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Sample records for phosphorylated creb torc2

  1. Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2.

    PubMed

    Du, Wei; Forte, Gabriella M; Smith, Duncan; Petersen, Janni

    2016-03-01

    Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKC(Pck2) expression prevented Gad8-TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress. PMID:26935949

  2. Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2

    PubMed Central

    Du, Wei; Forte, Gabriella M.; Smith, Duncan; Petersen, Janni

    2016-01-01

    Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKCPck2 expression prevented Gad8–TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress. PMID:26935949

  3. Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt.

    PubMed

    Zhang, Jinfang; Xu, Kai; Liu, Pengda; Geng, Yan; Wang, Bin; Gan, Wenjian; Guo, Jianping; Wu, Fei; Chin, Y Rebecca; Berrios, Christian; Lien, Evan C; Toker, Alex; DeCaprio, James A; Sicinski, Piotr; Wei, Wenyi

    2016-06-16

    The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell-cycle progression. However, it remains largely elusive whether the hyper-phosphorylated, non-E2F1-interacting form of Rb has any physiological role. Here we report that hyper-phosphorylated Rb directly binds to and suppresses the function of mTORC2 but not mTORC1. Mechanistically, Rb, but not p107 or p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D or using CDK4/6 inhibitors releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer. PMID:27237051

  4. TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids.

    PubMed

    Muir, Alexander; Ramachandran, Subramaniam; Roelants, Françoise M; Timmons, Garrett; Thorner, Jeremy

    2014-01-01

    Plasma membrane lipid composition must be maintained during growth and under environmental insult. In yeast, signaling mediated by TOR Complex 2 (TORC2)-dependent protein kinase Ypk1 controls lipid abundance and distribution in response to membrane stress. Ypk1, among other actions, alleviates negative regulation of L-serine:palmitoyl-CoA acyltransferase, upregulating production of long-chain base precursors to sphingolipids. To explore other roles for TORC2-Ypk1 signaling in membrane homeostasis, we devised a three-tiered genome-wide screen to identify additional Ypk1 substrates, which pinpointed both catalytic subunits of the ceramide synthase complex. Ypk1-dependent phosphorylation of both proteins increased upon either sphingolipid depletion or heat shock and was important for cell survival. Sphingolipidomics, other biochemical measurements and genetic analysis demonstrated that these modifications of ceramide synthase increased its specific activity and stimulated channeling of long-chain base precursors into sphingolipid end-products. Control at this branch point also prevents accumulation of intermediates that could compromise cell growth by stimulating autophagy. PMID:25279700

  5. TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae.

    PubMed

    Yerlikaya, Seda; Meusburger, Madeleine; Kumari, Romika; Huber, Alexandre; Anrather, Dorothea; Costanzo, Michael; Boone, Charles; Ammerer, Gustav; Baranov, Pavel V; Loewith, Robbie

    2016-01-15

    Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously. PMID:26582391

  6. CREB coactivator CRTC2/TORC2 and its regulator calcineurin crucially mediate follicle-stimulating hormone and transforming growth factor β1 upregulation of steroidogenesis.

    PubMed

    Fang, Wei-Ling; Lee, Ming-Ting; Wu, Leang-Shin; Chen, Yun-Ju; Mason, Jian; Ke, Ferng-Chun; Hwang, Jiuan-Jiuan

    2012-06-01

    In vitro and in vivo studies implicate that follicle-stimulating hormone (FSH) and transforming growth factor β1 (TGFβ1) play crucial physiological roles in regulating ovarian granulosa cell function essential to fertility control in females. FSH induces cAMP and calcium signaling, thereby activating transcription factor CREB to upregulate steroidogenic gene expression, and TGFβ1 greatly enhances FSH-stimulated steroidogenesis. A CREB coactivator CRTC2/TORC2 was identified to function as a cAMP and calcium-sensitive coincidence sensor. This led us to explore the role of CRTC2 and its regulator calcineurin in FSH and TGFβ1-stimulated steroidogenesis. Primary culture of granulosa cells from gonadotropin-primed immature rats was used. Immunoblotting analysis shows that FSH rapidly and transiently induced dephosphorylation/activation of CRTC2, and FSH + TGFβ1 additionally induced late-phase CRTC2 dephosphorylation. Immunofluorescence analysis further confirms FSH ± TGFβ1 promoted CRTC2 nuclear translocation. Using selective inhibitors, we demonstrate that FSH activated CRTC2 in a PKA- and calcineurin-dependent manner, and TGFβ1 acting through its type I receptor (TGFβRI)-modulated FSH action in a calcineurin-mediated and PKA-independent fashion. Next, we investigated the involvement of calcineurin and CRTC2 in FSH and TGFβ1-stimulated steroidogenesis. Calcineurin and TGFβRI inhibitor dramatically reduced the FSH ± TGFβ1-increased progesterone synthesis and protein levels of StAR, P450scc, and 3β-HSD enzyme. Furthermore, chromatin-immunoprecipitation and immunoprecipitation analyses demonstrate that FSH ± TGFβ1 differentially increased CRTC2, CREB, and CBP binding to these steroidogenic genes, and CREB nuclear association with CRTC2 and CBP. In all, this study reveals for the first time that CRTC2 and calcineurin are critical signaling mediators in FSH and TGFβ1-stimulated steroidogenesis in ovarian granulosa cells. PMID:21826657

  7. Metformin and Insulin Suppress Hepatic Gluconeogenesis by Inhibiting cAMP Signaling Through Phosphorylation of CREB Binding Protein (CBP)

    PubMed Central

    He, Ling; Sabet, Amin; Djedjos, Stephen; Miller, Ryan; Sun, Xiaojian; Hussain, Mehboob A.; Radovick, Sally; Wondisford, Fredric E.

    2009-01-01

    SUMMARY Insulin resistance and elevated glucagon levels result in non-suppressible hepatic glucose production and hyperglycemia in patients with type 2 diabetes. The CREB co-activator complex controls transcription of hepatic gluconeogenic enzyme genes. Here we show that both the antidiabetic agent metformin and insulin phosphorylate the transcriptional co-activator CBP at serine 436 via PKCι/λ. This event triggers the dissociation of the CREB-CBP-TORC2 transcription complex and reduces gluconeogenic enzyme gene expression. Mice carrying a germline mutation of this CBP phosphorylation site (S436A) demonstrate resistance to the hypoglycemic effect of both insulin and metformin. Obese, hyperglycemic mice display hepatic insulin resistance, but metformin is still effective in treating the hyperglycemia of these mice since it stimulates CBP phosphorylation by bypassing the block in insulin signaling. PMID:19450513

  8. mTORC2 Phosphorylation of Akt1: A Possible Mechanism for Hydrogen Sulfide-Induced Cardioprotection

    PubMed Central

    Zhou, Yue; Wang, Daying; Gao, Xiufang; Lew, Karsheng; Richards, Arthur Mark; Wang, Peipei

    2014-01-01

    Hydrogen sulfide (H2S) is known to have cardiac protective effects through Akt activation. Akt acts as a ‘central sensor’ for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i) normoxia, (ii) global ischemia and (iii) ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH) release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×103 vs. 10±3×103 mmHg/min, p<0.05) and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05) compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05). Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism. These data

  9. 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. PMID:26739493

  10. PI3K/mTORC2 regulates TGF-β/Activin signalling by modulating Smad2/3 activity via linker phosphorylation

    PubMed Central

    Yu, Jason S. L.; Ramasamy, Thamil Selvee; Murphy, Nick; Holt, Marie K.; Czapiewski, Rafal; Wei, Shi-Khai; Cui, Wei

    2015-01-01

    Crosstalk between the phosphatidylinositol 3-kinase (PI3K) and the transforming growth factor-β signalling pathways play an important role in regulating many cellular functions. However, the molecular mechanisms underpinning this crosstalk remain unclear. Here, we report that PI3K signalling antagonizes the Activin-induced definitive endoderm (DE) differentiation of human embryonic stem cells by attenuating the duration of Smad2/3 activation via the mechanistic target of rapamycin complex 2 (mTORC2). Activation of mTORC2 regulates the phosphorylation of the Smad2/3-T220/T179 linker residue independent of Akt, CDK and Erk activity. This phosphorylation primes receptor-activated Smad2/3 for recruitment of the E3 ubiquitin ligase Nedd4L, which in turn leads to their degradation. Inhibition of PI3K/mTORC2 reduces this phosphorylation and increases the duration of Smad2/3 activity, promoting a more robust mesendoderm and endoderm differentiation. These findings present a new and direct crosstalk mechanism between these two pathways in which mTORC2 functions as a novel and critical mediator. PMID:25998442

  11. PI3K/mTORC2 regulates TGF-β/Activin signalling by modulating Smad2/3 activity via linker phosphorylation.

    PubMed

    Yu, Jason S L; Ramasamy, Thamil Selvee; Murphy, Nick; Holt, Marie K; Czapiewski, Rafal; Wei, Shi-Khai; Cui, Wei

    2015-01-01

    Crosstalk between the phosphatidylinositol 3-kinase (PI3K) and the transforming growth factor-β signalling pathways play an important role in regulating many cellular functions. However, the molecular mechanisms underpinning this crosstalk remain unclear. Here, we report that PI3K signalling antagonizes the Activin-induced definitive endoderm (DE) differentiation of human embryonic stem cells by attenuating the duration of Smad2/3 activation via the mechanistic target of rapamycin complex 2 (mTORC2). Activation of mTORC2 regulates the phosphorylation of the Smad2/3-T220/T179 linker residue independent of Akt, CDK and Erk activity. This phosphorylation primes receptor-activated Smad2/3 for recruitment of the E3 ubiquitin ligase Nedd4L, which in turn leads to their degradation. Inhibition of PI3K/mTORC2 reduces this phosphorylation and increases the duration of Smad2/3 activity, promoting a more robust mesendoderm and endoderm differentiation. These findings present a new and direct crosstalk mechanism between these two pathways in which mTORC2 functions as a novel and critical mediator. PMID:25998442

  12. 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…

  13. Involvement of phosphorylated Apis mellifera CREB in gating a honeybee's behavioral response to an external stimulus.

    PubMed

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

    2016-05-01

    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 abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees' CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus. PMID:27084927

  14. Serine 133 phosphorylation is not required for hippocampal CREB-mediated transcription and behavior

    PubMed Central

    Briand, Lisa A.; Lee, Bridgin G.; Lelay, John; Kaestner, Klaus H.

