06 377 WHAT CAN THE HAIR FOLLICLES ...Award will test whether targeting PRMT5 can radiosensitize prostate cancer cells. In vitro and in vivo model systems developed 13 through current DoD...days after testosterone readministration to regressed tumors and tested gene specific mRNA expression. RESULTS: Overall AD induced expression of

  14. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  15. Advanced glycation end products induce moesin phosphorylation in murine brain endothelium.

    PubMed

    Li, Qiaoqin; Liu, Hongxia; Du, Jing; Chen, Bo; Li, Qiang; Guo, Xiaohua; Huang, Xuliang; Huang, Qiaobing

    2011-02-10

    Advanced glycation end products (AGEs) have been found to play an important role in the development of diabetes, and AGE levels are correlated with the severity of diabetic complications. We have demonstrated that moesin, a protein linker between actin filaments and the plasma membrane, undergoes phosphorylation of its threonine 558 residue by AGE stimulation in human dermal microvascular endothelial cells through activation of p38 and Rho kinase (ROCK) pathways. In this study, we observed in situ whether AGEs caused phosphorylation of vascular endothelial cells in the brains of AGE-stimulated mice. The animals were injected with AGE-modified mouse serum albumin (AGE-MSA) for 7 consecutive days. Immunohistochemistry was conducted to assess the phosphorylation of moesin in brain vessels. The level of moesin protein phosphorylation was also assessed in cerebral microvessels by western blotting. The effects of p38 and ROCK activation were determined by application of a p38 inhibitor (SB203580) and a ROCK inhibitor (Y27632) at 30 min before each AGE administration. The results showed specific expression of moesin in murine brain vascular endothelial cells. AGE treatment induced a significant increase of threonine 558 phosphorylation in moesin, while inhibition of p38 and ROCK remarkably attenuated the phosphorylation of moesin. The level of moesin protein phosphorylation was also increased in cerebral microvessels, along with an increased permeability of the blood-brain barrier, while inhibition of the p38 and ROCK attenuated these responses. These results demonstrate that AGEs cause the phosphorylation of moesin in murine brain microvascular endothelial cells, with p38 and ROCK being involved in this process.

  16. Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

    SciTech Connect

    Shawver, L.K.; Pierce, G.F.; Kawahara, R.S.; Deuel, T.F.

    1989-01-15

    The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation.

  17. Blue Light-Induced Phosphorylation of a Plasma Membrane-Associated Protein in Zea mays L.

    PubMed Central

    Palmer, J. M.; Short, T. W.; Gallagher, S.; Briggs, W. R.

    1993-01-01

    Blue light induces a variety of photomorphogenic responses in higher plants, among them phototropic curvature, the bending of seedlings toward a unidirectional light source. In dark-grown coleoptiles of maize (Zea mays L.) seedlings, blue light induces rapid phosphorylation of a 114-kD protein at fluence levels that are sufficient to stimulate phototropic curvature. Phosphorylation in response to blue light can be detected in vivo in coleoptile tips preincubated in 32Pi or in vitro in isolated membranes supplemented with [[gamma]-32P]ATP. Phosphorylation reaches a maximum level in vitro within 2 min following an inductive light pulse, but substantial labeling occurs within the first 15 s. Isolated membranes remain activated for several minutes following an in vitro blue light stimulus, even in the absence of exogenous ATP. Phosphoamino acid analysis of the 114-kD protein detected phosphoserine and a trace of phosphothreonine. The kinase involved in phosphorylating the protein in vitro is not dependent on calcium. The 114-kD protein itself has an apparent binding site for ATP, detected by incubating with the nonhydrolyzable analog, 5[prime]-p-fluorosulfonyl-benzoyladenosine. This result suggests that the 114-kD protein, which becomes phosphorylated in response to blue light, may also be capable of kinase activity. PMID:12231896

  18. Phosphorylation of Daxx by ATM Contributes to DNA Damage-Induced p53 Activation

    PubMed Central

    Cheng, Qian; Qu, Like; Brewer, Michael D.; Chen, Jiandong; Yang, Xiaolu

    2013-01-01

    p53 plays a central role in tumor suppression. It does so by inducing anti-proliferative processes as a response to various tumor-promoting stresses. p53 is regulated by the ubiquitin ligase Mdm2. The optimal function of Mdm2 requires Daxx, which stabilizes Mdm2 through the deubiquitinase Hausp/USP7 and also directly promotes Mdm2’s ubiquitin ligase activity towards p53. The Daxx-Mdm2 interaction is disrupted upon DNA damage. However, both the mechanisms and the consequence of the Daxx-Mdm2 dissociation are not understood. Here we show that upon DNA damage Daxx is phosphorylated in a manner that is dependent on ATM, a member of the PI 3-kinase family that orchestrates the DNA damage response. The main phosphorylation site of Daxx is identified to be Ser564, which is a direct target of ATM. Phosphorylation of endogenous Daxx at Ser564 occurs rapidly during the DNA damage response and precedes p53 activation. Blockage of this phosphorylation event prevents the separation of Daxx from Mdm2, stabilizes Mdm2, and inhibits DNA damage-induced p53 activation. These results suggest that phosphorylation of Daxx by ATM upon DNA damage disrupts the Daxx-Mdm2 interaction and facilitates p53 activation. PMID:23405218

  19. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    SciTech Connect

    Knabbe, C.; Lippman, M.E.; Greene, G.L.; Dickson, R.B.

    1986-05-01

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of (/sup 32/P)-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions.

  20. Cigarette smoke extracts induce overexpression of the proto-oncogenic gene interleukin-13 receptor α2 through activation of the PKA-CREB signaling pathway to trigger malignant transformation of lung vascular endothelial cells and angiogenesis.

    PubMed

    Meng, Mei; Liao, Huaidong; Zhang, Bin; Pan, Yanyan; Kong, Ying; Liu, Wenming; Yang, Ping; Huo, Zihe; Cao, Zhifei; Zhou, Quansheng

    2017-02-01

    Cigarette smoking is a major cause of lung cancer. Tumor-associated endothelial cells (TAECs) play important roles in tumor angiogenesis and metastasis. However, whether cigarette smoking can trigger genesis of lung TAECs has not been reported yet. In the current study, we used lung endothelial cell (EC) lines as a model to study the pathological effect of cigarette smoke extracts (CSEs) on human lung ECs, and found that a lower dose of 4% CSEs obviously caused abnormal morphological changes in ECs, increased the permeability of endothelial monolayer, while a higher concentration of 8% CSEs caused EC apoptosis. Strikingly, CSEs induced a 117-fold overexpression of a pro-tumorigenic interleukin-13 receptor α2 gene (IL-13Rα2, also named as CT-19) through activation of the protein kinase A (PKA) and cAMP response element-binding protein (CREB) signaling pathway. A PKA specific inhibitor H89 completely abolished CSEs-induced IL-13Rα2 overexpression. The overexpression of IL-13Rα2 in lung ECs significantly increased the tumorigenic, migratory, and angiogenic capabilities of the cells, suggesting that IL-13Rα2 promotes genesis of lung TAECs. Together, our data show that CSEs activate the PKA, CREB, and IL-13Rα2 axis in lung ECs, and IL-13Rα2 promotes the malignant transformation of lung ECs and genesis of TAECs with robust angiogenic and oncogenic capabilities. Our study provides new insight into the mechanism of CSEs-triggered lung cancer angiogenesis and tumorigenesis, suggesting that the PKA-CREB-IL-13Rα2 axis is a potential target for novel anti-lung tumor angiogenesis and anti-lung cancer drug discovery.

  1. Dorsal Hippocampal CREB Is Both Necessary and Sufficient for Spatial Memory

    ERIC Educational Resources Information Center

    Sekeres, Melanie J.; Neve, Rachael L.; Frankland, Paul W.; Josselyn, Sheena A.

    2010-01-01

    Although the transcription factor CREB has been widely implicated in memory, whether it is sufficient to produce spatial memory under conditions that do not normally support memory formation in mammals is unknown. We found that locally and acutely increasing CREB levels in the dorsal hippocampus using viral vectors is sufficient to induce robust…

  2. RACK1 promotes maintenance of morphine-associated memory via activation of an ERK-CREB dependent pathway in hippocampus.

    PubMed

    Liu, Litao; Zhu, Jiejun; Zhou, Liming; Wan, Lihong

    2016-02-02

    Existence of long-term drug-associated memories may be a crucial factor in drug cravings and relapse. RACK1 plays a critical role in morphine-induced reward. In the present study, we used conditioned place preference (CPP) to assess the acquisition and maintenance of morphine conditioned place preference memory. The hippocampal protein level of RACK1 and synaptic quantitation were evaluated by Western blotting, immunohistochemistry and electron microscopy, respectively. Additionally, shRACK1 (shGnb2l1) was used to silence RACK1 in vivo to evaluate the role and the underlying mechanism of RACK1 in maintenance of morphine CPP memory. We found that morphine induced CPP was maintained for at least 7 days after the last morphine treatment, which indicated a positive correlation with hippocampal RACK1 level, and was accompanied simultaneously by increases in the synapse density and hippocampal expression of synaptophysin (SYP), phosphorylation of extracellular signal-regulated kinase1/2 (pERK1/2) and the phosphorylation of cyclic adenosine monophosphate response element-binding (pCREB). ShGnb2l1 icv injection significantly suppressed the expression of all above proteins, decreased the synapse density in the hippocampus and attenuated the acquisition and maintenance of morphine CPP. Our present study highlights that RACK1 plays an important role in the maintenance of morphine CPP, likely via activation of ERK-CREB pathway in hippocampus.

  3. 2-Ethoxybenzamide stimulates melanin synthesis in B16F1 melanoma cells via the CREB signaling pathway.

    PubMed

    Sato, Kazuomi; Ando, Ryosuke; Kobayashi, Honoka; Nishio, Takashi

    2016-12-01

    Non-steroidal anti-inflammatory drugs are frequently used for the treatment of inflammation, pain, and fever. In this study, we found that 2-ethoxybenzamide (ETZ) significantly enhanced melanin synthesis in B16F1 melanoma cells, and also induced melanosome formation. Therefore, we investigated the mechanism by which ETZ up-regulated melanin synthesis. Western blot analysis demonstrated that ETZ increased melanogenic protein levels, except that for TRP-2. Moreover, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR analyses showed that ETZ enhanced the mRNA levels of melanogenic genes, including microphthalmia-associated transcription factor and melanocortin 1 receptor. We also observed phosphorylation of cAMP response element-binding protein (CREB) following ETZ treatment. However, ETZ did not affect intracellular cAMP levels. ERK was also activated by ETZ treatment, and melanin content was enhanced upon treatment with the specific ERK inhibitor PD98059. Together, our results indicate that ETZ induces melanin synthesis via CREB phosphorylation.

  4. Hepatic CREB3L3 controls whole-body energy homeostasis and improves obesity and diabetes.

    PubMed

    Nakagawa, Yoshimi; Satoh, Aoi; Yabe, Sachiko; Furusawa, Mika; Tokushige, Naoko; Tezuka, Hitomi; Mikami, Motoki; Iwata, Wakiko; Shingyouchi, Akiko; Matsuzaka, Takashi; Kiwata, Shiori; Fujimoto, Yuri; Shimizu, Hidehisa; Danno, Hirosuke; Yamamoto, Takashi; Ishii, Kiyoaki; Karasawa, Tadayoshi; Takeuchi, Yoshinori; Iwasaki, Hitoshi; Shimada, Masako; Kawakami, Yasushi; Urayama, Osamu; Sone, Hirohito; Takekoshi, Kazuhiro; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Yahagi, Naoya; Suzuki, Hiroaki; Yamada, Nobuhiro; Shimano, Hitoshi

    2014-12-01

    Transcriptional regulation of metabolic genes in the liver is the key to maintaining systemic energy homeostasis during starvation. The membrane-bound transcription factor cAMP-responsive element-binding protein 3-like 3 (CREB3L3) has been reported to be activated during fasting and to regulate triglyceride metabolism. Here, we show that CREB3L3 confers a wide spectrum of metabolic responses to starvation in vivo. Adenoviral and transgenic overexpression of nuclear CREB3L3 induced systemic lipolysis, hepatic ketogenesis, and insulin sensitivity with increased energy expenditure, leading to marked reduction in body weight, plasma lipid levels, and glucose levels. CREB3L3 overexpression activated gene expression levels and plasma levels of antidiabetic hormones, including fibroblast growth factor 21 and IGF-binding protein 2. Amelioration of diabetes by hepatic activation of CREB3L3 was also observed in several types of diabetic obese mice. Nuclear CREB3L3 mutually activates the peroxisome proliferator-activated receptor (PPAR) α promoter in an autoloop fashion and is crucial for the ligand transactivation of PPARα by interacting with its transcriptional regulator, peroxisome proliferator-activated receptor gamma coactivator-1α. CREB3L3 directly and indirectly controls fibroblast growth factor 21 expression and its plasma level, which contributes at least partially to the catabolic effects of CREB3L3 on systemic energy homeostasis in the entire body. Therefore, CREB3L3 is a therapeutic target for obesity and diabetes.

  5. NALP1 is a transcriptional target for cAMP-response-element-binding protein (CREB) in myeloid leukaemia cells

    PubMed Central

    2004-01-01

    NALP1 (also called DEFCAP, NAC, CARD7) has been shown to play a central role in the activation of inflammatory caspases and processing of pro-IL1β (pro-interleukin-1β). Previous studies showed that NALP1 is highly expressed in peripheral blood mononuclear cells. In the present study, we report that expression of NALP1 is absent from CD34+ haematopoietic blast cells, and its levels are upregulated upon differentiation of CD34+ cells into granulocytes and to a lesser extent into monocytes. In peripheral blood cells, the highest levels of NALP1 were observed in CD3+ (T-lymphocytes), CD15+ (granulocytes) and CD14+ (monocytes) cell populations. Notably, the expression of NALP1 was significantly increased in the bone marrow blast cell population of some patients with acute leukaemia, but not among tissue samples from thyroid and renal cancer. A search for consensus sites within the NALP1 promoter revealed a sequence for CREB (cAMP-response-element-binding protein) that was required for transcriptional activity. Moreover, treatment of TF1 myeloid leukaemia cells with protein kinase C and protein kinase A activators induced CREB phosphorylation and upregulated the mRNA and protein levels of NALP1. Conversely, ectopic expression of a dominant negative form of CREB in TF1 cells blocked the transcriptional activity of the NALP1 promoter and significantly reduced the expression of NALP1. Thus NALP1 is transcriptionally regulated by CREB in myeloid cells, a mechanism that may contribute to modulate the response of these cells to pro-inflammatory stimuli. PMID:15285719

  6. Tau phosphorylation and cleavage in ethanol-induced neurodegeneration in the developing mouse brain.

    PubMed

    Saito, Mariko; Chakraborty, Goutam; Mao, Rui-Fen; Paik, Sun-Mee; Vadasz, Csaba; Saito, Mitsuo

    2010-04-01

    Previous studies indicated that ethanol-induced neurodegeneration in postnatal day 7 (P7) mice, widely used as a model for the fetal alcohol spectrum disorders, was accompanied by glycogen synthase kinase-3beta (GSK-3beta) and caspase-3 activation. Presently, we examined whether tau, a microtubule associated protein, is modified by GSK-3beta and caspase-3 in ethanol-treated P7 mouse forebrains. We found that ethanol increased phosphorylated tau recognized by the paired helical filament (PHF)-1 antibody and by the antibody against tau phosphorylated at Ser199. Ethanol also generated tau fragments recognized by an antibody against caspase-cleaved tau (C-tau). C-tau was localized in neurons bearing activated caspase-3 and fragmented nuclei. Over time, cell debris and degenerated projections containing C-tau appeared to be engulfed by activated microglia. A caspase-3 inhibitor partially blocked C-tau formation. Lithium, a GSK-3beta inhibitor, blocked ethanol-induced caspase-3 activation, phosphorylated tau elevation, C-tau formation, and microglial activation. These results indicate that tau is phosphorylated by GSK-3beta and cleaved by caspase-3 during ethanol-induced neurodegeneration in the developing brain.

  7. A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase β

    PubMed Central

    Pei, De-Sheng; Yang, Xiao-Jie; Liu, Wei; Guikema, Jeroen E. J.; Schrader, Carol E.; Strauss, Phyllis R.

    2011-01-01

    DNA repair is required to maintain genome stability in stem cells and early embryos. At critical junctures, oxidative damage to DNA requires the base excision repair (BER) pathway. Since early zebrafish embryos lack the major polymerase in BER, DNA polymerase ß, repair proceeds via replicative polymerases, even though there is ample polb mRNA. Here, we report that Polb protein fails to appear at the appropriate time in development when AP endonuclease 1 (Apex), the upstream protein in BER, is knocked down. Because polb contains a Creb1 binding site, we examined whether knockdown of Apex affects creb1. Apex knockdown results in loss of Creb1 and Creb complex members but not Creb1 phosphorylation. This effect is independent of p53. Although both apex and creb1 mRNA rescue Creb1 and Polb after Apex knockdown, Apex is not a co-activator of creb1 transcription. This observation has broad significance, as similar results occur when Apex is inhibited in B cells from apex+/− mice. These results describe a novel regulatory circuit involving Apex, Creb1 and Polb and provide a mechanism for lethality of Apex loss in higher eukaryotes. PMID:21172930

  8. Oxidative stress–dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration

    PubMed Central

    Wakatsuki, Shuji; Furuno, Akiko; Ohshima, Makiko

    2015-01-01

    Oxidative stress is a well-known inducer of neuronal apoptosis and axonal degeneration. We previously showed that the E3 ubiquitin ligase ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B activation. We now demonstrate that oxidative stress serves as an activator of the ubiquitin ligase activity of ZNRF1 by inducing epidermal growth factor receptor (EGFR)–mediated phosphorylation at the 103rd tyrosine residue and that the up-regulation of ZNRF1 activity by oxidative stress leads to neuronal apoptosis and Wallerian degeneration. We also show that nicotinamide adenine dinucleotide phosphate–reduced oxidase activity is required for the EGFR-dependent phosphorylation-induced activation of ZNRF1 and resultant AKT degradation via the ubiquitin proteasome system to induce Wallerian degeneration. These results indicate the pathophysiological significance of the EGFR–ZNRF1 pathway induced by oxidative stress in the regulation of neuronal apoptosis and Wallerian degeneration. A deeper understanding of the regulatory mechanism for ZNRF1 catalytic activity via phosphorylation will provide a potential therapeutic avenue for neurodegeneration. PMID:26572622

  9. Extrasynaptic NMDA receptor-induced tau overexpression mediates neuronal death through suppressing survival signaling ERK phosphorylation

    PubMed Central

    Sun, Xu-Ying; Tuo, Qing-Zhang; Liuyang, Zhen-Yu; Xie, Ao-Ji; Feng, Xiao-Long; Yan, Xiong; Qiu, Mei; Li, Shen; Wang, Xiu-Lian; Cao, Fu-Yuan; Wang, Xiao-Chuan; Wang, Jian-Zhi; Liu, Rong

    2016-01-01

    Intracellular accumulation of the hyperphosphorylated tau is a pathological hallmark in the brain of Alzheimer disease. Activation of extrasynaptic NMDA receptors (E-NMDARs) induces excitatory toxicity that is involved in Alzheimer's neurodegeneration. However, the intrinsic link between E-NMDARs and the tau-induced neuronal damage remains elusive. In the present study, we showed in cultured primary cortical neurons that activation of E-NMDA receptors but not synaptic NMDA receptors dramatically increased tau mRNA and protein levels, with a simultaneous neuronal degeneration and decreased neuronal survival. Memantine, a selective antagonist of E-NMDARs, reversed E-NMDARs-induced tau overexpression. Activation of E-NMDARs in wild-type mouse brains resulted in neuron loss in hippocampus, whereas tau deletion in neuronal cultures and in the mouse brains rescued the E-NMDARs-induced neuronal death and degeneration. The E-NMDARs-induced tau overexpression was correlated with a reduced ERK phosphorylation, whereas the increased MEK activity, decreased binding and activity of ERK phosphatase to ERK, and increased ERK phosphorylation were observed in tau knockout mice. On the contrary, addition of tau proteins promoted ERK dephosphorylation in vitro. Taking together, these results indicate that tau overexpression mediates the excitatory toxicity induced by E-NMDAR activation through inhibiting ERK phosphorylation. PMID:27809304

  10. DFP initiated early alterations of PKA/p-CREB pathway and differential persistence of {beta}-tubulin subtypes in the CNS of hens contributes to OPIDN

    SciTech Connect

    Damodaran, Tirupapuliyur V.; Gupta, Ram P.; Attia, Moustafa K.; Abou-Donia, Mohamed B.

    2009-10-15

    Organophosphorus ester-induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken, which results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 mL/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were sacrificed at different time points such as 2, 4, and 8 h, as well as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum brainstem and spinal cord were quickly dissected and frozen for protein (western) and mRNA (northern) studies. Subcellular fractionation, SDS-PAGE and immunoblotting of the nuclear and supernatant fractions using standard protocols from spinal cord and cerebrum showed differential expression of protein levels of PKA, CREB and phosphorylated CREB (p-CREB). There was an increase in PKA level in spinal cord nuclear fraction after 4 h (130 {+-} 5%) and 8 h (133 {+-} 6 %), while cerebrum nuclear fraction showed decrease (77 {+-} 5%) at 4 h and remained at the same level at 8 h. No change was seen in either spinal cord or cerebrum soluble fraction at any time points. There was an increase in CREB level in the spinal cord supernatant (133 {+-} 3%) after 5 days, while nuclear and supernatant fraction of the cerebrum did not show any alterations at any time point. p-CREB was induced in the spinal cord nuclear fraction at 1 day (150 {+-} 3%) and 5 days (173{+-}{+-}7%) of treatment, in contrast to the decreased levels p-CREB (72 {+-} 4%) at 10 days in cerebrum nuclear fraction. Supernatant fraction of spinal cord and cerebrum did

  11. Microglia activation regulates GluR1 phosphorylation in chronic unpredictable stress-induced cognitive dysfunction.

    PubMed

    Liu, Mingchao; Li, Juan; Dai, Peng; Zhao, Fang; Zheng, Gang; Jing, Jinfei; Wang, Jiye; Luo, Wenjing; Chen, Jingyuan

    2015-01-01

    Chronic stress is considered to be a major risk factor in the development of psychopathological syndromes in humans. Cognitive impairments and long-term potentiation (LTP) impairments are increasingly recognized as major components of depression, anxiety disorders and other stress-related chronic psychological illnesses. It seems timely to systematically study the potentially underlying neurobiological mechanisms of altered cognitive and synaptic plasticity in the course of chronic stress. In the present study, a rat model of chronic unpredictable stress (CUS) induced a cognitive impairment in spatial memory in the Morris water maze (MWM) test and a hippocampal LTP impairment. CUS also induced hippocampal microglial activation and attenuated phosphorylation of glutamate receptor 1 (GluR1 or GluA1). Moreover, chronic treatment with the selective microglial activation blocker, minocycline (120 mg/kg per day), beginning 3 d before CUS treatment and continuing through the behavioral testing period, prevented the CUS-induced impairments of spatial memory and LTP induction. Additional studies showed that minocycline-induced inhibition of microglia activation was associated with increased phosphorylation of GluR1. These results suggest that hippocampal microglial activation modulates the level of GluR1 phosphorylation and might play a causal role in CUS-induced cognitive and LTP disturbances.

  12. Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

    SciTech Connect

    Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei

    2014-05-01

    Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

  13. Sodium phenylbutyrate enhances astrocytic neurotrophin synthesis via protein kinase C (PKC)-mediated activation of cAMP-response element-binding protein (CREB): implications for Alzheimer disease therapy.

    PubMed

    Corbett, Grant T; Roy, Avik; Pahan, Kalipada

    2013-03-22

    Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser(133)) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD.

  14. Phosphorylation of AMPA receptors is required for sensory deprivation-induced homeostatic synaptic plasticity.

    PubMed

    Goel, Anubhuti; Xu, Linda W; Snyder, Kevin P; Song, Lihua; Goenaga-Vazquez, Yamila; Megill, Andrea; Takamiya, Kogo; Huganir, Richard L; Lee, Hey-Kyoung

    2011-03-31

    Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+)-permeable AMPA receptors (CP-AMPARs). However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1) subunit at the serine 845 (S845) site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants), which is a substrate of cAMP-dependent kinase (PKA), show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

  15. Intracerebroventricular administration of okadaic acid induces hippocampal glucose uptake dysfunction and tau phosphorylation.

    PubMed

    Broetto, Núbia; Hansen, Fernanda; Brolese, Giovana; Batassini, Cristiane; Lirio, Franciane; Galland, Fabiana; Dos Santos, João Paulo Almeida; Dutra, Márcio Ferreira; Gonçalves, Carlos-Alberto

    2016-06-01

    Intraneuronal aggregates of neurofibrillary tangles (NFTs), together with beta-amyloid plaques and astrogliosis, are histological markers of Alzheimer's disease (AD). The underlying mechanism of sporadic AD remains poorly understood, but abnormal hyperphosphorylation of tau protein is suggested to have a role in NFTs genesis, which leads to neuronal dysfunction and death. Okadaic acid (OKA), a strong inhibitor of protein phosphatase 2A, has been used to induce dementia similar to AD in rats. We herein investigated the effect of intracerebroventricular (ICV) infusion of OKA (100 and 200ng) on hippocampal tau phosphorylation at Ser396, which is considered an important fibrillogenic tau protein site, and on glucose uptake, which is reduced early in AD. ICV infusion of OKA (at 200ng) induced a spatial cognitive deficit, hippocampal astrogliosis (based on GFAP increment) and increase in tau phosphorylation at site 396 in this model. Moreover, we observed a decreased glucose uptake in the hippocampal slices of OKA-treated rats. In vitro exposure of hippocampal slices to OKA altered tau phosphorylation at site 396, without any associated change in glucose uptake activity. Taken together, these findings further our understanding of OKA neurotoxicity, in vivo and vitro, particularly with regard to the role of tau phosphorylation, and reinforce the importance of the OKA dementia model for studying the neurochemical alterations that may occur in AD, such as NFTs and glucose hypometabolism.

  16. Regulatory light chain phosphorylation increases eccentric contraction-induced injury in skinned fast-twitch fibers.

    PubMed

    Childers, Martin K; McDonald, Kerry S

    2004-02-01

    During contraction, activation of Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) results in phosphorylation of myosin's regulatory light chain (RLC), which potentiates force and increases speed of force development over a wide range of [Ca(2+)]. We tested the hypothesis that RLC phosphorylation by MLCK mediates the extent of eccentric contraction-induced injury as measured by force deficit in skinned fast-twitch skeletal muscle fibers. Results indicated that RLC phosphorylation in single skinned rat psoas fibers significantly increased Ca(2+) sensitivity of isometric force; isometric force from 50 +/- 16 to 59 +/- 18 kN/m(2) during maximal Ca(2+) activation; peak absolute power output from 38 +/- 15 to 48 +/- 14 nW during maximal Ca(2+) activation; and the magnitude of contraction-induced force deficit during maximal (pCa 4.5) activation from 26 +/- 9.8 to 35 +/- 9.6%. We conclude that RLC phosphorylation increases force deficits following eccentric contractions, perhaps by increasing the number of force-generating cross-bridges.

  17. Human TNF-α induces differential protein phosphorylation in Schistosoma mansoni adult male worms.

    PubMed

    Oliveira, Katia C; Carvalho, Mariana L P; Bonatto, José Matheus C; Schechtman, Debora; Verjovski-Almeida, Sergio

    2016-02-01

    Schistosoma mansoni and its vertebrate host have a complex and intimate connection in which several molecular stimuli are exchanged and affect both organisms. Human tumor necrosis factor alpha (hTNF-α), a pro-inflammatory cytokine, is known to induce large-scale gene expression changes in the parasite and to affect several parasite biological processes such as metabolism, egg laying, and worm development. Until now, the molecular mechanisms for TNF-α activity in worms are not completely understood. Here, we aimed at exploring the effect of hTNF-α on S. mansoni protein phosphorylation by 2D gel electrophoresis followed by a quantitative analysis of phosphoprotein staining and protein identification by mass spectrometry. We analyzed three biological replicates of adult male worms exposed to hTNF-α and successfully identified 32 protein spots with a statistically significant increase in phosphorylation upon in vitro exposure to hTNF-α. Among the differentially phosphorylated proteins, we found proteins involved in metabolism, such as glycolysis, galactose metabolism, urea cycle, and aldehyde metabolism, as well as proteins related to muscle contraction and to cytoskeleton remodeling. The most differentially phosphorylated protein (30-fold increase in phosphorylation) was 14-3-3, whose function is known to be modulated by phosphorylation, belonging to a signal transduction protein family that regulates a variety of processes in all eukaryotic cells. Further, 75% of the identified proteins are known in mammals to be related to TNF-α signaling, thus suggesting that TNF-α response may be conserved in the parasite. We propose that this work opens new perspectives to be explored in the study of the molecular crosstalk between host and pathogen.

  18. 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.

  19. EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells

    PubMed Central

    Chun, Kyung-Soo; Shim, Minsub

    2015-01-01

    Prostaglandin E2 (PGE2), a major product of cyclooxygenase, binds to four different prostaglandin E2 receptors (EP1, EP2, EP3, and EP4) which are G-protein coupled transmembrane receptors (GPCRs). Although GPCRs including EP receptors have been shown to be associated with their specific G proteins, recent evidences suggest that GPCRs can regulate MAPK signaling via non-G protein coupled pathways including Src. EP2 is differentially expressed in various tissues and the expression of EP2 is induced by extracellular stimuli. We hypothesized that an increased level of EP2 expression may affect MAPK signaling. The overexpression of EP2 in HEK 293 cells resulted in significant increase in intracellular cAMP levels response to treatment with butaprost, a specific EP2 agonist, while overexpression of EP2 alone did not increase intracellular cAMP levels. However, EP2 overexpression in the absence of PGE2 induced an increase in the level of p38 phosphorylation as well as the kinase activity of p38, suggesting that up-regulation of EP2 may promote p38 activation via non-G protein coupled pathway. Inhibition of Src completely blocked EP2-induced p38 phosphorylation and overexpression of Src increased the level of p38 phosphorylation, indicating that Src is upstream kinase for EP2-induced p38 phosphorylation. EP2 overexpression also increased the Src activity and EP2 protein was co-immunoprecipitated with Src. Furthermore, sequential co-immunoprecipitation studies showed that EP2, Src, and β-arrestin can form a complex. Our study found a novel pathway in which EP2 is associated with Src, regulating p38 pathway. PMID:26535079

  20. Regulation of BRCA1 Function by DNA Damage-Induced Site-Specific Phosphorylation

    DTIC Science & Technology

    2005-06-01

    Rad51p proteins, the lat - end-joining. BRCA-2 complexed with machinery. ter a member of the RAD-52 epi- RAD-51 is active in strand exchange BRCA- 1 has...AD Award Number: DAMD17-02- 1 -0584 TITLE: Regulation of BRCAl Function by DNA Damage-Induced Site- Specific Phosphorylation PRINCIPAL INVESTIGATOR...ApprovedREPORT DOCUMENTATION PAGE OMB No. 0704-0188 Public reporting burden for this collection of Information Is estimated to average 1 hour per

  1. Advanced glycation end products induce moesin phosphorylation in murine retinal endothelium.

    PubMed

    Wang, Lingjun; Li, Qiaoqin; Du, Jing; Chen, Bo; Li, Qiang; Huang, Xuliang; Guo, Xiaohua; Huang, Qiaobing

    2012-02-01

    Increase in vascular permeability is the most important pathological event during the development of diabetic retinopathy. Deposition of advanced glycation end products (AGEs) plays a crucial role in the process of diabetes. This study was to investigate the role of moesin and its underlying signal transduction in retinal vascular hyper-permeability induced by AGE-modified mouse serum albumin (AGE-MSA). Female C57BL/6 mice were used to produce an AGE-treated model by intraperitoneal administration of AGE-MSA for seven consecutive days. The inner blood-retinal barrier was quantified by Evans blue leakage assay. Endothelial F-actin cytoskeleton in retinal vasculature was visualized by fluorescence probe staining. The expression and phosphorylation of moesin in retinal vessels were detected by RT-PCR and western blotting. Further studies were performed to explore the effects of Rho kinase (ROCK) and p38 MAPK pathway on the involvement of moesin in AGE-induced retinal vascular hyper-permeability response. Treatment with AGE-MSA significantly increased the permeability of the retinal microvessels and induced the disorganization of F-actin in retinal vascular endothelial cells. The threonine (T558) phosphorylation of moesin in retinal vessels was enhanced remarkably after AGE administration. The phosphorylation of moesin was attenuated by inhibitions of ROCK and p38 MAPK, while this treatment also prevented the dysfunction of inner blood-retinal barrier and the reorganization of F-actin in retinal vascular endothelial cells. These results demonstrate that moesin is involved in AGE-induced retinal vascular endothelial dysfunction and the phosphorylation of moesin is triggered via ROCK and p38 MAPK activation.