    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 activation of CREB. However, the importance of phosphorylation for CREB binding to DNA and subsequent gene transcription in vivo is controversial. To definitively address the role of CREB phosphorylation in gene transcription and learning and memory, we derived mutant mice lacking the Ser133 phosphorylation site. These mice exhibit normal CREB-mediated gene transcription for a number of genes implicated in learning and memory processes. Furthermore these mice have no deficits in hippocampus- or striatum-dependent learning. Strikingly, our findings show that CREB phosphorylation at Ser133 is not necessary for CREB binding to CRE sites, CREB-mediated transcription, or CREB-mediated behavioral phenotypes associated with learning and memory. PMID:25593297

  15. Phosphorylation of CREB at Ser-133 induces complex formation with CREB-binding protein via a direct mechanism.

    PubMed Central

    Parker, D; Ferreri, K; Nakajima, T; LaMorte, V J; Evans, R; Koerber, S C; Hoeger, C; Montminy, M R

    1996-01-01

    We have characterized a phosphoserine binding domain in the coactivator CREB-binding protein (CBP) which interacts with the protein kinase A-phosphorylated, and hence activated, form of the cyclic AMP-responsive factor CREB. The CREB binding domain, referred to as KIX, is alpha helical and binds to an unstructured kinase-inducible domain in CREB following phosphorylation of CREB at Ser-133. Phospho-Ser-133 forms direct contacts with residues in KIX, and these contacts are further stabilized by hydrophobic residues in the kinase-inducible domain which flank phospho-Ser-133. Like the src homology 2 (SH2) domains which bind phosphotyrosine-containing peptides, phosphoserine 133 appears to coordinate with a single arginine residue (Arg-600) in KIX which is conserved in the CBP-related protein P300. Since mutagenesis of Arg-600 to Gln severely reduces CREB-CBP complex formation, our results demonstrate that, as in the case of tyrosine kinase pathways, signal transduction through serine/threonine kinase pathways may also require protein interaction motifs which are capable of recognizing phosphorylated amino acids. PMID:8552098

  16. Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines

    PubMed Central

    2012-01-01

    Background Canine hemangiosarcoma (HSA) is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines. Results Seven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs), that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS) in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Ser473, mTOR complex 1 (mTORC1) at Ser2448, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) at Ser65 was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines. Conclusions Our findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in canine HSA formation both

  17. 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…

  18. 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

  19. TORC2 Structure and Function.

    PubMed

    Gaubitz, Christl; Prouteau, Manoel; Kusmider, Beata; Loewith, Robbie

    2016-06-01

    The target of rapamycin (TOR) kinase functions in two multiprotein complexes, TORC1 and TORC2. Although both complexes are evolutionarily conserved, only TORC1 is acutely inhibited by rapamycin. Consequently, only TORC1 signaling is relatively well understood; and, at present, only mammalian TORC1 is a validated drug target, pursued in immunosuppression and oncology. However, the knowledge void surrounding TORC2 is dissipating. Acute inhibition of TORC2 with small molecules is now possible and structural studies of both TORC1 and TORC2 have recently been reported. Here we review these recent advances as well as observations made from tissue-specific mTORC2 knockout mice. Together these studies help define TORC2 structure-function relationships and suggest that mammalian TORC2 may one day also become a bona fide clinical target. PMID:27161823

  20. mTORC2 mediates CXCL12-induced angiogenesis.

    PubMed

    Ziegler, Mary E; Hatch, Michaela M S; Wu, Nan; Muawad, Steven A; Hughes, Christopher C W

    2016-07-01

    The chemokine CXCL12, through its receptor CXCR4, positively regulates angiogenesis by promoting endothelial cell (EC) migration and tube formation. However, the relevant downstream signaling pathways in EC have not been defined. Similarly, the upstream activators of mTORC2 signaling in EC are also poorly defined. Here, we demonstrate for the first time that CXCL12 regulation of angiogenesis requires mTORC2 but not mTORC1. We find that CXCR4 signaling activates mTORC2 as indicated by phosphorylation of serine 473 on Akt and does so through a G-protein- and PI3K-dependent pathway. Significantly, independent disruption of the mTOR complexes by drugs or multiple independent siRNAs reveals that mTORC2, but not mTORC1, is required for microvascular sprouting in a 3D in vitro angiogenesis model. Importantly, in a mouse model, both tumor angiogenesis and tumor volume are significantly reduced only when mTORC2 is inhibited. Finally, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), which is a key regulator of glycolytic flux, is required for microvascular sprouting in vitro, and its expression is reduced in vivo when mTORC2 is targeted. Taken together, these findings identify mTORC2 as a critical signaling nexus downstream of CXCL12/CXCR4 that represents a potential link between mTORC2, metabolic regulation, and angiogenesis. PMID:27106789

  1. 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. PMID:26355639

  2. 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

  3. The DNA methylation inhibitor 5-azacytidine decreases melanin synthesis by inhibiting CREB phosphorylation.

    PubMed

    Shin, Jun Seob; Jeong, Hyo-Soon; Kim, Myo-Kyoung; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Kim, Dong-Seok

    2015-10-01

    Here we examined the effects of a DNA methylation inhibitor, 5-azacytidine, on melanogenesis in Mel-Ab cells. We found that 5-azacytidine decreased the melanin content and tyrosinase activity in these cells in a dose-dependent manner; importantly, 5-azacytidine was not cytotoxic at the concentrations used in these experiments. On the other hand, 5-azacytidine did not affect tyrosinase activity in a cell-free system, indicating that 5-azacytidine is not a direct tyrosinase inhibitor. Instead, 5-azacytidine decreased the protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Thus, we investigated the effects of 5-azacytidine on signal transduction pathways related to melanogenesis. However, 5-azacytidine did not have any effect on either Akt or glycogen synthase kinase 3β (GSK3β) phosphorylation. The phosphorylation of cAMP response element-binding protein (CREB) is well known to regulate MITF expression, thereby also regulating tyrosinase expression. We found that 5-azacytidine decreased the phosphorylation of CREB. Therefore, we propose that 5-azacytidine may decrease melanin synthesis by downregulating MITF and tyrosinase via CREB inactivation. PMID:26601420

  4. Transmembrane Inhibitor of RICTOR/mTORC2 in Hematopoietic Progenitors.

    PubMed

    Lee, Dongjun; Sykes, Stephen M; Kalaitzidis, Demetrios; Lane, Andrew A; Kfoury, Youmna; Raaijmakers, Marc H G P; Wang, Ying-Hua; Armstrong, Scott A; Scadden, David T

    2014-11-11

    Central to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1) or are upstream and regulatory of it (mTORC2). Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2) negatively regulates mTORC2 enzymatic activity, reducing AKT(S473), PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL) model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway. PMID:25418727

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

    PubMed

    Bocarsly, M E; Avena, N M

    2013-09-17

    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 paraventricular nucleus (PVN), or consumption of a HFD, would be associated with a decrease in NAc CREB. In Exp. 1, GAL in the PVN of naïve rats decreased phosphorylated-CREB (pCREB) which is the activated form of CREB, in the NAc compared to saline-injected controls. In Exp. 2, rats fed ad libitum HFD for 4 weeks 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 HFD, thereby providing a potential mechanism underlying behavioral differences seen with addiction-like overconsumption of different types of palatable foods. PMID:23747305

  6. 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...

  7. CIP2A regulates cancer metabolism and CREB phosphorylation in non-small cell lung cancer.

    PubMed

    Peng, Bo; Lei, Ningjing; Chai, Yurong; Chan, Edward K L; Zhang, Jian-Ying

    2015-01-01

    The cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently characterized endogenous inhibitor of the phosphatase activity of protein phosphatase 2A (PP2A), which extends the half-life of oncogenic protein c-myc and promotes in vivo tumor growth. The function of CIP2A in cancer progression is still poorly understood. To uncover the underlying mechanism of CIP2A-mediated cell proliferation, we implemented a two-dimensional electrophoresis (2DE)-based proteomic approach to examine lung cancer cell H1299 with and without CIP2A. We found 47 proteins differentially expressed where 19 proteins were upregulated and 28 proteins were downregulated. These were categorized into functional groups such as metabolism (25%), transcriptional and translational control (23%), and the signaling pathway and protein degradation (20%). On one hand, we validate our proteomic work by measuring the metabolic change. The knockdown of CIP2A decreased the expression of LDH-A as well as the enzymatic activity, accompanying with a decreased lactate production, an increased NADH/NAD+ ratio and ROS production. On the other hand, we found that CIP2A may regulate CREB activity through bioinformatics analysis. Our following experiments showed that, CIP2A positively regulated the phosphorylation of CREB in response to the serum treatment. Therefore, our proteomic study suggested that CIP2A mediates cancer progression through the metabolic pathway and intracellular signaling cascade. PMID:25325377

  8. 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

  9. Scopoletin from Cirsium setidens Increases Melanin Synthesis via CREB Phosphorylation in B16F10 Cells

    PubMed Central

    Ahn, Mi-Ja; Hur, Sun-Jung; Kim, Eun-Hyun; Lee, Seung Hoon; Shin, Jun Seob; Kim, Myo-Kyoung; Uchizono, James A.; Whang, Wan-Kyunn

    2014-01-01

    In this study, we isolated scopoletin from Cirsium setidens Nakai (Compositae) and tested its effects on melanogenesis. Scopoletin was not toxic to cells at concentrations less than 50 µM and increased melanin synthesis in a dose-dependent manner. As melanin synthesis increased, scopoletin stimulated the total tyrosinase activity, the rate-limiting enzyme of melanogenesis. In a cell-free system, however, scopoletin did not increase tyrosinase activity, indicating that scopoletin is not a direct activator of tyrosinase. Furthermore, Western blot analysis showed that scopoletin stimulated the production of microphthalmia-associated transcription factor (MITF) and tyrosinase expression via cAMP response element-binding protein (CREB) phosphorylation in a dose-dependent manner. Based on these results, preclinical and clinical studies are needed to assess the use of scopoletin for the treatment of vitiligo. PMID:25177162

  10. Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation

    PubMed Central

    Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.

    2013-01-01

    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK and phospho-CREB are higher in rapid eye movement (REM) sleep compared to awake mice but are not elevated in non-rapid eye movement (NREM) sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

  11. Increases in cAMP, MAPK activity, and CREB phosphorylation during REM sleep: implications for REM sleep and memory consolidation.

    PubMed

    Luo, Jie; Phan, Trongha X; Yang, Yimei; Garelick, Michael G; Storm, Daniel R

    2013-04-10

    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK), and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Because mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity, and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK, and phospho-CREB are higher in rapid eye movement (REM) sleep compared with awake mice but are not elevated in non-REM sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity, and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

  12. Phosphorylation of CREB, a cyclic AMP responsive element binding protein, contributes partially to lysophosphatidic acid-induced fibroblast cell proliferation

    SciTech Connect

    Kwon, Yong-Jun; Sun, Yuanjie; Kim, Nam-Ho; Huh, Sung-Oh

    2009-03-13

    Lysophospholipids regulate a wide array of biological processes including cell survival and proliferation. In our previous studies, we found that in addition to SRE, CRE is required for maximal c-fos promoter activation triggered by lysophosphatidic acid (LPA). c-fos is an early indicator of various cells into the cell cycle after mitogenic stimulation. However, role of CREB activation in LPA-stimulated proliferation has not been elucidated yet. Here, we investigate how LPA induces proliferation in Rat-2 fibroblast cell via CREB activation. We found that total cell number and BrdU-positive cells were increased by LPA. Moreover, levels of c-fos mRNA and cyclin D1 protein were increased via LPA-induced CREB phosphorylation. Furthermore, LPA-induced Rat-2 cell proliferation was decreased markedly by ERK inhibitor (U0126) and partially by MSK inhibitor (H89). Taken together, these results suggest that CREB activation could partially up-regulate accumulation of cyclin D1 protein level and proliferation of LPA-stimulated Rat-2 fibroblast cells.