  2. Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy*

    PubMed Central

    Puente, Cindy; Hendrickson, Ronald C.; Jiang, Xuejun

    2016-01-01

    Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation. PMID:26801615

  3. AKAP12 mediates PKA-induced phosphorylation of ATR to enhance nucleotide excision repair

    PubMed Central

    Jarrett, Stuart G.; Wolf Horrell, Erin M.; D'Orazio, John A.

    2016-01-01

    Loss-of-function in melanocortin 1 receptor (MC1R), a GS protein-coupled receptor that regulates signal transduction through cAMP and protein kinase A (PKA) in melanocytes, is a major inherited melanoma risk factor. Herein, we report a novel cAMP-mediated response for sensing and responding to UV-induced DNA damage regulated by A-kinase-anchoring protein 12 (AKAP12). AKAP12 is identified as a necessary participant in PKA-mediated phosphorylation of ataxia telangiectasia mutated and Rad3-related (ATR) at S435, a post-translational event required for cAMP-enhanced nucleotide excision repair (NER). Moreover, UV exposure promotes ATR-directed phosphorylation of AKAP12 at S732, which promotes nuclear translocation of AKAP12–ATR-pS435. This complex subsequently recruits XPA to UV DNA damage and enhances 5′ strand incision. Preventing AKAP12's interaction with PKA or with ATR abrogates ATR-pS435 accumulation, delays recruitment of XPA to UV-damaged DNA, impairs NER and increases UV-induced mutagenesis. Our results define a critical role for AKAP12 as an UV-inducible scaffold for PKA-mediated ATR phosphorylation, and identify a repair complex consisting of AKAP12–ATR-pS435-XPA at photodamage, which is essential for cAMP-enhanced NER. PMID:27683220

  4. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    SciTech Connect

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  5. Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy.

    PubMed

    Puente, Cindy; Hendrickson, Ronald C; Jiang, Xuejun

    2016-03-11

    Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.

  6. Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.

    PubMed

    Abushahba, Walid; Olabisi, Oyenike O; Jeong, Byeong-Seon; Boregowda, Rajeev K; Wen, Yu; Liu, Fang; Goydos, James S; Lasfar, Ahmed; Cohen-Solal, Karine A

    2012-01-01

    Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.

  7. The effects of in vitro phosphorylation and dephosphorylation on the thrombin-induced gelation and plasmin degradation of fibrinogen.

    PubMed

    Martin, S C; Forsberg, P O; Eriksson, S D

    1991-02-01

    The alpha-chain of human fibrinogen was found to be phosphorylated in EDTA-anticoagulated whole blood when trace amounts of (gamma-32P)ATP and 7.5 mM Mg2+ ions were added. Fibrinogen was not phosphorylated if only the ATP was added. The thrombin-induced gelation of fibrinogen phosphorylated by protein kinase A, casein kinase I or II was studied spectrophotomerically. It was found that phosphorylation by protein kinase A caused the formation of thinner fibrin fibres, whereas phosphorylation by casein kinase II resulted in fibres slightly thicker than those of the control fibrinogen (equivalent to a 20% increase in the control fibrinogen concentration). Phosphorylation with casein kinase I did not significantly affect the fibrin fibre thickness. Dephosphorylation by alkaline phosphatase removed 50% of the 32P-labelled phosphate from protein kinase A-phosphorylated fibrinogen and over 90% from the casein kinase I or II-phosphorylated fibrinogens. This dephosphorylation resulted in a general increase in fibre thickness in the gelation assay in all samples, although the fibres of the phosphorylated fibrinogens remained substantially thinner than the dephosphorylated control fibrinogen. Plasmin digestion of the phosphorylated fibrinogens showed that they were more resistant to cleavage, being cleaved at only 30% to 70% of the rate of control fibrinogen and that this resistance was unaltered by dephosphorylation, in contrast to the thrombin gelation experiments.

  8. Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

    PubMed

    Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2014-09-19

    The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7.

  9. Ligand-induced IFN gamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91).

    PubMed Central

    Greenlund, A C; Farrar, M A; Viviano, B L; Schreiber, R D

    1994-01-01

    Herein we report that interferon-gamma (IFN gamma) induces the rapid and reversible tyrosine phosphorylation of the IFN gamma receptor. Using a panel of receptor intracellular domain mutants, we show that a membrane-proximal LPKS sequence (residues 266-269) is required for ligand-induced tyrosine kinase activation and/or kinase-receptor association and biological responsiveness, and a functionally critical membrane-distal tyrosine residue (Y440) is a target of the activated enzyme. The biological significance of Y440 phosphorylation was demonstrated by showing that a receptor-derived nonapeptide corresponding to receptor residues 436-444 and containing phosphorylated Y440 bound specifically to p91, blocked p91 phosphorylation and inhibited the generation of an active p91-containing transcription factor complex. In contrast, nonphosphorylated wild-type, phosphorylated mutant, or phosphorylated irrelevant peptides did not. Moreover, the phosphorylated Y440-containing peptide did not interact with a related but distinct latent transcription factor (p113) which is activatible by IFN alpha but not IFN gamma. These results thus document the specific and inducible association of p91 with the phosphorylated IFN gamma receptor and thereby elucidate the mechanism by which ligand couples the IFN gamma receptor to its signal transduction system. Images PMID:8156998

  10. Calcium/calmodulin dependent protein kinase II regulates the phosphorylation of cyclic AMP-responsive element-binding protein of spinal cord in rats following noxious stimulation.

    PubMed

    Fang, Li; Wu, Jing; Zhang, Xuan; Lin, Qing; Willis, William D

    2005-02-01

    We have previously reported that intradermal capsaicin injection causes the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein (CREB) in the spinal cord of rats. The present study was designed to investigate the role of calcium/camodulin protein dependent protein kinase II (CaM kinase II) in the regulation of phosphorylation of CREB after capsaicin injection. We found that capsaicin injection produces a significant upregulation of phosphorylated CREB in the spinal cord of rat. Intrathecal treatment with a CaM kinase II inhibitor, KN-93, significantly blocked the increased phosphorylation of CREB, but did not affect the CREB protein itself. These results suggest that increased phosphorylation of CREB protein may contribute to central sensitization following acute peripheral noxious stimuli, and the effect may be regulated through the activation of CaM kinase cascades.

  11. p38 MAPK downregulates phosphorylation of Bad in doxorubicin-induced endothelial apoptosis

    SciTech Connect

    Grethe, Simone; Coltella, Nadia; Di Renzo, Maria Flavia; Poern-Ares, M. Isabella . E-mail: isabella.ares@helsinki.fi

    2006-09-01

    Doxorubicin is the anthracycline with the widest spectrum of antitumor activity, and it has been shown that the antitumor activity is mediated in vivo by selective triggering of apoptosis in proliferating endothelial cells. We studied cultured human endothelial cells and observed that doxorubicin-induced apoptosis was mediated by p38 mitogen-activated protein kinase (MAPK). Doxorubicin-provoked apoptosis was significantly inhibited by expression of dominant negative p38 MAPK or pharmacological inhibition with SB203580. Furthermore, blocking phosphatidylinositol-3-kinase/Akt signaling significantly increased doxorubicin-induced caspase-3 activity and cell death, indicating that Akt is a survival factor in this system. Notably, we also found that doxorubicin-provoked apoptosis included p38 MAPK-mediated inhibition of Akt and Bad phosphorylation. Furthermore, doxorubicin-stimulated phosphorylation of Bad in cells expressing dominant negative p38 MAPK was impeded by the inhibition of PI3-K. In addition to the impact on Bad phosphorylation, doxorubicin-treatment caused p38 MAPK-dependent downregulation of Bcl-xL protein.

  12. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio)

    PubMed Central

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-01-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine’s metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol’s locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug. PMID:26366782

  13. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio).

    PubMed

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-11-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

  14. Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance

    PubMed Central

    Petersen, Max C.; Madiraju, Anila K.; Gassaway, Brandon M.; Marcel, Michael; Nasiri, Ali R.; Butrico, Gina; Marcucci, Melissa J.; Zhang, Dongyan; Abulizi, Abudukadier; Zhang, Xian-Man; Philbrick, William; Hubbard, Stevan R.; Samuel, Varman T.; Rinehart, Jesse

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet–induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance. PMID:27760050

  15. Genome-wide functional analysis of CREB/long-term memory-dependent transcription reveals distinct basal and memory gene expression programs.

    PubMed

    Lakhina, Vanisha; Arey, Rachel N; Kaletsky, Rachel; Kauffman, Amanda; Stein, Geneva; Keyes, William; Xu, Daniel; Murphy, Coleen T

    2015-01-21

    Induced CREB activity is a hallmark of long-term memory, but the full repertoire of CREB transcriptional targets required specifically for memory is not known in any system. To obtain a more complete picture of the mechanisms involved in memory, we combined memory training with genome-wide transcriptional analysis of C. elegans CREB mutants. This approach identified 757 significant CREB/memory-induced targets and confirmed the involvement of known memory genes from other organisms, but also suggested new mechanisms and novel components that may be conserved through mammals. CREB mediates distinct basal and memory transcriptional programs at least partially through spatial restriction of CREB activity: basal targets are regulated primarily in nonneuronal tissues, while memory targets are enriched for neuronal expression, emanating from CREB activity in AIM neurons. This suite of novel memory-associated genes will provide a platform for the discovery of orthologous mammalian long-term memory components.

  16. MeCP2 phosphorylation limits psychostimulant-induced behavioral and neuronal plasticity.

    PubMed

    Deng, Jie V; Wan, Yehong; Wang, Xiaoting; Cohen, Sonia; Wetsel, William C; Greenberg, Michael E; Kenny, Paul J; Calakos, Nicole; West, Anne E

    2014-03-26

    The methyl-DNA binding protein MeCP2 is emerging as an important regulator of drug reinforcement processes. Psychostimulants induce phosphorylation of MeCP2 at Ser421; however, the functional significance of this posttranslational modification for addictive-like behaviors was unknown. Here we show that MeCP2 Ser421Ala knock-in mice display both a reduced threshold for the induction of locomotor sensitization by investigator-administered amphetamine and enhanced behavioral sensitivity to the reinforcing properties of self-administered cocaine. These behavioral differences were accompanied in the knock-in mice by changes in medium spiny neuron intrinsic excitability and nucleus accumbens gene expression typically observed in association with repeated exposure to these drugs. These data show that phosphorylation of MeCP2 at Ser421 functions to limit the circuit plasticities in the nucleus accumbens that underlie addictive-like behaviors.

  17. MeCP2 Phosphorylation Limits Psychostimulant-Induced Behavioral and Neuronal Plasticity

    PubMed Central

    Deng, Jie V.; Wan, Yehong; Wang, Xiaoting; Cohen, Sonia; Wetsel, William C.; Greenberg, Michael E.; Kenny, Paul J.; Calakos, Nicole

    2014-01-01

    The methyl-DNA binding protein MeCP2 is emerging as an important regulator of drug reinforcement processes. Psychostimulants induce phosphorylation of MeCP2 at Ser421; however, the functional significance of this posttranslational modification for addictive-like behaviors was unknown. Here we show that MeCP2 Ser421Ala knock-in mice display both a reduced threshold for the induction of locomotor sensitization by investigator-administered amphetamine and enhanced behavioral sensitivity to the reinforcing properties of self-administered cocaine. These behavioral differences were accompanied in the knock-in mice by changes in medium spiny neuron intrinsic excitability and nucleus accumbens gene expression typically observed in association with repeated exposure to these drugs. These data show that phosphorylation of MeCP2 at Ser421 functions to limit the circuit plasticities in the nucleus accumbens that underlie addictive-like behaviors. PMID:24671997

  18. Tyrosine hydroxylase phosphorylation after naloxone-induced morphine withdrawal in the left ventricle.

    PubMed

    Almela, Pilar; Victoria Milanés, Maria; Luisa Laorden, Maria

    2009-07-01

    Our previous studies have shown that morphine withdrawal induced hyperactivity of cardiac noradrenergic pathways. The purpose of the present study was to evaluate the effects of morphine withdrawal on site-specific tyrosine hydroxylase (TH) phosphorylation in the rat left ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg/kg, s.c.). TH phosphorylation was determined by quantitative blot immunolabelling using phosphorylation state-specific antibodies. Ninety min after naloxone administration to morphine-dependent rats there was an increase in phospho-Ser40-TH (139.0 +/- 13%, P < 0.05) and Ser31-TH (135.5 +/- 11%, P < 0.05) in the left ventricle which is associated with both an increase in total TH levels (114.4 +/- 4.6%, P < 0.05, P < 0.01) and an enhancement of TH activity (51.0 +/- 11 dm/microg protein, P < 0.001). When HA-1004 (40 nmol/day), inhibitor of cyclic AMP dependent protein kinase (PKA) was infused, concomitantly with morphine, it diminished the increase in noradrenaline (NA) turnover, total TH expression (95.76 +/- 4.1 %, P < 0.01) and TH phosphorylation at Ser40 (85.5 +/- 11%, P < 0.01) in morphine-withdrawn rats. In addition, we showed that the ability of morphine withdrawal to stimulate phosphorylation at serine 31 is reduced (101.7 +/- 7.7%, P < 0.05) by SL327 (100 mg/kg, i.p.), an inhibitor of extracellular signal-regulated kinase (ERK) activation. The present findings demonstrate that the enhancement of total TH expression and the increase of the phosphorylation state of TH during morphine withdrawal are dependent on PKA and ERK and suggest that these transduction pathways might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine- withdrawal.

  19. Intermittent hypoxic exposure during light phase induces changes in cAMP response element binding protein activity in the rat CA1 hippocampal region: water maze performance correlates.

    PubMed

    Goldbart, A; Row, B W; Kheirandish, L; Schurr, A; Gozal, E; Guo, S Z; Payne, R S; Cheng, Z; Brittian, K R; Gozal, D

    2003-01-01

    Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.

  20. p38α MAPK is required for arsenic-induced cell transformation.

    PubMed

    Kim, Hong-Gyum; Shi, Chengcheng; Bode, Ann M; Dong, Zigang

    2016-05-01

    Arsenic exposure has been reported to cause neoplastic transformation through the activation of PcG proteins. In the present study, we show that activation of p38α mitogen-activated protein kinase (MAPK) is required for arsenic-induced neoplastic transformation. Exposure of cells to 0.5 μM arsenic increased CRE and c-Fos promoter activities that were accompanied by increases in p38α MAPK and CREB phosphorylation and expression levels concurrently with AP-1 activation. Introduction of short hairpin (sh) RNA-p38α into BALB/c 3T3 cells markedly suppressed arsenic-induced colony formation compared with wildtype cells. CREB phosphorylation and AP-1 activation were decreased in p38α knockdown cells after arsenic treatment. Arsenic-induced AP-1 activation, measured as c-Fos and CRE promoter activities, and CREB phosphorylation were attenuated by p38 inhibition in BALB/c 3T3 cells. Thus, p38α MAPK activation is required for arsenic-induced neoplastic transformation mediated through CREB phosphorylation and AP-1 activation.

  1. Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels.

    PubMed

    Murbartián, Janet; Lei, Qiubo; Sando, Julianne J; Bayliss, Douglas A

    2005-08-26

    Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G alpha(q)-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R(p))-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.

  2. MTOR inhibition attenuates DNA damage and apoptosis through autophagy-mediated suppression of CREB1.

    PubMed

    Wang, Ying; Hu, Zhongdong; Liu, Zhibo; Chen, Rongrong; Peng, Haiyong; Guo, Jing; Chen, Xinxin; Zhang, Hongbing

    2013-12-01

    Hyperactivation of mechanistic target of rapamycin (MTOR) is a common feature of human cancers, and MTOR inhibitors, such as rapamycin, are thus becoming therapeutics in targeting certain cancers. However, rapamycin has also been found to compromise the efficacy of chemotherapeutics to cells with hyperactive MTOR. Here, we show that loss of TSC2 or PTEN enhanced etoposide-induced DNA damage and apoptosis, which was blunted by suppression of MTOR with either rapamycin or RNA interference. cAMP response element-binding protein 1 (CREB1), a nuclear transcription factor that regulates genes involved in survival and death, was positively regulated by MTOR in mouse embryonic fibroblasts (MEFs) and cancer cell lines. Silencing Creb1 expression with siRNA protected MTOR-hyperactive cells from DNA damage-induced apoptosis. Furthermore, loss of TSC2 or PTEN impaired either etoposide or nutrient starvation-induced autophagy, which in turn, leads to CREB1 hyperactivation. We further elucidated an inverse correlation between autophagy activity and CREB1 activity in the kidney tumor tissue obtained from a TSC patient and the mouse livers with hepatocyte-specific knockout of PTEN. CREB1 induced DNA damage and subsequent apoptosis in response to etoposide in autophagy-defective cells. Reactivation of CREB1 or inhibition of autophagy not only improved the efficacy of rapamycin but also alleviated MTOR inhibition-mediated chemoresistance. Therefore, autophagy suppression of CREB1 may underlie the MTOR inhibition-mediated chemoresistance. We suggest that inhibition of MTOR in combination with CREB1 activation may be used in the treatment of cancer caused by an abnormal PI3K-PTEN-AKT-TSC1/2-MTOR signaling pathway. CREB1 activators should potentiate the efficacy of chemotherapeutics in treatment of these cancers.

  3. Effects of acute cocaine on ERK and DARPP-32 phosphorylation pathways in the caudate-putamen of Fischer rats

    PubMed Central

    Sun, Wei-Lun; Zhou, Luyi; Hazim, Ruhal; Quinones-Jenab, Vanya; Jenab, Shirzad

    2010-01-01

    Activation of extracellular signal-regulated kinase (ERK) and dopamine- and cAMP-regulated phosphoprotein (DARPP-32) pathways has been implicated in biochemical and behavioral effects induced by various drugs of abuse. In this study, we investigated the phosphorylation pathways of these two proteins in response to acute cocaine administration. A single cocaine administration (30 mg/kg) increased ERK-mediated signaling proteins, phosphoryation of cAMP response element-binding protein (CREB) kinase, pp90 ribosomal S6 kinase (RSK), and c-Fos protein levels in the caudate/putamen of Fischer rats. Acute cocaine administration also induced phosphorylation of the striatal-enriched protein tyrosine phosphatase (STEP) and decreased the phosphorylation of DARPP-32 protein at the Thr-75 site. The phosphorylation states of these inhibitors of ERK and DARPP-32 proteins may thus contribute to the effects of cocaine on ERK- and DARPP-32-mediated cascades, on gene expression and on behaviors. PMID:17920048

  4. Short-Chain Fatty Acids Inhibit Growth Hormone and Prolactin Gene Transcription via cAMP/PKA/CREB Signaling Pathway in Dairy Cow Anterior Pituitary Cells

    PubMed Central

    Wang, Jian-Fa; Fu, Shou-Peng; Li, Su-Nan; Hu, Zhong-Ming; Xue, Wen-Jing; Li, Zhi-Qiang; Huang, Bing-Xu; Lv, Qing-Kang; Liu, Ju-Xiong; Wang, Wei

    2013-01-01

    Short-chain fatty acids (SCFAs) play a key role in altering carbohydrate and lipid metabolism, influence endocrine pancreas activity, and as a precursor of ruminant milk fat. However, the effect and detailed mechanisms by which SCFAs mediate bovine growth hormone (GH) and prolactin (PRL) gene transcription remain unclear. In this study, we detected the effects of SCFAs (acetate, propionate, and butyrate) on the activity of the cAMP/PKA/CREB signaling pathway, GH, PRL, and Pit-1 gene transcription in dairy cow anterior pituitary cells (DCAPCs). The results showed that SCFAs decreased intracellular cAMP levels and a subsequent reduction in PKA activity. Inhibition of PKA activity decreased CREB phosphorylation, thereby inhibiting GH and PRL gene transcription. Furthermore, PTX blocked SCFAs- inhibited cAMP/PKA/CREB signaling pathway. These data showed that the inhibition of GH and PRL gene transcription induced by SCFAs is mediated by Gi activation and that propionate is more potent than acetate and butyrate in inhibiting GH and PRL gene transcription. In conclusion, this study identifies a biochemical mechanism for the regulation of SCFAs on bovine GH and PRL gene transcription in DCAPCs, which may serve as one of the factors that regulate pituitary function in accordance with dietary intake. PMID:24177567

  5. Mixture of Peanut Skin Extract and Fish Oil Improves Memory in Mice via Modulation of Anti-Oxidative Stress and Regulation of BDNF/ERK/CREB Signaling Pathways

    PubMed Central

    Xiang, Lan; Cao, Xue-Li; Xing, Tian-Yan; Mori, Daisuke; Tang, Rui-Qi; Li, Jing; Gao, Li-Juan; Qi, Jian-Hua

    2016-01-01

    Long-term use of fish oil (FO) is known to induce oxidative stress and increase the risk of Alzheimer’s disease in humans. In the present study, peanut skin extract (PSE), which has strong antioxidant capacity, was mixed with FO to reduce its side effects while maintaining its beneficial properties. Twelve-week Institute of Cancer Research (ICR) mice were used to conduct animal behavior tests in order to evaluate the memory-enhancing ability of the mixture of peanut skin extract and fish oil (MPF). MPF significantly increased alternations in the Y-maze and cognitive index in the novel object recognition test. MPF also improved performance in the water maze test. We further sought to understand the mechanisms underlying these effects. A significant decrease in superoxide dismutase (SOD) activity and an increase in malonyldialdehyde (MDA) in plasma were observed in the FO group. The MPF group showed reduced MDA level and increased SOD activity in the plasma, cortex and hippocampus. Furthermore, the gene expression levels of brain-derived neurotrophic factor (BDNF) and cAMP responsive element-binding protein (CREB) in the hippocampus were increased in the MPF group, while phosphorylation of protein kinase B (AKT), extracellular signal-regulated kinase (ERK) and CREB in the hippocampus were enhanced. MPF improves memory in mice via modulation of anti-oxidative stress and activation of BDNF/ERK/CREB signaling pathways. PMID:27136583

  6. The Dictyostelium prestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes

    PubMed Central

    Sugden, Chris; Urbaniak, Michael D.; Araki, Tsuyoshi; Williams, Jeffrey G.

    2015-01-01

    Differentiation-inducing factor-1 (DIF-1) is a polyketide that induces Dictyostelium amoebae to differentiate as prestalk cells. We performed a global quantitative screen for phosphorylation changes that occur within the first minutes after addition of DIF-1, using a triple-label SILAC approach. This revealed a new world of DIF-1–controlled signaling, with changes in components of the MAPK and protein kinase B signaling pathways, components of the actinomyosin cytoskeletal signaling networks, and a broad range of small GTPases and their regulators. The results also provide evidence that the Ca2+/calmodulin–dependent phosphatase calcineurin plays a role in DIF-1 signaling to the DimB prestalk transcription factor. At the global level, DIF-1 causes a major shift in the phosphorylation/dephosphorylation equilibrium toward net dephosphorylation. Of interest, many of the sites that are dephosphorylated in response to DIF-1 are phosphorylated in response to extracellular cAMP signaling. This accords with studies that suggest an antagonism between the two inducers and also with the rapid dephosphorylation of the cAMP receptor that we observe in response to DIF-1 and with the known inhibitory effect of DIF-1 on chemotaxis to cAMP. All MS data are available via ProteomeXchange with identifier PXD001555. PMID:25518940

  7. CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation

    PubMed Central

    Xu, Shangcheng; Wang, Pei; Zhang, Huiliang; Gong, Guohua; Gutierrez Cortes, Nicolas; Zhu, Weizhong; Yoon, Yisang; Tian, Rong; Wang, Wang

    2016-01-01

    Mitochondrial permeability transition pore (mPTP) is involved in cardiac dysfunction during chronic β-adrenergic receptor (β-AR) stimulation. The mechanism by which chronic β-AR stimulation leads to mPTP openings is elusive. Here, we show that chronic administration of isoproterenol (ISO) persistently increases the frequency of mPTP openings followed by mitochondrial damage and cardiac dysfunction. Mechanistically, this effect is mediated by phosphorylation of mitochondrial fission protein, dynamin-related protein 1 (Drp1), by Ca2+/calmodulin-dependent kinase II (CaMKII) at a serine 616 (S616) site. Mutating this phosphorylation site or inhibiting Drp1 activity blocks CaMKII- or ISO-induced mPTP opening and myocyte death in vitro and rescues heart hypertrophy in vivo. In human failing hearts, Drp1 phosphorylation at S616 is increased. These results uncover a pathway downstream of chronic β-AR stimulation that links CaMKII, Drp1 and mPTP to bridge cytosolic stress signal with mitochondrial dysfunction in the heart. PMID:27739424

  8. Phosphorylation of p53 by LRRK2 induces microglial tumor necrosis factor α-mediated neurotoxicity.

    PubMed

    Ho, Dong Hwan; Seol, Wongi; Eun, Jin Hwan; Son, Il-Hong

    2017-01-22

    Leucine-rich repeat kinase (LRRK2), a major causal gene of Parkinson's disease (PD), functions as a kinase. The most prevalent mutation of LRRK2 is G2019S. It exhibits increased kinase activity compared to the wildtype LRRK2. Previous studies have shown that LRRK2 can phosphorylate p53 at T304 and T377 of threonine-X-arginine (TXR) motif in neurons. Reduction of LRRK2 expression or inhibition of LRRK2 kinase activity has been shown to be able to alleviate LPS-induced neuroinflammation in microglia cells. In this study, we found that LRRK2 could also phosphorylate p53 in microglia model BV2 cells. Transfection of BV2 with phosphomimetic p53 T304/377D significantly increased the secretion of pro-inflammatory cytokine TNFα compared to BV2 transfected with p53 wild type after LPS treatment. In addition, conditioned media from these transfected cells increased the death of dopaminergic neuronal SN4741 cells. Moreover, such neurotoxic effect was rescued by co-treatment with the conditioned media and etanercept, a TNFα blocking antibody. Furthermore, TNFα secretion was significantly increased in primary microglia derived from G2019S transgenic mice treated with LPS compared to that in cells derived from their littermates. These results suggest that LRRK2 kinase activity in microglia can contribute to neuroinflammation in PD via phosphorylating p53 at T304 and T377 site.

  9. Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids.

    PubMed

    Werner, David F; Porcu, Patrizia; Boyd, Kevin N; O'Buckley, Todd K; Carter, Jenna M; Kumar, Sandeep; Morrow, A Leslie

    2016-04-01

    GABAA receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABAA receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABAA receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABAA receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABAA receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.

  10. Herpes simplex virus type 1 infection activates the Epstein-Barr virus replicative cycle via a CREB-dependent mechanism.

    PubMed

    Wu, Hongling; Li, Ting; Zeng, Musheng; Peng, Tao

    2012-04-01

    The reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation of EBV is not yet fully understood. In the present study, herpes simplex virus type 1 (HSV-1) was shown to induce the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post HSV-1 infection in virus entry-dependent and replication-independent manners. Using a panel of Zp deletion mutants, HSV-1 was shown to promote Zp through a cyclic adenosine monophosphate (cAMP) response element (CRE) located in ZII. The phosphorylated cAMP response element-binding (phos-CREB) protein, the cellular transactivator that binds to CRE, also increased after HSV-1 infection. By transient transfection, cAMP-dependent protein kinase A and HSV-1 US3 protein were found to be capable of activating Zp in CREB- and CRE-dependent manners. The relationship between EBV activation and HSV-1 infection revealed a possible common mechanism that stimulated latent EBV into lytic cycles in vivo.

  11. Mineral induced phosphorylation of glycolate ion--a metaphor in chemical evolution

    NASA Technical Reports Server (NTRS)

    Kolb, V.; Zhang, S.; Xu, Y.; Arrhenius, G.

    1997-01-01

    Bilateral surface-active minerals with excess positive charge concentrate glycolate and trimetaphosphate ion from l0(-3) m aqueous solution to half-saturation of the internal surface sites, and induce phosphorylation of glycolate ion in the mineral with trimetaphosphate, sorbed from l0(-2) m solution. By utilizing reactants from dilute solution at near-neutral pH, and eliminating the need for participating organic nitrogen compounds, the reaction comprises several elements considered necessary for geochemical realism in models for molecular evolution.

  12. Mineral induced phosphorylation of glycolate ion--a metaphor in chemical evolution.

    PubMed

    Kolb, V; Zhang, S; Xu, Y; Arrhenius, G

    1997-12-01

    Bilateral surface-active minerals with excess positive charge concentrate glycolate and trimetaphosphate ion from l0(-3) m aqueous solution to half-saturation of the internal surface sites, and induce phosphorylation of glycolate ion in the mineral with trimetaphosphate, sorbed from l0(-2) m solution. By utilizing reactants from dilute solution at near-neutral pH, and eliminating the need for participating organic nitrogen compounds, the reaction comprises several elements considered necessary for geochemical realism in models for molecular evolution.

  13. Inflammatory cytokines induce phosphorylation and ubiquitination of prostate suppressor protein NKX3.1.

    PubMed

    Markowski, Mark C; Bowen, Cai; Gelmann, Edward P

    2008-09-01

    Inflammation of the prostate is a risk factor for the development of prostate cancer. In the aging prostate, regions of inflammatory atrophy are foci for prostate epithelial cell transformation. Expression of the suppressor protein NKX3.1 is reduced in regions of inflammatory atrophy and in preinvasive prostate cancer. Inflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin-1beta accelerate NKX3.1 protein loss by inducing rapid ubiquitination and proteasomal degradation. The effect of TNF-alpha is mediated via the COOH-terminal domain of NKX3.1 where phosphorylation of serine 196 is critical for cytokine-induced degradation. Mutation of serine 196 to alanine abrogates phosphorylation at that site and the effect of TNF-alpha on NKX3.1 ubiquitination and protein loss. This is in contrast to control of steady-state NKX3.1 turnover, which is mediated by serine 185. Mutation of serine 185 to alanine increases NKX3.1 protein stability by inhibiting ubiquitination and doubling the protein half-life. A third COOH-terminal serine at position 195 has a modulating effect on both steady-state protein turnover and on ubiquitination induced by TNF-alpha. Thus, cellular levels of the NKX3.1 tumor suppressor are affected by inflammatory cytokines that target COOH-terminal serine residues to activate ubiquitination and protein degradation. Our data suggest that strategies to inhibit inflammation or to inhibit effector kinases may be useful approaches to prostate cancer prevention.

  14. Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

    PubMed

    Hough, Chris; Radu, Maria; Doré, Jules J E

    2012-01-01

    The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

  15. Requirement for tyrosine phosphorylation in lipopolysaccharide-induced murine B-cell proliferation.

    PubMed Central

    Dearden-Badet, M T; Revillard, J P

    1993-01-01

    Bacterial lipopolysaccharide (LPS) induces a strong B-cell proliferative response with subsequent differentiation, through a complex signal transduction pathway. This process is known to be mediated through protein kinase C (PKC) translocation without Ca2+ mobilization. Here, we show that B-cell proliferative responses induced by five different LPS preparations, as well as by F(ab')2 anti-IgM antibodies, are inhibited by the tyrosine kinase inhibitors, genistein and herbimycin A. In contrast, B-cell proliferation induced by the combination of phorbol 12-myristate 13-acetate (PMA) plus ionomycin was not influenced by treatment with either herbimycin A or genistein. These data indicate that tyrosine phosphorylation is required to initiate B-cell proliferation by LPS. PMID:8307617

  16. Pesticides induce spatial memory deficits with synaptic impairments and an imbalanced tau phosphorylation in rats.

    PubMed

    Chen, Ning-Ning; Luo, Dan-Ju; Yao, Xiu-Qing; Yu, Cong; Wang, Yi; Wang, Qun; Wang, Jian-Zhi; Liu, Gong-Ping

    2012-01-01

    Pesticides are widely used in agriculture, and epidemiological studies suggest that pesticide exposure is a risk factor for Alzheimer's disease (AD), but the mechanisms are elusive. Here, we studied the effects of pesticide exposure on the cognitive ability and the underlying mechanisms in rats. Deltamethrin and carbofuran were administered respectively into the rats once a day for 28 days by gavage. We found that pesticide exposure induced spatial learning and memory deficits with a simultaneous decrease of N-methyl-D-aspartate receptor 1, synaptophysin, and synapsin I, all of which are memory-related synaptic proteins. Pesticide exposure also induced tau hyperphosphorylation at multiple AD-related phosphorylation sites with activation of glycogen synthase kinase-3β and inhibition of protein phosphatase-2A. Additionally, neuron loss in the hippocampus and cortex was observed upon administration of the pesticides. These results indicate that the pesticides exposure could induce AD-like pathology and cognitive abnormality in rats.

  17. Involvement of tyrosine phosphorylation in HMG-CoA reductase inhibitor-induced cell death in L6 myoblasts.

    PubMed

    Mutoh, T; Kumano, T; Nakagawa, H; Kuriyama, M

    1999-02-05

    Our previous studies have shown that the HMG-CoA reductase (HCR) inhibitor (HCRI), simvastatin, causes myopathy in rabbits and kills L6 myoblasts. The present study was designed to elucidate the molecular mechanism of HCRI-induced cell death. We have demonstrated that simvastatin induces the tyrosine phosphorylation of several cellular proteins within 10 min. These phosphorylations were followed by apoptosis, as evidenced by the occurrence of internucleosomal DNA fragmentation and by morphological changes detected with Nomarski optics. Simvastatin-induced cell death was prevented by tyrosine kinase inhibitors. The MTT assay revealed that the addition of mevalonic acid into the culture medium partially inhibited simvastatin-induced cell death. Thus, these results suggested that protein tyrosine phosphorylation might play an important role in the intracellular signal transduction pathway mediating the HCRI-induced death of myoblasts.

  18. Wnt3A Induces GSK-3β Phosphorylation and β-Catenin Accumulation Through RhoA/ROCK.

    PubMed

    Kim, Jae-Gyu; Kim, Myoung-Ju; Choi, Won-Ji; Moon, Mi-Young; Kim, Hee-Jun; Lee, Jae-Yong; Kim, Jaebong; Kim, Sung-Chan; Kang, Seung Goo; Seo, Goo-Young; Kim, Pyeung-Hyeun; Park, Jae-Bong

    2017-05-01

    In canonical pathway, Wnt3A has been known to stabilize β-catenin through the dissociation between β-catenin and glycogen synthase kinase-3β (GSK-3β) that suppresses the phosphorylation and degradation of β-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3β and accumulation of β-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3β and accumulation of β-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3β phosphorylation and β-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3β in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3β phosphorylation and β-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 232: 1104-1113, 2017. © 2016 Wiley Periodicals, Inc.

  19. ERK phosphorylation of MED14 in promoter complexes during mitogen-induced gene activation by Elk-1.

    PubMed

    Galbraith, Matthew D; Saxton, Janice; Li, Li; Shelton, Samuel J; Zhang, Hongmei; Espinosa, Joaquin M; Shaw, Peter E

    2013-12-01

    The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression.

  20. Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

    SciTech Connect

    Pan, Xiao; Whitten, Douglas A.; Wu, Ming; Chan, Christina; Wilkerson, Curtis G.; Pestka, James J.

    2013-04-15

    Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

  1. COMPARATIVE ANALYSES OF DIFFERENTIALLY-INDUCED TCR-MEDIATED PHOSPHORYLATION PATHWAYS IN T LYMPHOMA CELLS

    PubMed Central

    Ortiz, Serina; Lee, Wenhui; Smith, David; Forman, Stephen J.; Lee, Terry D.; Liu, Chih-Pin

    2011-01-01

    Activation of T lymphoma cells expressing Syk, but not ZAP-70 tyrosine kinase, has been shown to negatively regulate cell activation and activation induced cell death (AICD), perhaps due to differential induction of tyrosine phosphorylation modified proteins. To better understand the role of these proteins and their associated molecules/pathways, we studied a previously described model of T lymphoma cells expressing either a kinase-activated chimeric Syk or ZAP-70 genetically linked to TCR ζ chain (Z/Syk or Z/ZAP cells, respectively). To help identify molecules and pathways linked to cell activation or AICD, a comparative semi-quantitative proteomics-based approach was utilized to analyze tyrosine phosphorylated protein immunoprecipitates from 2 min short-term activated Z/Syk or Z/ZAP cells. Using the resulting bioinformatics datasets, we identified several differentially immunoprecipitated proteins that could be validated biochemically. More tyrosine-phosphorylated and phosphotyrosine-associated proteins were found in Z/Syk than in Z/ZAP cells. Proteins involved in different unique functional pathways were induced in these cells and showed altered intermolecular interactions in varied pathways. Remarkably, 41% of differentially identified proteins in Z/Syk cells belonged to cell cycle or vesicle/trafficking pathways. In contrast, 21% of such proteins in Z/ZAP cells belonged to metabolism pathways. Therefore, molecular pathways involved in post-translational modifications linked to distinct cellular/physiological functions are differentially activated, which may contribute to varied activation and AICD responses of these cells. In summary, we identified proteins belonging to novel differentially activated pathways involved in TCR-mediated signaling, which may be targets for regulating activation and AICD of T lymphoma cells and for potential cancer therapy. PMID:21127342

  2. Phosphorylation of Tyr245 in the open-inhibited state of Abelson kinase does not induce downstream signaling.

    PubMed

    Skora, Lukasz; Kempf, Dominique; Mestan, Jürgen; D'Orazio, Daniel; Jahnke, Wolfgang

    2016-05-01

    Binding of tyrosine kinase inhibitors such as imatinib was shown to induce a novel open-inhibited conformation of BCR-ABL, in which Tyr245 is exposed and prone to phosphorylation. To evaluate whether this leads to priming of the kinase in cellular systems, we probed activation of downstream signaling as a result of Tyr245 phosphorylation in a series of cellular washout experiments. While a spike in Tyr245 phosphorylation was observed both in overexpression and endogenous settings, no induction of downstream signaling was detected, showing that the priming hypothesis is not relevant for the therapeutic situation.

  3. CREB Regulates Experience-Dependent Spine Formation and Enlargement in Mouse Barrel Cortex

    PubMed Central

    Pignataro, Annabella; Borreca, Antonella; Ammassari-Teule, Martine; Middei, Silvia

    2015-01-01

    Experience modifies synaptic connectivity through processes that involve dendritic spine rearrangements in neuronal circuits. Although cAMP response element binding protein (CREB) has a key function in spines changes, its role in activity-dependent rearrangements in brain regions of rodents interacting with the surrounding environment has received little attention so far. Here we studied the effects of vibrissae trimming, a widely used model of sensory deprivation-induced cortical plasticity, on processes associated with dendritic spine rearrangements in the barrel cortex of a transgenic mouse model of CREB downregulation (mCREB mice). We found that sensory deprivation through prolonged whisker trimming leads to an increased number of thin spines in the layer V of related barrel cortex (Contra) in wild type but not mCREB mice. In the barrel field controlling spared whiskers (Ipsi), the same trimming protocol results in a CREB-dependent enlargement of dendritic spines. Last, we demonstrated that CREB regulates structural rearrangements of synapses that associate with dynamic changes of dendritic spines. Our findings suggest that CREB plays a key role in dendritic spine dynamics and synaptic circuits rearrangements that account for new brain connectivity in response to changes in the environment. PMID:26075101

  4. CREB decreases astrocytic excitability by modifying subcellular calcium fluxes via the sigma-1 receptor.

    PubMed

    Eraso-Pichot, A; Larramona-Arcas, R; Vicario-Orri, E; Villalonga, R; Pardo, L; Galea, E; Masgrau, R

    2017-03-01

    Astrocytic excitability relies on cytosolic calcium increases as a key mechanism, whereby astrocytes contribute to synaptic transmission and hence learning and memory. While it is a cornerstone of neurosciences that experiences are remembered, because transmitters activate gene expression in neurons, long-term adaptive astrocyte plasticity has not been described. Here, we investigated whether the transcription factor CREB mediates adaptive plasticity-like phenomena in astrocytes. We found that activation of CREB-dependent transcription reduced the calcium responses induced by ATP, noradrenaline, or endothelin-1. As to the mechanism, expression of VP16-CREB, a constitutively active CREB mutant, had no effect on basal cytosolic calcium levels, extracellular calcium entry, or calcium mobilization from lysosomal-related acidic stores. Rather, VP16-CREB upregulated sigma-1 receptor expression thereby increasing the release of calcium from the endoplasmic reticulum and its uptake by mitochondria. Sigma-1 receptor was also upregulated in vivo upon VP16-CREB expression in astrocytes. We conclude that CREB decreases astrocyte responsiveness by increasing calcium signalling at the endoplasmic reticulum-mitochondria interface, which might be an astrocyte-based form of long-term depression.

  5. Interactions between CBP, NF-kappaB, and CREB in the lungs after hemorrhage and endotoxemia.

    PubMed

    Shenkar, R; Yum, H K; Arcaroli, J; Kupfner, J; Abraham, E

    2001-08-01

    The transcriptional regulatory factor nuclear factor (NF)-kappaB has a central role in modulating expression of proinflammatory mediators that are important in acute lung injury. In vitro studies have shown that competition between NF-kappaB and cAMP response element binding protein (CREB) for binding to the coactivator CREB-binding protein (CBP) is important in regulating transcriptional activity of these factors. In the present study, we examined in vivo interactions between CBP, CREB, and NF-kappaB in hemorrhage- or endotoxemia-induced acute lung injury. Association of CBP with CREB or the p65 subunit of NF-kappaB increased in the lungs after hemorrhage or endotoxemia. Inhibition of xanthine oxidase before hemorrhage, but not before endotoxemia, decreased p65-CBP interactions while increasing those between CREB and CBP. These alterations in CREB-CBP and p65-CBP interactions were functionally significant because xanthine oxidase inhibition before hemorrhage resulted in increased expression of the CREB-dependent gene c-Fos and decreased expression of macrophage inflammatory protein-2, a NF-kappaB-dependent gene. The present results show that the coactivator CBP has an important role in modulating transcription in vivo under clinically relevant pathophysiological conditions.

  6. Zn2+-dependent Activation of the Trk Signaling Pathway Induces Phosphorylation of the Brain-enriched Tyrosine Phosphatase STEP

    PubMed Central

    Poddar, Ranjana; Rajagopal, Sathyanarayanan; Shuttleworth, C. William; Paul, Surojit

    2016-01-01

    Excessive release of Zn2+ in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn2+-induced neurotoxicity, a potential contribution of tyrosine phosphatases in this process has not been well explored. Here we show that exposure to high concentrations of Zn2+ led to a progressive increase in phosphorylation of the striatal-enriched phosphatase (STEP), a component of the excitotoxic-signaling pathway that plays a role in neuroprotection. Zn2+-mediated phosphorylation of STEP61 at multiple sites (hyperphosphorylation) was induced by the up-regulation of brain-derived neurotropic factor (BDNF), tropomyosin receptor kinase (Trk) signaling, and activation of cAMP-dependent PKA (protein kinase A). Mutational studies further show that differential phosphorylation of STEP61 at the PKA sites, Ser-160 and Ser-221 regulates the affinity of STEP61 toward its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn2+-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn2+-dependent neurotoxicity. The strong correlation between the temporal profile of STEP61 hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP61 through phosphorylation is necessary for maintaining sustained ERK2 phosphorylation. This interpretation is further supported by the findings that deletion of the STEP gene led to a rapid and sustained increase in ERK2 phosphorylation within minutes of exposure to Zn2+. The study provides further insight into the mechanisms of regulation of STEP61 and also offers a molecular basis for the Zn2+-induced sustained activation of ERK2. PMID:26574547

  7. Osmotin attenuates amyloid beta-induced memory impairment, tau phosphorylation and neurodegeneration in the mouse hippocampus

    PubMed Central

    Ali, Tahir; Yoon, Gwang Ho; Shah, Shahid Ali; Lee, Hae Young; Kim, Myeong Ok

    2015-01-01

    The pathological hallmarks of Alzheimer’s disease (AD) include amyloid beta (Aβ) accumulation, neurofibrillary tangle formation, synaptic dysfunction and neuronal loss. In this study, we investigated the neuroprotection of novel osmotin, a plant protein extracted from Nicotiana tabacum that has been considered to be a homolog of mammalian adiponectin. Here, we observed that treatment with osmotin (15 μg/g, intraperitoneally, 4 hr) at 3 and 40 days post-intracerebroventricular injection of Aβ1-42 significantly ameliorated Aβ1-42-induced memory impairment in mice. These results revealed that osmotin reverses Aβ1-42 injection-induced synaptic deficits, Aβ accumulation and BACE-1 expression. Treatment with osmotin also alleviated the Aβ1-42-induced hyperphosphorylation of the tau protein at serine 413 through the regulation of the aberrant phosphorylation of p-PI3K, p-Akt (serine 473) and p-GSK3β (serine 9). Moreover, our western blots and immunohistochemical results indicated that osmotin prevented Aβ1-42-induced apoptosis and neurodegeneration in the Aβ1-42-treated mice. Furthermore, osmotin attenuated Aβ1-42-induced neurotoxicity in vitro. To our knowledge, this study is the first to investigate the neuroprotective effect of a novel osmotin against Aβ1-42-induced neurotoxicity. Our results demonstrated that this ubiquitous plant protein could potentially serve as a novel, promising, and accessible neuroprotective agent against progressive neurodegenerative diseases such as AD. PMID:26118757

  8. Insulin-induced myosin light-chain phosphorylation during receptor capping in IM-9 human B-lymphoblasts.

    PubMed Central

    Majercik, M H; Bourguignon, L Y

    1988-01-01

    We have examined further the interaction between insulin surface receptors and the cytoskeleton of IM-9 human lymphoblasts. Using immunocytochemical techniques, we determined that actin, myosin, calmodulin and myosin light-chain kinase (MLCK) are all accumulated directly underneath insulin-receptor caps. In addition, we have now established that the concentration of intracellular Ca2+ (as measured by fura-2 fluorescence) increases just before insulin-induced receptor capping. Most importantly, we found that the binding of insulin to its receptor induces phosphorylation of myosin light chain in vivo. Furthermore, a number of drugs known to abolish the activation properties of calmodulin, such as trifluoperazine (TFP) or W-7, strongly inhibit insulin-receptor capping and myosin light-chain phosphorylation. These data imply that an actomyosin cytoskeletal contraction, regulated by Ca2+/calmodulin and MLCK, is involved in insulin-receptor capping. Biochemical analysis in vitro has revealed that IM-9 insulin receptors are physically associated with actin and myosin; and most interestingly, the binding of insulin-receptor/cytoskeletal complex significantly enhances the phosphorylation of the 20 kDa myosin light chain. This insulin-induced phosphorylation is inhibited by calmodulin antagonists (e.g. TFP and W-7), suggesting that the phosphorylation is catalysed by MLCK. Together, these results strongly suggest that MLCK-mediated myosin light-chain phosphorylation plays an important role in regulating the membrane-associated actomyosin contraction required for the collection of insulin receptors into caps. Images Fig. 2. Fig. 4. PMID:3048249

  9. Egg jelly induces the phosphorylation of histone H3 in spermatozoa of the sea urchin Arbacia punctulata.

    PubMed

    Vacquier, V D; Porter, D C; Keller, S H; Aukerman, M

    1989-05-01

    When spermatozoa of Arbacia punctulata are labeled with 32P and treated with soluble egg jelly, radiolabel is incorporated into histone H3. The time course of labeling correlates with the period of chromatin decondensation of sperm pronuclei in eggs. Phosphorylation is on serine and may result from increased turnover of phosphate on H3. The macromolecular fraction of egg jelly (and not the peptide fraction) is the inducer of H3 phosphorylation. The reaction is dependent on external Ca2+ and is induced by monensin and A23187. H3 phosphorylation is not induced by the phosphodiesterase inhibitor IBMX and relatively high (250 microM) concentrations of the protein kinase inhibitor H8 are needed to block the reaction, suggesting that it is cAMP independent. A surprising finding is that merely diluting the cells into Na+ free media is the most effective method to induce the radiolabeling of H3. These results are in contrast to findings on the egg jelly induced phosphorylation of histone H1 in S. purpuratus spermatozoa. These species differences must reflect the great evolutionary divergence between these two sea urchin species in the mechanism of regulation of the phosphorylation of nuclear proteins during fertilization.

  10. Dipeptidyl Peptidase IV Inhibition Activates CREB and Improves Islet Vascularization through VEGF-A/VEGFR-2 Signaling Pathway

    PubMed Central

    Samikannu, Balaji; Chen, Chunguang; Lingwal, Neelam; Padmasekar, Manju; Engel, Felix B.; Linn, Thomas

    2013-01-01

    Substitution of pancreatic islets is a potential therapy to treat diabetes and it depends on reconstitution of islet’s capillary network. In this study, we addressed the question whether stabilization of Glucagon-Like-Peptide-1 (GLP-1) by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) increases β-cell mass by modulating vascularization. Mouse or porcine donor islets were implanted under kidney capsule of diabetic mice treated with DPP-IV inhibitor sitagliptin. Grafts were analyzed for insulin production, β-cell proliferation and vascularization. In addition, the effect of sitagliptin on sprouting and Vascular Endothelial Growth Factor (VEGF)-A expression was examined ex vivo. The cAMP response element-binding (CREB) and VEGF-A/ Vascular Endothelial Growth Factor Receptor (VEGFR)-2 signaling pathway leading to islet vascularization was explored. Sitagliptin increased mean insulin content of islet grafts and area of insulin-positive tissue as well as β-cell proliferation. Interestingly, sitagliptin treatment also markedly increased endothelial cell proliferation, microvessel density and blood flow. Finally, GLP-1 (7-36) stimulated sprouting and VEGF expression, which was significantly enhanced by sitagliptin- mediated inhibition of DPP-IV. Our in vivo data demonstrate that sitagliptin treatment phosphorylated CREB and induced islet vascularization through VEGF-A/VEGFR-2 signaling pathway. This study paves a new pathway for improvement of islet transplantation in treating diabetes mellitus. PMID:24349326

  11. Adaptation of oxidative phosphorylation to photoperiod-induced seasonal metabolic states in migratory songbirds.

    PubMed

    Trivedi, Amit Kumar; Malik, Shalie; Rani, Sangeeta; Kumar, Vinod

    2015-06-01

    Eukaryotic cells produce chemical energy in the form of ATP by oxidative phosphorylation of metabolic fuels via a series of enzyme mediated biochemical reactions. We propose that the rates of these reactions are altered, as per energy needs of the seasonal metabolic states in avian migrants. To investigate this, blackheaded buntings were photoperiodically induced with non-migratory, premigratory, migratory and post-migratory phenotypes. High plasma levels of free fatty acids, citrate (an intermediate that begins the TCA cycle) and malate dehydrogenase (mdh, an enzyme involved at the end of the TCA cycle) confirmed increased availability of metabolic reserves and substrates to the TCA cycle during the premigratory and migratory states, respectively. Further, daily expression pattern of genes coding for enzymes involved in the oxidative decarboxylation of pyruvate to acetyl-CoA (pdc and pdk) and oxidative phosphorylation in the TCA cycle (cs, odgh, sdhd and mdh) was monitored in the hypothalamus and liver. Reciprocal relationship between pdc and pdk expressions conformed with the altered requirements of acetyl-CoA for the TCA cycle in different metabolic states. Except for pdk, all genes had a daily expression pattern, with high mRNA expression during the day in the premigratory/migratory phenotypes, and at night (cs, odhg, sdhd and mdh) in the nonmigratory phenotype. Differences in mRNA expression patterns of pdc, sdhd and mdh, but not of pdk, cs and odgh, between the hypothalamus and liver indicated a tissue dependent metabolism in buntings. These results suggest the adaptation of oxidative phosphorylation pathway(s) at gene levels to the seasonal alternations in metabolism in migratory songbirds.

  12. Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

    SciTech Connect

    Bauerschmidt, Christina; Helleday, Thomas

    2011-02-01

    Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.

  13. Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

    PubMed

    Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph R; O'Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay K; Yadava, Nagendra; Chandra, Dhyan

    2016-01-01

    We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins.

  14. Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

    PubMed

    Madhavarao, Chikkathur N; Agarabi, Cyrus D; Wong, Lily; Müller-Loennies, Sven; Braulke, Thomas; Khan, Mansoor; Anderson, Howard; Johnson, Gibbes R

    2014-01-01

    Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant β-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The β-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased β-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in β-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased β-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.

  15. Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme

    PubMed Central

    Alkharusi, Amira; Yu, Shengze; Landázuri, Natalia; Zadjali, Fahad; Davodi, Belghis; Nyström, Thomas; Gräslund, Torbjörn; Rahbar, Afsar; Norstedt, Gunnar

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans and is characterized with poor outcome. In this study, we investigated components of prolactin (Prl) system in cell models of GBM and in histological tissue sections obtained from GBM patients. Expression of Prolactin receptor (PrlR) was detected at high levels in U251-MG, at low levels in U87-MG and barely detectable in U373 cell lines and in 66% of brain tumor tissues from 32 GBM patients by immunohistochemical technique. In addition, stimulation of U251-MG and U87-MG cells but not U373 with Prl resulted in increased STAT5 phosphorylation and only in U251-MG cells with increased cellular invasion. Furthermore, STAT5 phosphorylation and cellular invasion induced in Prl stimulated cells were significantly reduced by using a Prl receptor antagonist that consists of Prl with four amino acid replacements. We conclude that Prl receptor is expressed at different levels in the majority of GBM tumors and that blocking of PrlR in U251-MG cells significantly reduce cellular invasion. PMID:27788487

  16. 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) Induces Phosphorylation of Eukaryotic Elongation Factor-2 (eEF2)

    PubMed Central

    Chen, Zehan; Gopalakrishnan, Sujatha M.; Bui, Mai-Ha; Soni, Niru B.; Warrior, Usha; Johnson, Eric F.; Donnelly, Jennifer B.; Glaser, Keith B.

    2011-01-01

    Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies. PMID:22020937

  17. Xylaria nigripes mitigates spatial memory impairment induced by rapid eye movement sleep deprivation.

    PubMed

    Zhao, Zhengqing; Li, Yanpeng; Chen, Haiyan; Huang, Liuqing; Zhao, Fei; Yu, Qiang; Xiang, Zhenghua; Zhao, Zhongxin

    2014-01-01

    We aimed to investigate the effects of Xylaria nigripes (XN) extracts on the rapid eye movement sleep deprivation (REMSD)-induced memory impairment, and explore related mechanism. Male Sprague Dawley rats were randomly divided into 6 groups: cage control (CC)-NaCl group; tank control (TC)-NaCl group; sleep deprivation (SD)-NaCl group; CC-XN group; TC-XN group and SD-XN group. The rats were administered with intragastric XN and 0.9% of sodium chloride. SD group rats were deprived of REM sleep for 72 h. Morris water maze (MWM) was used to assess the effects of XN on spatial learning and memory. The expression of cAMP-response element binding protein (CREB) and p-CREB were also investigated in all groups. Result showed rats in SD-NaCl group had significantly longer mean escape latencies in finding the platform as compared to the control rats (p<0.05) in MWM test. The SD-NaCl group spent significantly less time in goal quadrant compared with the SD-XN group. REMSD and XN did not alter CREB expression in the hippocampus, while sleep deprivation resulted in reduced phosphorylation of CREB in the hippocampus, which was reversed by XN. XN mitigates spatial memory impairment induced by REMSD in rat. Phosphorylation of CREB in hippocampus might be one of the mechanisms.

  18. Promotion of PDGF-induced endothelial cell migration by phosphorylated VASP depends on PKA anchoring via AKAP.

    PubMed

    Zhang, Deling; Ouyang, Jingping; Wang, Nian; Zhang, Yahui; Bie, Jinghua; Zhang, Yemin

    2010-02-01

    Vasodilator-stimulated phosphoprotein (VASP), an important substrate of PKA, plays a critical role in remodeling of actin cytoskeleton and actin-based cell motility. However, how PKA accurately transfers extracellular signals to VASP and then how phosphorylation of VASP regulates endothelial cell migration have not been clearly defined. Protein kinase A anchoring proteins (AKAPs) are considered to regulate intracellular-specific signal targeting of PKA via AKAP-mediated PKA anchoring. Thus, our study investigated the relationship among AKAP anchoring of PKA, PKA activity, and VASP phosphorylation, which is to clarify the exact role of VASP and its upstream regulatory mechanism in PKA-dependent migration. Our results show that chemotactic factor PDGF activated PKA, increased phosphorylation of VASP at Ser157, and enhanced ECV304 endothelial cell migration. However, phosphorylation site-directed mutation of VASP at Ser157 attenuated the chemotactic effect of PDGF on endothelial cells, suggesting phosphorylation of VASP at Ser157 promotes PKA-mediated endothelial cell migration. Furthermore, disrupting PKA anchoring to AKAP or PKA activity significantly attenuated the PKA activity, VASP phosphorylation, and subsequent cell migration. Meanwhile, disrupting PKA anchoring to AKAP abolished PDGF-induced lamellipodia formation and special VASP accumulation at leading edge of lamellipodia. These results indicate that PKA activation and PKA-mediated substrate responses in VASP phosphorylation and localization depend on PKA anchoring via AKAP in PDGF-induced endothelial cell migration. In conclusion, AKAP anchoring of PKA is an essential upstream event in regulation of PKA-mediated VASP phosphorylation and subsequent endothelial cell migration, which contributes to explore new methods for controlling endothelial cell migration related diseases and angiogenesis.

  19. Retinoblastoma protein promotes oxidative phosphorylation through upregulation of glycolytic genes in oncogene-induced senescent cells.

    PubMed

    Takebayashi, Shin-Ichiro; Tanaka, Hiroshi; Hino, Shinjiro; Nakatsu, Yuko; Igata, Tomoka; Sakamoto, Akihisa; Narita, Masashi; Nakao, Mitsuyoshi

    2015-08-01

    Metabolism is closely linked with cellular state and biological processes, but the mechanisms controlling metabolic properties in different contexts remain unclear. Cellular senescence is an irreversible growth arrest induced by various stresses, which exhibits active secretory and metabolic phenotypes. Here, we show that retinoblastoma protein (RB) plays a critical role in promoting the metabolic flow by activating both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) in cells that have undergone oncogene-induced senescence (OIS). A combination of real-time metabolic monitoring, and metabolome and gene expression analyses showed that OIS-induced fibroblasts developed an accelerated metabolic flow. The loss of RB downregulated a series of glycolytic genes and simultaneously reduced metabolites produced from the glycolytic pathway, indicating that RB upregulates glycolytic genes in OIS cells. Importantly, both mitochondrial OXPHOS and glycolytic activities were abolished in RB-depleted or downstream glycolytic enzyme-depleted OIS cells, suggesting that RB-mediated glycolytic activation induces a metabolic flux into the OXPHOS pathway. Collectively, our findings reveal that RB essentially functions in metabolic remodeling and the maintenance of the active energy production in OIS cells.

  20. Retinoblastoma protein promotes oxidative phosphorylation through upregulation of glycolytic genes in oncogene-induced senescent cells

    PubMed Central

    Takebayashi, Shin-ichiro; Tanaka, Hiroshi; Hino, Shinjiro; Nakatsu, Yuko; Igata, Tomoka; Sakamoto, Akihisa; Narita, Masashi; Nakao, Mitsuyoshi

    2015-01-01

    Metabolism is closely linked with cellular state and biological processes, but the mechanisms controlling metabolic properties in different contexts remain unclear. Cellular senescence is an irreversible growth arrest induced by various stresses, which exhibits active secretory and metabolic phenotypes. Here, we show that retinoblastoma protein (RB) plays a critical role in promoting the metabolic flow by activating both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) in cells that have undergone oncogene-induced senescence (OIS). A combination of real-time metabolic monitoring, and metabolome and gene expression analyses showed that OIS-induced fibroblasts developed an accelerated metabolic flow. The loss of RB downregulated a series of glycolytic genes and simultaneously reduced metabolites produced from the glycolytic pathway, indicating that RB upregulates glycolytic genes in OIS cells. Importantly, both mitochondrial OXPHOS and glycolytic activities were abolished in RB-depleted or downstream glycolytic enzyme-depleted OIS cells, suggesting that RB-mediated glycolytic activation induces a metabolic flux into the OXPHOS pathway. Collectively, our findings reveal that RB essentially functions in metabolic remodeling and the maintenance of the active energy production in OIS cells. PMID:26009982

  1. Defective CFTR-dependent CREB activation results in impaired spermatogenesis and azoospermia.

    PubMed

    Xu, Wen Ming; Chen, Jing; Chen, Hui; Diao, Rui Ying; Fok, Kin Lam; Dong, Jian Da; Sun, Ting Ting; Chen, Wen Ying; Yu, Mei Kuen; Zhang, Xiao Hu; Tsang, Lai Ling; Lau, Ann; Shi, Qi Xian; Shi, Qing Hua; Huang, Ping Bo; Chan, Hsiao Chang

    2011-01-01

    Cystic fibrosis (CF) is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) with over 95% male patients infertile. However, whether CFTR mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element binding protein (CREB) expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO(3) (-) transport and the expression of the HCO(3) (-) sensor, soluble adenylyl cyclase (sAC), are demonstrated for the first time in the primary culture of rat Sertoli cells. Inhibition of CFTR or depletion of HCO(3) (-) could reduce FSH-stimulated, sAC-dependent cAMP production and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related diseases.

  2. Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting

    PubMed Central

    Bazou, Despina; Ng, Mei Rosa; Song, Jonathan W.; Chin, Shan Min; Maimon, Nir; Munn, Lance L.

    2016-01-01

    Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), with each EC residing in the vessel wall integrating local signals to determine whether to remain quiescent or undergo morphogenesis. These signals include vascular endothelial growth factor (VEGF) and flow-induced mechanical stimuli such as interstitial flow, which are both elevated in the tumor microenvironment. However, it is not clear how VEGF signaling and mechanobiological activation due to interstitial flow cooperate during angiogenesis. Here, we show that endothelial morphogenesis is histone deacetylase-1- (HDAC1) dependent and that interstitial flow increases the phosphorylation of HDAC1, its activity, and its export from the nucleus. Furthermore, we show that HDAC1 inhibition decreases endothelial morphogenesis and matrix metalloproteinase-14 (MMP14) expression. Our results suggest that HDAC1 modulates angiogenesis in response to flow, providing a new target for modulating vascularization in the clinic. PMID:27669993

  3. Dehydroepiandrosterone sulfate mediates activation of transcription factors CREB and ATF-1 via a Gα11-coupled receptor in the spermatogenic cell line GC-2.