  13. Regulation of insulin receptor substrate-1 by mTORC2 (mammalian target of rapamycin complex 2)

    PubMed Central

    DeStefano, Michael A.; Jacinto, Estela

    2016-01-01

    mTOR (mammalian target of rapamycin) responds to the presence of nutrients, energy and growth factors to link cellular metabolism, growth and proliferation. The rapamycin-sensitive mTORC (mTOR complex) 1 activates the translational regulator S6K (S6 kinase), leading to increased protein synthesis in the presence of nutrients. On the other hand, the rapamycin-insensitive mTORC2 responds to the presence of growth factors such as insulin by phosphorylating Akt to promote its maturation and allosteric activation. We recently found that mTORC2 can also regulate insulin signalling at the level of IRS-1 (insulin receptor substrate-1). Whereas mTORC1 promotes IRS-1 serine phosphorylation that is linked to IRS-1 down-regulation, we uncovered that mTORC2 mediates its degradation. In mTORC2-disrupted cells, inactive IRS-1 accumulated despite undergoing phosphorylation at the mTORC1-mediated serine sites. Defective IRS-1 degradation was due to attenuated expression of the CUL7 (Cullin 7) ubiquitin ligase substrate-targeting subunit Fbw8. mTORC2 and Fbw8 co-localize at the membrane where mTORC2 phosphorylates Ser86 to stabilize Fbw8 and promotes its cytosolic localization upon insulin stimulation. Under conditions of chronic insulin exposure, inactive serine-phosphorylated IRS-1 and Fbw8 co-localize to the cytosol where the former becomes ubiquitylated via CUL7/Fbw8. Thus mTORC2 negatively feeds back to IRS-1 via control of Fbw8 stability and localization. Our findings reveal that, in addition to persistent mTORC1 signalling, increased mTORC2 signals can promote insulin resistance due to mTORC2-mediated degradation of IRS-1. PMID:23863152

  14. NKCC1 mediates traumatic brain injury-induced hippocampal neurogenesis through CREB phosphorylation and HIF-1α expression.

    PubMed

    Lu, Kwok-Tung; Huang, Tai-Chun; Wang, Jia-Yi; You, Ya-Shen; Chou, Jian-Liang; Chan, Michael W Y; Wo, Peter Y Y; Amstislavskaya, Tamara G; Tikhonova, Maria A; Yang, Yi-Ling

    2015-08-01

    Traumatic brain injury (TBI) is one of the most prevalent causes of worldwide mortality and morbidity. We previously had evidenced that TBI induced Na-K-2Cl co-transporter (NKCC1) upregulation in hippocampus. Here, we aim to investigate the role of NKCC1 in TBI-induced neurogenesis and the detailed mechanisms. The TBI-associated alternations in the expression of NKCC1, HIF-1α, VEGF, MAPK cascade, and CREB phosphorylation were analyzed by Western blot. TBI-induced neurogenesis was determined by immuno-fluorescence labeling. Chromatin immunoprecipitation was used to elucidate whether HIF-1α would activate VEGF gene after TBI. We found that the level of hippocampal NKCC1 and VEGF began to rise 8 h after TBI, and both of them reached maxima at day 7. Along with the upregulation of NKCC1 and VEGF, MAPK cascade was activated and hippocampal neurogenesis was promoted. Administration of CREB antisense oligonucleotide significantly attenuated the expression of HIF-1α, while HIF-1α antisense oligonucleotide exhibited little effect on the expression of CREB. However, HIF-1α antisense oligonucleotide administration did effectively suppress the expression of VEGF. Our results of the chromosome immunoprecipitation also indicated that HIF-1α could directly act on the VEGF promoter and presumably would elevate the VEGF expression after TBI. All these results have illustrated the correlation between NKCC1 upregulation and TBI-associated neurogenesis. The pathway involves the activation of Raf/MEK/ERK cascade, CREB phosphorylation, and HIF-1α upregulation, and finally leads to the stimulation of VEGF expression and the induction of neurogenesis. PMID:25201604

  15. mTORC2 critically regulates renal potassium handling

    PubMed Central

    Grahammer, Florian; Nesterov, Viatcheslav; Ahmed, Azaz; Steinhardt, Frederic; Sandner, Lukas; Arnold, Frederic; Cordts, Tomke; Negrea, Silvio; Ruegg, Marcus A.; Hall, Michael N.; Walz, Gerd; Korbmacher, Christoph; Artunc, Ferruh; Huber, Tobias B.

    2016-01-01

    The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1) in the kidney has been widely studied; however, mTORC2 function in renal tubules is poorly characterized. Here, we generated mice lacking mTORC2 in the distal tubule (Rictorfl/fl Ksp-Cre mice), which were viable and had no obvious phenotype, except for a 2.5-fold increase in plasma aldosterone. Challenged with a low-Na+ diet, these mice adequately reduced Na+ excretion; however, Rictorfl/fl Ksp-Cre mice rapidly developed hyperkalemia on a high-K+ diet, despite a 10-fold increase in serum aldosterone levels, implying that mTORC2 regulates kaliuresis. Phosphorylation of serum- and glucocorticoid-inducible kinase 1 (SGK1) and PKC-α was absent in Rictorfl/fl Ksp-Cre mice, indicating a functional block in K+ secretion activation via ROMK channels. Indeed, patch-clamp experiments on split-open tubular segments from the transition zone of the late connecting tubule and early cortical collecting duct demonstrated that Ba2+-sensitive apical K+ currents were barely detectable in the majority of Rictorfl/fl Ksp-Cre mice. Conversely, epithelial sodium channel (ENaC) activity was largely preserved, suggesting that the reduced ability to maintain K+ homeostasis is the result of impaired apical K+ conductance and not a reduced electrical driving force for K+ secretion. Thus, these data unravel a vital and nonredundant role of mTORC2 for distal tubular K+ handling. PMID:27043284

  16. mTORC2 critically regulates renal potassium handling.

    PubMed

    Grahammer, Florian; Nesterov, Viatcheslav; Ahmed, Azaz; Steinhardt, Frederic; Sandner, Lukas; Arnold, Frederic; Cordts, Tomke; Negrea, Silvio; Bertog, Marko; Ruegg, Marcus A; Hall, Michael N; Walz, Gerd; Korbmacher, Christoph; Artunc, Ferruh; Huber, Tobias B

    2016-05-01

    The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1) in the kidney has been widely studied; however, mTORC2 function in renal tubules is poorly characterized. Here, we generated mice lacking mTORC2 in the distal tubule (Rictorfl/fl Ksp-Cre mice), which were viable and had no obvious phenotype, except for a 2.5-fold increase in plasma aldosterone. Challenged with a low-Na+ diet, these mice adequately reduced Na+ excretion; however, Rictorfl/fl Ksp-Cre mice rapidly developed hyperkalemia on a high-K+ diet, despite a 10-fold increase in serum aldosterone levels, implying that mTORC2 regulates kaliuresis. Phosphorylation of serum- and glucocorticoid-inducible kinase 1 (SGK1) and PKC-α was absent in Rictorfl/fl Ksp-Cre mice, indicating a functional block in K+ secretion activation via ROMK channels. Indeed, patch-clamp experiments on split-open tubular segments from the transition zone of the late connecting tubule and early cortical collecting duct demonstrated that Ba2+-sensitive apical K+ currents were barely detectable in the majority of Rictorfl/fl Ksp-Cre mice. Conversely, epithelial sodium channel (ENaC) activity was largely preserved, suggesting that the reduced ability to maintain K+ homeostasis is the result of impaired apical K+ conductance and not a reduced electrical driving force for K+ secretion. Thus, these data unravel a vital and nonredundant role of mTORC2 for distal tubular K+ handling. PMID:27043284

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

    PubMed

    Yan, Xiaodong; Liu, Juanfang; 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

  18. 17ß-Estradiol Regulates mTORC2 Sensitivity to Rapamycin in Adaptive Cardiac Remodeling

    PubMed Central

    Kusch, Angelika; Schmidt, Maria; Gürgen, Dennis; Postpieszala, Daniel; Catar, Rusan; Hegner, Björn; Davidson, Merci M.; Mahmoodzadeh, Shokoufeh; Dragun, Duska

    2015-01-01

    Adaptive cardiac remodeling is characterized by enhanced signaling of mTORC2 downstream kinase Akt. In females, 17ß-estradiol (E2), as well as Akt contribute essentially to sex-related premenopausal cardioprotection. Pharmacologic mTOR targeting with rapamycin is increasingly used for various clinical indications, yet burdened with clinical heterogeneity in therapy responses. The drug inhibits mTORC1 and less-so mTORC2. In male rodents, rapamycin decreases maladaptive cardiac hypertrophy whereas it leads to detrimental dilative cardiomyopathy in females. We hypothesized that mTOR inhibition could interfere with 17β-estradiol (E2)-mediated sexual dimorphism and adaptive cell growth and tested responses in murine female hearts and cultured female cardiomyocytes. Under physiological in vivo conditions, rapamycin compromised mTORC2 function only in female, but not in male murine hearts. In cultured female cardiomyocytes, rapamycin impaired simultaneously IGF-1 induced activation of both mTOR signaling branches, mTORC1 and mTORC2 only in presence of E2. Use of specific estrogen receptor (ER)α- and ERβ-agonists indicated involvement of both estrogen receptors (ER) in rapamycin effects on mTORC1 and mTORC2. Classical feedback mechanisms common in tumour cells with upregulation of PI3K signaling were not involved. E2 effect on Akt-pS473 downregulation by rapamycin was independent of ERK as shown by sequential mTOR and MEK-inhibition. Furthermore, regulatory mTORC2 complex defining component rictor phosphorylation at Ser1235, known to interfere with Akt-substrate binding to mTORC2, was not altered. Functionally, rapamycin significantly reduced trophic effect of E2 on cell size. In addition, cardiomyocytes with reduced Akt-pS473 under rapamycin treatment displayed decreased SERCA2A mRNA and protein expression suggesting negative functional consequences on cardiomyocyte contractility. Rictor silencing confirmed regulation of SERCA2A expression by mTORC2 in E2-cultured

  19. PI3K/Akt promotes feedforward mTORC2 activation through IKKα

    PubMed Central

    Dan, Han C.; Antonia, Ricardo J.; Baldwin, Albert S.

    2016-01-01

    The ser-thr Akt plays a critical role in the regulation of cell survival, cell growth and proliferation, as well as energy metabolism and is dysregulated in many cancers. The regulation of Akt activity depends on the phosphorylation at two sites: (i) Thr308 in the activation loop by phosphoinositide-dependent kinase-1 (PDK1) and (ii) Ser473 hydrophobic motif at the carboxyl terminus by a second activity termed PDK2, which is the mTORC2 complex composed of mTOR, rictor, and Sin1. Previously we demonstrated that IKKα, a component of the IKK complex that controls NF-κB activation, participates in the Akt-dependent regulation of mTORC1. Here we have explored a potential involvement of IKKα in controlling Akt activity and whether this may involve mTORC2. The experiments show that IKKα associates with mTORC2 in several cancer cells in a manner dependent on PI3K/Akt activity and that IKKα positively promotes Akt phosphorylation at Ser473 and at Thr308. Moreover, IKKα enhances mTORC2 kinase activity directed to Akt on Ser473 and Akt-mediated phosphorylation of FOXO3a and GSK3β, but not other Akt-associated targets such as TSC2 and PRAS40, indicating the existence of multiple mechanisms of Akt activation in cells. In addition, loss of IKKα suppresses growth factor-induced Akt activation associated with mTORC1 inhibition. These results indicate that IKKα serves as a feedforward regulator of mTORC2 and that IKKα could serve as a key therapeutic target to block mTORC2 and Akt activation in some cancers. PMID:27027448

  20. mTORC2 regulates cardiac response to stress by inhibiting MST1

    PubMed Central

    Sciarretta, Sebastiano; Zhai, Peiyong; Maejima, Yasuhiro; Del Re, Dominic P.; Nagarajan, Narayani; Yee, Derek; Liu, Tong; Magnuson, Mark A.; Volpe, Massimo; Frati, Giacomo; Li, Hong; Sadoshima, Junichi

    2015-01-01

    Summary The mTOR and Hippo pathways have recently emerged as the major signaling transduction cascades regulating organ size and cellular homeostasis. However, direct crosstalk between two pathways is yet to be determined. Here we demonstrate that mTORC2 is a direct negative regulator of the MST1 kinase, a key component of the Hippo pathway. mTORC2 phosphorylates MST1 at serine 438 in the SARAH domain, thereby reducing its homodimerization and activity. We found that Rictor/mTORC2 preserves cardiac structure and function by restraining the activity of MST1 kinase. Cardiac-specific mTORC2 disruption through rictor deletion leads to a marked activation of MST1 that, in turn, promotes cardiac dysfunction and dilation, impairing cardiac growth and adaptation in response to pressure overload. In conclusion, our study demonstrates the existence of a direct crosstalk between mTORC2 and MST1 that is critical for cardiac cell survival and growth. PMID:25843706

  1. Conantokins inhibit NMDAR-dependent calcium influx in developing rat hippocampal neurons in primary culture with resulting effects on CREB phosphorylation

    PubMed Central

    Huang, Luoxiu; Balsara, Rashna D.; Sheng, Zhenyu; Castellino, Francis J.

    2010-01-01

    The effects of conantokin (con)-G, con-R[1-17], and con-T on ion flow through N-methyl-D-aspartate receptor (NMDAR) ion channels were determined in cultured primary rat hippocampal neurons. The potency of con-G diminished, whereas inhibition by con-R[1-17] and con-T did not change, as the neurons matured. Con-G, con-R[1-17], and con-T effectively diminished NMDA-induced Ca2+ influx into the cells. A similar age-dependent decrease in con-G-mediated inhibition of the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) was observed, compared to con-R[1-17] and con-T. The effects of the conantokins on NMDA-induced cAMP response element-binding protein (CREB) phosphorylation in immature (DIV 9) and mature (DIV 16) neurons showed that, at DIV 9, con-G, con-R[1-17], and con-T inhibited NMDA-mediated P-CREB levels, whereas in DIV 16 neurons the conantokins did not inhibit overall levels of NMDA-induced P-CREB. In contrast, P-CREB levels were enhanced through inhibition of the protein phosphatases, PP1 and PP2B (calcineurin). This ability of conantokins to sustain CREB phosphorylation can thus enhance neuronal survival and plasticity. PMID:20600930

  2. mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

    PubMed Central

    Zou, Zhipeng; Chen, Juan; Liu, Anling; Zhou, Xuan; Song, Qiancheng; Jia, Chunhong; Chen, Zhenguo; Lin, Jun; Yang, Cuilan; Li, Ming; Jiang, Yu

    2015-01-01

    Previous studies have reported that mTORC2 promotes cell survival through phosphorylating AKT and enhancing its activity. We reveal another mechanism by which mTORC2 controls apoptosis. Inactivation of mTORC2 promotes binding of CIP2A to PP2A, leading to reduced PP2A activity toward c-Myc serine 62 and, consequently, enhancement of c-Myc phosphorylation and expression. Increased c-Myc activity induces transcription of pri-miR-9-2/miR-9-3p, in turn inhibiting expression of E2F1, a transcriptional factor critical for cancer cell survival and tumor progression, resulting in enhanced apoptosis. In vivo experiments using B cell–specific mTORC2 (rapamycin-insensitive companion of mTOR) deletion mice and a xenograft tumor model confirmed that inactivation of mTORC2 causes up-regulation of c-Myc and miR-9-3p, down-regulation of E2F1, and consequent reduction in cell survival. Conversely, Antagomir-9-3p reversed mTORC1/2 inhibitor–potentiated E2F1 suppression and resultant apoptosis in xenograft tumors. Our in vitro and in vivo findings collectively demonstrate that mTORC2 promotes cell survival by stimulating E2F1 expression through a c-Myc– and miR-9-3p–dependent mechanism. PMID:26459601

  3. Transmembrane Protease Serine 4 Promotes Thyroid Cancer Proliferation via CREB Phosphorylation

    PubMed Central

    Guan, Hongyu; Liang, Weiwei; Liu, Juan; Wei, Guohong; Li, Hai; Xiu, Lingling; Xiao, Haipeng

    2015-01-01

    Background: Transmembrane protease serine 4 (TMPRSS4), one of the type II transmembrane serine proteases (TTSPs), is elevated in various cancers and is associated with multiple malignant phenotypes. However, the expression pattern and biologic significance of TMPRSS4 in thyroid cancer are largely unknown. In this study, we investigated the expression of TMPRSS4 in thyroid cancer and assessed the pro-proliferative role of TMPRSS4 in thyroid cancer. Methods: Immunohistochemistry and real-time reverse transcription-polymerase chain reaction (RT-PCR) assays were performed to assess the expression of TMPRSS4 in thyroid cancer. We evaluated in vitro cell proliferation using MTT, colony formation, anchorage-independent growth, flow cytometry analysis, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. Western blot, real-time RT-PCR, and luciferase assays were conducted to reveal the underlying mechanisms. Results: TMPRSS4 is overexpressed in thyroid cancer and is associated with the grade of malignancy. Depletion of TMPRSS4 in thyroid cancer cells significantly suppressed proliferation. Moreover, the proliferation of thyroid cancer cells with TMPRSS4 overexpression was significantly enhanced. We also show that cyclic adenosine monophosphate response element-binding protein (CREB)-cyclin D1 signaling mediates, at least partially, the role of TMPRSS4 in thyroid cancer cell proliferation. Conclusions: TMPRSS4 is overexpressed in thyroid cancer and TMPRSS4-CREB signaling is needed to sustain thyroid cancer cell proliferation. PMID:25244400

  4. TSC1/TSC2 and Rheb have different effects on TORC1 and TORC2 activity

    PubMed Central

    Yang, Qian; Inoki, Ken; Kim, Eunjung; Guan, Kun-Liang

    2006-01-01

    Target of rapamycin (TOR) plays a central role in cell growth regulation by integrating signals from growth factors, nutrients, and cellular energy levels. TOR forms two distinct physical and functional complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1, which is sensitive to rapamycin, regulates translation and cell growth, whereas TORC2, which is insensitive to rapamycin, regulates cell morphology and cell growth. The Ras homology enriched in brain (Rheb) small GTPase is known to be a key upstream activator of TORC1, although the mechanism of Rheb in TORC1 activation remains to be determined. However, the function of Rheb in the TORC2 regulation has not been elucidated. By measuring Akt and S6K phosphorylation as a functional assay for TORC1 and -2, here, we report that dRheb has an inhibitory effect on dTORC2 activity in Drosophila S2 cells. This negative effect of dRheb on dTORC2 is possibly due to a feedback mechanism involving dTORC1 and dS6K. We also observed that Rheb does not activate TORC2 in human embryonic kidney 293 cells, although it potently stimulates TORC1. Furthermore, tuberous sclerosis complex 1 (TSC1) and TSC2, which are negative regulators of Rheb, have negative and positive effects on TORC1 and -2, respectively. Our observations suggest that TSC1/2 and Rheb have different effects on the activity of TORC1 and -2, further supporting the complexity of TOR regulation. PMID:16627617

  5. Dysregulation of epithelial Na+ absorption induced by inhibition of the kinases TORC1 and TORC2

    PubMed Central

    Mansley, Morag K; Wilson, Stuart M

    2010-01-01

    BACKGROUND AND PURPOSE Although the serum and glucocorticoid-inducible protein kinase 1 (SGK1) appears to be involved in controlling epithelial Na+ absorption, its role in this physiologically important ion transport process is undefined. As SGK1 activity is dependent upon target of rapamycin complex 2 (TORC2)-catalysed phosphorylation of SGK1-Ser422, we have explored the effects of inhibiting TORC2 and/or TORC1 upon the hormonal control of Na+ absorption. EXPERIMENTAL APPROACH Na+ absorption was quantified electrometrically in mouse cortical collecting duct cells (mpkCCD) grown to confluence on permeable membranes. Kinase activities were assessed by monitoring endogenous protein phosphorylation, with or without TORC1/2 inhibitors (TORIN1 and PP242) and the TORC1 inhibitor: rapamycin. KEY RESULTS Inhibition of TORC1/2 (TORIN1, PP242) suppressed basal SGK1 activity, prevented insulin- and dexamethasone-induced SGK1 activation, and caused modest (10–20%) inhibition of basal Na+ absorption and substantial (∼80%) inhibition of insulin/dexamethasone-induced Na+ transport. Inhibition of TORC1 did not impair SGK1 activation or insulin-induced Na+ transport, but did inhibit (∼80%) dexamethasone-induced Na+ absorption. Arginine vasopressin stimulated Na+ absorption via a TORC1/2-independent mechanism. CONCLUSION AND IMPLICATIONS Target of rapamycin complex 2, but not TORC1, is important to SGK1 activation. Signalling via phosphoinositide-3-kinase/TORC2/SGK1 can explain insulin-induced Na+ absorption. TORC2, but not TORC1, is also involved in glucocorticoid-induced SGK1 activation but its role is permissive. Glucocorticoid-induced Na+ transport displayed a requirement for TORC1 activity. Therefore, TORC1 and TORC2 contribute to the regulation of Na+ absorption. Pharmacological manipulation of TORC1/2 signalling may provide novel therapies for Na+-sensitive hypertension. PMID:20735411

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

    PubMed Central

    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. PMID:26313266

  7. 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.