    PubMed

    Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2013-12-01

    Dehydroepiandrosterone sulfate (DHEAS) is a circulating steroid produced in the adrenal cortex, brain, and gonads. Whereas a series of investigations attest to neuroprotective effects of the steroid in the brain, surprisingly little is known about the physiological effects of DHEAS on cells of the reproductive system. Here we demonstrate that DHEAS acting on the spermatogenic cell line GC-2 induces a time- and concentration-dependent phosphorylation of c-Src and Erk1/2 and activates the transcription factors activating transforming factor-1 (ATF-1) and cyclic AMP-responsive element binding protein (CREB). These actions are consistent with the non-classical signaling pathway of testosterone and suggest that DHEAS is a pro-androgen that is converted into testosterone in order to exert its biological activity. The fact, however, that steroid sulfatase mRNA was not detected in the GC-2 cells and the clear demonstration of DHEAS-induced activation of Erk1/2, ATF-1 and CREB after silencing the androgen receptor by small interfering RNA (siRNA) clearly contradict this assumption and make it appear unlikely that DHEAS has to be converted in the cytosol into a different steroid in order to activate the kinases and transcription factors mentioned. Instead, it is likely that the DHEAS-induced signaling is mediated through the interaction of the steroid with a membrane-bound G-protein-coupled receptor, since silencing of Guanine nucleotide-binding protein subunit alpha-11 (Gnα11) leads to the abolition of the DHEAS-induced stimulation of Erk1/2, ATF-1, and CREB. The investigation presented here shows a hormone-like activity of DHEAS on a spermatogenic cell line. Since DHEAS is produced in male and female reproductive organs, these findings could help to define new roles for DHEAS in the physiology of reproduction.

  4. Increased Hippocampal Neurogenesis and Accelerated Response to Antidepressants in Mice with Specific Deletion of CREB in the Hippocampus: Role of cAMP Response-Element Modulator τ

    PubMed Central

    Gundersen, Brigitta B.; Briand, Lisa A.; Onksen, Jennifer L.; LeLay, John; Kaestner, Klaus H.

    2013-01-01

    The transcription factor cAMP response element-binding protein (CREB) has been implicated in the pathophysiology of depression as well as in the efficacy of antidepressant treatment. However, altering CREB levels appears to have differing effects on anxiety- and depression-related behaviors, depending on which brain region is examined. Furthermore, many manipulations of CREB lead to corresponding changes in other CREB family proteins, and the impact of these changes has been largely ignored. To further investigate the region-specific importance of CREB in depression-related behavior and antidepressant response, we used CrebloxP/loxP mice to localize CREB deletion to the hippocampus. In an assay sensitive to chronic antidepressant response, the novelty-induced hypophagia procedure, hippocampal CREB deletion, did not alter the response to chronic antidepressant treatment. In contrast, mice with hippocampal CREB deletion responded to acute antidepressant treatment in this task, and this accelerated response was accompanied by an increase in hippocampal neurogenesis. Upregulation of the CREB-family protein cAMP response-element modulator (CREM) was observed after CREB deletion. Viral overexpression of the activator isoform of CREM, CREMτ, in the hippocampus also resulted in an accelerated response to antidepressants as well as increased hippocampal neurogenesis. This is the first demonstration of CREMτ within the brain playing a role in behavior and specifically in behavioral outcomes following antidepressant treatment. The current results suggest that activation of CREMτ may provide a means to accelerate the therapeutic efficacy of current antidepressant treatment. PMID:23966689

  5. Mitomycin C treatment induces resistance and enhanced migration via phosphorylated Akt in aggressive lung cancer cells

    PubMed Central

    Lai, Liang-Chuan; Chuang, Eric Y.; Tsai, Mong-Hsun

    2016-01-01

    Since 1984, mitomycin C (MMC) has been applied in the treatment of non-small-cell lung cancer (NSCLC). MMC-based chemotherapeutic regimens are still under consideration owing to the efficacy and low cost as compared with other second-line regimens in patients with advanced NSCLC. Hence, it is important to investigate whether MMC induces potential negative effects in NSCLC. Here, we found that the malignant lung cancer cells, CL1-2 and CL1-5, were more resistant to MMC than were the parental CL1-0 cells and pre-malignant CL1-1 cells. CL1-2 and CL1-5 cells consistently showed lower sub-G1 fractions post MMC treatment. DNA repair-related proteins were not induced more in CL1-5 than in CL1-0 cells, but the levels of endogenous and MMC-induced phosphorylated Akt (p-Akt) were higher in CL1-5 cells. Administering a p-Akt inhibitor reduced the MMC resistance, demonstrating that p-Akt is important in the MMC resistance of CL1-5 cells. Furthermore, we revealed that cell migration was enhanced by MMC but lowered by a p-Akt inhibitor in CL1-5 cells. This study suggests that in CL1-5 cells, the activity of p-Akt, rather than DNA repair mechanisms, may underlie the resistance to MMC and enhance the cells' migration abilities after MMC treatment. PMID:27833080

  6. The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.

    PubMed

    Valenti, G; Procino, G; Carmosino, M; Frigeri, A; Mannucci, R; Nicoletti, I; Svelto, M

    2000-06-01

    Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient P(f) by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 microM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 microM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in P(f).

  7. Suppression of ERK phosphorylation through oxidative stress is involved in the mechanism underlying sevoflurane-induced toxicity in the developing brain

    PubMed Central

    Yufune, Shinya; Satoh, Yasushi; Akai, Ryosuke; Yoshinaga, Yosuke; Kobayashi, Yasushi; Endo, Shogo; Kazama, Tomiei

    2016-01-01

    In animal models, neonatal exposure to general anesthetics significantly increased neuronal apoptosis with subsequent behavioral deficits in adulthood. Although the underlying mechanism is largely unknown, involvement of extracellular signal-regulated kinases (ERKs) is speculated since ERK phosphorylation is decreased by neonatal anesthetic exposure. Importance of ERK phosphorylation for neuronal development is underscored by our recent finding that transient suppression of ERK phosphorylation during the neonatal period significantly increased neuronal apoptosis and induced behavioral deficits. However, it is still unknown as to what extent decreased ERK phosphorylation contributes to the mechanism underlying anesthetic-induced toxicity. Here we investigated the causal relationship of decreased ERK phosphorylation and anesthetic-induced toxicity in the developing brain. At postnatal day 6 (P6), mice were exposed to sevoflurane (2%) or the blood-brain barrier-penetrating MEK inhibitor, α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile (SL327) (50 mg/kg). Transient suppression of ERK phosphorylation by an intraperitoneal injection of SL327 at P6 significantly increased apoptosis similar to sevoflurane-induced apoptosis. Conversely, SL327 administration at P14 or P21 did not induce apoptosis, even though ERK phosphorylation was inhibited. Restoring ERK phosphorylation by administration of molecular hydrogen ameliorated sevoflurane-induced apoptosis. Together, our results strongly suggests that suppressed ERK phosphorylation is critically involved in the mechanism underlying anesthetic-induced toxicity in the developing brain. PMID:26905012

  8. Olprinone and colforsin daropate alleviate septic lung inflammation and apoptosis through CREB-independent activation of the Akt pathway.

    PubMed

    Oishi, Hirofumi; Takano, Ken-ichi; Tomita, Kengo; Takebe, Mariko; Yokoo, Hiroki; Yamazaki, Mitsuaki; Hattori, Yuichi

    2012-07-01

    Olprinone, a specific phosphodiesterase III inhibitor, and corforsin daropate, a direct adenylate cyclase activator, are now being used in critical conditions. We investigated whether their therapeutic use provides protection against septic acute lung injury (ALI) and mortality. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in BALB/c mice. Olprinone or colforsin daropate was continuously given through an osmotic pump that was implanted into the peritoneal cavity immediately following CLP. These treatments prevented the ALI development in CLP mice, as indicated by the findings that severe hypoxemia, increased pulmonary vascular permeability, and histological lung damage were strikingly remedied. Furthermore, continued administration of olprinone or colforsin daropate suppressed apoptosis induction in septic lungs and improved the survival of CLP mice. Olprinone and corforsin daropate enhanced Akt phosphorylation in septic lungs. Wortmannin, which inhibits the Akt upstream regulator phosphatidylinositol 3-kinase, abrogated the protective effects of olprinone and corforsin daropate on sepsis-associated lung inflammation and apoptosis. In vivo transfection of cyclic AMP response element binding protein (CREB) decoy oligodeoxynucleotide failed to negate the abilities of these agents to increase Akt phosphorylation and to inhibit IκBα degradation in septic lungs. These results demonstrate for the first time that CREB-independent Akt-mediated signaling is a critical mechanism contributing to the therapeutic effects of olprinone and corforsin daropate on septic ALI. Moreover, our data also suggest that these cyclic AMP-related agents, by blocking both nuclear factor-κB activation and apoptosis induction, may represent an effective therapeutic approach to the treatment of the septic syndrome.

  9. The Effects of Insulin-Induced Hypoglycaemia on Tyrosine Hydroxylase Phosphorylation in Rat Brain and Adrenal Gland.

    PubMed

    Senthilkumaran, Manjula; Johnson, Michaela E; Bobrovskaya, Larisa

    2016-07-01

    In this study we investigated the effects of insulin-induced hypoglycaemia on tyrosine hydroxylase (TH) protein and TH phosphorylation in the adrenal gland, C1 cell group, locus coeruleus (LC) and midbrain dopaminergic cell groups that are thought to play a role in response to hypoglycaemia and compared the effects of different concentrations of insulin in rats. Insulin (1 and 10 U/kg) treatment caused similar reductions in blood glucose concentration (from 7.5-9 to 2-3 mmol/L); however, plasma adrenaline concentration was increased 20-30 fold in response to 10 U/kg insulin and only 14 fold following 1 U/kg. Time course studies (at 10 U/kg insulin) revealed that in the adrenal gland, Ser31 phosphorylation was increased between 30 and 90 min (4-5 fold), implying that TH was activated to increase catecholamine synthesis in adrenal medulla to replenish the stores. In the brain, Ser19 phosphorylation was limited to certain dopaminergic groups in the midbrain, while Ser31 phosphorylation was increased in most catecholaminergic regions at 60 min (1.3-2 fold), suggesting that Ser31 phosphorylation may be an important mechanism to maintain catecholamine synthesis in the brain. Comparing the effects of 1 and 10 U/kg insulin revealed that Ser31 phosphorylation was increased to similar extent in the adrenal gland and C1 cell group in response to both doses whereas Ser31 and Ser19 phosphorylation were only increased in response to 1 U/kg insulin in LC and in response to 10 U/kg insulin in most midbrain regions. Thus, the adrenal gland and some catecholaminergic brain regions become activated in response to insulin administration and brain catecholamines may be important for initiation of physiological defences against insulin-induced hypoglycaemia.

  10. Osmotic stress induces the phosphorylation of WNK4 Ser575 via the p38MAPK-MK pathway

    PubMed Central

    Maruyama, Junichi; Kobayashi, Yumie; Umeda, Tsuyoshi; Vandewalle, Alain; Takeda, Kohsuke; Ichijo, Hidenori; Naguro, Isao

    2016-01-01

    The With No lysine [K] (WNK)-Ste20-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway has been reported to be a crucial signaling pathway for triggering pseudohypoaldosteronism type II (PHAII), an autosomal dominant hereditary disease that is characterized by hypertension. However, the molecular mechanism(s) by which the WNK-SPAK/OSR1 pathway is regulated remain unclear. In this report, we identified WNK4 as an interacting partner of a recently identified MAP3K, apoptosis signal-regulating kinase 3 (ASK3). We found that WNK4 is phosphorylated in an ASK3 kinase activity-dependent manner. By exploring the ASK3-dependent phosphorylation sites, we identified Ser575 as a novel phosphorylation site in WNK4 by LC-MS/MS analysis. ASK3-dependent WNK4 Ser575 phosphorylation was mediated by the p38MAPK-MAPK-activated protein kinase (MK) pathway. Osmotic stress, as well as hypotonic low-chloride stimulation, increased WNK4 Ser575 phosphorylation via the p38MAPK-MK pathway. ASK3 was required for the p38MAPK activation induced by hypotonic stimulation but was not required for that induced by hypertonic stimulation or hypotonic low-chloride stimulation. Our results suggest that the p38MAPK-MK pathway might regulate WNK4 in an osmotic stress-dependent manner but its upstream regulators might be divergent depending on the types of osmotic stimuli. PMID:26732173

  11. Cyclooxygenase-2-dependent phosphorylation of the pro-apoptotic protein Bad inhibits tonicity-induced apoptosis in renal medullary cells.

    PubMed

    Küper, Christoph; Bartels, Helmut; Beck, Franz-X; Neuhofer, Wolfgang

    2011-11-01

    During antidiuresis, cell survival in the renal medulla requires cyclooxygenase-2 (COX-2) activity. We have recently found that prostaglandin E2 (PGE2) promotes cell survival by phosphorylation and, hence, inactivation of the pro-apoptotic protein Bad during hypertonic stress in Madin-Darby canine kidney (MDCK) cells in vitro. Here we determine the role of COX-2-derived PGE(2) on phosphorylation of Bad and medullary apoptosis in vivo using COX-2-deficient mice. Both wild-type and COX-2-knockout mice constitutively expressed Bad in tubular epithelial cells of the renal medulla. Dehydration caused a robust increase in papillary COX-2 expression, PGE2 excretion, and Bad phosphorylation in wild-type, but not in the knockout mice. The abundance of cleaved caspase-3, a marker of apoptosis, was significantly higher in papillary homogenates, especially in tubular epithelial cells of the knockout mice. Knockdown of Bad in MDCK cells decreased tonicity-induced caspase-3 activation. Furthermore, the addition of PGE2 to cells with knockdown of Bad had no effect on caspase-3 activation; however, PGE2 caused phosphorylation of Bad and substantially improved cell survival in mock-transfected cells. Thus, tonicity-induced COX-2 expression and PGE2 synthesis in the renal medulla entails phosphorylation and inactivation of the pro-apoptotic protein Bad, thereby counteracting apoptosis in renal medullary epithelial cells.

  12. PDGF-RECEPTOR ACTIVATION INDUCES p120-CATENIN PHOSPHORYLATION AT SERINE 879 VIA A PKCα-DEPENDENT PATHWAY

    PubMed Central

    Brown, Meredith V.; Burnett, Patrick E.; Denning, Mitchell F.; Reynolds, Albert B.

    2010-01-01

    p120-catenin (p120) is required for cadherin stability and is thought to have a central role in modulating cell-cell adhesion. Several lines of evidence suggest that S/T phosphorylation may regulate p120 activity, but the upstream kinases involved have not been established, nor has a discreet measurable function been assigned to an individual site. To approach these issues, we have generated p120 phospho-specific monoclonal antibodies to several individual phosphorylation sites and are using them to pinpoint upstream kinases and signaling pathways that control p120 activity. Protein Kinase C (PKC) has been implicated as a signaling intermediate in several cadherin-associated cellular activities. Signaling events that activate PKC induce rapid phosphorylation at p120 Serine 879 (S879), suggesting that p120 activity is regulated, in part, by one or more PKC isoforms. Here, we find that physiologic activation of a G-protein coupled receptor (i.e., endothelin receptor), as well as several Receptor Tyrosine Kinases, induce rapid and robust p120 phosphorylation at S879, suggesting that these pathways crosstalk to cadherin complexes via p120. Using Va2 cells and PDGF stimulation, we show for the first time that PDGFR-mediated phosphorylation at this site is dependent on PKCα, a conventional PKC isoform implicated previously in disruption of adherens junctions. PMID:18950621

  13. ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

    EPA Science Inventory

    ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)
    Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

  14. Protective effect of wogonin on endotoxin-induced acute lung injury via reduction of p38 MAPK and JNK phosphorylation.

    PubMed

    Wei, Cheng-Yu; Sun, Hai-Lun; Yang, Ming-Ling; Yang, Ching-Ping; Chen, Li-You; Li, Yi-Ching; Lee, Chien-Ying; Kuan, Yu-Hsiang

    2017-02-01

    Acute lung injury (ALI) is a serious inflammatory disorder which remains the primary cause of incidence and mortality in patients with acute pulmonary inflammation. However, there is still no effective medical strategy available clinically for the improvement of ALI. Wogonin, isolated from roots of Scutellaria baicalensis Georgi, is a common medicinal herb which presents biological and pharmacological effects, including antioxidation, anti-inflammation, and anticancer. Preadministration of wogonin inhibited not only lung edema but also protein leakage into the alveolar space in murine model of lipopolysaccharide (LPS)-induced ALI. Moreover, wogonin not only reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 but also inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) induced by LPS. We further found wogonin inhibited the phosphorylation of p38 MAPK and JNK at a concentration lower than ERK. In addition, inhibition of lung edema, protein leakage, expression of iNOS and COX-2, and phosphorylation of p38 MAPK and JNK were all observed in a parallel concentration-dependent manner. These results suggest that wogonin possesses potential protective effect against LPS-induced ALI via downregulation of iNOS and COX-2 expression by blocking phosphorylation of p38 MAPK and JNK. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 397-403, 2017.

  15. Stress-induced nuclear translocation of CDK5 suppresses neuronal death by downregulating ERK activation via VRK3 phosphorylation

    PubMed Central

    Song, Haengjin; Kim, Wanil; Choi, Jung-Hyun; Kim, Sung-Hoon; Lee, Dohyun; Park, Choon-Ho; Kim, Sangjune; Kim, Do-Yeon; Kim, Kyong-Tai

    2016-01-01

    Although extracellular signal-related kinase 1/2 (ERK 1/2) activity is generally associated with cell survival, prolonged ERK activation induced by oxidative stress also mediates neuronal cell death. Here we report that oxidative stress-induced cyclin-dependent kinase 5 (CDK5) activation stimulates neuroprotective signaling via phosphorylation of vaccinia-related kinase 3 (VRK3) at Ser 108. The binding of vaccinia H1-related (VHR) phosphatase to phosphorylated VRK3 increased its affinity for phospho-ERK and subsequently downregulated ERK activation. Overexpression of VRK3 protected human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced apoptosis. However the CDK5 was unable to phosphorylate mutant VRK3, and thus the mutant forms of VRK3 could not attenuate apoptotic process. Suppression of CDK5 activity results in increase of ERK activation and elevation of proapoptotic protein Bak expression in mouse cortical neurons. Results from VRK3-deficient neurons were further confirmed the role of VRK3 phosphorylation in H2O2-evoked ERK regulation. Importantly, we showed an association between phospho-VRK3 levels and the progression of human Alzheimer’s disease (AD) and Parkinson’s disease (PD). Together our work reveals endogenous protective mechanism against oxidative stress-induced neuronal cell death and suggest VRK3 as a potential therapeutic target in neurodegenerative diseases. PMID:27346674

  16. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    EPA Science Inventory

    Prolactin-Induced Tyrosine Phosphorylation, Activation and Receptor
    Association of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells.
    Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental Protection
    Agency, MD-72, Research Triangle Park, NC 27711, and

  17. Determinants of CREB degradation and KChIP2 gene transcription in cardiac memory

    PubMed Central

    Özgen, Nazira; Lau, David H.; Shlapakova, Iryna N.; Sherman, Warren; Feinmark, Steven J.; Danilo, Peter; Rosen, Michael R.

    2010-01-01

    Background Left ventricular pacing (LVP) to induce cardiac memory (CM) in dogs results in a decreased transient outward K current (Ito) and reduced mRNA and protein of the Ito channel accessory subunit, KChIP2. The KChIP2 decrease is attributed to a decrease in its transcription factor CREB (cAMP response element binding protein). Objective To determine the mechanisms responsible for the CREB decrease that is initiated by LVP. Methods CM was quantified as T wave vector displacement in 18 LVP dogs. In 5 dogs, Ang II receptor blocker, saralasin, was infused before and during pacing. In 3 dogs, proteasomal inhibitor, lactacystin, was injected into the left anterior descending artery before LVP. Epicardial biopsies were taken before and after LVP. Neonatal rat cardiomyocytes (NRCM) were incubated with H2O2 (50 μmol/L) for 1h with or without lactacystin. Results LVP significantly displaced the T wave vector and was associated with increased lipid peroxidation and increased tissue Ang II levels. Saralasin prevented T vector displacement and lipid peroxidation. CREB was significantly decreased after 2h of LVP and was comparably decreased in H2O2–treated NRM. Lactacystin inhibited the CREB decrease in LVP dogs and H2O2 -treated NRM. LVP and H2O2 both induced CREB ubiquitination and the H2O2-induced CREB decrease was prevented by knocking down ubiquitin. Conclusion LVP initiates myocardial Ang II production and ROS synthesis leading to CREB ubiquitination and its proteasomal degradation. This sequence of events would explain the pacing-induced reduction in KChIP2, and contribute to altered repolarization and the T wave changes of cardiac memory. PMID:20346417

  18. Naloxone's pentapeptide binding site on filamin A blocks Mu opioid receptor-Gs coupling and CREB activation of acute morphine.

    PubMed

    Wang, Hoau-Yan; Burns, Lindsay H

    2009-01-01

    Chronic morphine causes the mu opioid receptor (MOR) to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Galphas and its Gbetagamma dimer. Ultra-low-dose naloxone (NLX) prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA), a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show that acute morphine induces a dose-dependent Go-to-Gs coupling switch at 5 and 15 min that resolves by 1 hr. The acute Gs coupling induced by 100 microM morphine was completely prevented by co-treatment with 100 pM NLX, (+)NLX, or naltrexone (NTX), or their pentapeptide binding site (FLNA(2561-2565)), which we show can act as a decoy for MOR or bind to FLNA itself. All of these co-treatments presumably prevent the MOR-FLNA interaction. Since ultra-low-dose NTX also attenuates the addictive properties of opioids, we assessed striatal cAMP production and CREB phosphorylation at S(133). Correlating with the Gs coupling, acute morphine induced elevated cAMP levels and a several-fold increase in pS(133)CREB that were also completely blocked by NLX, NTX or the FLNA pentapeptide. We propose that acute, robust stimulation of MOR causes an interaction with FLNA that allows an initially transient MOR-Gs coupling, which recovers with receptor recycling but persists when MOR stimulation is repeated or prolonged. The complete prevention of this acute, morphine-induced MOR-Gs coupling by 100 pM NLX/NTX or 10 microM pentapeptide segment of FLNA further elucidates both MOR signaling and the mechanism of action of ultra-low-dose NLX or NTX in attenuating opioid tolerance, dependence and addictive potential.

  19. Stress Induces p38 MAPK-mediated Phosphorylation and Inhibition of Drosha-dependent Cell Survival

    PubMed Central

    Yang, Qian; Li, Wenming; She, Hua; Dou, Juan; Duong, Duc M; Du, Yuhong; Yang, Shao-Hua; Seyfried, Nicholas T.; Fu, Haian; Gao, Guodong; Mao, Zixu

    2015-01-01

    SUMMARY MicroRNAs (miRNAs) regulate the translational potential of their mRNA targets and control many cellular processes. The key step in canonical miRNA biogenesis is the cleavage of the primary transcripts by the nuclear RNase III enzyme Drosha. Emerging evidence suggests that the miRNA biogenic cascade is tightly controlled. However, little is known whether Drosha is regulated. Here we show that Drosha is targeted by stress. Under stress, p38 MAPK directly phosphorylates Drosha at its N-terminus. This reduces its interaction with DiGeorge syndrome critical region 8, and promotes its nuclear export and degradation by calpain. This regulatory mechanism mediates stress-induced inhibition of Drosha function. Reduction of Drosha sensitizes cells to stress and increases death. In contrast, increase in Drosha attenuates stress-induced death. These findings reveal a critical regulatory mechanism by which stress engages p38 MAPK pathway to destabilize Drosha and inhibit Drosha-mediated cellular survival. PMID:25699712

  20. SCO2 Mediates Oxidative Stress-Induced Glycolysis to Oxidative Phosphorylation Switch in Hematopoietic Stem Cells.

    PubMed

    Du, Wei; Amarachintha, Surya; Wilson, Andrew F; Pang, Qishen

    2016-04-01

    Fanconi anemia (FA) is an inherited bone marrow (BM) failure syndrome, presumably resulting from defects in hematopoietic stem cells (HSCs). Normal HSCs depend more on glycolysis than on oxidative phosphorylation (OXPHOS) for energy production. Here, we show that FA HSCs are more sensitive to the respiration inhibitor NaN3 treatment than to glycolytic inhibitor 2-deoxy-d-glucose (2-DG), indicating more dependence on OXPHOS. FA HSCs undergo glycolysis-to-OXPHOS switch in response to oxidative stress through a p53-dependent mechanism. Metabolic stresses induce upregulation of p53 metabolic targets in FA HSCs. Inactivation of p53 in FA HSCs prevents glycolysis-to-OXPHOS switch. Furthermore, p53-deficient FA HSCs are more sensitive to 2-DG-mediated metabolic stress. Finally, oxidative stress-induced glycolysis-to-OXPHOS switch is mediated by synthesis of cytochrome c oxidase 2 (SCO2). These findings demonstrate p53-mediated OXPHOS function as a compensatory alteration in FA HSCs to ensure a functional but mildly impaired energy metabolism and suggest a cautious approach to manipulating p53 signaling in FA.

  1. Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

    SciTech Connect

    Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra; Galadari, Sehamuddin

    2010-04-09

    Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

  2. Expression of Iron Regulatory Protein 1 Is Regulated not only by HIF-1 but also pCREB under Hypoxia

    PubMed Central

    Luo, Qian-Qian; Qian, Zhong-Ming; Zhou, Yu-Fu; Zhang, Meng-Wan; Wang, Dang; Zhu, Li; Ke, Ya

    2016-01-01

    The inconsistent of responses of IRP1 and HIF-1 alpha to hypoxia and the similar tendencies in the changes of IRP1 and pCREB contents led us to hypothesize that pCREB might be involved in the regulation of IRP1 under hypoxia. Here, we investigated the role of pCREB in IRP1 expression in HepG2 cells under hypoxia using quantitative PCR, western blot, immunofluorescence, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). We demonstrated that 1) Hypoxia increased pCREB levels inside of the nucleus; 2) Putative CREs were found in the IRP1 gene; 3) Nuclear extracts of HepG2 cells treated with hypoxia could bind to CRE1 and CRE3, and 100-fold competitor of putative CREs could abolish the binding activity to varying degrees; 4) pCREB was found in the CRE1 and CRE3 DNA-protein complexes of EMSA; 5) CRE1 and CRE3 binding activity of IRP1 depended on CREB activation but not on HIF-1; 6) Increased IRP1 expression under hypoxia could be prevented by LY294002; 7) ChIP assays demonstrated that pCREB binds to IRP1 promoter; and 8) HIF-1 and/or HIF-2 siRNA had no effect on the expression of pCREB and IRP1 proteins in cells treated with hypoxia for 8 hours. Our findings evidenced for the involvement of pCREB in IRP1 expression and revealed a dominant role of PI3K/Akt pathway in CREB activation under hypoxia and also suggested that dual-regulation of IRP1 expression by HIF-1 and pCERB or other transcription factor(s) under hypoxia might be a common mechanism in most if not all of hypoxia-inducible genes. PMID:27766034

  3. CREB1 directly activates the transcription of ribonucleotide reductase small subunit M2 and promotes the aggressiveness of human colorectal cancer

    PubMed Central

    Fang, Zejun; Lin, Aifen; Chen, Jiaoe; Zhang, Xiaomin; Liu, Hong; Li, Hongzhang; Hu, Yanyan; Zhang, Xia; Zhang, Jiangang; Qiu, Lanlan; Mei, Lingming; Shao, Jimin; Chen, Xiang

    2016-01-01

    As the small subunit of Ribonucleotide reductase (RR), RRM2 displays a very important role in various critical cellular processes such as cell proliferation, DNA repair, and senescence, etc. Importantly, RRM2 functions like a tumor driver in most types of cancer but little is known about the regulatory mechanism of RRM2 in cancer development. In this study, we found that the cAMP responsive element binding protein 1 (CREB1) acted as a transcription factor of RRM2 gene in human colorectal cancer (CRC). CREB1 directly bound to the promoter of RRM2 gene and induced its transcriptional activation. Knockdown of CREB1 decreased the expression of RRM2 at both mRNA and protein levels. Moreover, knockdown of RRM2 attenuated CREB1-induced aggressive phenotypes of CRC cells in vitro and in vivo. Analysis of the data from TCGA database and clinical CRC specimens with immunohistochemical staining also demonstrated a strong correlation between the co-expression of CREB1 and RRM2. Decreased disease survivals were observed in CRC patients with high expression levels of CREB1 or RRM2. Our results indicate CREB1 as a critical transcription factor of RRM2 which promotes tumor aggressiveness, and imply a significant correlation between CREB1 and RRM2 in CRC specimens. These may provide the possibility that CREB1 and RRM2 could be used as biomarkers or targets for CRC diagnosis and treatment. PMID:27801665

  4. Nitric oxide and brassinosteroids mediated fungal endophyte-induced volatile oil production through protein phosphorylation pathways in Atractylodes lancea plantlets.

    PubMed

    Ren, Cheng-Gang; Dai, Chuan-Chao

    2013-11-01

    Fungal endophytes have been isolated from almost every plant, infecting their hosts without causing visible disease symptoms, and yet have still proved to be involved in plant secondary metabolites accumulation. To decipher the possible physiological mechanisms of the endophytic fungus-host interaction, the role of protein phosphorylation and the relationship between endophytic fungus-induced kinase activity and nitric oxide (NO) and brassinolide (BL) in endophyte-enhanced volatile oil accumulation in Atractylodes lancea plantlets were investigated using pharmacological and biochemical approaches. Inoculation with the endophytic fungus Gilmaniella sp. AL12 enhanced the activities of total protein phosphorylation, Ca²⁺-dependent protein kinase, and volatile oil accumulation in A. lancea plantlets. The upregulation of protein kinase activity could be blocked by the BL inhibitor brassinazole. Furthermore, pretreatments with the NO-specific scavenger cPTIO significantly reduced the increased activities of protein kinases in A. lancea plantlets inoculated with endophytic fungus. Pretreatments with different protein kinase inhibitors also reduced fungus-induced NO production and volatile oil accumulation, but had barely no effect on the BL level. These data suggest that protein phosphorylation is required for endophyte-induced volatile oil production in A. lancea plantlets, and that crosstalk between protein phosphorylation and the NO pathway may occur and act as a downstream signaling event of the BL pathway.