  8. Phosphorylation of ETS Transcription Factor ER81 in a Complex with Its Coactivators CREB-Binding Protein and p300

    PubMed Central

    Papoutsopoulou, Stamatia; Janknecht, Ralf

    2000-01-01

    The ETS protein ER81 is a DNA-binding factor capable of enhancing gene transcription and is implicated in cellular transformation, but presently the mechanisms of its actions are unclear. In this report, ER81 is shown to coimmunoprecipitate with the transcriptional coactivator CREB-binding protein (CBP) and the related p300 protein (together referred to as CBP/p300). Moreover, confocal laser microscopic studies demonstrated that ER81 and p300 colocalized to nuclear speckles. In vitro and in vivo interaction studies revealed that ER81 amino acids 249 to 429, which encompass the ETS DNA-binding domain, are responsible for binding to CBP/p300. However, mutation of a putative protein-protein interaction motif, LXXLL, in the ETS domain of ER81 did not affect interaction with CBP/p300, whereas DNA binding of ER81 was abolished. Furthermore, two regions within CBP, amino acids 451 to 721 and 1891 to 2175, are capable of binding to ER81. Consistent with the physical interaction between ER81 and the coactivators CBP and p300, ER81 transcriptional activity was potentiated by CBP/p300 overexpression. Moreover, an ER81-associated protein kinase activity was enhanced upon p300 overexpression. This protein kinase phosphorylates ER81 on serines 191 and 216, and mutation of these phosphorylation sites increased ER81 transcriptional activity in Mv1Lu cells but not in HeLa cells. Altogether, our data elucidate the mechanism of how ER81 regulates gene transcription, through interaction with the coactivators CBP and p300 and an associated kinase that may cell type specifically modulate the ability of ER81 to activate gene transcription. PMID:10982847

  9. TRIB3 suppresses tumorigenesis by controlling mTORC2/AKT/FOXO signaling

    PubMed Central

    Salazar, María; Lorente, Mar; García-Taboada, Elena; Gómez, Eduardo Pérez; Dávila, David; Zúñiga-García, Patricia; Flores, Juana M; Rodríguez, Antonio; Hegedus, Zoltan; Mosén-Ansorena, David; Aransay, Ana M; Hernández-Tiedra, Sonia; López-Valero, Israel; Quintanilla, Miguel; Sánchez, Cristina; Iovanna, Juan L; Dusetti, Nelson; Guzmán, Manuel; Francis, Sheila E; Carracedo, Arkaitz; Kiss-Toth, Endre; Velasco, Guillermo

    2015-01-01

    In a recent article, we found that Tribbles pseudokinase 3 (TRIB3) plays a tumor suppressor role and that this effect relies on the dysregulation of the phosphorylation of v-akt murine thymoma viral oncogene homolog (AKT) by the mammalian target of rapamycin complex 2 (mTORC2 complex), and the subsequent hyperphosphorylation and inactivation of the transcription factor Forkhead box O3 (FOXO3). PMID:27308456

  10. Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation.

    PubMed

    Morón, José A; Gullapalli, Srinivas; Taylor, Chirisse; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A

    2010-03-01

    Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior. PMID:19956087

  11. Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway*

    PubMed Central

    Cohen, Adiel; Kupiec, Martin; Weisman, Ronit

    2014-01-01

    The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling. PMID:24928510

  12. Neuroprotective effects of 3,6'-disinapoyl sucrose through increased BDNF levels and CREB phosphorylation via the CaMKII and ERK1/2 pathway.

    PubMed

    Hu, Yuan; Liu, Ming-Yue; Liu, Ping; Dong, XianZhe; Boran, Aislyn D W

    2014-08-01

    3,6'-Disinapoyl sucrose (DISS) is an oligosaccharide ester natural product originating from the root of wild Polygala tenuifolia. Our previous reports suggested that DISS can have neuroprotective effects and antidepressive activity in rats, at least in part, by increased expression of cyclic AMP response element (CRE)-binding protein (CREB) and its downstream target protein, brain-derived neurotrophic factor (BDNF). The aim of the present study was to explore the mechanism of DISS-modulated BDNF and CREB expression. In this study, we confirmed its neuroprotective effect by showing that DISS, at concentrations above 30 μM, could promote the neuron cell viability and protected the glutamate and H2O2-induced toxicity in the human neuroblastoma (SH-SY5Y) cell line. DISS treatment also increased acute (from 15 to 30 min) BDNF expression and CREB phosphorylation in a dose-dependent manner. Pharmacological inhibition of mitogen-activated protein kinase 1 (ERK1/2), CaMKII, and Trk (with U0126, KN93, or K252a, respectively) partially attenuated the stimulatory effect of DISS on phospho-CREB and BDNF expression; however, it was not inhibited by pharmacological inhibition of PKA or PI3K (with H89 and LY294002, respectively). The results are consistent with the effects of DISS on CRE-directed gene transcription, as U0126 and KN-93 treatment also blocked the DISS-induced expression of the CRE-luciferase reporter gene. The results from the present study suggest that DISS-mediated regulation of BDNF gene expression is associated with CREB-mediated transcription of BDNF and upstream activation of ERK1/2 and CaMKII. Finally, DISS may exert neuroprotective and antidepressant effects through these signaling pathways in neuronal cells. PMID:24488601

  13. Negative regulation of the FOXO3a transcription factor by mTORC2 induces a pro-survival response following exposure to ultraviolet-B irradiation.

    PubMed

    Feehan, Robert P; Shantz, Lisa M

    2016-08-01

    Exposure to ultraviolet-B (UVB) irradiation, the principal cause of non-melanoma skin cancer (NMSC), activates both the rapamycin-sensitive mammalian target of rapamycin complex 1 (mTORC1) and the rapamycin-resistant mTORC2. We have previously reported that UVB-induced keratinocyte survival is dependent on mTORC2, though the specific mechanism is not well understood. FOXO3a is an important transcription factor involved in regulating cell survival. The activity of FOXO3a is reduced as a result of protein kinase B (AKT/PKB) activation, which is downstream of mTORC2; however, the specific function of FOXO3a during UVB-induced apoptosis is unclear. In this study, we establish that in cells with wild-type mTORC2 activity, FOXO3a is quickly phosphorylated in response to UVB and sequestered in the cytoplasm. In contrast, loss of mTORC2 causes FOXO3a to be localized to the nucleus and sensitizes cells to UVB-induced apoptosis. Furthermore, this sensitization is rescued by knockdown of FOXO3a. Taken together, these studies provide strong evidence that inhibition of mTORC2 enhances UVB-induced apoptosis in a FOXO3a-dependent manner, and suggest that FOXO3a activation by mTORC2 inhibitors may be a valuable chemopreventive target in NMSC. PMID:27058291

  14. Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels.

    PubMed

    Williams, Claire M; El Mohsen, Manal Abd; Vauzour, David; Rendeiro, Catarina; Butler, Laurie T; Ellis, Judi A; Whiteman, Matthew; Spencer, Jeremy P E

    2008-08-01

    Phytochemical-rich foods have been shown to be effective at reversing age-related deficits in memory in both animals and humans. We show that a supplementation with a blueberry diet (2% w/w) for 12 weeks improves the performance of aged animals in spatial working memory tasks. This improvement emerged within 3 weeks and persisted for the remainder of the testing period. Memory performance correlated well with the activation of cAMP-response element-binding protein (CREB) and increases in both pro- and mature levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. Changes in CREB and BDNF in aged and blueberry-supplemented animals were accompanied by increases in the phosphorylation state of extracellular signal-related kinase (ERK1/2), rather than that of calcium calmodulin kinase (CaMKII and CaMKIV) or protein kinase A. Furthermore, age and blueberry supplementation were linked to changes in the activation state of Akt, mTOR, and the levels of Arc/Arg3.1 in the hippocampus, suggesting that pathways involved in de novo protein synthesis may be involved. Although causal relationships cannot be made among supplementation, behavior, and biochemical parameters, the measurement of anthocyanins and flavanols in the brain following blueberry supplementation may indicate that changes in spatial working memory in aged animals are linked to the effects of flavonoids on the ERK-CREB-BDNF pathway. PMID:18457678

  15. Deletion of dopamine D1 and D3 receptors differentially affects spontaneous behaviour and cocaine-induced locomotor activity, reward and CREB phosphorylation.

    PubMed

    Karasinska, Joanna M; George, Susan R; Cheng, Regina; O'Dowd, Brian F

    2005-10-01

    Co-localization of dopamine D1 and D3 receptors in striatal neurons suggests that these two receptors interact at a cellular level in mediating dopaminergic function including psychostimulant-induced behaviour. To study D1 and D3 receptor interactions in cocaine-mediated effects, cocaine-induced locomotion and reward in mice lacking either D1, D3 or both receptors were analysed. Spontaneous locomotor activity was increased in D1-/- and D1-/-D3-/- mice and D1-/-D3-/- mice did not exhibit habituation of spontaneous rearing activity. Cocaine (20 mg/kg) increased locomotor activity in wild-type and D3-/- mice, failed to stimulate activity in D1-/- mice and reduced activity in D1-/-D3-/- mice. In the conditioned place preference, all groups exhibited reward at 5, 10 and 20 mg/kg of cocaine. D1-/-D3-/- mice did not demonstrate preference at 2.5 mg/kg of cocaine although preference was observed in wild-type, D1-/- and D3-/- mice. The transcription factor cAMP-responsive element binding protein (CREB) is activated by phosphorylation in striatal regions following dopamine receptor activation. Striatal pCREB levels following acute cocaine were increased in wild-type and D3-/- mice and decreased in D1-/- and D1-/-D3-/- mice. After repeated administration of 2.5 mg/kg of cocaine, D1-/- mice had lower pCREB levels in caudate-putamen and nucleus accumbens. Our findings suggest that, although spontaneous and cocaine-induced horizontal activity depended mainly on the presence of the D1 receptor, there may be crosstalk between D1 and D3 receptors in rearing habituation and the perception of cocaine reward at low doses of the drug. Furthermore, alterations in pCREB levels were associated with changes in cocaine-induced locomotor activity but not reward. PMID:16197514

  16. Dynamic Adipocyte Phosphoproteome Reveals that Akt Directly Regulates mTORC2

    PubMed Central

    Humphrey, Sean J.; Yang, Guang; Yang, Pengyi; Fazakerley, Daniel J.; Stöckli, Jacqueline; Yang, Jean Y.; James, David E.

    2013-01-01

    Summary A major challenge of the post-genomics era is to define the connectivity of protein phosphorylation networks. Here, we quantitatively delineate the insulin signaling network in adipocytes by high-resolution mass spectrometry-based proteomics. These data reveal the complexity of intracellular protein phosphorylation. We identified 37,248 phosphorylation sites on 5,705 proteins in this single-cell type, with approximately 15% responding to insulin. We integrated these large-scale phosphoproteomics data using a machine learning approach to predict physiological substrates of several diverse insulin-regulated kinases. This led to the identification of an Akt substrate, SIN1, a core component of the mTORC2 complex. The phosphorylation of SIN1 by Akt was found to regulate mTORC2 activity in response to growth factors, revealing topological insights into the Akt/mTOR signaling network. The dynamic phosphoproteome described here contains numerous phosphorylation sites on proteins involved in diverse molecular functions and should serve as a useful functional resource for cell biologists. PMID:23684622

  17. Phospholipase D regulates myogenic differentiation through the activation of both mTORC1 and mTORC2 complexes.

    PubMed

    Jaafar, Rami; Zeiller, Caroline; Pirola, Luciano; Di Grazia, Antonio; Naro, Fabio; Vidal, Hubert; Lefai, Etienne; Némoz, Georges

    2011-06-24

    How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis. PMID:21525000

  18. TORC2-a new player in genome stability.

    PubMed

    Weisman, Ronit; Cohen, Adiel; Gasser, Susan M

    2014-01-01

    The inhibition of the central growth regulatory kinase TOR, which participates in two complexes, TORC1 and TORC2, has been a focus of metabolic and cancer studies for many years. Most studies have dealt with TORC1, the canonical target of rapamycin, and the role of this complex in autophagy, protein synthesis, and cell growth control. Recent work on TORC2 in budding and fission yeast species points to a conserved role of this lesser-known TOR complex in the survival of DNA damage. In budding yeast, TORC2 controls lipid biosynthesis and actin cytoskeleton through downstream AGC kinases, which are now, surprisingly, implicated in the survival of oxidative DNA damage. Preliminary data from mTORC2 modulation in cancer cells suggest that an extension to human chemotherapy is worth exploring. PMID:24992933

  19. TORC2—a new player in genome stability

    PubMed Central

    Weisman, Ronit; Cohen, Adiel; Gasser, Susan M

    2014-01-01

    The inhibition of the central growth regulatory kinase TOR, which participates in two complexes, TORC1 and TORC2, has been a focus of metabolic and cancer studies for many years. Most studies have dealt with TORC1, the canonical target of rapamycin, and the role of this complex in autophagy, protein synthesis, and cell growth control. Recent work on TORC2 in budding and fission yeast species points to a conserved role of this lesser-known TOR complex in the survival of DNA damage. In budding yeast, TORC2 controls lipid biosynthesis and actin cytoskeleton through downstream AGC kinases, which are now, surprisingly, implicated in the survival of oxidative DNA damage. Preliminary data from mTORC2 modulation in cancer cells suggest that an extension to human chemotherapy is worth exploring. PMID:24992933

  20. Basal mTORC2 activity and expression of its components display diurnal variation in mouse perivascular adipose tissue.