  5. Phosphorylation of GSK-3β mediates Intralipid-induced cardioprotection against Ischemia/Reperfusion injury

    PubMed Central

    Rahman, Siamak; Li, Jingyuan; Bopassa, Jean Chrisostome; Umar, Soban; Iorga, Andrea; Partownavid, Parisa; Eghbali, Mansoureh

    2012-01-01

    Background Intralipid, a brand name for the first safe fat emulsion for human use, has been shown to be cardioprotective. However, the mechanism of this protection is not known. Here we investigated the molecular mechanism(s) of Intralipid-induced cardioprotection against ischemia/reperfusion injury, particularly the role of GSK-3β and mitochondiral permeability transition pore in this protective action. Methods In-vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with Intralipid (1% in ex-vivo and one bolus of 20% in in-vivo) or vehicle. The hemodynamic function, infarct size, threshold for the opening of mitochondiral permeability transition pore and phosphorylation levels of Akt/ERK/GSK-3β were measured. Results Administration of Intralipid at the onset of reperfusion resulted in ~70% reduction in infarct size in the in-vivo rat model. Intralipid also significantly improved functional recovery of isolated Langendorff-perfused mouse hearts as the rate pressure product was increased from 2999±863mmHg*beats/min in control to 13676±611 mmHg*beats/min (Mean±SEM) and the infarct size was markedly smaller (18.3±2.4% vs. 54.8±2.9% in control, P<0.01). The Intralipid-induced cardioprotection was fully abolished by LY294002, a specific inhibitor of PI3K, but only partially by PD98059, a specific ERK inhibitor. Intralipid also increased the phosphorylation levels of Akt/ERK1/GSK-3β by 8, 3 and 9 fold, respectively. The opening of mitochondiral permeability transition pore was inhibited by Intralipid as calcium retention capacity was higher in Intralipid group (274.3±8.4nM/mg vs. 168.6±9.6nM/mg control). Conclusions Postischemic treatment with Intralipid inhibits the opening of mitochondiral permeability transition pore and protects the heart through GSK-3β via PI3K/Akt/ERK pathways. PMID:21691195

  6. Kindling alters neurosteroid-induced modulation of phasic and tonic GABAA receptor-mediated currents: role of phosphorylation.

    PubMed

    Kia, Arash; Ribeiro, Fabiola; Nelson, Renee; Gavrilovici, Cezar; Ferguson, Stephen S G; Poulter, Michael O

    2011-03-01

    We have previously shown that after kindling (a model of temporal lobe epilepsy), the neuroactive steroid tetrahydrodeoxycorticosterone (THDOC) was unable to augment GABA type A receptor (GABA(A))-mediated synaptic currents occurring on pyramidal cells of the piriform cortex. Phosphorylation of GABA(A) receptors has been shown previously to alter the activity of THDOC, so we tested the hypothesis that kindling induces changes in the phosphorylation of GABA(A) receptors and this accounts for the loss in efficacy. To assay whether GABA(A) receptors are more phosphorylated after kindling, we examined the phosphorylation state of the β3 subunit and found that it was increased. Incubation of brain slices with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (100 nM) also increased phosphorylation in the same assay. In patch clamp, recordings from non-kindled rat brain slices PMA also reduced the activity of THDOC in a manner that was identical to what is observed after kindling. We also found that the tonic current was no longer augmented by THODC after kindling and PMA treatment. The protein kinase C (PKC) antagonist bisindolylmaleimide I blocked the effects PMA on the synaptic but not the tonic currents. However, the broad spectrum PKC antagonist staurosporine blocked the effects of PMA on the tonic currents, implying that different PKC isoforms phosphorylate GABA(A) receptors responsible for phasic and tonic currents. The phosphatase activator Li(+) palmitate restored the 'normal' activity of THDOC on synaptic currents in kindled brain slices but not the tonic currents. These data demonstrate that kindling enhances the phosphorylation state of GABA(A) receptors expressed in pyramidal neurons reducing THDOC efficacy.

  7. Molecular dynamics simulation of phosphorylation-induced conformational transitions in the mycobacterium tuberculosis response regulator PrrA

    SciTech Connect

    Chen, Guo; Mcmahon, Benjamin H; Tung, Chang - Shung

    2008-01-01

    Phosphorylation-activated modulation of response regulators (RR) is predominantly used by bacteria as a strategy in regulating their two-component signaling (TCS) systems, the underlying molecular mechanisms are however far from fully understood. In this work we have conducted a molecular dynamics (MD) simulation of the phosphorylation-induced conformational transitions of RRs with the Mycobacterium Tuberculosis PrrA as a particular example. Starting from the full-length inactive structure of PrrA we introduced a local disturbance by phosphorylating the conserved aspartic acid residue, Asp-58, in the regulatory domain. A Go-model-type algorithm packaged with AMBER force fields was then applied to simulate the dynamics upon phosphorylation. The MD simulation shows that the phosphorylation of Asp-58 facilitates PrrA, whose inactive state has a compact conformation with a closed interdomain interface, to open up with its interdomain separation being increased by an average of about 1.5 {angstrom} for a simulation of 20 ns. The trans-activation loop, which is completely buried within the interdomain interface in the inactive PrrA, is found to become more exposed with the phosphorylated structure as well. These results provide more structural details of how the phosphorylation of a local aspartate activates PrrA to undergo a global conformational rearrangement toward its extended active state. This work also indicates that MD simulations can serve as a fast tool to unravel the regulation mechanisms of all RRs, which is especially valuable when the structures of full-length active RRs are currently unavailable.

  8. Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB.

    PubMed Central

    Deak, M; Clifton, A D; Lucocq, L M; Alessi, D R

    1998-01-01

    We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB. PMID:9687510

  9. Insulin Resistance Prevents AMPK-induced Tau Dephosphorylation through Akt-mediated Increase in AMPKSer-485 Phosphorylation*

    PubMed Central

    Kim, Bhumsoo; Figueroa-Romero, Claudia; Pacut, Crystal; Backus, Carey; Feldman, Eva L.

    2015-01-01

    Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including obesity, diabetes, and dyslipidemia, and insulin resistance (IR) is the central feature of MetS. Recent studies suggest that MetS is a risk factor for Alzheimer disease (AD). AMP-activated kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme and a key player in regulating energy metabolism. In this report, we examined the role of IR on the regulation of AMPK phosphorylation and AMPK-mediated Tau phosphorylation. We found that AMPKSer-485, but not AMPKThr-172, phosphorylation is increased in the cortex of db/db and high fat diet-fed obese mice, two mouse models of IR. In vitro, treatment of human cortical stem cell line (HK-5320) and primary mouse embryonic cortical neurons with the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), induced AMPK phosphorylation at both Thr-172 and Ser-485. AMPK activation also triggered Tau dephosphorylation. When IR was mimicked in vitro by chronically treating the cells with insulin, AICAR specifically induced AMPKSer-485, but not AMPKThr-172, hyperphosphorylation whereas AICAR-induced Tau dephosphorylation was inhibited. IR also resulted in the overactivation of Akt by AICAR treatment; however, preventing Akt overactivation during IR prevented AMPKSer-485 hyperphosphorylation and restored AMPK-mediated Tau dephosphorylation. Transfection of AMPKS485A mutant caused similar results. Therefore, our results suggest the following mechanism for the adverse effect of IR on AD pathology: IR → chronic overactivation of Akt → AMPKSer-485 hyperphosphorylation → inhibition of AMPK-mediated Tau dephosphorylation. Together, our results show for the first time a possible contribution of IR-induced AMPKSer-485 phosphorylation to the increased risk of AD in obesity and diabetes. PMID:26100639

  10. Antidepressant effects of crocin and its effects on transcript and protein levels of CREB, BDNF, and VGF in rat hippocampus

    PubMed Central

    2014-01-01

    Background Antidepressants have been shown to affect levels of brain-derived neurotrophic factor (BDNF) and VGF (non-acronymic) whose transcriptions are dependent on cAMP response element binding protein (CREB) in long term treatment. The aim of this study was to verify the subacute antidepressant effects of crocin, an active constituent of saffron (Crocus sativus L.), and its effects on CREB, BDNF, and VGF proteins, transcript levels and amount of active, phosphorylated CREB (P-CREB) protein in rat hippocampus. Methods Crocin (12.5, 25, and 50 mg/kg), imipramine (10 mg/kg; positive control) and saline (1 mL/kg; neutral control) were administered intraperitoneally (IP) to male Wistar rats for 21 days. The antidepressant effects were studied using the forced swimming test (FST) on day 21 after injection. Protein expression and transcript levels of genes in the rat hippocampus were evaluated using western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively. Results Crocin significantly reduced the immobility time in the FST. Western blot analysis showed that 25 and 50 mg/kg of crocin increased the levels of CREB and BDNF significantly and dose dependently. All doses of crocin increased the VGF levels in a dose-dependent manner. Levels of p-CREB increased significantly by 50 mg/kg dose of crocin. Only 12.5 mg/kg crocin could significantly increase the transcript levels of BDNF. No changes in CREB and VGF transcript levels were observed in all groups. Conclusions These results suggest that crocin has antidepressant-like action by increasing CREB, BDNF and VGF levels in hippocampus. PMID:24401376

  11. Jade-1S phosphorylation induced by CK1α contributes to cell cycle progression.

    PubMed

    Borgal, Lori; Rinschen, Markus M; Dafinger, Claudia; Liebrecht, Valérie I; Abken, Hinrich; Benzing, Thomas; Schermer, Bernhard

    2016-01-01

    The PHD zinc finger protein Jade-1S is a component of the HBO1 histone acetyltransferase complex and binds chromatin in a cell cycle-dependent manner. Jade-1S also acts as an E3 ubiquitin ligase for the canonical Wnt effector protein β-catenin and is influenced by CK1α-mediated phosphorylation. To further elucidate the functional impact of this phosphorylation, we used a stable, low-level expression system to express either wild-type or mutant Jade-1S lacking the N-terminal CK1α phosphorylation motif. Interactome analyses revealed that the Jade-1S mutant unable to be phosphorylated by CK1α has an increased binding affinity to proteins involved in chromatin remodelling, histone deacetylation, transcriptional repression, and ribosome biogenesis. Interestingly, cells expressing the mutant displayed an elongated cell shape and a delay in cell cycle progression. Finally, phosphoproteomic analyses allowed identification of a Jade-1S site phosphorylated in the presence of CK1α but closely resembling a PLK1 phosphorylation motif. Our data suggest that Jade-1S phosphorylation at an N-terminal CK1α motif creates a PLK1 phospho-binding domain. We propose CK1α phosphorylation of Jade 1S to serve as a molecular switch, turning off chromatin remodelling functions of Jade-1S and allowing timely cell cycle progression. As Jade-1S protein expression in the kidney is altered upon renal injury, this could contribute to understanding mechanisms underlying epithelial injury repair.

  12. Dexmedetomidine-Induced Contraction in the Isolated Endothelium-Denuded Rat Aorta Involves PKC-δ-mediated JNK Phosphorylation.

    PubMed

    Yu, Jongsun; Ok, Seong-Ho; Kim, Won Ho; Cho, Hyunhoo; Park, Jungchul; Shin, Il-Woo; Lee, Heon Keun; Chung, Young-Kyun; Choi, Mun-Jeoung; Kwon, Seong-Chun; Sohn, Ju-Tae

    2015-01-01

    Vasoconstriction mediated by the highly selective alpha-2 adrenoceptor agonist dexmedetomidine leads to transiently increased blood pressure and severe hypertension. The dexmedetomidine-induced contraction involves the protein kinase C (PKC)-mediated pathway. However, the main PKC isoform involved in the dexmedetomidine-induced contraction remains unknown. The goal of this in vitro study was to examine the specific PKC isoform that contributes to the dexmedetomidine-induced contraction in the isolated rat aorta. The endothelium-denuded rat aorta was suspended for isometric tension recording. Dexmedetomidine dose-response curves were generated in the presence or absence of the following inhibitors: the pan-PKC inhibitor, chelerythrine; the PKC-α and -β inhibitor, Go6976; the PKC-α inhibitor, safingol; the PKC-β inhibitor, ruboxistaurin; the PKC-δ inhibitor, rottlerin; the c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125; and the myosin light chain kinase inhibitor, ML-7 hydrochloride. Western blot analysis was used to examine the effect of rottlerin on dexmedetomidine-induced PKC-δ expression and JNK phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) and to investigate the effect of dexmedetomidine on PKC-δ expression in VSMCs transfected with PKC-δ small interfering RNA (siRNA) or control siRNA. Chelerythrine as well as SP600125 and ML-7 hydrochloride attenuated the dexmedetomidine-induced contraction. Go6976, safingol, and ruboxistaurin had no effect on the dexmedetomidine-induced contraction, whereas rottlerin inhibited the dexmedetomidine-induced contraction. Dexmedetomidine induced PKC-δ expression, whereas rottlerin and PKC-δ siRNA transfection inhibited dexmedetomidine-induced PKC-δ expression. Dexmedetomidine also induced JNK phosphorylation, which was inhibited by rottlerin. Taken together, these results suggest that the dexmedetomidine-induced contraction involves PKC-δ-dependent JNK phosphorylation in the isolated rat aorta.

  13. Activation of cannabinoid CB2 receptor-mediated AMPK/CREB pathway reduces cerebral ischemic injury.

    PubMed

    Choi, In-Young; Ju, Chung; Anthony Jalin, Angela M A; Lee, Da In; Prather, Paul L; Kim, Won-Ki

    2013-03-01

    The type 2 cannabinoid receptor (CB2R) was recently shown to mediate neuroprotection in ischemic injury. However, the role of CB2Rs in the central nervous system, especially neuronal and glial CB2Rs in the cortex, remains unclear. We, therefore, investigated anti-ischemic mechanisms of cortical CB2R activation in various ischemic models. In rat cortical neurons/glia mixed cultures, a CB2R agonist, trans-caryophyllene (TC), decreased neuronal injury and mitochondrial depolarization caused by oxygen-glucose deprivation/re-oxygenation (OGD/R); these effects were reversed by the selective CB2R antagonist, AM630, but not by a type 1 cannabinoid receptor antagonist, AM251. Although it lacked free radical scavenging and antioxidant enzyme induction activities, TC reduced OGD/R-evoked mitochondrial dysfunction and intracellular oxidative stress. Western blot analysis demonstrated that TC enhanced phosphorylation of AMP-activated protein kinase (AMPK) and cAMP responsive element-binding protein (CREB), and increased expression of the CREB target gene product, brain-derived neurotrophic factor. However, TC failed to alter the activity of either Akt or extracellular signal-regulated kinase, two major CB2R signaling pathways. Selective AMPK and CREB inhibitors abolished the neuroprotective effects of TC. In rats, post-ischemic treatment with TC decreased cerebral infarct size and edema, and increased phosphorylated CREB and brain-derived neurotrophic factor expression in neurons. All protective effects of TC were reversed by co-administration with AM630. Collectively, these data demonstrate that cortical CB2R activation by TC ameliorates ischemic injury, potentially through modulation of AMPK/CREB signaling, and suggest that cortical CB2Rs might serve as a putative therapeutic target for cerebral ischemia.

  14. Hydrophobic motif site-phosphorylated protein kinase CβII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

    PubMed

    Das, Falguni; Ghosh-Choudhury, Nandini; Mariappan, Meenalakshmi M; Kasinath, Balakuntalam S; Choudhury, Goutam Ghosh

    2016-04-01

    PKCβII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCβII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCβII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCβII, dominant negative PKCβII, and PKCβII hydrophobic motif phosphorylation-deficient mutant, we found that PKCβII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCβII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCβII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCβII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCβII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCβII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.

  15. The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

    PubMed

    Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

    2014-09-01

    Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

  16. Arcuate Src activation-induced phosphorylation of NR2B NMDA subunit contributes to inflammatory pain in rats.

    PubMed

    Xu, Longsheng; Pan, Yanyan; Zhu, Qi; Gong, Shan; Tao, Jin; Xu, Guang-Yin; Jiang, Xinghong

    2012-12-01

    The tyrosine kinases of Src family play an important role in the central sensitization following peripheral inflammation. However, whether the Src family in the arcuate nucleus (ARC) of mediobasal hypothalamus is involved in central sensitization remains unknown. The aim of this study was to investigate the role and mechanisms of tyrosine kinases of Src family in N-methyl-d-aspartate (NMDA) receptor activity in the ARC following peripheral inflammation. Peripheral inflammation was induced by unilateral injection of complete Freund's adjuvant (CFA) into rat hindpaw. The neuronal activities of the ARC were recorded using electrophysiological field recording from the in vitro mediobasal hypothalamic slices from control and CFA rats. Expression of total and phosphorylated Src and NR2B subunit protein was analyzed by Western blot and immuoprecipitation. Our results showed that CFA injection resulted in an increase in mechanical and thermal sensitivity, which was partially blocked by neonatal monosodium glutamate treatment. CFA injection also enhanced spontaneous firings of ARC neurons, which were reversed by the NMDA receptor NR2B subunit specific antagonist Ro25-6981 and by PP2, an Src family tyrosine kinase inhibitor. In addition, peripheral inflammation enhanced Src phosphorylation and NMDA receptor NR2B subunit phosphorylation without alteration of total NR2B subunit expression in the ARC. Peripheral inflammation also increased the association of NR2B protein with p-Src protein in the ARC. Administration of PP2 blocked the upregulation of NR2B phosphorylation induced by CFA injection. Taken together, our present results suggest that the arcuate Src activation-induced tyrosine phosphorylation of NR2B NMDA subunit may contribute to inflammatory pain.

  17. Modifications of myofilament protein phosphorylation and function in response to cardiac arrest induced in a swine model

    PubMed Central

    Woodward, Mike; Previs, Michael J.; Mader, Timothy J.; Debold, Edward P.

    2015-01-01

    Cardiac arrest is a prevalent condition with a poor prognosis, attributable in part to persistent myocardial dysfunction following resuscitation. The molecular basis of this dysfunction remains unclear. We induced cardiac arrest in a porcine model of acute sudden death and assessed the impact of ischemia and reperfusion on the molecular function of isolated cardiac contractile proteins. Cardiac arrest was electrically induced, left untreated for 12 min, and followed by a resuscitation protocol. With successful resuscitations, the heart was reperfused for 2 h (IR2) and the muscle harvested. In failed resuscitations, tissue samples were taken following the failed efforts (IDNR). Actin filament velocity, using myosin isolated from IR2 or IDNR cardiac tissue, was nearly identical to myosin from the control tissue in a motility assay. However, both maximal velocity (25% faster than control) and calcium sensitivity (pCa50 6.57 ± 0.04 IDNR vs. 6.34 ± 0.07 control) were significantly (p < 0.05) enhanced using native thin filaments (actin+troponin+tropomyosin) from IDNR samples, suggesting that the enhanced velocity is mediated through an alteration in muscle regulatory proteins (troponin+tropomyosin). Mass spectrometry analysis showed that only samples from the IR2 had an increase in total phosphorylation levels of troponin (Tn) and tropomyosin (Tm), but both IR2 and IDNR samples demonstrated a significant shift from mono-phosphorylated to bis-phosphorylated forms of the inhibitory subunit of Tn (TnI) compared to control. This suggests that the shift to bis-phosphorylation of TnI is associated with the enhanced function in IDNR, but this effect may be attenuated when phosphorylation of Tm is increased in tandem, as observed for IR2. There are likely many other molecular changes induced following cardiac arrest, but to our knowledge, these data provide the first evidence that this form cardiac arrest can alter the in vitro function of the cardiac contractile proteins

  18. Stress-induced apoptosis in Spodoptera frugiperda (Sf9) cells: baculovirus p35 mitigates eIF2 alpha phosphorylation.

    PubMed

    Aparna, Gunda; Bhuyan, Abani K; Sahdev, Sudhir; Hasnain, Seyed E; Kaufman, Randal J; Ramaiah, Kolluru V A

    2003-12-30

    Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.

  19. Lectin-induced activation of platelets may require only limited phosphorylation of the 47K protein

    SciTech Connect

    Ganguly, C.; Chelladurai, M.; Ganguly, P.

    1986-05-01

    Wheat germ agglutinin (WGA) is an N-acetylglucosamine (Glc-NAc) specific lectin which can activate platelets. Like thrombin, stimulation of platelets by WGA is accompanied by enhanced phosphorylation of two polypeptides of M/sub r/ 47K and 20K. Addition of GlcNAc at different time intervals arrested that aggregation of platelets by WGA and paralleled the modification of phosphorylation of the 47K polypeptide. So, the phosphorylation of the 47K polypeptide may regulate the WGA-receptor mediated stimulation of platelets. However, the ratio of phosphoserine to phosphothreonine in the 47K protein was markedly different in WGA-activated than thrombin-stimulated platelets. Thus, the molecular mechanism of action of thrombin and WGA could be different. To explore this idea, /sup 32/P/sub i/-labeled platelets were stimulated with WGA and the activation arrested with N-acetyl-glucosamine at different times. Two-dimensional gel electrophoresis of total protein at 5s showed only two phosphorylated species of 47K protein. At 60s, maximally four phosphorylated species were noted. In contrast, with thrombin using the same technique, seven to nine phosphorylated components have been reported. These results suggest that the different activators of platelets may act by different mechanisms. In addition, activation of platelets may require only limited levels of phosphorylation of the 47K polypeptide.

  20. Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation.

    PubMed

    Yamada, Aya; Futagi, Masaharu; Fukumoto, Emiko; Saito, Kan; Yoshizaki, Keigo; Ishikawa, Masaki; Arakaki, Makiko; Hino, Ryoko; Sugawara, Yu; Ishikawa, Momoko; Naruse, Masahiro; Miyazaki, Kanako; Nakamura, Takashi; Fukumoto, Satoshi

    2016-01-08

    Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43(-/-) salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43(-/-) samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43(-/-) phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

  1. Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation*

    PubMed Central

    Yamada, Aya; Futagi, Masaharu; Fukumoto, Emiko; Saito, Kan; Yoshizaki, Keigo; Ishikawa, Masaki; Arakaki, Makiko; Hino, Ryoko; Sugawara, Yu; Ishikawa, Momoko; Naruse, Masahiro; Miyazaki, Kanako; Nakamura, Takashi; Fukumoto, Satoshi

    2016-01-01

    Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis. PMID:26565022

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

    PubMed Central

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

    2016-01-01

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

  3. Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues. [Zea mays

    SciTech Connect

    Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

    1990-11-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of ({sup 32}P) ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of {sup 32}P incorporation and the electrophoretic patterns were dependent on {sup 32}P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K{sub m} values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins.

  4. The CREB coactivator CRTC2 controls hepatic lipid metabolism by regulating SREBP1.

    PubMed

    Han, Jinbo; Li, Erwei; Chen, Liqun; Zhang, Yuanyuan; Wei, Fangchao; Liu, Jieyuan; Deng, Haiteng; Wang, Yiguo

    2015-08-13

    Abnormal accumulation of triglycerides in the liver, caused in part by increased de novo lipogenesis, results in non-alcoholic fatty liver disease and insulin resistance. Sterol regulatory element-binding protein 1 (SREBP1), an important transcriptional regulator of lipogenesis, is synthesized as an inactive precursor that binds to the endoplasmic reticulum (ER). In response to insulin signalling, SREBP1 is transported from the ER to the Golgi in a COPII-dependent manner, processed by proteases in the Golgi, and then shuttled to the nucleus to induce lipogenic gene expression; however, the mechanisms underlying enhanced SREBP1 activity in insulin-resistant obesity and diabetes remain unclear. Here we show in mice that CREB regulated transcription coactivator 2 (CRTC2) functions as a mediator of mTOR signalling to modulate COPII-dependent SREBP1 processing. CRTC2 competes with Sec23A, a subunit of the COPII complex, to interact with Sec31A, another COPII subunit, thus disrupting SREBP1 transport. During feeding, mTOR phosphorylates CRTC2 and attenuates its inhibitory effect on COPII-dependent SREBP1 maturation. As hepatic overexpression of an mTOR-defective CRTC2 mutant in obese mice improved the lipogenic program and insulin sensitivity, these results demonstrate how the transcriptional coactivator CRTC2 regulates mTOR-mediated lipid homeostasis in the fed state and in obesity.

  5. Interleukin-18 directly protects cortical neurons by activating PI3K/AKT/NF-κB/CREB pathways.

    PubMed

    Zhou, Jia; Ping, Feng-feng; Lv, Wen-ting; Feng, Jun-yi; Shang, Jing

    2014-09-01

    Interleukin-18 (IL-18), a member of the IL-1 family of cytokines, was initially identified as an interferon (IFN)-γ-inducing factor. IL-18 is expressed in both immune and non-immune cells and participates in the adjustment of multitude cellular functions. Nonetheless, the effects of IL-18 on cortical neurons have not been explored. The present study was conducted to investigate the influence of IL-18 on rat primary cortical neurons and elucidate the underlying mechanisms. We proved that rrIL-18 increased the brain-derived neurotrophic factor (BDNF) expression in a time-dependent manner. Treatment with rrIL-18 (50 ng/ml) deactivated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by facilitating its phosphorylation, enhanced the expression of Phosphoinositide 3-OH kinase (PI3K) and p-Akt, standing for the activation of the PI3K/Akt pathway. As its pivotal downstream pathways, nuclear factor-kappa B (NF-κB), cAMP-responsive element binding protein (CREB)/Bcl-2 and glycogen synthase kinase-3β (GSK-3β) were examined in further steps. Our data revealed that rrIL-18 stimulated NF-κB activation, improved p-CREB and anti-apoptotic Bcl-2 expression levels. But rrIL-18 had little or no effect on GSK-3β pathway. Besides, rrIL-18 increased levels of BDNF and Bcl-2/Bax ratio and decreased cleaved caspase-3 expression to protect cortical neurons from damage induced by oxygen-glucose deprivation (OGD). These results in vitro showed the protection of IL-18 on cortical neurons. And this direct neuroprotective effect of IL-18 is crippled by PI3K inhibitor wortmannin.

  6. CREB is a key negative regulator of carbonic anhydrase IX (CA9) in gastric cancer.

    PubMed

    Wang, Guanqiao; Cheng, Zhenguo; Liu, Funan; Zhang, Hongyan; Li, Jiabin; Li, Feng

    2015-07-01

    Carbonic anhydrase IX(CA9)is a member of the carbonic anhydrase family that catalyzes the reversible hydration of carbon dioxide, and plays a key role in the regulation of pH. Although a large number of studies have shown that CA9 is strongly up-regulated by HIF1-α, little is known about the negative regulation mechanism of CA9 in cancer cells. Here we find that CREB is a key negative regulator of CA9 in gastric cancer. Over-expression of CREB can significantly repress the expression of CA9. Treating with anisomycin (ANS), an activator of p38, the phosphorylation and nuclear translocation of CREB are both promoted, while the transcription of CA9 is repressed. Besides, our results firstly identify that CREB can recruit SIRT1 (class III HDACS) by adaptor protein p300, then repress the expression of CA9. These findings may contribute to understand the negative regulation mechanisms of CA9 in gastric cancer.

  7. MYB75 Phosphorylation by MPK4 Is Required for Light-Induced Anthocyanin Accumulation in Arabidopsis[OPEN

    PubMed Central

    Li, Shengnan; Wang, Wenyi; Gao, Jinlan; Yin, Kangquan; Wang, Rui; Wang, Chengcheng; Mundy, John

    2016-01-01

    Light is a major environmental cue affecting various physiological and metabolic processes in plants. Although plant photoreceptors are well characterized, the mechanisms by which light regulates downstream responses are less clear. In Arabidopsis thaliana, the accumulation of photoprotective anthocyanin pigments is light dependent, and the R2R3 MYB transcription factor MYB75/PAP1 regulates anthocyanin accumulation. Here, we report that MYB75 interacts with and is phosphorylated by MAP KINASE4 (MPK4). Their interaction is dependent on MPK4 kinase activity and is required for full function of MYB75. MPK4 can be activated in response to light and is involved in the light-induced accumulation of anthocyanins. We show that MPK4 phosphorylation of MYB75 increases its stability and is essential for light-induced anthocyanin accumulation. Our findings reveal an important role for a MAPK pathway in light signal transduction. PMID:27811015

  8. A Comprehensive Proteomic Survey of ABA-Induced Protein Phosphorylation in Rice (Oryza sativa L.)

    PubMed Central

    Qiu, Jiehua; Hou, Yuxuan; Wang, Yifeng; Li, Zhiyong; Zhao, Juan; Tong, Xiaohong; Lin, Haiyan; Wei, Xiangjin; Ao, Hejun; Zhang, Jian

    2017-01-01

    abscisic acid (ABA) is a key phytohormone regulating plant development and stress response. The signal transduction of ABA largely relies on protein phosphorylation. However; little is known about the phosphorylation events occurring during ABA signaling in rice thus far. By employing a label-free; MS (Mass Spectrometry)-based phosphoproteomic approach; we identified 2271 phosphosites of young rice seedlings and their intensity dynamics in response to ABA; during which 1060 proteins were found to be differentially phosphorylated. Western-blot analysis verified the differential phosphorylation pattern of D1, SMG1 and SAPK9 as indicated by the MS result; suggesting the high reliability of our phosphoproteomic data. The DP (differentially phosphorylated) proteins are extensively involved in ABA as well as other hormone signaling pathways. It is suggested that ABA antagonistically regulates brassinosteroid (BR) signaling via inhibiting BR receptor activity. The result of this study not only expanded our knowledge of rice phosphoproteome, but also shed more light on the pattern of protein phosphorylation in ABA signaling. PMID:28054942

  9. Protein Kinase D1 (PKD1) Phosphorylation Promotes Dopaminergic Neuronal Survival during 6-OHDA-Induced Oxidative Stress

    PubMed Central

    Asaithambi, Arunkumar; Ay, Muhammet; Jin, Huajun; Gosh, Anamitra; Anantharam, Vellareddy; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2014-01-01

    Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1WT) or constitutively active PKD1 (PKD1S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD. PMID:24806360

  10. Brevetoxin-induced phosphorylation of Pyk2 and Src in murine neocortical neurons involves distinct signaling pathways

    PubMed Central

    Cao, Zhengyu; George, Joju; Baden, Daniel G.; Murray, Thomas F.

    2009-01-01

    Brevetoxins (PbTx-1 to PbTx-10) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis. Brevetoxins bind to site 5 of the α-subunit of voltage-gated sodium channels (VGSCs) and augment Na+ influx. In neocortical neurons brevetoxins elevate intracellular Ca2+ and augment NMDA receptor signaling. In this study, we explored the effects of PbTx-2 on Pyk2 and Src activation in neocortical neurons. We found that both Pyk2 and Src were activated following PbTx-2 exposure. PbTx-2-induced Pyk2 Tyr402 phosphorylation was dependent on elevation of Ca2+ influx through NMDA receptors. Moreover, Pyk2 Tyr402 phosphorylation was also found to require PKC activation inasmuch as RO-31-8425 and GF 109203x both attenuated the response. In contrast, PbTx-2-induced Src Tyr416 phosphorylation involved a Gq-coupled receptor inasmuch as U73122, a specific PLC inhibitor, abolished the response. This Gq-coupled receptor appears to be mGluR 5. The PKCδ inhibitor rottlerin abolished PbTx-2-induced Src activation demonstrating that this isoform of PKC is involved in the activation of Src by PbTx-2. Considered together these data suggest that although activation of neuronal Pyk2 and Src result from PbTx-2 stimulation of VGSC, engagement of these two non-receptor tyrosine kinases involves distinct signaling pathways. PMID:17963734

  11. Acute Footshock Stress Induces Time-Dependent Modifications of AMPA/NMDA Protein Expression and AMPA Phosphorylation.

    PubMed

    Bonini, Daniela; Mora, Cristina; Tornese, Paolo; Sala, Nathalie; Filippini, Alice; La Via, Luca; Milanese, Marco; Calza, Stefano; Bonanno, Gianbattista; Racagni, Giorgio; Gennarelli, Massimo; Popoli, Maurizio; Musazzi, Laura; Barbon, Alessandro

    2016-01-01

    Clinical studies on patients with stress-related neuropsychiatric disorders reported functional and morphological changes in brain areas where glutamatergic transmission is predominant, including frontal and prefrontal areas. In line with this evidence, several preclinical works suggest that glutamate receptors are targets of both rapid and long-lasting effects of stress. Here we found that acute footshock- (FS-) stress, although inducing no transcriptional and RNA editing alterations of ionotropic AMPA and NMDA glutamate receptor subunits, rapidly and transiently modulates their protein expression, phosphorylation, and localization at postsynaptic spines in prefrontal and frontal cortex. In total extract, FS-stress increased the phosphorylation levels of GluA1 AMPA subunit at Ser(845) immediately after stress and of GluA2 Ser(880) 2 h after start of stress. At postsynaptic spines, stress induced a rapid decrease of GluA2 expression, together with an increase of its phosphorylation at Ser(880), suggesting internalization of GluA2 AMPA containing receptors. GluN1 and GluN2A NMDA receptor subunits were found markedly upregulated in postsynaptic spines, 2 h after start of stress. These results suggest selected time-dependent changes in glutamatergic receptor subunits induced by acute stress, which may suggest early and transient enhancement of AMPA-mediated currents, followed by a transient activation of NMDA receptors.

  12. HDAC Inhibitor-Mediated Beta-Cell Protection Against Cytokine-Induced Toxicity Is STAT1 Tyr701 Phosphorylation Independent

    PubMed Central

    Dahllöf, Mattias S.; Christensen, Dan P.; Harving, Mette; Wagner, Bridget K.; Mandrup-Poulsen, Thomas

    2015-01-01

    Histone deacetylase (HDAC) inhibition protects pancreatic beta-cells against apoptosis induced by the combination of the proinflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ. Decreased expression of cell damage-related genes is observed on the transcriptional level upon HDAC inhibition using either IL-1β or IFN-γ alone. Whereas HDAC inhibition has been shown to regulate NFκB-activity, related primarily to IL-1β signaling, it is unknown whether the inhibition of HDACs affect IFN-γ signaling in beta-cells. Further, in non-beta-cells, there is a dispute whether HDAC inhibition regulates IFN-γ signaling at the level of STAT1 Tyr701 phosphorylation. Using different small molecule HDAC inhibitors with varying class selectivity, INS-1E wild type and stable HDAC1-3 knockdown pancreatic INS-1 cell lines, we show that IFN-γ-induced Cxcl9 and iNos expression as well as Cxcl9 and GAS reporter activity were decreased by HDAC inhibition in a STAT1 Tyr701 phosphorylation-independent fashion. In fact, knockdown of HDAC1 increased IFN-γ-induced STAT1 phosphorylation. PMID:25062500

  13. Acute Footshock Stress Induces Time-Dependent Modifications of AMPA/NMDA Protein Expression and AMPA Phosphorylation

    PubMed Central

    Bonini, Daniela; Mora, Cristina; Tornese, Paolo; Sala, Nathalie; Filippini, Alice; La Via, Luca; Milanese, Marco; Calza, Stefano; Bonanno, Gianbattista; Racagni, Giorgio; Gennarelli, Massimo; Popoli, Maurizio; Musazzi, Laura; Barbon, Alessandro

    2016-01-01

    Clinical studies on patients with stress-related neuropsychiatric disorders reported functional and morphological changes in brain areas where glutamatergic transmission is predominant, including frontal and prefrontal areas. In line with this evidence, several preclinical works suggest that glutamate receptors are targets of both rapid and long-lasting effects of stress. Here we found that acute footshock- (FS-) stress, although inducing no transcriptional and RNA editing alterations of ionotropic AMPA and NMDA glutamate receptor subunits, rapidly and transiently modulates their protein expression, phosphorylation, and localization at postsynaptic spines in prefrontal and frontal cortex. In total extract, FS-stress increased the phosphorylation levels of GluA1 AMPA subunit at Ser845 immediately after stress and of GluA2 Ser880 2 h after start of stress. At postsynaptic spines, stress induced a rapid decrease of GluA2 expression, together with an increase of its phosphorylation at Ser880, suggesting internalization of GluA2 AMPA containing receptors. GluN1 and GluN2A NMDA receptor subunits were found markedly upregulated in postsynaptic spines, 2 h after start of stress. These results suggest selected time-dependent changes in glutamatergic receptor subunits induced by acute stress, which may suggest early and transient enhancement of AMPA-mediated currents, followed by a transient activation of NMDA receptors. PMID:26966584

  14. AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

    PubMed Central

    Johanns, M.; Lai, Y.-C.; Hsu, M.-F.; Jacobs, R.; Vertommen, D.; Van Sande, J.; Dumont, J. E.; Woods, A.; Carling, D.; Hue, L.; Viollet, B.; Foretz, M; Rider, M H

    2016-01-01

    Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation. PMID:26952277

  15. Spermidine-induced improvement of memory involves a cross-talk between protein kinases C and A.

    PubMed

    Guerra, Gustavo P; Mello, Carlos F; Bochi, Guilherme V; Pazini, Andréia M; Rosa, Michelle M; Ferreira, Juliano; Rubin, Maribel A

    2012-07-01

    Spermidine (SPD) is an endogenous aliphatic amine with polycationic structure that modulates NMDA receptor activity and improves memory. Recent evidence suggests that cAMP-dependent protein kinase (PKA) and cAMP response element-binding protein (CREB) play a role in SPD-induced improvement of memory. In the current study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in SPD-induced facilitation of memory of inhibitory avoidance task in adult rats. The post-training administration of the PKC inhibitor, 3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride [GF 109203X, 2.5 ρmol, intrahippocampal (ih)] with SPD (0.2 nmol, ih) prevented memory improvement induced by SPD. Intrahippocampal administration of SPD (0.2 nmol) facilitated PKC phosphorylation in the hippocampus, 30 min after administration. GF 109203X prevented not only the stimulatory effect of SPD on PKC but also PKA and CREB phosphorylation. These results suggest that memory enhancement induced by the ih administration of SPD involves the cross-talk between PKC and PKA/CREB, with sequential activation of PKC and PKA/CREB pathways, in rats.

  16. Involvement of AMPA/Kainate Glutamate Receptor in the Extinction and Reinstatement of Morphine-Induced Conditioned Place Preference: A Behavioral and Molecular Study.

    PubMed

    Siahposht-Khachaki, Ali; Fatahi, Zahra; Yans, Asal; Khodagholi, Fariba; Haghparast, Abbas

    2017-03-01

    Glutamate receptors in mesolimbic areas such as the nucleus accumbens, ventral tegmental area, prefrontal cortex (PFC), and hippocampus (HIP) are a component of the mechanisms of drug-induced reward and can modulate the firing pattern of dopaminergic neurons in the reward system. In addition, several lines of study have indicated that cAMP response element-binding protein (CREB) and c-fos have important role in morphine-induced conditioned place preference (CPP) induced by drugs of abuse, such as morphine, cocaine, nicotine, and alcohol. Therefore, in the present study, we investigated the changes in phosphorylated CREB (p-CREB) and c-fos induction within the nucleus accumbens (NAc), HIP, and PFC after intracerebroventricular (ICV) administration of different doses of CNQX or vehicle during extinction period or reinstatement of morphine-induced CPP. In all groups, the CPP procedure was done; afterward, the conditioning scores were recorded by Ethovision software. After behavioral test recording, we dissected out the NAc, HIP, and PFC regions and measured the p-CREB/CREB ratio and c-fos level by Western blot analysis. Our results showed that administration of CNQX significantly shortened the extinction of morphine CPP. Besides, ICV microinjection of CNQX following extinction period decreased the reinstatement of morphine CPP in extinguished rats. In molecular section, in treatment group, all mentioned factors were dose-dependently decreased in comparison with vehicle group (DMSO) after ICV microinjection of different doses of CNQX but not in pre-extinction microinjection. These findings suggested that antagonism of AMPA receptor decreased p-CREB/CREB ratio and c-fos level in the PFC, NAc, and HIP. Modulation of the drug memory reconsolidation may be useful for faster extinction of drug-induced reward and attenuation of drug-seeking behavior.

  17. Effects of curcumin (Curcuma longa) on learning and spatial memory as well as cell proliferation and neuroblast differentiation in adult and aged mice by upregulating brain-derived neurotrophic factor and CREB signaling.

    PubMed

    Nam, Sung Min; Choi, Jung Hoon; Yoo, Dae Young; Kim, Woosuk; Jung, Hyo Young; Kim, Jong Whi; Yoo, Miyoung; Lee, Sanghee; Kim, Chul Jung; Yoon, Yeo Sung; Hwang, In Koo

    2014-06-01

    Aging is a progressive process, and it may lead to the initiation of neurological diseases. In this study, we investigated the effects of wild Indian Curcuma longa using a Morris water maze paradigm on learning and spatial memory in adult and D-galactose-induced aged mice. In addition, the effects on cell proliferation and neuroblast differentiation were assessed by immunohistochemistry for Ki67 and doublecortin (DCX) respectively. The aging model in mice was induced through the subcutaneous administration of D-galactose (100 mg/kg) for 10 weeks. C. longa (300 mg/kg) or its vehicle (physiological saline) was administered orally to adult and D-galactose-treated mice for the last three weeks before sacrifice. The administration of C. longa significantly shortened the escape latency in both adult and D-galactose-induced aged mice and significantly ameliorated D-galactose-induced reduction of cell proliferation and neuroblast differentiation in the subgranular zone of hippocampal dentate gyrus. In addition, the administration of C. longa significantly increased the levels of phosphorylated CREB and brain-derived neurotrophic factor in the subgranular zone of dentate gyrus. These results indicate that C. longa mitigates D-galactose-induced cognitive impairment, associated with decreased cell proliferation and neuroblast differentiation, by activating CREB signaling in the hippocampal dentate gyrus.

  18. Spinal serum-inducible and glucocorticoid-inducible kinase 1 mediates neuropathic pain via kalirin and downstream PSD-95-dependent NR2B phosphorylation in rats.

    PubMed

    Peng, Hsien-Yu; Chen, Gin-Den; Lai, Cheng-Yuan; Hsieh, Ming-Chun; Lin, Tzer-Bin

    2013-03-20

    The coupling of the spinal postsynaptic density-95 (PSD-95) with the glutamatergic N-methyl-d-aspartate receptor NR2B subunit and the subsequent NR2B phosphorylation contribute to pain-related plasticity. Increasing evidence reveals that kalirin, a Rho-guanine nucleotide exchange factor, modulates PSD-95-NR2B-dependent neuroplasticity. Our laboratory recently demonstrated that serum-inducible and glucocorticoid-inducible kinase 1 (SGK1) participates in inflammation-associated pain hypersensitivity by modulating spinal glutamatergic neurotransmission. Because kalirin is one of the proteins in PSD that is highly phosphorylated by various kinases, we tested whether kalirin could be a downstream target of spinal SGK1 that participates in neuropathic pain development via regulation of the PSD-95-NR2B coupling-dependent phosphorylation of NR2B. We observed that spinal nerve ligation (SNL, L5) in male Sprague Dawley rats resulted in behavioral allodynia, which was associated with phosphorylated SGK1 (pSGK1), kalirin, and phosphorylated NR2B (pNR2B) expression and an increase in pSGK1-kalirin-PSD-95-pNR2B coprecipitation in the ipsilateral dorsal horn (L4-L5). SNL-enhanced kalirin immunofluorescence was coincident with pSGK1, PSD-95, and pNR2B immunoreactivity. Small-interfering RNA (siRNA) that targeted spinal kalirin mRNA expression (10 μg, 10 μl; i.t.) reduced SNL-induced allodynia, kalirin and pNR2B expression, as well as kalirin-PSD-95 and PSD-95-pNR2B coupling and costaining without affecting SGK1 phosphorylation. Daily administration of GSK-650394 (an SGK1 antagonist; 100 nm, 10 μl, i.t.) not only exhibited effects similar to the kalirin mRNA-targeting siRNA but also attenuated pSGK1-kalirin costaining and SGK1-kalirin coupling. We suggest that nerve injury could induce spinal SGK1 phosphorylation that subsequently interacts with and upregulates kalirin to participate in neuropathic pain development via PSD-95-NR2B coupling-dependent NR2B phosphorylation.

  19. CREB controls hepatic lipid metabolism through nuclear hormone receptor PPAR-gamma.

    PubMed

    Herzig, Stephan; Hedrick, Susan; Morantte, Ianessa; Koo, Seung-Hoi; Galimi, Francesco; Montminy, Marc

    2003-11-13

    Fasting triggers a series of hormonal cues that promote energy balance by inducing glucose output and lipid breakdown in the liver. In response to pancreatic glucagon and adrenal cortisol, the cAMP-responsive transcription factor CREB activates gluconeogenic and fatty acid oxidation programmes by stimulating expression of the nuclear hormone receptor coactivator PGC-1 (refs 2-5). In parallel, fasting also suppresses lipid storage and synthesis (lipogenic) pathways, but the underlying mechanism is unknown. Here we show that mice deficient in CREB activity have a fatty liver phenotype and display elevated expression of the nuclear hormone receptor PPAR-gamma, a key regulator of lipogenic genes. CREB inhibits hepatic PPAR-gamma expression in the fasted state by stimulating the expression of the Hairy Enhancer of Split (HES-1) gene, a transcriptional repressor that is shown here to be a mediator of fasting lipid metabolism in vivo. The coordinate induction of PGC-1 and repression of PPAR-gamma by CREB during fasting provides a molecular rationale for the antagonism between insulin and counter-regulatory hormones, and indicates a potential role for CREB antagonists as therapeutic agents in enhancing insulin sensitivity in the liver.

  20. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

    PubMed

    Takayama, S; White, M F; Kahn, C R

    1988-03-05

    The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

  1. Alpha particles induce pan-nuclear phosphorylation of H2AX in primary human lymphocytes mediated through ATM.

    PubMed

    Horn, Simon; Brady, Darren; Prise, Kevin

    2015-10-01

    The use of high linear energy transfer radiations in the form of carbon ions in heavy ion beam lines or alpha particles in new radionuclide treatments has increased substantially over the past decade and will continue to do so due to the favourable dose distributions they can offer versus conventional therapies. Previously it has been shown that exposure to heavy ions induces pan-nuclear phosphorylation of several DNA repair proteins such as H2AX and ATM in vitro. Here we describe similar effects of alpha particles on ex vivo irradiated primary human peripheral blood lymphocytes. Following alpha particle irradiation pan-nuclear phosphorylation of H2AX and ATM, but not DNA-PK and 53BP1, was observed throughout the nucleus. Inhibition of ATM, but not DNA-PK, resulted in the loss of pan-nuclear phosphorylation of H2AX in alpha particle irradiated lymphocytes. Pan-nuclear gamma-H2AX signal was rapidly lost over 24h at a much greater rate than foci loss. Surprisingly, pan-nuclear gamma-H2AX intensity was not dependent on the number of alpha particle induced double strand breaks, rather the number of alpha particles which had traversed the cell nucleus. This distinct fluence dependent damage signature of particle radiation is important in both the fields of radioprotection and clinical oncology in determining radionuclide biological dosimetry and may be indicative of patient response to new radionuclide cancer therapies.

  2. Phosphorylation of Ser8 promotes zinc-induced dimerization of the amyloid-β metal-binding domain.

    PubMed

    Kulikova, Alexandra A; Tsvetkov, Philipp O; Indeykina, Maria I; Popov, Igor A; Zhokhov, Sergey S; Golovin, Andrey V; Polshakov, Vladimir I; Kozin, Sergey A; Nudler, Evgeny; Makarov, Alexander A

    2014-10-01

    Zinc-induced aggregation of the amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease (AD). Recently it was shown that phosphorylation of Aβ at Ser8 promotes the formation of toxic aggregates. In this work, we have studied the impact of Ser8 phosphorylation on the mode of zinc interaction with the Aβ metal-binding domain 1-16 using isothermal titration calorimetry, electrospray ionization mass spectrometry and NMR spectroscopy. We have discovered a novel zinc binding site ((6)HDpS(8)) in the phosphorylated peptide, in which the zinc ion is coordinated by the imidazole ring of His6, the phosphate group attached to Ser8 and a backbone carbonyl group of His6 or Asp7. Interaction of the zinc ion with this site involves His6, thereby withdrawing it from the interaction pattern observed in the non-modified peptide. This event was found to stimulate dimerization of peptide chains through the (11)EVHH(14) site, where the zinc ion is coordinated by the two pairs of Glu11 and His14 in the two peptide subunits. The proposed molecular mechanism of zinc-induced dimerization could contribute to the understanding of initiation of pathological Aβ aggregation, and the (11)EVHH(14) tetrapeptide can be considered as a promising drug target for the prevention of amyloidogenesis.

  3. Accumulation of Phosphorylated β-Catenin Enhances ROS-Induced Cell Death in Presenilin-Deficient Cells

    PubMed Central

    Boo, Jung H.; Song, Hyundong; Kim, Ji E.; Kang, David E.; Mook-Jung, Inhee

    2009-01-01

    Presenilin (PS) is involved in many cellular events under physiological and pathological conditions. Previous reports have revealed that PS deficiency results in hyperproliferation and resistance to apoptotic cell death. In the present study, we investigated the effects of PS on β-catenin and cell mortality during serum deprivation. Under these conditions, PS1/PS2 double-knockout MEFs showed aberrant accumulation of phospho-β-catenin, higher ROS generation, and notable cell death. Inhibition of β-catenin phosphorylation by LiCl reversed ROS generation and cell death in PS deficient cells. In addition, the K19/49R mutant form of β-catenin, which undergoes normal phosphorylation but not ubiquitination, induced cytotoxicity, while the phosphorylation deficient S37A β-catenin mutant failed to induce cytotoxicity. These results indicate that aberrant accumulation of phospho-β-catenin underlies ROS-mediated cell death in the absence of PS. We propose that the regulation of β-catenin is useful for identifying therapeutic targets of hyperproliferative diseases and other degenerative conditions. PMID:19137062

  4. Ethanol Exposure Induces Neonatal Neurodegeneration by Enhancing CB1R Exon1 Histone H4K8 Acetylation and Up-regulating CB1R Function causing Neurobehavioral Abnormalities in Adult Mice

    PubMed Central

    Subbanna, Shivakumar; Nagre, Nagaraja N.; Umapathy, Nagavedi S.; Pace, Betty S.

    2015-01-01

    Background: Ethanol exposure to rodents during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces long-term potentiation and memory deficits. However, the molecular mechanisms underlying these deficits are still poorly understood. Methods: In the present study, we explored the potential role of epigenetic changes at cannabinoid type 1 (CB1R) exon1 and additional CB1R functions, which could promote memory deficits in animal models of fetal alcohol spectrum disorder. Results: We found that ethanol treatment of P7 mice enhances acetylation of H4 on lysine 8 (H4K8ace) at CB1R exon1, CB1R binding as well as the CB1R agonist-stimulated GTPγS binding in the hippocampus and neocortex, two brain regions that are vulnerable to ethanol at P7 and are important for memory formation and storage, respectively. We also found that ethanol inhibits cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation and activity-regulated cytoskeleton-associated protein (Arc) expression in neonatal and adult mice. The blockade or genetic deletion of CB1Rs prior to ethanol treatment at P7 rescued CREB phosphorylation and Arc expression. CB1R knockout mice exhibited neither ethanol-induced neurodegeneration nor inhibition of CREB phosphorylation or Arc expression. However, both neonatal and adult mice did exhibit enhanced CREB phosphorylation and Arc protein expression. P7 ethanol-treated adult mice exhibited impaired spatial and social recognition memory, which were prevented by the pharmacological blockade or deletion of CB1Rs at P7. Conclusions: Together, these findings suggest that P7 ethanol treatment induces CB1R expression through epigenetic modification of the CB1R gene, and that the enhanced CB1R function induces pCREB, Arc, spatial, and social memory deficits in adult mice. PMID:25609594

  5. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induces initiation factor 2 alpha phosphorylation and translation inhibition in PC12 cells.

    PubMed

    Muñoz, F; Martín, M E; Salinas, M; Fando, J L

    2001-03-09

    We have investigated the effect of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on protein synthesis rate and initiation factor 2 (eIF2) phosphorylation in PC12 cells differentiated with nerve growth factor. FCCP treatment induced a very rapid 2-fold increase in intracellular Ca(2+) concentration that was accompanied by a strong protein synthesis rate inhibition (68%). The translation inhibition correlated with an increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) (25% vs. 7%, for FCCP-treated and control cells, respectively) and a 1.7-fold increase in the double-stranded RNA-dependent protein kinase activity. No changes in the PKR endoplasmic reticulum-related kinase or eIF2 alpha phosphatase were found. Translational regulation may play a significant role in the process triggered by mitochondrial calcium mobilization.

  6. Proatherogenic Abnormalities Of Lipid Metabolism In SirT1 Transgenic Mice Are Mediated Through Creb Deacetylation

    PubMed Central

    Qiang, Li; Lin, Hua V.; Kim-Muller, Ja Young; Welch, Carrie L.; Gu, Wei; Accili, Domenico

    2011-01-01

    SUMMARY Dyslipidemia and atherosclerosis are associated with reduced insulin sensitivity and diabetes, but the mechanism is unclear. Gain-of-function of the gene encoding deacetylase SirT1 improves insulin sensitivity, and could be expected to protect against lipid abnormalities. Surprisingly, when transgenic mice overexpressing SirT1 (SirBACO) are placed on atherogenic diet, they maintain better glucose homeostasis, but develop worse lipid profiles and larger atherosclerotic lesions than controls. We show that transcription factor cAMP response element binding protein (Creb) is deacetylated in SirBACO mice. We identify Lys136 is a substrate for SirT1-dependent deacetylation that affects Creb activity by preventing its cAMP-dependent phosphorylation, leading to reduced expression of glucogenic genes, and promoting hepatic lipid accumulation and secretion. Expression of constitutively acetylated Creb (K136Q) in SirBACO mice mimics Creb activation and abolishes the dyslipidemic and insulin-sensitizing effects of SirT1 gain-of-function. We propose that SirT1-dependent Creb deacetylation regulates the balance between glucose and lipid metabolism, integrating fasting signals. PMID:22078933

  7. cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.

    PubMed

    Ahn, J I; Yoo, J-Y; Kim, T H; Kim, Y I; Ferguson, S D; Fazleabas, A T; Young, S L; Lessey, B A; Ahn, J Y; Lim, J M; Jeong, J-W

    2016-01-01

    Endometriosis is a major cause of infertility and pelvic pain, affecting more than 10% of reproductive-aged women. Progesterone resistance has been observed in the endometrium of women with this disease, as evidenced by alterations in progesterone-responsive gene and protein expression. cAMPResponse Element-Binding 3-like protein 1 (Creb3l1) has previously been identified as a progesterone receptor (PR) target gene in mouse uterus via high density DNA microarray analysis. However, CREB3L1 function has not been studied in the context of endometriosis and uterine biology. In this study, we validated progesterone (P4) regulation of Creb3l1 in the uteri of wild-type and progesterone receptor knockout (PRKO) mice. Furthermore, we observed that CREB3L1 expression was significantly higher in secretory phase human endometrium compared to proliferative phase and that CREB3L1 expression was significantly decreased in the endometrium of women with endometriosis. Lastly, by transfecting CREB3L1 siRNA into cultured human endometrial stromal cells (hESCs) prior to hormonal induction of in vitro decidualization, we showed that CREB3L1 is required for the decidualization process. Interestingly, phosphorylation of ERK1/2, critical factor for decidualization, was also significantly reduced in CREB3L1-silenced hESCs. It is known that hESCs from patients with endometriosis show impaired decidualization and that dysregulation of the P4-PR signaling axis is linked to a variety of endometrial diseases including infertility and endometriosis. Therefore, these results suggest that CREB3L1 is required for decidualization in mice and humans and may be linked to the pathogenesis of endometriosis in a P4-dependent manner.

  8. Parallel activation of Ca(2+)-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress.

    PubMed

    Chiong, M; Parra, V; Eisner, V; Ibarra, C; Maldonado, C; Criollo, A; Bravo, R; Quiroga, C; Contreras, A; Vicencio, J M; Cea, P; Bucarey, J L; Molgó, J; Jaimovich, E; Hidalgo, C; Kroemer, G; Lavandero, S

    2010-08-01

    Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways.

  9. The Med1 Subunit of the Mediator Complex Induces Liver Cell Proliferation and Is Phosphorylated by AMP Kinase*

    PubMed Central

    Viswakarma, Navin; Jia, Yuzhi; Bai, Liang; Gao, Qian; Lin, Bingliang; Zhang, Xiaohong; Misra, Parimal; Rana, Ajay; Jain, Sanjay; Gonzalez, Frank J.; Zhu, Yi-Jun; Thimmapaya, Bayar; Reddy, Janardan K.

    2013-01-01

    Mediator, a large multisubunit protein complex, plays a pivotal role in gene transcription by linking gene-specific transcription factors with the preinitiation complex and RNA polymerase II. In the liver, the key subunit of the Mediator complex, Med1, interacts with several nuclear receptors and transcription factors to direct gene-specific transcription. Conditional knock-out of Med1 in the liver showed that hepatocytes lacking Med1 did not regenerate following either partial hepatectomy or treatment with certain nuclear receptor activators and failed to give rise to tumors when challenged with carcinogens. We now report that the adenovirally driven overexpression of Med1 in mouse liver stimulates hepatocyte DNA synthesis with enhanced expression of DNA replication, cell cycle control, and liver-specific genes, indicating that Med1 alone is necessary and sufficient for liver cell proliferation. Importantly, we demonstrate that AMP-activated protein kinase (AMPK), an important cellular energy sensor, interacts with, and directly phosphorylates, Med1 in vitro at serine 656, serine 756, and serine 796. AMPK also phosphorylates Med1 in vivo in mouse liver and in cultured primary hepatocytes and HEK293 and HeLa cells. In addition, we demonstrate that PPARα activators increase AMPK-mediated Med1 phosphorylation in vivo. Inhibition of AMPK by compound C decreased hepatocyte proliferation induced by Med1 and also by the PPARα activators fenofibrate and Wy-14,643. Co-treatment with compound C attenuated PPARα activator-inducible fatty acid β-oxidation in liver. Our results suggest that Med1 phosphorylation by its association with AMPK regulates liver cell proliferation and fatty acid oxidation, most likely as a downstream effector of PPARα and AMPK. PMID:23943624

  10. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

    PubMed

    Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L; Dephoure, Noah; Ballif, Bryan A; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M; Xavier, Ramnik J; Gygi, Steven P; Elledge, Stephen J

    2016-06-28

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast.

  11. Identification of okadaic acid-induced phosphorylation events by a mass spectrometry approach

    SciTech Connect

    Hill, Jennifer J. . E-mail: Jennifer.Hill@nrc.gc.ca; Callaghan, Deborah A.; Ding Wen; Kelly, John F.; Chakravarthy, Balu R.

    2006-04-14

    Okadaic acid (OA) is a widely used small-molecule phosphatase inhibitor that is thought to selectively inhibit protein phosphatase 2A (PP2A). Multiple studies have demonstrated that PP2A activity is compromised in Brains of Alzheimer's disease patients. Thus, we set out to determine changes in phosphorylation that occur upon OA treatment of neuronal cells. Utilizing isotope-coded affinity tags and mass spectrometry analysis, we determined the relative abundance of proteins in a phosphoprotein enriched fraction from control and OA-treated primary cortical neurons. We identified many proteins whose phosphorylation state is regulated by OA, including glycogen synthase kinase 3{beta}, collapsin-response mediator proteins (DRP-2, DPYSL-5, and CRMP-4), and the B subunit of PP2A itself. Most interestingly, we have found that complexin 2, an important regulator of neurotransmitter release and synaptic plasticity, is phosphorylated at serine 93 upon OA treatment of neurons. This is First report of a phosphorylation site on complexin 2.

  12. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks

    PubMed Central

    Zhou, Chunshui; Elia, Andrew E. H.; Naylor, Maria L.; Ballif, Bryan A.; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M.; Xavier, Ramnik J.; Gygi, Steven P.; Elledge, Stephen J.

    2016-01-01

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast. PMID:27298372

  13. The Role of VDR in Phosphorylation in Vitamin D-Induced Apoptosis

    DTIC Science & Technology

    2001-07-01

    Terpening , C.A. Haussler, M.R. Haussler and G.K. Whitfield (1993) Phosphorylation of serine 208 in the human vitamin D receptor. The predominant amino acid...fragmentation, MCF-7 cells were harvested by destained with H 20, and solubilized with 0.2% Triton X-100. Absorb - trypsinization, collected by centrifugation

  14. Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner.

    PubMed

    Fan, Junkai; Liu, Yajuan; Jia, Jianhang

    2012-06-15

    The activation of Smoothened (Smo) requires phosphorylation at three clusters of Serine residues in Drosophila Hedgehog (Hh) signaling. However, the mechanism by which phosphorylation promotes Smo conformational change and subsequently activates Smo in response to Hh gradient remains unclear. Here, we show that the conformational states of Smo are determined by not only the amount but also the position of the negative charges provided by phosphorylation. By using a Smo phospho-specific antibody, we demonstrate that Smo is differentially phosphorylated at three clusters of serine residues in response to levels of Hh activity. Mutating the first cluster, compared to mutating the other clusters, impairs Smo activity more severely, whereas mutating the last cluster prohibits C-terminus dimerization. In addition, phosphorylation of the membrane proximal cluster promotes phosphorylation of the distal cluster. We propose a zipper-lock model in which the gradual phosphorylation at these clusters induces a gradual conformational change in the Smo cytoplasmic tail, which promotes the interaction between Smo and Costal2 (Cos2). Moreover, we show that Hh regulates both PKA and CK1 phosphorylation of Smo. Thus, the differential phosphorylation of Smo mediates the thresholds of Hh activity.

  15. NMDA Receptor Subunit Composition Controls Dendritogenesis of Hippocampal Neurons through CAMKII, CREB-P and H3K27ac.

    PubMed

    Bustos, Fernando J; Jury, Nur; Martinez, Pablo; Ampuero, Estibaliz; Campos, Matias; Abarzúa, Sebastian; Jaramillo, Karen; Ibing, Susanne; Mardones, Muriel D; Haensgen, Henny; Kzhyshkowska, Julia; Tevy, Maria Florencia; Neve, Rachael; Sanhueza, Magdalena; Varela-Nallar, Lorena; Montecino, Martín; van Zundert, Brigitte

    2017-02-04

    Dendrite arbor growth, or dendritogenesis, is choreographed by a diverse set of cues, including the NMDA receptor (NMDAR) subunits NR2A and NR2B. While NR1NR2B receptors are predominantly expressed in immature neurons and promote plasticity, NR1NR2A receptors are mainly expressed in mature neurons and induce circuit stability. How the different subunits regulate these processes is unclear, but this is likely related to the presence of their distinct C-terminal sequences that couple different signaling proteins. Calcium-calmodulin-dependent protein kinase II (CaMKII) is an interesting candidate as this protein can be activated by calcium influx through NMDARs. CaMKII triggers a series of biochemical signaling cascades, involving the phosphorylation of diverse targets. Among them, the activation of cAMP response element-binding protein (CREB-P) pathway triggers a plasticity-specific transcriptional program through unknown epigenetic mechanisms. Here we found that dendritogenesis in hippocampal neurons is impaired by several well-characterized constructs (i.e. NR2B-RS/QD) and peptides (i.e. tatCN21) that specifically interfere with the recruitment and interaction of CaMKII with the NR2B C-terminal domain. Interestingly, we found that transduction of NR2AΔIN, a mutant NR2A construct with increased interaction to CaMKII, reactivates dendritogenesis in mature hippocampal neurons in vitro and in vivo. To gain insights into the signaling and epigenetic mechanisms underlying NMDAR-mediated dendritogenesis, we used immunofluorescence staining to detect CREB-P and acetylated lysine 27 of histone H3 (H3K27ac), an activation-associated histone tail mark. In contrast to control mature neurons, our data shows that activation of the NMDAR/CaMKII/ERK-P/CREB-P signaling axis in neurons expressing NR2AΔIN is not correlated with increased nuclear H3K27ac levels. This article is protected by copyright. All rights reserved.