    PubMed

    Drägert, Katja; Bhattacharya, Indranil; Hall, Michael N; Humar, Rok; Battegay, Edouard; Haas, Elvira

    2016-04-22

    In adipose tissue mTOR complex 2 (mTORC2) contributes to the regulation of glucose/lipid metabolism and inflammatory molecule expression. Both processes display diurnal variations during the course of the day. RICTOR and mSIN1 are unique and essential components of mTORC2, which is activated by growth factors including insulin. To assess whether mTORC2 components display diurnal variations, we analyzed steady state mRNA expression levels of Rictor, mSin1, and mTor in various adipose tissues during a 24 h period. Diurnally regulated expression of Rictor was detected in brown adipose tissues displaying highest mRNA expression levels at the beginning of the 12 h light period (zeitgeber time 2, ZT2). Gene expression patterns of mSin1 and mTor displayed a similar diurnal regulation as Rictor in PVAT while smaller changes were detected for these genes in aorta during the course of the day. Basal mTORC2 activity was measured by phosphorylation of protein kinase C (PKC) α at serine 657 was higher at ZT14 as compared with ZT2 in PVAT. In line, gene expression of inflammatory molecules nitric oxide synthase 2 and tumor necrosis factor α was lower at ZT 14 compared to ZT2. Our findings provide evidence for a diurnal regulation of expression of mTORC2 components and activity. Hence, mTORC2 is possibly an integral part of diurnally regulated signaling pathways in PVAT and possibly in other adipose tissues. PMID:27016480

  1. mTORC2/Rictor Signaling Disrupts Dopamine-Dependent Behaviors via Defects in Striatal Dopamine Neurotransmission

    PubMed Central

    Dadalko, Olga I.; Siuta, Michael; Poe, Amanda; Erreger, Kevin; Matthies, Heinrich J.G.; Niswender, Kevin

    2015-01-01

    Disrupted neuronal protein kinase B (Akt) signaling has been associated with dopamine (DA)-related neuropsychiatric disorders, including schizophrenia, a devastating mental illness. We hypothesize that proper DA neurotransmission is therefore dependent upon intact neuronal Akt function. Akt is activated by phosphorylation of two key residues: Thr308 and Ser473. Blunted Akt phosphorylation at Ser473 (pAkt-473) has been observed in lymphocytes and postmortem brains of schizophrenia patients, and psychosis-prone normal individuals. Mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multiprotein complex that is responsible for phosphorylation of Akt at Ser473 (pAkt-473). We demonstrate that mice with disrupted mTORC2 signaling in brain exhibit altered striatal DA-dependent behaviors, such as increased basal locomotion, stereotypic counts, and exaggerated response to the psychomotor effects of amphetamine (AMPH). Combining in vivo and ex vivo pharmacological, electrophysiological, and biochemical techniques, we demonstrate that the changes in striatal DA neurotransmission and associated behaviors are caused, at least in part, by elevated D2 DA receptor (D2R) expression and upregulated ERK1/2 activation. Haloperidol, a typical antipsychotic and D2R blocker, reduced AMPH hypersensitivity and elevated pERK1/2 to the levels of control animals. By viral gene delivery, we downregulated mTORC2 solely in the dorsal striatum of adult wild-type mice, demonstrating that striatal mTORC2 regulates AMPH-stimulated behaviors. Our findings implicate mTORC2 signaling as a novel pathway regulating striatal DA tone and D2R signaling. PMID:26063917

  2. Rictor/mTORC2 Pathway in Oocytes Regulates Folliculogenesis, and Its Inactivation Causes Premature Ovarian Failure*

    PubMed Central

    Chen, Zhenguo; Kang, Xiangjin; Wang, Liping; Dong, Heling; Wang, Caixia; Xiong, Zhi; Zhao, Wanlu; Jia, Chunhong; Lin, Jun; Zhang, Wen; Yuan, Weiping; Zhong, Mei; Du, Hongzi; Bai, Xiaochun

    2015-01-01

    Molecular basis of ovarian folliculogenesis and etiopathogenesis of premature ovarian failure (POF), a common cause of infertility in women, are not fully understood. Mechanistic target of rapamycin complex 2 (mTORC2) is emerging as a central regulator of cell metabolism, proliferation, and survival. However, its role in folliculogenesis and POF has not been reported. Here, we showed that the signaling activity of mTORC2 is inhibited in a 4-vinylcyclohexene diepoxide (VCD)-induced POF mouse model. Notably, mice with oocyte-specific ablation of Rictor, a key component of mTORC2, demonstrated POF phenotypes, including massive follicular death, excessive loss of functional ovarian follicles, abnormal gonadal hormone secretion, and consequently, secondary subfertility in conditional knock-out (cKO) mice. Furthermore, reduced levels of Ser-473-phosphorylated Akt and Ser-253-phosphorylated Foxo3a and elevated pro-apoptotic proteins, Bad, Bax, and cleaved poly ADP-ribose polymerase (PARP), were observed in cKO mice, replicating the signaling alterations in 4-VCD-treated ovaries. These results indicate a critical role of the Rictor/mTORC2/Akt/Foxo3a pro-survival signaling axis in folliculogenesis. Interestingly, loss of maternal Rictor did not cause obvious developmental defects in embryos or placentas from cKO mice, suggesting that maternal Rictor is dispensable for preimplantation embryonic development. Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis, follicle survival, and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POF. PMID:25564616

  3. Rictor/mTORC2 pathway in oocytes regulates folliculogenesis, and its inactivation causes premature ovarian failure.

    PubMed

    Chen, Zhenguo; Kang, Xiangjin; Wang, Liping; Dong, Heling; Wang, Caixia; Xiong, Zhi; Zhao, Wanlu; Jia, Chunhong; Lin, Jun; Zhang, Wen; Yuan, Weiping; Zhong, Mei; Du, Hongzi; Bai, Xiaochun

    2015-03-01

    Molecular basis of ovarian folliculogenesis and etiopathogenesis of premature ovarian failure (POF), a common cause of infertility in women, are not fully understood. Mechanistic target of rapamycin complex 2 (mTORC2) is emerging as a central regulator of cell metabolism, proliferation, and survival. However, its role in folliculogenesis and POF has not been reported. Here, we showed that the signaling activity of mTORC2 is inhibited in a 4-vinylcyclohexene diepoxide (VCD)-induced POF mouse model. Notably, mice with oocyte-specific ablation of Rictor, a key component of mTORC2, demonstrated POF phenotypes, including massive follicular death, excessive loss of functional ovarian follicles, abnormal gonadal hormone secretion, and consequently, secondary subfertility in conditional knock-out (cKO) mice. Furthermore, reduced levels of Ser-473-phosphorylated Akt and Ser-253-phosphorylated Foxo3a and elevated pro-apoptotic proteins, Bad, Bax, and cleaved poly ADP-ribose polymerase (PARP), were observed in cKO mice, replicating the signaling alterations in 4-VCD-treated ovaries. These results indicate a critical role of the Rictor/mTORC2/Akt/Foxo3a pro-survival signaling axis in folliculogenesis. Interestingly, loss of maternal Rictor did not cause obvious developmental defects in embryos or placentas from cKO mice, suggesting that maternal Rictor is dispensable for preimplantation embryonic development. Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis, follicle survival, and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POF. PMID:25564616

  4. PKC-α-dependent augmentation of cAMP and CREB phosphorylation mediates the angiotensin II stimulation of renin in the collecting duct.

    PubMed

    Gonzalez, Alexis A; Liu, Liu; Lara, Lucienne S; Bourgeois, Camille R T; Ibaceta-Gonzalez, Cristobal; Salinas-Parra, Nicolas; Gogulamudi, Venkateswara R; Seth, Dale M; Prieto, Minolfa C

    2015-11-15

    In contrast to the negative feedback of angiotensin II (ANG II) on juxtaglomerular renin, ANG II stimulates renin in the principal cells of the collecting duct (CD) in rats and mice via ANG II type 1 (AT1R) receptor, independently of blood pressure. In vitro data indicate that CD renin is augmented by AT1R activation through protein kinase C (PKC), but the exact mechanisms are unknown. We hypothesize that ANG II stimulates CD renin synthesis through AT1R via PKC and the subsequent activation of cAMP/PKA/CREB pathway. In M-1 cells, ANG II increased cAMP, renin mRNA (3.5-fold), prorenin, and renin proteins, as well as renin activity in culture media (2-fold). These effects were prevented by PKC inhibition with calphostin C, PKC-α dominant negative, and by PKA inhibition. Forskolin-induced increases in cAMP and renin expression were prevented by calphostin C. PKC inhibition and Ca2+ depletion impaired ANG II-mediated CREB phosphorylation and upregulation of renin. Adenylate cyclase 6 (AC) siRNA remarkably attenuated the ANG II-dependent upregulation of renin mRNA. Physiological activation of AC with vasopressin increased renin expression in M-1 cells. The results suggest that the ANG II-dependent upregulation of renin in the CD depends on PKC-α, which allows the augmentation of cAMP production and activation of PKA/CREB pathway via AC6. This study defines the intracellular signaling pathway involved in the ANG II-mediated stimulation of renin in the CD. This is a novel mechanism responsible for the regulation of local renin-angiotensin system in the distal nephron. PMID:26268270

  5. Phosphoinositide-Dependent Kinase 1 and mTORC2 Synergistically Maintain Postnatal Heart Growth and Heart Function in Mice

    PubMed Central

    Zhao, Xia; Lu, Shuangshuang; Nie, Junwei; Hu, Xiaoshan; Luo, Wen; Wu, Xiangqi; Liu, Hailang; Feng, Qiuting; Chang, Zai; Liu, Yaoqiu; Cao, Yunshan; Sun, Haixiang; Li, Xinli; Hu, Yali

    2014-01-01

    The protein kinase Akt plays a critical role in heart function and is activated by phosphorylation of threonine 308 (T308) and serine 473 (S473). While phosphoinositide-dependent kinase 1 (PDK1) is responsible for Akt T308 phosphorylation, the identities of the kinases for Akt S473 phosphorylation in the heart remain controversial. Here, we disrupted mTOR complex 2 (mTORC2) through deletion of Rictor in the heart and found normal heart growth and function. Rictor deletion caused significant reduction of Akt S473 phosphorylation but enhanced Akt T308 phosphorylation, suggesting that a high level of Akt T308 phosphorylation maintains Akt activity and heart function. Deletion of Pdk1 in the heart caused significantly enhanced Akt S473 phosphorylation that was suppressed by removal of Rictor, leading to worsened dilated cardiomyopathy (DCM) and accelerated heart failure in Pdk1-deficient mice. In addition, we found that increasing Akt S473 phosphorylation through deletion of Pten or chemical inhibition of PTEN reversed DCM and heart failure in Pdk1-deficient mice. Investigation of heart samples from human DCM patients revealed changes similar to those in the mouse models. These results demonstrated that PDK1 and mTORC2 synergistically promote postnatal heart growth and maintain heart function in postnatal mice. PMID:24662050

  6. PTEN negatively regulates mTORC2 formation and signaling in grade IV glioma via Rictor hyperphosphorylation at Thr1135 and direct the mode of action of an mTORC1/2 inhibitor.

    PubMed

    Bhattacharya, K; Maiti, S; Mandal, C

    2016-01-01

    To investigate the role of PTEN (phosphatase and tensin homolog) in mammalian target of rapamycin complex 2 (mTORC2) signaling in glioblastoma multiforme (GBM), we found higher activation of mTORC2 in PTEN(mu) cells, as evidenced by enhanced phosphorylation of mTOR (Ser2481), AKT (Ser473) and glycogen synthase kinase 3 beta (GSK3β) (Ser9) as compared with PTEN(wt) cells. In addition, PTEN(wt) cells upon PTEN depletion showed mTORC2 activation. The reduced mTORC2 signaling in PTEN(wt) cells was related to higher Rictor phosphorylation at Thr1135 residue. Phosphorylation of Rictor at Thr1135 inhibited its association with mTORC and thus there was a reduction in mTORC2 complex formation. In addition, PTEN(wt) cells expressing mutated Rictor in which Thr1135 was substituted with alanine, showed enhanced mTORC2 formation and signaling. This enhanced mTORC2 signaling promoted inactivation of GSK3β. Thus, we established the reciprocal activation of mTORC2 and GSK3β in GBM. To the best of our knowledge, this is the first report describing role of PTEN in mTORC2 formation by promoting Rictor phosphorylation (Thr1135) in GBM. Furthermore, the drug sensitivity of mTORC2 was evaluated. A newly identified carbazole alkaloid, mahanine, showed cytotoxicity in both PTEN(mu) and PTEN(wt) cells. It inhibited both mTORC1/2 and AKT completely in PTEN(mu) cells, whereas it inhibited only mTORC1 in PTEN(wt) cells. Cytotoxity and AKT-inhibitory activity of the mTORC1/2 inhibitor was increased either by depleting PTEN or in combination with phosphatidylinositol 3 kinase inhibitors in PTEN(wt) cells. In contrast, depletion of Rictor decreased the cytotoxicity of the mTORC1/2 inhibitor in PTEN(mu) cells. Thus, PTEN has an important role in mTORC2 formation and also influences the effectiveness of an mTORC1/2 inhibitor in GBM. PMID:27239959

  7. mTORC2 Puts Its Shoulder to Krebs' Wheel.

    PubMed

    Arriola Apelo, Sebastian I; Lamming, Dudley W

    2016-09-01

    In this issue of Molecular Cell, Moloughney et al. (2016) find that mTORC2 responds to falling levels of glucose and glutamine catabolites, promoting glutaminolysis and preserving the TCA cycle and hexosamine biosynthesis. PMID:27588599

  8. Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

    PubMed

    Li, P; Rial, D; Canas, P M; Yoo, J-H; Li, W; Zhou, X; Wang, Y; van Westen, G J P; Payen, M-P; Augusto, E; Gonçalves, N; Tomé, A R; Li, Z; Wu, Z; Hou, X; Zhou, Y; IJzerman, A P; PIJzerman, Ad; Boyden, E S; Cunha, R A; Qu, J; Chen, J-F

    2015-11-01

    Human and animal studies have converged to suggest that caffeine consumption prevents memory deficits in aging and Alzheimer's disease through the antagonism of adenosine A2A receptors (A2ARs). To test if A2AR activation in the hippocampus is actually sufficient to impair memory function and to begin elucidating the intracellular pathways operated by A2AR, we have developed a chimeric rhodopsin-A2AR protein (optoA2AR), which retains the extracellular and transmembrane domains of rhodopsin (conferring light responsiveness and eliminating adenosine-binding pockets) fused to the intracellular loop of A2AR to confer specific A2AR signaling. The specificity of the optoA2AR signaling was confirmed by light-induced selective enhancement of cAMP and phospho-mitogen-activated protein kinase (p-MAPK) (but not cGMP) levels in human embryonic kidney 293 (HEK293) cells, which was abolished by a point mutation at the C terminal of A2AR. Supporting its physiological relevance, optoA2AR activation and the A2AR agonist CGS21680 produced similar activation of cAMP and p-MAPK signaling in HEK293 cells, of p-MAPK in the nucleus accumbens and of c-Fos/phosphorylated-CREB (p-CREB) in the hippocampus, and similarly enhanced long-term potentiation in the hippocampus. Remarkably, optoA2AR activation triggered a preferential p-CREB signaling in the hippocampus and impaired spatial memory performance, while optoA2AR activation in the nucleus accumbens triggered MAPK signaling and modulated locomotor activity. This shows that the recruitment of intracellular A2AR signaling in the hippocampus is sufficient to trigger memory dysfunction. Furthermore, the demonstration that the biased A2AR signaling and functions depend on intracellular A2AR loops prompts the possibility of targeting the intracellular A2AR-interacting partners to selectively control different neuropsychiatric behaviors. PMID:25687775

  9. The effects of ginsenoside Rg1 on chronic stress induced depression-like behaviors, BDNF expression and the phosphorylation of PKA and CREB in rats.

    PubMed

    Liu, Z; Qi, Y; Cheng, Z; Zhu, X; Fan, C; Yu, S Y

    2016-05-13

    Depression is a common neuropsychiatric disorder which has been associated with a wide range of structural and functional changes within specific brain regions. Ginsenoside Rg1 has been shown to exert a number of neuroprotective effects as demonstrated in various in vivo and in vitro studies. However, little information is available regarding the site and mechanisms of ginsenoside Rg1 in promoting antidepressant effects. The present study aimed to investigate the neuroprotective and ameliorating effects of ginsenoside Rg1 on depression-like behavior as induced by chronic unpredictable mild stress (CUMS). The results showed that CUMS was effective in producing depression-like behaviors in rats as indicated by decreased responses in sucrose preference and forced swim tests which were associated with ultrastructural changes in neurons within the amygdala. Moreover, levels of PKA and CREB phosphorylation and the expression of brain-derived neurotrophic factor (BDNF) were decreased in the amygdala of CUMS rats. Remarkably, chronic ginsenoside Rg1 (40mg/kg, i.p., 5weeks) treatment significantly ameliorated these behavioral and biochemical alterations associated with CUMS-induced depression. Taken together, the results of the present study demonstrate that ginsenoside Rg1 exhibits antidepressant-like effects against CUMS-induced depression. This amelioration of depression-like behaviors by ginsenoside Rg1 appears to be mediated, at least in part, by a CREB-regulated increase of BDNF expression in the amygdala of rats. Therefore, these findings reveal the therapeutic potential of ginsenoside Rg1 for use in clinical trials in the treatment of depression. PMID:26926964

  10. Sestrin 3 Protein Enhances Hepatic Insulin Sensitivity by Direct Activation of the mTORC2-Akt Signaling

    PubMed Central

    Tao, Rongya; Xiong, Xiwen; Liangpunsakul, Suthat

    2015-01-01

    Sestrin proteins have been implicated in multiple biological processes including resistance to oxidative and genotoxic stresses, protection against aging-related pathologies, and promotion of metabolic homeostasis; however, the underlying mechanisms are incompletely understood. Some evidence suggests that sestrins may inhibit mTORC1 (mechanistic target of rapamycin complex 1) through inhibition of RagA/B GTPases or activation of AMPK; however, whether sestrins are also involved in mTORC2 regulation and function is unclear. To investigate the functions and mechanisms of Sestrin 3 (Sesn3), we generated Sesn3 liver-specific transgenic and knockout mice. Our data show that Sesn3 liver-specific knockout mice exhibit insulin resistance and glucose intolerance, and Sesn3 transgenic mice were protected against insulin resistance induced by a high-fat diet. Using AMPK liver-specific knockout mice, we demonstrate that the Sesn3 insulin-sensitizing effect is largely independent of AMPK. Biochemical analysis reveals that Sesn3 interacts with and activates mTORC2 and subsequently stimulates Akt phosphorylation at Ser473. These findings suggest that Sesn3 can activate Akt via mTORC2 to regulate hepatic insulin sensitivity and glucose metabolism. PMID:25377878

  11. mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR.

    PubMed

    Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

    2016-01-01

    Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation. PMID:26584640

  12. Mechanistic Target of Rapamycin Complex 1 (mTORC1) and mTORC2 as Key Signaling Intermediates in Mesenchymal Cell Activation.

    PubMed

    Walker, Natalie M; Belloli, Elizabeth A; Stuckey, Linda; Chan, Kevin M; Lin, Jules; Lynch, William; Chang, Andrew; Mazzoni, Serina M; Fingar, Diane C; Lama, Vibha N

    2016-03-18

    Fibrotic diseases display mesenchymal cell (MC) activation with pathologic deposition of matrix proteins such as collagen. Here we investigate the role of mTOR complex 1 (mTORC1) and mTORC2 in regulating MC collagen expression, a hallmark of fibrotic disease. Relative to normal MCs (non-Fib MCs), MCs derived from fibrotic human lung allografts (Fib-MCs) demonstrated increased phosphoinositide-3kinase (PI3K) dependent activation of both mTORC1 and mTORC2, as measured by increased phosphorylation of S6K1 and 4E-BP1 (mTORC1 substrates) and AKT (an mTORC2 substrate). Dual ATP-competitive TORC1/2 inhibitor AZD8055, in contrast to allosteric mTORC1-specific inhibitor rapamycin, strongly inhibited 4E-BP1 phosphorylation and collagen I expression in Fib-MCs. In non-Fib MCs, increased mTORC1 signaling was shown to augment collagen I expression. mTORC1/4E-BP1 pathway was identified as an important driver of collagen I expression in Fib-MCs in experiments utilizing raptor gene silencing and overexpression of dominant-inhibitory 4E-BP1. Furthermore, siRNA-mediated knockdown of rictor, an mTORC2 partner protein, reduced mTORC1 substrate phosphorylation and collagen expression in Fib-, but not non-Fib MCs, revealing a dependence of mTORC1 signaling on mTORC2 function in activated MCs. Together these studies suggest a novel paradigm where fibrotic activation in MCs increases PI3K dependent mTORC1 and mTORC2 signaling and leads to increased collagen I expression via the mTORC1-dependent 4E-BP1/eIF4E pathway. These data provide rationale for targeting specific components of mTORC pathways in fibrotic states and underscore the need to further delineate mTORC2 signaling in activated cell states. PMID:26755732

  13. 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

    2016-09-01

    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. PMID:27461790

  14. Calpain-2 activates Akt via TGF-β1-mTORC2 pathway in pulmonary artery smooth muscle cells.

    PubMed

    Abeyrathna, Prasanna; Kovacs, Laszlo; Han, Weihong; Su, Yunchao

    2016-07-01

    Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 small interfering RNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308), as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable-specific transforming growth factor (TGF)-β receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down activin receptor-like kinase-5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF-β1 using a cell-impermeable TGF-β1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Furthermore, inhibition of mammalian target of rapamycin complex 2 (mTORC2) by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 upregulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGF-β1/mTORC2 mechanism. PMID:27099352

  15. Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress.