  16. Myeloid differentiation factor-2 interacts with Lyn kinase and is tyrosine phosphorylated following lipopolysaccharide-induced activation of the TLR4 signaling pathway.

    PubMed

    Gray, Pearl; Dagvadorj, Jargalsaikhan; Michelsen, Kathrin S; Brikos, Constantinos; Rentsendorj, Altan; Town, Terrence; Crother, Timothy R; Arditi, Moshe

    2011-10-15

    Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrated that myeloid differentiation factor-2 (MD-2) is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific; it is blocked by the tyrosine kinase inhibitor, herbimycin A, as well as by an inhibitor of endocytosis, cytochalasin D, suggesting that MD-2 phosphorylation occurs during trafficking of MD-2 and not on the cell surface. Furthermore, we identified two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine had reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD-2 coprecipitated and colocalized with Lyn kinase, most likely in the endoplasmic reticulum. A Lyn-binding peptide inhibitor abolished MD-2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phosphorylation. Our study demonstrated that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response.

  17. Vitamin C suppresses TNF alpha-induced NF kappa B activation by inhibiting I kappa B alpha phosphorylation.

    PubMed

    Cárcamo, Juan M; Pedraza, Alicia; Bórquez-Ojeda, Oriana; Golde, David W

    2002-10-29

    Extracellular stimuli signal for activation of the transcription factor NFkappaB, leading to gene expression regulating processes involved in immune responses, inflammation, and cell survival. Tumor necrosis factor-alpha (TNFalpha) activates NFkappaB via a well-defined kinase pathways involving NFkappaB-inducing kinase (NIK), which activates downstream multisubunit IkappaB kinases (IKK). IKK in turn phosphorylates IkappaB, the central regulator of NFkappaB function. We found that intracellular vitamin C inhibits TNFalpha-induced activation of NFkappaB in human cell lines (HeLa, monocytic U937, myeloid leukemia HL-60, and breast MCF7) and primary endothelial cells (HUVEC) in a dose-dependent manner. Vitamin C is an important antioxidant, and most cells accumulate ascorbic acid (AA) intracellularly by transporting the oxidized form of the vitamin, dehydroascorbic acid (DHA). Because ascorbic acid is a strong pro-oxidant in the presence of transition metals in vitro, we loaded cells with vitamin C by incubating them with DHA. Vitamin C-loaded cells showed significantly decreased TNFalpha-induced nuclear translocation of NFkappaB, NFkappaB-dependent reporter transcription, and IkappaBalpha phosphorylation. Our data point to a mechanism of vitamin C suppression of NFkappaB activation by inhibiting TNFalpha-induced activation of NIK and IKKbeta kinases independent of p38 MAP kinase. These results suggest that intracellular vitamin C can influence inflammatory, neoplastic, and apoptotic processes via inhibition of NFkappaB activation.

  18. BMP-2 induces Osterix expression through up-regulation of Dlx5 and its phosphorylation by p38.

    PubMed

    Ulsamer, Arnau; Ortuño, María José; Ruiz, Silvia; Susperregui, Antonio R G; Osses, Nelson; Rosa, José Luis; Ventura, Francesc

    2008-02-15

    Osterix, a zinc-finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osterix null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that bone morphogenetic protein-2 (BMP-2) induces an increase in Osterix expression, which is mediated through a homeodomain sequence located in the proximal region of the Osterix promoter. Our results demonstrate that induction of Dlx5 by BMP-2 mediates Osterix transcriptional activation. First, BMP-2 induction of Dlx5 precedes the induction of Osterix. Second, Dlx5 binds to the BMP-responsive homeodomain sequences both in vitro and in vivo. Third, Dlx5 overexpression and knock-down assays demonstrate its role in activating Osterix expression in response to BMP-2. Furthermore, we show that Dlx5 is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-34 and Ser-217 are the sites phosphorylated by p38. Phosphorylation at Ser-34/217 increases the transactivation potential of Dlx5. Thus, we propose that BMP activates expression of Osterix through the induction of Dlx5 and its further transcriptional activation by p38-mediated phosphorylation.

  19. Nitric oxide attenuates hydrogen peroxide-induced barrier disruption and protein tyrosine phosphorylation in monolayers of intestinal epithelial cell.

    PubMed

    Katsube, Takanori; Tsuji, Hideo; Onoda, Makoto

    2007-06-01

    The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H(2)O(2), Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and beta-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H(2)O(2) and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H(2)O(2) alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells.

  20. Prostaglandin F2 alpha administered in vivo induces Ca2+-dependent protein phosphorylation in rat luteal tissue

    SciTech Connect

    Baum, M.S.

    1989-01-01

    The present study was performed in order to further elucidate the mechanism of action of PGF2 alpha in luteolysis in the rat ovary. Seven days after priming with superovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin to induce luteal tissue formation, the rats were injected with a luteolytic dose of the prostaglandin F2 alpha analogue cloprostenol. The ovaries were then homogenized, a 30,000 x g supernatant and pellet were prepared, whereafter aliquots of the preparations were incubated in the presence of (gamma-/sup 32/P)ATP with or without Ca2+. The phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and localized by autoradiography. The presence of Ca2+ caused an increased phosphorylation of a 45 kDa protein band in the particulate, but not in the cytosol, fraction. Furthermore, PGF2 alpha rapidly increased the /sup 32/P incorporation into the same protein band of 45 kDa. Thus, the PGF2 alpha-stimulated /sup 32/P incorporation was Ca2+-dependent and seen only in the particulate fraction. These results suggest that PGF2 alpha in its role as a luteolytic agent stimulates a Ca2+-dependent phosphorylation of a specific protein in luteal membranes of the rat ovary.

  1. Cholecystokinin (CCK) stimulates S6 phosphorylation and induced activation of S6 protein kinase in rat pancreatic acini

    SciTech Connect

    Sung, C.; Okabayashi, Y.; Williams, J.

    1987-05-01

    CCK and insulin stimulate pancreatic protein synthesis at a post transcriptional step. To better understand this regulation the authors evaluated the phosphorylation state of ribosomal protein S6 and the presence of a specific S6 protein kinase in pancreatic acini from diabetic rats. Both CCK and insulin increased S6 phosphorylation by up to 400% in intact TSP-labelled acini. The phorbol ester 12-0-tetradecanoylphorbol 13-acetate also stimulated both protein synthesis and S6 phosphorlyation suggesting a role for protein kinase C in mediating the effect of CCK. By contrast, the CaS ionophore ionomycin had no effect on either parameter. Recently, insulin has been shown to activate a unique S6 kinase in various cells. To test for its presence, cytosolic extracts were prepared from acini stimulated with CCK and insulin by homogenization in US -glycerophosphate buffer and assayed for the kinase using el-TSP ATP and rat pancreatic ribosomes followed by SDS-polyacrylamide gel electrophoresis. CCK and insulin both increased S6 kinase activity which required neither CaS or phospholipid. The dose response for CCk was similar to S6 phosphorlyation in the intact acini. TPA did not stimulate the S6 kinase. Thus, CCK may induce S6 phosphorylation both via C kinase and by activation of a unique S6 kinase.

  2. Gamma-radiation-induced phosphorylation of p53 on serine 15 is dose-dependent in MOLT-4 leukaemia cells.

    PubMed

    Tichý, A; Záskodová, D; Zoelzer, F; Vávrová, J; Sinkorová, Z; Pejchal, J; Osterreicher, J; Rezácová, M

    2009-01-01

    Molecular indicators of the absorbed dose of ionizing radiation are powerful tools in biodosimetry. The studies reported here were undertaken with the motivation to find such a marker among the mo lecules involved in ataxia-telangiectasia mutated kinase- dependent signalling induced by ionizing radiation (ATM-kinase, checkpoint kinase-2, protein p53, and oncoprotein Mdm2). In our previous work on T-lymphocyte leukaemia MOLT-4 cells we described the mentioned molecules of ATM-dependent pathway and none of them showed a pronounced dosedependent response. Here we employed Western blotting and ELISA assay to investigate the response of post-translationally modified p53 (particularly phosphorylated on serine 15) after gamma-irradiation. We have found the amount of phosphorylated p53 to be homogenously increased after irradiation by the doses of 0.5 to 7.5 Gy. The dose-dependent response was pronounced especially after the doses up to 3.0 Gy. The presented data indicate that p53 phosphorylated on serine 15 might be used as a potential biodosimetric marker.

  3. High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation

    PubMed Central

    Trejo Bittar, Humberto E.; Welch, Lynn C.; Vagin, Olga; Deiss-Yehiely, Nimrod; Kelly, Aileen M.; Baker, Mairead R.; Capri, Joseph; Cohn, Whitaker; Whitelegge, Julian P.; Vadász, István; Gruenbaum, Yosef; Sznajder, Jacob I.

    2015-01-01

    The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells. PMID:26370512

  4. High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation.

    PubMed

    Dada, Laura A; Trejo Bittar, Humberto E; Welch, Lynn C; Vagin, Olga; Deiss-Yehiely, Nimrod; Kelly, Aileen M; Baker, Mairead R; Capri, Joseph; Cohn, Whitaker; Whitelegge, Julian P; Vadász, István; Gruenbaum, Yosef; Sznajder, Jacob I

    2015-12-01

    The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells.

  5. Compressive Stress Induces Dephosphorylation of the Myosin Regulatory Light Chain via RhoA Phosphorylation by the Adenylyl Cyclase/Protein Kinase A Signaling Pathway

    PubMed Central

    Takemoto, Kenji; Ishihara, Seiichiro; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2015-01-01

    Mechanical stress that arises due to deformation of the extracellular matrix (ECM) either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC) via the RhoA/RhoA-associated protein kinase (ROCK) pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853). Because myosin phosphatase targeting subunit 1 (Thr853) is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form) increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188) that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188) induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188). Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells. PMID:25734240

  6. Potent homocysteine-induced ERK phosphorylation in cultured neurons depends on self-sensitization via system Xc{sup -}

    SciTech Connect

    Gu Li; Hu Xiaoling; Xue Zhanxia; Yang Jun; Wan Lishu; Ren Yan; Hertz, Leif; Peng Liang

    2010-01-15

    Homocysteine is increased during pathological conditions, endangering vascular and cognitive functions, and elevated homocysteine during pregnancy may be correlated with an increased incidence of schizophrenia in the offspring. This study showed that millimolar homocysteine concentrations in saline medium cause phosphorylation of extracellular-signal regulated kinases 1 and 2 (ERK{sub 1/2}) in cerebellar granule neurons, inhibitable by metabotropic but not ionotropic glutamate receptor antagonists. These findings are analogous to observations by , that similar concentrations cause neuronal death. However, these concentrations are much higher than those occurring clinically during hyperhomocysteinemia. It is therefore important that a approx 10-fold increase in potency occurred in the presence of the glutamate precursor glutamine, when ERK{sub 1/2} phosphorylation became inhibitable by NMDA or non-NMDA antagonists and dependent upon epidermal growth factor (EGF) receptor transactivation. However, glutamate release to the medium was reduced, suggesting that reversal of the cystine/glutamate antiporter, system X{sub c}{sup -} could be involved in potentiation of the response by causing a localized release of initially accumulated homocysteine. In agreement with this hypothesis further enhancement of ERK{sub 1/2} phosphorylation occurred in the additional presence of cystine. Pharmacological inhibition of system X{sub c}{sup -} prevented the effect of micromolar homocysteine concentrations, and U0126-mediated inhibition of ERK{sub 1/2} phosphorylation enhanced homocysteine-induced death. In conclusion, homocysteine interacts with system X{sub c}{sup -} like quisqualate (Venkatraman et al. 1994), by 'self-sensitization' with initial accumulation and subsequent release in exchange with cystine and/or glutamate, establishing high local homocysteine concentrations, which activate adjacent ionotropic glutamate receptors and cause neurotoxicity.

  7. The MEK1 inhibitors UO126 and PD98059 block PDGF-AB induced phosphorylation of threonine 292 in porcine smooth muscle cells.

    PubMed

    Kubin, Thomas; Cetinkaya, Ayse; Schönburg, Markus; Beiras-Fernandez, Andres; Walther, Thomas; Richter, Manfred

    2017-02-21

    PDGF-AB and FGF-2 (GFs) induce smooth muscle cell (SMC) proliferation which is indispensible for arteriogenesis. While there is common agreement that GFs stimulate SMC proliferation through phosphorylation (P-) of MEK1/2 at Ser218/222, we previously demonstrated that the MEK inhibitors PD98059 and UO126 did not inhibit P-Ser218/222 as originally proposed but caused strong hyperphosphorylation. Here, we demonstrate that GFs increased phosphorylation of MEK1 at Thr292 while UO126 and PD98059 blocked this phosphorylation. This was again surprising since phosphorylation of Thr292 is regarded as a negative feedback loop. Our findings suggest that inhibition of Thr292 phosphorylation in combination with hyperphosphorylation of Ser218/222 serves as an "off" switch of SMC proliferation and potentially of arteriogenesis.

  8. Hormone-induced protein phosphorylation. I. Relationship between secretagogue action and endogenous protein phosphorylation in intact cells from the exocrine pancreas and parotid

    PubMed Central

    1982-01-01

    We undertook studies to determine whether secretagogue action on the exocrine pancreas and parotid is accompanied by phosphorylation of proteins in intact cells. For this purpose, rat pancreatic, and parotid lobules were preincubated with 32Pi for 45 min at 37 degrees C, washed, and then incubated at 37 degrees C in the presence or absence of secretagogues that effect discharge through different second messengers. Among a variety of polypeptides exhibiting enhanced phosphorylation in pancreatic lobules upon a 30-s incubation in the presence of the secretagogues carbamylcholine, cholecystokinin octapeptide, or secretin, one species with an Mr of 29,000 was especially notable for three reasons: (a) its enhanced level of phosphorylation was dependent on the dose of secretagogue used and was still apparent after incubation for 30 min at 37 degrees C; (b) an analogous phosphorylated polypeptide was observed in isoproterenol- stimulated parotid lobules; and (c) in both tissues its selective dephosphorylation was observed upon termination of stimulation by administration of atropine to carbamylcholine-stimulated pancreatic lobules and propranolol to isoproterenol-stimulated parotid lobules. These results suggest that the phosphorylation of one protein with an Mr of 29,000 is closely correlated both temporally and in a dose- dependent fashion with secretagogue action in both the exocrine pancreas and parotid. PMID:6296160

  9. A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization.

    PubMed Central

    Short, T. W.; Reymond, P.; Briggs, W. R.

    1993-01-01

    Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase. PMID:12231721

  10. Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

    SciTech Connect

    Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth; Bourand, Alexa; Darbon, Emmanuelle; Joly, Nathalie; Briozzo, Pierre; Deutscher, Josef

    2009-09-11

    Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase

  11. A Large-Scale Quantitative Proteomic Approach To Identifying Sulfur Mustard-Induced Protein Phosphorylation Cascades

    DTIC Science & Technology

    2009-07-31

    with immobilized metal affinity chromatography to study the large-scale protein phosphorylation changes resulting from SM exposure in a human...medium, resulting in isotopically “light” and “ heavy ” cell populations, respectively. Protein samples collected from control (light-labeled) and...experimental ( heavy -labeled) cells can then be mixed in equal ratios, digested with trypsin, and analyzed by high-resolution mass spectrometry. The

  12. CREB- and NF-κB-Regulated CXC Chemokine Gene Expression in Lung Carcinogenesis

    PubMed Central

    Sun, Hongxia; Chung, Wen-Cheng; Ryu, Seung-Hee; Ju, Zhenlin; Tran, Hai T.; Kim, Edward; Kurie, Jonathan M.; Koo, Ja Seok

    2009-01-01

    The recognition of the importance of angiogenesis in tumor progression has led to the development of antiangiogenesis as a new strategy for cancer treatment and prevention. By modulating tumor microenvironment and inducing angiogenesis, the proinflammatory cytokine interleukine (IL)-1 β has been reported to promote tumor development. However, the factors mediating IL-1β-induced angiogenesis in non-small cell lung cancer (NSCLC) and the regulation of these angiogenic factors by IL-1β are less clear. Here, we report that IL-1β upregulated an array of proangiogenic CXC chemokine genes in NSCLC cell line A549 and in normal human tracheobronchial epithelium (NHTBE) cells, as determined by microarray analysis. Further analysis revealed that IL-1β induced much higher protein levels of CXC chemokines in NSCLC cells than in NHTBE cells. Conditioned medium from IL-1β treated A549 cells markedly increased endothelial cell migration, which was suppressed by neutralizing antibodies against CXCL5 and CXCR2. We also found that IL-1β-induced CXC chemokine gene overexpression in NSCLC cells was abrogated with the knockdown of CREB or NF-κB. Moreover, the expression of the CXC chemokine genes as well as CREB and NF-κB activities were greatly increased in tumorigenic NSCLC cell line compared with normal, premalignant immortalized or non-tumorigenic cell lines. A disruptor of the interaction between CREB-binding protein (CBP) and transcription factors such as CREB and NF-κB, 2-naphthol-AS-E-phosphate (KG-501), inhibited IL-1β-induced CXC chemokine gene expression and angiogenic activity in NSCLC. We propose that targeting CREB or NF-κB using small molecule inhibitors, such as KG-501, holds promise as a preventive and/or therapeutic approach for NSCLC. PMID:19138976

  13. Phosphorylation of DGCR8 increases its intracellular stability and induces a progrowth miRNA profile.

    PubMed

    Herbert, Kristina M; Pimienta, Genaro; DeGregorio, Suzanne J; Alexandrov, Andrei; Steitz, Joan A

    2013-11-27

    During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8's ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.

  14. Phosphorylation of DGCR8 Increases Its Intracellular Stability and Induces a Progrowth miRNA Profile

    PubMed Central

    Herbert, Kristina M.; Pimienta, Genaro; DeGregorio, Suzanne J.; Alexandrov, Andrei; Steitz, Joan A.

    2014-01-01

    SUMMARY During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8’s ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8. PMID:24239349

  15. Altered protein phosphorylation in sciatic nerve from rats with streptozocin-induced diabetes

    SciTech Connect

    Schrama, L.H.; Berti-Mattera, L.N.; Eichberg, J.

    1987-11-01

    The effect of experimental diabetes on the phosphorylation of proteins in the rat sciatic nerve was studied. Nerves from animals made diabetic with streptozocin were incubated in vitro with (/sup 32/P)orthophosphate and divided into segments from the proximal to the distal end, and proteins from each segment were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The principal labeled species were the major myelin proteins, P0, and the basic proteins. After 6 wk of diabetes, the incorporation of isotope into these proteins rose as a function of distance along the nerve in a proximal to distal direction and was significantly higher at the distal end compared with incorporation into nerves from age-matched controls. The overall level of isotope uptake was similar in nerves from diabetic animals and weight-matched controls. The distribution of /sup 32/P among proteins also differed in diabetic nerve compared with both control groups in that P0 and the small basic protein accounted for a greater proportion of total label incorporated along the entire length of nerve. In contrast to intact nerve, there was no significant difference in protein phosphorylation when homogenates from normal and diabetic nerve were incubated with (/sup 32/P)-gamma-ATP. The results suggest that abnormal protein phosphorylation, particularly of myelin proteins, is a feature of experimental diabetic neuropathy and that the changes are most pronounced in the distal portion of the nerve.

  16. Aloe-emodin suppressed NMDA-induced apoptosis of retinal ganglion cells through regulation of ERK phosphorylation.

    PubMed

    Lin, Hui-Ju; Chao, Pei-Dawn Lee; Huang, Shiuan-Yi; Wan, Lai; Wu, Chen-Ju; Tsai, Fuu-Jen

    2007-11-01

    A high concentration of glutamate in the vitreous body and optic nerves of the eyes activates N-methyl-D-aspartate (NMDA) receptors and is toxic to retina ganglion cells (RGCs) in glaucomatous patients. Aloe-emodin sulfates/glucuronides (s/g), the major metabolites of aloe-emodin, was found to be effective in decreasing NMDA-induced apoptosis in RGCs. In order to elucidate the mechanisms, an in vitro optic neuropathy model adding NMDA to N18 RGCs was used in this study. The phosphorylation level of extra-cellular signal-regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 kinase (cytokines-suppressive antiinflammatory drug binding protein kinase) were measured by western blotting and luciferase reporter assay. The results showed that aloe-emodin metabolites significantly decreased the activation of three major mitogen-activated protein (MAP) kinase pathways and the activation of downstream genes in nucleus induced by NMDA, which were verified by the addition of the respective inhibitors. Comparing the effect of the inhibitors of the three MAP kinase pathways, the ERK pathway was found to be the major route of aloe-emodin metabolites in decreasing the apoptosis of NMDA-treated RGCs. Besides, cfos rather then cjun was the target downstream gene. Aloe-emodin emodin metabolites could regulate the phosphorylation of ERK kinases and it was a promising candidate for NMDA-induced apoptosis of RGCs.

  17. Cobalt chloride-mediated protein kinase Cα (PKCα) phosphorylation induces hypoxia-inducible factor 1α (HIF1α) in the nucleus of gastric cancer cell.

    PubMed

    Rath, Suvasmita; Anand, Aditya; Ghosh, Nilabh; Das, Lopamudra; Kokate, Shrikant B; Dixit, Pragyesh; Majhi, Swetapadma; Rout, Niranjan; Singh, Shivaram P; Bhattacharyya, Asima

    2016-02-26

    Hypoxia promotes cancer progression, and metastasis. The major protein expressed in hypoxic solid cancer is hypoxia-inducible factor 1 (HIF1). We show that enhanced phosphorylation of a conventional protein kinase C isoform, PKCα, at threonine 638 (T(638)) by hypoxia-mimetic cobalt chloride induces HIF1α in nuclei of gastric epithelial cells (GECs). Moreover, phospho-T(638)-PKCα (P-PKCα) interacts with p300-HIF1α complex in the nuclei of hypoxic GECs and PKCα phosphorylation at T(638) enhances transcriptional activity of HIF1α. High P-PKCα expression in neoplastic gastric cancer biopsy samples as compared to nonneoplastic samples suggests that P-PKCα might act as an indicator of gastric cancer progression.

  18. Hippocampal Overexpression of Mutant CREB Blocks Long-Term, but Not Short-Term Memory for a Socially Transmitted Food Preference

    ERIC Educational Resources Information Center

    Brightwell, Jennifer J.; Countryman, Renee A.; Neve, Rachael L.; Colombo, Paul J.; Smith, Clayton A.

    2005-01-01

    Phosphorylation of the transcription factor CREB on Ser133 is implicated in the establishment of long-term memory for hippocampus-dependent tasks, including spatial learning and contextual fear conditioning. We reported previously that training on a hippocampus-dependent social transmission of food preference (STFP) task increases CREB…

  19. H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib.

    PubMed

    Dong, Yaqiong; Xiong, Min; Duan, Lianning; Liu, Ze; Niu, Tianhui; Luo, Yuan; Wu, Xinpin; Xu, Chengshan; Lu, Chengrong

    2014-08-01

    Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.

  20. The Roles of Phosphorylation and SHAGGY-Like Protein Kinases in Geminivirus C4 Protein Induced Hyperplasia

    PubMed Central

    Mills-Lujan, Katherine; Andrews, David L.; Chou, Chau-wen; Deom, C. Michael

    2015-01-01

    Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control. PMID:25815729

  1. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

    PubMed Central

    Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation. PMID:28072855

  2. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

    PubMed

    Ohtsuka, Hiroko; Iguchi, Tomohiro; Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

  3. Enhancement of UV-induced nucleotide excision repair activity upon forskolin treatment is cell growth-dependent

    PubMed Central

    Lee, Jeong-Min; Park, Jeong-Min; Kang, Tae-Hong

    2016-01-01

    Forskolin (FSK), an adenylyl cyclase activator, has recently been shown to enhance nucleotide excision repair (NER) upon UV exposure. However, our study revealed that this effect was detected in human skin epithelial ARPE19 cells only in growing cells, but not in non-cycling cells. When the cells were grown at low density (70% confluence), FSK was capable of stimulating cAMP responsive element binding (CREB) phosphorylation, a marker for FSK-stimulated PKA activation, and resulted in a significant increase of NER activity compared to control treatment. However, cells grown under 100% confluent conditions showed neither FSK-induced CREB phosphorylation nor the resulting NER enhancement. These findings indicate that cellular growth is critical for FSK-induced NER enhancement and suggest that cellular growth conditions should be considered as a variable while evaluating a reagent’s pharmacotherapeutic efficacy. [BMB Reports 2016; 49(10): 566-571] PMID:27470212

  4. Enhancement of UV-induced nucleotide excision repair activity upon forskolin treatment is cell growth-dependent.

    PubMed

    Lee, Jeong-Min; Park, Jeong-Min; Kang, Tae-Hong

    2016-10-01

    Forskolin (FSK), an adenylyl cyclase activator, has recently been shown to enhance nucleotide excision repair (NER) upon UV exposure. However, our study revealed that this effect was detected in human skin epithelial ARPE19 cells only in growing cells, but not in non-cycling cells. When the cells were grown at low density (70% confluence), FSK was capable of stimulating cAMP responsive element binding (CREB) phosphorylation, a marker for FSK-stimulated PKA activation, and resulted in a significant increase of NER activity compared to control treatment. However, cells grown under 100% confluent conditions showed neither FSK-induced CREB phosphorylation nor the resulting NER enhancement. These findings indicate that cellular growth is critical for FSK-induced NER enhancement and suggest that cellular growth conditions should be considered as a variable while evaluating a reagent's pharmacotherapeutic efficacy. [BMB Reports 2016; 49(10): 566-571].

  5. Nocturnal activation of aurora C in rat pineal gland: its role in the norepinephrine-induced phosphorylation of histone H3 and gene expression.

    PubMed

    Price, D M; Kanyo, R; Steinberg, N; Chik, C L; Ho, A K

    2009-05-01

    We have shown previously that Ser10 phosphorylation of histone H3 occurs in rat pinealocytes after stimulation with norepinephrine (NE) and that histone modifications such as acetylation appear to play an important role in pineal gene transcription. Here we report the nocturnal phosphorylation of a Ser10 histone H3 kinase, Aurora C, in the rat pineal gland. The time profile of this phosphorylation parallels the increase in the level of phospho-Ser10 histone H3. Studies with cultured pinealocytes indicate that Aurora C phosphorylation is induced by NE and this induction can be blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor. Moreover, only treatment with dibutyryl cAMP, but not other kinase activators, mimics the effect of NE on Aurora C phosphorylation. These results indicate that Aurora C is phosphorylated primarily by a beta-adrenergic/protein kinase A-mediated mechanism. Treatment with an Aurora C inhibitor reduces the NE-induced histone H3 phosphorylation and suppresses the NE-stimulated induction of arylalkylamine N-acetyltransferase (AA-NAT), the rhythm-controlling enzyme of melatonin synthesis, and melatonin production. The effects of Aurora C inhibitors on adrenergic-induced genes in rat pinealocytes are gene specific: inhibitory for Aa-nat and inducible cAMP repressor but stimulatory for c-fos. Together our results support a role for the NE-stimulated phosphorylation of Aurora C and the subsequent remodeling of chromatin in NE-stimulated Aa-nat transcription. This phenomenon suggests that activation of this mitotic kinase can be induced by extracellular signals to participate in the transcriptional induction of a subset of genes in the rat pineal gland.

  6. The role of nitric-oxide synthase in the regulation of UVB light-induced phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

    PubMed

    Lu, Wei; László, Csaba F; Miao, Zhixin; Chen, Hao; Wu, Shiyong

    2009-09-04

    UV light induces phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2alpha in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2alpha phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2alpha phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2alpha after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2alpha phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N(G)-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2alpha phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2alpha phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.

  7. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    SciTech Connect

    Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  8. MD-2 interacts with Lyn kinase and is tyrosine phosphorylated following LPS-induced activation of the Toll-like receptor 4 signaling pathway

    PubMed Central

    Gray, Pearl; Dagvadorj, Jargalsaikhan; Michelsen, Kathrin S.; Brikos, Constantinos; Rentsendorj, Altan; Town, Terrence; Crother, Timothy R.; Arditi, Moshe

    2011-01-01

    Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrate that MD-2 is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific, it is blocked by the tyrosine kinase inhibitor, Herbimycin A, and by an inhibitor of endocytosis, Cytochalsin-D, suggesting that MD-2 phosphorylation occurs during trafficking of MD2 and not on cell surface. Furthermore, we identify two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine have reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD2 co-precipitates and colocalizes with Lyn kinase, most likely in ER. A Lyn-binding peptide inhibitor abolished MD2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phophorylation. Our study demonstrates that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response. PMID:21918188

  9. Involvement of myristoylated alanine-rich C kinase substrate phosphorylation and translocation in cholecystokinin-induced amylase release in rat pancreatic acini.

    PubMed

    Satoh, Keitaro; Narita, Takanori; Katsumata-Kato, Osamu; Sugiya, Hiroshi; Seo, Yoshiteru

    2016-03-15

    Cholecystokinin (CCK) is a gastrointestinal hormone that induces exocytotic amylase release in pancreatic acinar cells. The activation of protein kinase C (PKC) is involved in the CCK-induced pancreatic amylase release. Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed substrate of PKC. MARCKS has been implicated in membrane trafficking in several cell types. The phosphorylation of MARCKS by PKC results in the translocation of MARCKS from the membrane to the cytosol. Here, we studied the involvement of MARCKS in the CCK-induced amylase release in rat pancreatic acini. Employing Western blotting, we detected MARCKS protein in the rat pancreatic acini. CCK induced MARCKS phosphorylation. A PKC-δ inhibitor, rottlerin, inhibited the CCK-induced MARCKS phosphorylation and amylase release. In the translocation assay, we also observed CCK-induced PKC-δ activation. An immunohistochemistry study showed that CCK induced MARCKS translocation from the membrane to the cytosol. When acini were lysed by a detergent, Triton X-100, CCK partially induced displacement of the MARCKS from the GM1a-rich detergent-resistant membrane fractions (DRMs) in which Syntaxin2 is distributed. A MARCKS-related peptide inhibited the CCK-induced amylase release. These findings suggest that MARCKS phosphorylation by PKC-δ and then MARCKS translocation from the GM1a-rich DRMs to the cytosol are involved in the CCK-induced amylase release in pancreatic acinar cells.

  10. Multiple phosphorylated forms of the Saccharomyces cerevisiae Mcm1 protein include an isoform induced in response to high salt concentrations.

    PubMed Central

    Kuo, M H; Nadeau, E T; Grayhack, E J

    1997-01-01

    The Saccharomyces cerevisiae Mcm1 protein is an essential multifunctional transcription factor which is highly homologous to human serum response factor. Mcm1 protein acts on a large number of distinctly regulated genes: haploid cell-type-specific genes, G2-cell-cycle-regulated genes, pheromone-induced genes, arginine metabolic genes, and genes important for cell wall and cell membrane function. We show here that Mcm1 protein is phosphorylated in vivo. Several (more than eight) isoforms of Mcm1 protein, resolved by isoelectric focusing, are present in vivo; two major phosphorylation sites lie in the N-terminal 17 amino acids immediately adjacent to the conserved MADS box DNA-binding domain. The implications of multiple species of Mcm1, particularly the notion that a unique Mcm1 isoform could be required for regulation of a specific set of Mcm1's target genes, are discussed. We also show here that Mcm1 plays an important role in the response to stress caused by NaCl. G. Yu, R. J. Deschenes, and J. S. Fassler (J. Biol. Chem. 270:8739-8743, 1995) showed that Mcm1 function is affected by mutations in the SLN1 gene, a signal transduction component implicated in the response to osmotic stress. We find that mcm1 mutations can confer either reduced or enhanced survival on high-salt medium; deletion of the N terminus or mutation in the primary phosphorylation site results in impaired growth on high-salt medium. Furthermore, Mcm1 protein is a target of a signal transduction system responsive to osmotic stress: a new isoform of Mcm1 is induced by NaCl or KCl; this result establishes that Mcm1 itself is regulated. PMID:9001236

  11. Neuronal activity controls Bdnf expression via Polycomb de-repression and CREB/CBP/JMJD3 activation in mature neurons

    PubMed Central

    Palomer, Ernest; Carretero, Javier; Benvegnù, Stefano; Dotti, Carlos G.; Martin, Mauricio G.

    2016-01-01

    It has been recently described that in embryonic stem cells, the expression of some important developmentally regulated genes is repressed, but poised for fast activation under the appropriate stimuli. In this work we show that Bdnf promoters are repressed by Polycomb Complex 2 in mature hippocampal neurons, and basal expression is guaranteed by the coexistence with activating histone marks. Neuronal stimulation triggered by N-methyl-D-aspartate application induces the transcription of these promoters by H3K27Me3 demethylation and H3K27Me3 phosphorylation at Serine 28 leading to displacement of EZH2, the catalytic subunit of Polycomb Repressor Complex 2. Our data show that the fast transient expression of Bdnf promoters II and VI after neuronal stimulation is dependent on acetylation of histone H3K27 by CREB-p/CBP. Thus, regulatory mechanisms established during development seem to remain after differentiation controlling genes induced by different stimuli, as would be the case of early memory genes in mature neurons. PMID:27010597

  12. 6,7-di-O-acetylsinococuline (FK-3000) induces G2/M phase arrest in breast carcinomas through p38 MAPK phosphorylation and CDC25B dephosphorylation

    PubMed Central

    LI, YONG-CHUN; KIM, BONG-HEE; CHO, SOON-CHANG; BANG, MI-AE; KIM, SUNMIN; PARK, DAE-HUN

    2015-01-01

    We evaluated the cytostatic effect of 6,7-di-O-acetylsinococuline (FK-3000) isolated from Stephania delavayi Diels. against breast carcinoma cell lines MDA-MB-231 and MCF-7. FK-3000 suppressed CDC25B phosphorylation directly and indirectly via p38 MAPK phosphorylation. CDC25B dephosphorylation decreased levels of cyclin B and phospho-CDC-2, and ultimately induced cell cycle arrest at the G2/M phase. The p38 MAPK inhibitor, SB 239063 blocked FK-3000-induced p38 MAPK phosphorylation and nuclear accumulation, but did not completely rescue cell death. Conclusively FK-3000 exerts its antiproliferative effect through two pathways: i) G2/M cell cycle arrest via downregulation of cyclin B and phospho-CDC2 by p38 MAPK phosphorylation and CDC25B dephosphorylation, and ii) p38 MAPK-independent induction of apoptosis. PMID:25384584

  13. Inhibition of Akt2 phosphorylation abolishes the calorie restriction-induced improvement in insulin-stimulated glucose uptake by rat soleus muscle

    PubMed Central

    Sharma, Naveen; Arias, Edward B.; Cartee, Gregory D.

    2017-01-01

    Calorie restriction (CR; ~60–65% of ad libitum, AL, consumption) can enhance insulin-stimulated glucose uptake (ISGU) in predominantly slow-twitch skeletal muscles (e.g., soleus) by an incompletely understood mechanism. We used an Akt inhibitor (MK-2206) to eliminate CR’s effect on insulin-stimulated Akt2 phosphorylation in isolated rat soleus muscles. We found long-term CR-enhanced ISGU was abolished by eliminating the CR-effect on Akt2 phosphorylation, suggesting the CR-induced benefit on ISGU in the predominantly slow-twitch soleus relies on enhanced Akt2 phosphorylation. PMID:27786542

  14. ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

    SciTech Connect

    Colonna, Cecilia . E-mail: ccolonna@fmed.uba.ar; Podesta, Ernesto J.

    2005-04-01

    Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.

  15. STIM1 Phosphorylation at Y361 Recruits Orai1 to STIM1 Puncta and Induces Ca2+ Entry

    PubMed Central

    Yazbeck, Pascal; Tauseef, Mohammad; Kruse, Kevin; Amin, Md-Ruhul; Sheikh, Rayees; Feske, Stefan; Komarova, Yulia; Mehta, Dolly

    2017-01-01

    Store-operated Ca2+ entry (SOCE) mediates the increase in intracellular calcium (Ca2+) in endothelial cells (ECs) that regulates several EC functions including tissue-fluid homeostasis. Stromal-interaction molecule 1 (STIM1), upon sensing the depletion of (Ca2+) from the endoplasmic reticulum (ER) store, organizes as puncta that trigger store-operated Ca2+ entry (SOCE) via plasmalemmal Ca2+-selective Orai1 channels. While the STIM1 and Orai1 binding interfaces have been mapped, signaling mechanisms activating STIM1 recruitment of Orai1 and STIM1-Orai1 interaction remains enigmatic. Here, we show that ER Ca2+-store depletion rapidly induces STIM1 phosphorylation at Y361 via proline-rich kinase 2 (Pyk2) in ECs. Surprisingly, the phospho-defective STIM1-Y361F mutant formed puncta but failed to recruit Orai1, thereby preventing. SOCE Furthermore, studies in mouse lungs, expression of phosphodefective STIM1-Y361F mutant in ECs prevented the increase in vascular permeability induced by the thrombin receptor, protease activated receptor 1 (PAR1). Hence, Pyk2-dependent phosphorylation of STIM1 at Y361 is a critical phospho-switch enabling recruitment of Orai1 into STIM1 puncta leading to SOCE. Therefore, Y361 in STIM1 represents a novel target for limiting SOCE-associated vascular leak. PMID:28218251

  16. ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells.

    PubMed

    Colonna, Cecilia; Podestá, Ernesto J

    2005-04-01

    Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.

  17. β-Adrenergic stimulation activates protein kinase Cε and induces extracellular signal-regulated kinase phosphorylation and cardiomyocyte hypertrophy.

    PubMed

    Li, Lin; Cai, Hongyan; Liu, Hua; Guo, Tao

    2015-06-01

    The cardiac adrenergic signaling pathway is important in the induction of cardiac hypertrophy. The cardiac adrenergic pathway involves two main branches, phospholipase C (PLC)/protein kinase C (PKC) and the adenylate cyclase (cAMPase)/protein kinase A (PKA) signaling pathways. It is hypothesized that PLC/PKC and cAMPase/PKA are activated by the α‑adrenergic receptor (αAR) and the β‑adrenergic receptor (βAR), respectively. Previous studies have demonstrated that exchange protein directly activated by cAMP (Epac), a guanine exchange factor, activates phospholipase Cε. It is possible that there are βAR‑activated PKC pathways mediated by Epac and PLC. In the present study, the role of Epac and PLC in βAR activated PKC pathways in cardiomyocytes was investigated. It was found that PKCε activation and translocation were induced by the βAR agonist, isoproterenol (Iso). Epac agonist 8‑CPT‑2'OMe‑cAMP also enhanced PKCε activation. βAR stimulation activated PKCε in the cardiomyocytes and was regulated by Epac. Iso‑induced change in PKCε was not affected in the cardiomyocytes, which were infected with adenovirus coding rabbit muscle cAMP‑dependent protein kinase inhibitor. However, Iso‑induced PKCε activation was inhibited by the PLC inhibitor, U73122. The results suggested that Iso‑induced PKCε activation was independent of PKA, but was regulated by PLC. To further investigate the downstream signal target of PKCε activation, the expression of phosphorylated extracellular signal‑regulated kinase (pERK)1/2 and the levels of ERK phosphorylation was analyzed. The results revealed that Iso‑induced PKCε activation led to an increase in the expression of pERK1/2. ERK phosphorylation was inhibited by the PKCε inhibitor peptide. Taken together, these data demonstrated that the βAR is able to activate PKCε dependent on Epac and PLC, but independent of PKA.

  18. Phosphorylation-triggered CUEDC2 degradation promotes UV-induced G1 arrest through APC/C(Cdh1) regulation.

    PubMed

    Zhang, Wei-Na; Zhou, Jie; Zhou, Tao; Li, Ai-Ling; Wang, Na; Xu, Jin-Jing; Chang, Yan; Man, Jiang-Hong; Pan, Xin; Li, Tao; Li, Wei-Hua; Mu, Rui; Liang, Bing; Chen, Liang; Jin, Bao-Feng; Xia, Qing; Gong, Wei-Li; Zhang, Xue-Min; Wang, Li; Li, Hui-Yan

    2013-07-02

    DNA damage triggers cell cycle arrest to provide a time window for DNA repair. Failure of arrest could lead to genomic instability and tumorigenesis. DNA damage-induced G1 arrest is generally achieved by the accumulation of Cyclin-dependent kinase inhibitor 1 (p21). However, p21 is degraded and does not play a role in UV-induced G1 arrest. The mechanism of UV-induced G1 arrest thus remains elusive. Here, we have identified a critical role for CUE domain-containing protein 2 (CUEDC2) in this process. CUEDC2 binds to and inhibits anaphase-promoting complex/cyclosome-Cdh1 (APC/C(Cdh1)), a critical ubiquitin ligase in G1 phase, thereby stabilizing Cyclin A and promoting G1-S transition. In response to UV irradiation, CUEDC2 undergoes ERK1/2-dependent phosphorylation and ubiquitin-dependent degradation, leading to APC/C(Cdh1)-mediated Cyclin A destruction, Cyclin-dependent kinase 2 inactivation, and G1 arrest. A nonphosphorylatable CUEDC2 mutant is resistant to UV-induced degradation. Expression of this stable mutant effectively overrides UV-induced G1-S block. These results establish CUEDC2 as an APC/C(Cdh1) inhibitor and indicate that regulated CUEDC2 degradation is critical for UV-induced G1 arrest.

  19. Aurora Kinases Phosphorylate Lgl to Induce Mitotic Spindle Orientation in Drosophila Epithelia

    PubMed Central

    Bell, Graham P.; Fletcher, Georgina C.; Brain, Ruth; Thompson, Barry J.

    2015-01-01

    Summary The Lethal giant larvae (Lgl) protein was discovered in Drosophila as a tumor suppressor in both neural stem cells (neuroblasts) and epithelia. In neuroblasts, Lgl relocalizes to the cytoplasm at mitosis, an event attributed to phosphorylation by mitotically activated aPKC kinase and thought to promote asymmetric cell division. Here we show that Lgl also relocalizes to the cytoplasm at mitosis in epithelial cells, which divide symmetrically. The Aurora A and B kinases directly phosphorylate Lgl to promote its mitotic relocalization, whereas aPKC kinase activity is required only for polarization of Lgl. A form of Lgl that is a substrate for aPKC, but not Aurora kinases, can restore cell polarity in lgl mutants but reveals defects in mitotic spindle orientation in epithelia. We propose that removal of Lgl from the plasma membrane at mitosis allows Pins/LGN to bind Dlg and thus orient the spindle in the plane of the epithelium. Our findings suggest a revised model for Lgl regulation and function in both symmetric and asymmetric cell divisions. PMID:25484300

  20. Susceptibility to simvastatin-induced toxicity is partly determined by mitochondrial respiration and phosphorylation state of Akt.

    PubMed

    Mullen, Peter J; Zahno, Anja; Lindinger, Peter; Maseneni, Swarna; Felser, Andrea; Krähenbühl, Stephan; Brecht, Karin

    2011-12-01

    Statins are widely used to prevent cardiovascular diseases. They are well-tolerated, with side-effects mainly seen in skeletal muscle. How these side-effects are caused is unknown. We compared isolated primary mouse skeletal muscle myocytes, C2C12 myotubes and liver HepG2 cells to detect differences that could uncover why statins are toxic in skeletal muscle but less so in the liver. 10μM simvastatin caused a decrease in mitochondrial respiration in the primary mouse myocytes and C2C12 myotubes, but had no effect in the HepG2 cells. Mitochondrial integrity is maintained by multiple signaling pathways. One of these pathways, Igf-1/Akt signaling, is also heavily implicated in causing statin-induced toxicity by upregulating atrogin-1. We found that phosphorylated Akt was reduced in C2C12 myotubes but not in HepG2 cells. HepG2 mitochondrial respiration became susceptible to simvastatin-treatment after Akt inhibition, and mitochondrial respiration was rescued in Igf-1-treated C2C12 myotubes. These results suggest that disruption of Igf-1/Akt signaling is a causative factor in simvastatin-induced mitochondrial dysfunction in C2C12 myotubes, whereas HepG2 cells are protected by maintaining Igf-1/Akt signaling. We conclude that phosphorylation of Akt is a key indicator of susceptibility to statin-induced toxicity. How statins can disrupt Igf-1/Akt signaling is unknown. Statins reduce geranylgeranylation of small GTPases, such as Rap1. Previous studies implicate Rap1 as a link between cAMP/Epac and Igf-1/Akt signaling. Transient transfection of constitutively active Rap1 into C2C12 myotubes led to a partial rescue of simvastatin-induced inhibition of mitochondrial respiration, providing a novel link between signaling and respiration.

  1. Inhibition of p38 MAPK Phosphorylation Is Critical for Bestatin to Enhance ATRA-Induced Cell Differentiation in Acute Promyelocytic Leukemia NB4 Cells.

    PubMed

    Qian, Xijun; He, Jingsong; Zhao, Yi; Lin, Maofang

    2016-01-01

    Bestatin has been known as an immunomodulating agent in anti-leukemia treatment. The mechanism by which Bestatin enhances all-trans retinoic acid (ATRA)-induced cell differentiation of acute promyelocytic leukemia (APL) cells is generally attributed to inhibition of cell surface CD13/aminopeptidase N activity. Bestatin also exerts its biological activities besides its ability to inhibit aminopeptidase N enzymatic activity. This article provides data to support an alternative mechanism regarding an important role of inhibition of p38 mitogen-activated protein kinase (MAPK) signal pathway in Bestatin's anti-leukemia effect. Bestatin enhanced ATRA-induced differentiation and inhibited ATRA-driven phosphorylation of p38 MAPK in ATRA-sensitive APL NB4 cells. In contrast, Bestatin could not reverse the differentiation block in ATRA-resistant APL MR2 cells, in which ATRA was unable to induce phosphorylation of p38 MAPK. Moreover, CD13 ligation with anti-CD13 antibody WM-15 resulted in phosphorylation of p38 MAPK, reduced the inhibition of Bestatin on the phosphorylation of p38 MAPK, and completely abolished the enhancement of Bestatin on ATRA-inducing differentiation in NB4 cells. This study shows that inhibition of p38 MAPK phosphorylation is critical for Bestatin to enhance ATRA-induced cell differentiation in ATRA-sensitive APL NB4 cells. Results suggested that pharmacological inhibition of the p38 MAPK pathway might enhance ATRA-dependent differentiation.

  2. The adrenergic-regulated CRTC1 and CRTC2 phosphorylation and cellular distribution is independent of endogenous SIK1 in the male rat pinealocyte.

    PubMed

    McTague, J; Ferguson, M; Chik, C L; Ho, A K

    2015-10-15

    Salt inducible kinase 1 (SIK1) has been reported to repress cAMP-response element binding protein (CREB)-mediated gene transcription by causing the nuclear export of CREB-regulated transcription coactivators (CRTCs) through phosphorylation. Although the repressor role of SIK1 in suppressing the expression of arylalkylamine N-acetyltransferase, the enzyme that controls the daily rhythm in melatonin production in the rat pineal gland, has been established, whether SIK1 regulates the phosphorylation and localization of CRTC1 and CRTC2 in this tissue remains unclear. The present study found that overexpressing SIK1 in NE-stimulated rat pinealocytes could increase the phosphorylation of CRTC1 and CRTC2, reduced selectively the nuclear level of CRTC2 (but not that of CRTC1), and elevated the cytosolic levels of both CRTC1 and CRTC2. In contrast, transient knockdown of endogenous SIK1 had no effect on the phosphorylation or distribution of CRTC1 and CRTC2 in norepinephrine (NE)-stimulated pinealocytes. Our results also showed that adrenergic blockade during NE stimulation led to a rapid rephosphorylation and decline in the nucleus levels of CRTC1 and CRTC2; however SIK1 knockdown had no effect on this rapid rephosphorylation. Moreover, studies with kinase inhibitors revealed that kinase(s) sensitive to KT5823 appeared to be involved in this rapid rephosphorylation. Together, these results indicate that although overexpressing SIK1 can phosphorylate CRTC1 and CRTC2 in the NE-stimulated pinealocyte, the endogenous SIK1, in spite of its induction by NE, does not appear to be the main regulator of the phosphorylation and intracellular localization of these two coactivators.

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

    PubMed

    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

    2015-05-10

    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 expression 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 kinase(T172) [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.

  4. Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr{sup 286} in squamous cell carcinoma

    SciTech Connect

    Mori, Jun; Takahashi-Yanaga, Fumi . E-mail: yanaga@clipharm.med.kyushu-u.ac.jp; Miwa, Yoshikazu; Watanabe, Yutaka; Hirata, Masato; Morimoto, Sachio; Shirasuna, Kanemitsu; Sasaguri, Toshiyuki

    2005-11-01

    Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G{sub 0}/G{sub 1} phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3{beta} (GSK-3{beta}). Depletion of endogenous GSK-3{beta} by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3{beta} and found that DIF-1 dephosphorylated GSK-3{beta} on Ser{sup 9} and induced the nuclear translocation of GSK-3{beta}, suggesting that DIF-1 activated GSK-3{beta}. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr{sup 286} was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3{beta}-mediated phosphorylation of Thr{sup 286}.

  5. Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

    PubMed Central

    Nantajit, Danupon; Fan, Ming; Duru, Nadire; Wen, Yunfei; Reed, John C.; Li, Jian Jian

    2010-01-01

    The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53. PMID:20808790

  6. Chronic restraint stress induces sperm acrosome reaction and changes in testicular tyrosine phosphorylated proteins in rats

    PubMed Central

    Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Maneenin, Chanwit; Iamsaard, Sitthichai

    2016-01-01

    Background: Stress is a cause of male infertility. Although sex hormones and sperm quality have been shown to be low in stress, sperm physiology and testicular functional proteins, such as phosphotyrosine proteins, have not been documented. Objective: To investigate the acrosome status and alterations of testicular proteins involved in spermatogenesis and testosterone synthesis in chronic stress in rats. Materials and Methods: In this experimental study, male rats were divided into 2 groups (control and chronic stress (CS), n=7). CS rats were immobilized (4 hr/day) for 42 consecutive days. The blood glucose level (BGL), corticosterone, testosterone, acrosome status, and histopathology were examined. The expressions of testicular steroidogenic acute regulatory (StAR), cytochrome P450 side chain cleavage (CYP11A1), and phosphorylated proteins were analyzed. Results: Results showed that BGL (71.25±2.22 vs. 95.60±3.36 mg/dl), corticosterone level (24.33±4.23 vs. 36.9±2.01 ng/ml), acrosome reacted sperm (3.25±1.55 vs. 17.71±5.03%), and sperm head abnormality (3.29±0.71 vs. 6.21±1.18%) were significantly higher in CS group in comparison with control. In contrast, seminal vesicle (0.41±0.05 vs. 0.24±0.07 g/100g), testosterone level (3.37±0.79 vs. 0.61±0.29 ng/ml), and sperm concentration (115.33±7.70 vs. 79.13±3.65×106 cells/ml) of CS were significantly lower (p<0.05) than controls. Some atrophic seminiferous tubules and low sperm mass were apparent in CS rats. The expression of CYP11A1 except StAR protein was markedly decreased in CS rats. In contrast, a 55 kDa phosphorylated protein was higher in CS testes. Conclusion: CS decreased the expression of CYP11A, resulting in decreased testosterone, and increased acrosome-reacted sperm, assumed to be the result of an increase of 55 kDa phosphorylated protein. PMID:27525328

  7. Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation

    SciTech Connect

    Okazaki, Hideki; Tokumaru, Sho; Hanakawa, Yasushi; Shiraishi, Ken; Shirakata, Yuji; Dai, Xiuju; Yang, Lijun; Tohyama, Mikiko; Hashimoto, Koji; Sayama, Koji

    2011-09-02

    Highlights: {yields} VEGF-A enhanced lymphatic endothelial cell migration and increased tube formation. {yields} VEGF-A treated lymphatic endothelial cell showed activation of STAT3. {yields} Dominant-negative STAT3 inhibited VEGF-A-induced lymphatic endothelial cell migration and tube formation. -- Abstract: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube length by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.

  8. Senescence may mediate conversion of tau phosphorylation-induced apoptotic escape to neurodegeneration.

    PubMed

    Wang, Jian-Zhi; Wang, Zhi-Hao

    2015-08-01

    Neurodegeneration is the characteristic pathology in the brains of Alzheimer's disease (AD). However, the nature and molecular mechanism leading to the degeneration are not clarified. Given that only the neurons filled with neurofibrillary tangles survive to the end stage of the disease and the major component of the tangles is the hyperphosphorylated tau proteins, it is conceivable that tau hyperphosphorylation must play a crucial role in AD neurodegeneration. We have demonstrated that tau hyperphosphorylation renders the cells more resistant to the acute apoptosis. The molecular mechanisms involve substrate competition of tau and β-catenin for glycogen synthase kinase 3β (GSK-3β); activation of Akt; preservation of Bcl-2 and suppression of Bax, cytosolic cytochrome-c, and caspase-3 activity; and upregulation of unfolded protein response (UPR), i.e., up-regulating phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. On the other hand, tau hyperphosphorylation promotes its intracellular accumulation and disrupts axonal transport; hyperphosphorylated tau also impairs cholinergic function and inhibits proteasome activity. These findings indicate that tau hyperphosphorylation and its intracellular accumulation play dual role in the evolution of AD. We speculate that transient tau phosphorylation helps cells abort from an acute apoptosis, while persistent tau hyperphosphorylation/accumulation may trigger cell senescence that eventually causes a chronic neurodegeneration. Therefore, the nature of "AD neurodegeneration" may represent a new type of tau-regulated chronic neuron death; and the stage of cell senescence may provide a broad window for the intervention of AD.

  9. Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate.

    PubMed

    Kumaresan, A; Siqueira, A P; Hossain, M S; Bergqvist, A S

    2011-12-01

    Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P<0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca(2+)]i between P1 and SRF-P1 in fresh as well as in frozen-thawed semen. A higher (P<0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.

  10. Semaphorin3A-induced axonal transport mediated through phosphorylation of Axin-1 by GSK3β.

    PubMed

    Hida, Tomonobu; Nakamura, Fumio; Usui, Hiroshi; Takeuchi, Kan; Yamashita, Naoya; Goshima, Yoshio

    2015-02-19

    The establishment of neuronal polarity is necessary for proper neuronal wiring. Semaphorin3A (Sema3A), originally identified as a repulsive axon guidance molecule, exerts a wide variety of biological functions through signaling pathways including sequential phosphorylation of collapsin response mediator protein by cyclin-dependent kinase-5 (Cdk5) and glycogen synthase kinase-3β (GSK3β). Sema3A acts on its receptor neuropilin-1 to regulate axonal transport. To delineate mechanism by which Sema3A induces axonal transport, we investigate whether GSK3β is involved in mediating Sema3A-induced axonal transport. 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, an inhibitor of GSK3β, suppressed Sema3A-induced antero- and retrograde axonal transport. Introduction of either GSK3β mutants, GSK3β-L128A or K85M, suppressed Sema3A-induced axonal transport. On the other hand, introduction of GSK3β-R96A did not affect the Sema3A effect, suggesting that unprimed substrates are primarily involved in Sema3A-induced axonal transport. Overexpression of a partial fragment of frequently rearranged in advanced T-cell lymphomas 1 (FRATtide), which interferes the interaction between GSK3β and Axis inhibitor-1 (Axin-1), also suppressed Sema3A-induced transport. siRNA knockdown of Axin-1, an unprimed substrate of GSK3β, suppressed Sema3A-induced antero- and retrograde axonal transport. These results indicate that GSK3β and Axin-1 are involved in Sema3A-induced bidirectional axonal transport. This finding should provide a clue for understanding of mechanisms of a wide variety of biological activities of Sema3A.

  11. RhoA Phosphorylation Induces Rac1 Release from Guanine Dissociation Inhibitor α and Stimulation of Vascular Smooth Muscle Cell Migration▿

    PubMed Central

    Rolli-Derkinderen, Malvyne; Toumaniantz, Gilles; Pacaud, Pierre; Loirand, Gervaise

    2010-01-01

    Although overactivation of RhoA is recognized as a common component of vascular disorders, the molecular mechanisms regulating RhoA activity in vascular smooth muscle cells (VSMC) are still unclear. We have previously shown that in VSMC, RhoA is phosphorylated on Ser188 by nitric oxide (NO)-stimulated cGMP-dependent kinase (PKG), which leads to RhoA-Rho kinase pathway inhibition. In this study, we showed that expression of phosphoresistant RhoA mutants prevented the stimulation of VSMC migration and adhesion induced by NO-PKG pathway activation. In contrast, under basal conditions, phosphomimetic RhoA mutants stimulated VSMC adhesion and migration through a signaling pathway requiring Rac1 and the Rho exchange factor Vav3. RhoA phosphorylation or phosphomimetic RhoA mutants induced Rac1 activation but did not activate Vav3. Indeed, phosphorylated RhoA or phosphomimetic mutants trapped guanine dissociation inhibitor α (GDIα), leading to the release of Rac1 and its translocation to the membrane, where it was then activated by the basal Vav3 nucleotide exchange activity. In vivo, RhoA phosphorylation induced by PKG activation in the aortas of rats treated with sildenafil induced dissociation of Rac1 from GDIα and activation of the Rac1 signaling pathway. These results suggest that the phosphorylation of RhoA represents a novel potent and physiological GDIα displacement factor that leads to Rac1 activation and regulation of Rac1-dependent VSMC functions. PMID:20696841

  12. Cyclical compressive stress induces differentiation of rat primary mandibular condylar chondrocytes through phosphorylated myosin light chain II.

    PubMed

    Liu, Limin; Chen, Lin; Mai, Zhihui; Peng, Zhuli; Yu, Kafung; Liu, Guanqi; Ai, Hong

    2016-11-01

    The role of myosin light chain II (MLC‑II) in cellular differentiation of rat mandibular condylar chondrocytes (MCCs) induced by cyclical uniaxial compressive stress (CUCS) remains unclear. In the current study, a four‑point bending system was used to apply CUCS to primary cultured MCCs from rats. It was identified that CUCS stimulated features of cellular differentiation including morphological alterations, cytoskeleton rearrangement and overproduction of proteoglycans. Furthermore, CUCS promoted runt‑related transcription factor‑2 (RUNX2) expression at mRNA (P<0.01) and protein levels (P<0.05) and elevated alkaline phosphatase (ALP) activity (P<0.01), which are both markers of osteogenic differentiation. Under conditions of stress, western blotting indicated that the ratio of phosphorylated MLC‑II to total MLC‑II was increased significantly (P<0.05). Silencing MLC‑II by RNA interference reduced ALP activity (P<0.01), and eliminated RUNX2 mRNA expression (P<0.01). Addition of the MLC kinase inhibitor, ML‑7, reduced the CUCS‑associated upregulation of RUNX2 expression (P<0.01) and ALP activity (P<0.01). The data indicated that CUCS promoted cellular differentiation of rat primary MCCs, and this was suggested to be via the phosphorylation of MLC‑II.

  13. Vitronectin induces phosphorylation of ezrin/radixin/moesin actin-binding proteins through binding to its novel neuronal receptor telencephalin.

    PubMed

    Furutani, Yutaka; Kawasaki, Miwa; Matsuno, Hitomi; Mitsui, Sachiko; Mori, Kensaku; Yoshihara, Yoshihiro

    2012-11-09

    Vitronectin (VN) is an extracellular matrix protein abundantly present in blood and a wide variety of tissues and plays important roles in a number of biological phenomena mainly through its binding to αV integrins. However, its definite function in the brain remains largely unknown. Here we report the identification of telencephalin (TLCN/ICAM-5) as a novel VN receptor on neuronal dendrites. VN strongly binds to TLCN, a unique neuronal member of the ICAM family, which is specifically expressed on dendrites of spiny neurons in the mammalian telencephalon. VN-coated microbeads induce the formation of phagocytic cup-like plasma membrane protrusions on dendrites of cultured hippocampal neurons and trigger the activation of TLCN-dependent intracellular signaling cascade including the phosphorylation of ezrin/radixin/moesin actin-binding proteins and recruitment of F-actin and phosphatidylinositol 4,5-bisphosphate for morphological transformation of the dendritic protrusions. These results suggest that the extracellular matrix molecule VN and its neuronal receptor TLCN play a pivotal role in the phosphorylation of ezrin/radixin/moesin proteins and the formation of phagocytic cup-like structures on neuronal dendrites.

  14. Rat Aquaporin-5 Is pH-Gated Induced by Phosphorylation and Is Implicated in Oxidative Stress

    PubMed Central

    Rodrigues, Claudia; Mósca, Andreia Filipa; Martins, Ana Paula; Nobre, Tatiana; Prista, Catarina; Antunes, Fernando; Cipak Gasparovic, Ana; Soveral, Graça

    2016-01-01

    Aquaporin-5 (AQP5) is a membrane water channel widely distributed in human tissues that was found up-regulated in different tumors and considered implicated in carcinogenesis in different organs and systems. Despite its wide distribution pattern and physiological importance, AQP5 short-term regulation was not reported and mechanisms underlying its involvement in cancer are not well defined. In this work, we expressed rat AQP5 in yeast and investigated mechanisms of gating, as well as AQP5’s ability to facilitate H2O2 plasma membrane diffusion. We found that AQP5 can be gated by extracellular pH in a phosphorylation-dependent manner, with higher activity at physiological pH 7.4. Moreover, similar to other mammalian AQPs, AQP5 is able to increase extracellular H2O2 influx and to affect oxidative cell response with dual effects: whereas in acute oxidative stress conditions AQP5 induces an initial higher sensitivity, in chronic stress AQP5 expressing cells show improved cell survival and resistance. Our findings support the involvement of AQP5 in oxidative stress and suggest AQP5 modulation by phosphorylation as a novel tool for therapeutics. PMID:27983600

  15. Aging-induced alterations in gene transcripts and functional activity of mitochondrial oxidative phosphorylation complexes in the heart.

    PubMed

    Preston, Claudia C; Oberlin, Andrew S; Holmuhamedov, Ekhson L; Gupta, Anu; Sagar, Sandeep; Syed, Rashad H Khazi; Siddiqui, Sabeeh A; Raghavakaimal, Sreekumar; Terzic, Andre; Jahangir, Arshad

    2008-06-01

    Aging is associated with progressive decline in energetic reserves compromising cardiac performance and tolerance to injury. Although deviations in mitochondrial functions have been documented in senescent heart, the molecular bases for the decline in energy metabolism are only partially understood. Here, high-throughput transcription profiles of genes coding for mitochondrial proteins in ventricles from adult (6-months) and aged (24-months) rats were compared using microarrays. Out of 614 genes encoding for mitochondrial proteins, 94 were differentially expressed with 95% downregulated in the aged. The majority of changes affected genes coding for proteins involved in oxidative phosphorylation (39), substrate metabolism (14) and tricarboxylic acid cycle (6). Compared to adult, gene expression changes in aged hearts translated into a reduced mitochondrial functional capacity, with decreased NADH-dehydrogenase and F(0)F(1) ATPase complex activities and capacity for oxygen-utilization and ATP synthesis. Expression of genes coding for transcription co-activator factors involved in the regulation of mitochondrial metabolism and biogenesis were downregulated in aged ventricles without reduction in mitochondrial density. Thus, aging induces a selective decline in activities of oxidative phosphorylation complexes I