    PubMed

    Muir, Alexander; Roelants, Françoise M; Timmons, Garrett; Leskoske, Kristin L; Thorner, Jeremy

    2015-01-01

    In eukaryotes, exposure to hypertonic conditions activates a MAPK (Hog1 in Saccharomyces cerevisiae and ortholog p38 in human cells). In yeast, intracellular glycerol accumulates to counterbalance the high external osmolarity. To prevent glycerol efflux, Hog1 action impedes the function of the aquaglyceroporin Fps1, in part, by displacing channel co-activators (Rgc1/2). However, Fps1 closes upon hyperosmotic shock even in hog1∆ cells, indicating another mechanism to prevent Fps1-mediated glycerol efflux. In our prior proteome-wide screen, Fps1 was identified as a target of TORC2-dependent protein kinase Ypk1 (Muir et al., 2014). We show here that Fps1 is an authentic Ypk1 substrate and that the open channel state of Fps1 requires phosphorylation by Ypk1. Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress. These events are all Hog1-independent. Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions. PMID:26274562

  16. Rictor/mTORC2Loss in the Myf5-lineage Reprograms Brown Fat Metabolism and Protects Mice against Obesity and Metabolic Disease

    PubMed Central

    Hung, Chien-Min; Calejman, Camila Martinez; Sanchez-Gurmaches, Joan; Li, Huawei; Clish, Clary B.; Hettmer, Simone; Wagers, Amy J.; Guertin, David A.

    2014-01-01

    Summary The in vivo functions of mTORC2, and the signaling mechanisms that control brown adipose tissue (BAT) fuel utilization and activity, are not well understood. Here, by conditionally deleting Rictorin the Myf5-lineage, we provide in vivo evidence that mTORC2 is dispensable for skeletal muscle development and regeneration but essential for BAT growth. Furthermore, deleting RictorinMyf5 precursors shifts BAT metabolism to a more oxidative and less lipogenic state and protects mice from obesity and metabolic disease at thermoneutrality. We additionally find that Rictor is required for brown adipocyte differentiation in vitro, that the mechanismspecifically requires AKT1 hydrophobic motif phosphorylation but is independent of pan-AKT signaling, and is rescued with BMP7. Our findings provide new insights into the signaling circuitry that regulates brown adipocytes and could have important implications for developing therapies aimed at increasing energy expenditure as a means to combat human obesity. PMID:25001283

  17. Imaging CREB Activation in Living Cells*

    PubMed Central

    Friedrich, Michael W.; Aramuni, Gayane; Mank, Marco; Mackinnon, Jonathan A. G.; Griesbeck, Oliver

    2010-01-01

    The Ca2+- and cAMP-responsive element-binding protein (CREB) and the related ATF-1 and CREM are stimulus-inducible transcription factors that link certain forms of cellular activity to changes in gene expression. They are attributed to complex integrative activation characteristics, but current biochemical technology does not allow dynamic imaging of CREB activation in single cells. Using fluorescence resonance energy transfer between mutants of green fluorescent protein we here develop a signal-optimized genetically encoded indicator that enables imaging activation of CREB due to phosphorylation of the critical serine 133. The indicator of CREB activation due to phosphorylation (ICAP) was used to investigate the role of the scaffold and anchoring protein AKAP79/150 in regulating signal pathways converging on CREB. We show that disruption of AKAP79/150-mediated protein kinase A anchoring or knock-down of AKAP150 dramatically reduces the ability of protein kinase A to activate CREB. In contrast, AKAP79/150 regulation of CREB via L-type channels may only have minor importance. ICAP allows dynamic and reversible imaging in living cells and may become useful in studying molecular components and cell-type specificity of activity-dependent gene expression. PMID:20484048

  18. TORC2 Is Required to Maintain Genome Stability during S Phase in Fission Yeast*

    PubMed Central

    Schonbrun, Miriam; Kolesnikov, Masha; Kupiec, Martin; Weisman, Ronit

    2013-01-01

    DNA damage can occur due to environmental insults or intrinsic metabolic processes and is a major threat to genome stability. The DNA damage response is composed of a series of well coordinated cellular processes that include activation of the DNA damage checkpoint, transient cell cycle arrest, DNA damage repair, and reentry into the cell cycle. Here we demonstrate that mutant cells defective for TOR complex 2 (TORC2) or the downstream AGC-like kinase, Gad8, are highly sensitive to chronic replication stress but are insensitive to ionizing radiation. We show that in response to replication stress, TORC2 is dispensable for Chk1-mediated cell cycle arrest but is required for the return to cell cycle progression. Rad52 is a DNA repair and recombination protein that forms foci at DNA damage sites and stalled replication forks. TORC2 mutant cells show increased spontaneous nuclear Rad52 foci, particularly during S phase, suggesting that TORC2 protects cells from DNA damage that occurs during normal DNA replication. Consistently, the viability of TORC2-Gad8 mutant cells is dependent on the presence of the homologous recombination pathway and other proteins that are required for replication restart following fork replication stalling. Our findings indicate that TORC2 is required for genome integrity. This may be relevant for the growing amount of evidence implicating TORC2 in cancer development. PMID:23703609

  19. The small GTPases Ras and Rap1 bind to and control TORC2 activity.

    PubMed

    Khanna, Ankita; Lotfi, Pouya; Chavan, Anita J; Montaño, Nieves M; Bolourani, Parvin; Weeks, Gerald; Shen, Zhouxin; Briggs, Steven P; Pots, Henderikus; Van Haastert, Peter J M; Kortholt, Arjan; Charest, Pascale G

    2016-01-01

    Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration. PMID:27172998

  20. The small GTPases Ras and Rap1 bind to and control TORC2 activity

    PubMed Central

    Khanna, Ankita; Lotfi, Pouya; Chavan, Anita J.; Montaño, Nieves M.; Bolourani, Parvin; Weeks, Gerald; Shen, Zhouxin; Briggs, Steven P.; Pots, Henderikus; Van Haastert, Peter J. M.; Kortholt, Arjan; Charest, Pascale G.

    2016-01-01

    Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration. PMID:27172998

  1. Constitutive activation with overexpression of the mTORC2-phospholipase D1 pathway in uterine leiomyosarcoma and STUMP: morphoproteomic analysis with therapeutic implications.

    PubMed

    Dhingra, Sadhna; Rodriguez, Michelle E; Shen, Qi; Duan, Xuizhen; Stanton, Melissa L; Chen, Lei; Zhang, Rongzhen; Brown, Robert E

    2011-01-01

    The mammalian target of rapamycin (mTOR) is centrally involved in growth, survival and metabolism. In cancer, mTOR is frequently hyperactivated and is a clinically validated target for therapy and drug development. Biologically, mTOR acts as the catalytic subunit of two functionally distinct complexes, called mTOR complex 1 (mTORC1) which is predominantly cytoplasmic in subcellular localization and mTOR complex 2 (mTORC2) which is both cytoplasmic and nuclear. mTORC1 is sensitive to the selective inhibitor rapamycin. By contrast, mTORC2 is relatively resistant to rapamycin. Moreover, its putative downstream effector, Akt phosphorylated on serine 473 represents a signal transduction pathway for tumor survival. Phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as an activator of mTOR signaling, including the direct phosphorylative activation of p70S6K atthreonine 389. The latter promotes cell cycle progression. In this study, we investigated the activation status and subcellular localization of mTOR and the relative expression of PLD1, as well as their downstream effectors in a spectrum of uterine smooth muscle tumors using normal myometria as controls. The results show significant activation with overexpression of phosphorylated mTORC2 complex in uterine leiomyosarcoma (ULMS) and smooth muscle tumors of uncertain malignant potential (STUMP) as evidenced by nuclear localization of p-mTOR (Ser 2448) in ULMS>STUMP>uterine leiomyoma and normal myometria (p<0.05) and with overexpression of PLD1(p<0.05). Cor-relatively, there are overexpressions of nuclear p-Akt (Ser 473) and nuclear p-p70S6K (Thr 389) in ULMS and STUMP (p<0.05). The activation with overexpression of components of the mTORC2-PLD1 pathway in ULMS and to a lesser degree in STUMP provides insight into their tumorigenic mechanisms. Thus the development of therapies designed to target mTORC2 and PLD1 activity may be beneficial in treating ULMS. PMID:21326806

  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. Oct-1 Potentiates CREB-Driven Cyclin D1 Promoter Activation via a Phospho-CREB- and CREB Binding Protein-Independent Mechanism

    PubMed Central

    Boulon, Séverine; Dantonel, Jean-Christophe; Binet, Virginie; Vié, Annick; Blanchard, Jean-Marie; Hipskind, Robert A.; Philips, Alexandre

    2002-01-01

    Cyclin D1, the regulatory subunit for mid-G1 cyclin-dependent kinases, controls the expression of numerous cell cycle genes. A cyclic AMP-responsive element (CRE), located upstream of the cyclin D1 mRNA start site, integrates mitogenic signals that target the CRE-binding factor CREB, which can recruit the transcriptional coactivator CREB-binding protein (CBP). We describe an alternative mechanism for CREB-driven cyclin D1 induction that involves the ubiquitous POU domain protein Oct-1. In the breast cancer cell line MCF-7, overexpression of Oct-1 or its POU domain strongly increases transcriptional activation of cyclin D1 and GAL4 reporter genes that is specifically dependent upon CREB but independent of Oct-1 DNA binding. Gel retardation and chromatin immunoprecipitation assays confirm that POU forms a complex with CREB bound to the cyclin D1 CRE. In solution, CREB interaction with POU requires the CREB Q2 domain and, notably, occurs with CREB that is not phosphorylated on Ser 133. Accordingly, Oct-1 also potently enhances transcriptional activation mediated by a Ser133Ala CREB mutant. Oct-1/CREB synergy is not diminished by the adenovirus E1A 12S protein, a repressor of CBP coactivator function. In contrast, E1A strongly represses CBP-enhanced transactivation by CREB phosphorylated on Ser 133. Our observation that Oct-1 potentiates CREB-dependent cyclin D1 transcriptional activity independently of Ser 133 phosphorylation and E1A-sensitive coactivator function offers a new paradigm for the regulation of cyclin D1 induction by proliferative signals. PMID:12391146

  4. Hepatic Sirt1 deficiency in mice impairs mTorc2/Akt signaling and results in hyperglycemia, oxidative damage, and insulin resistance

    PubMed Central

    Wang, Rui-Hong; Kim, Hyun-Seok; Xiao, Cuiying; Xu, Xiaoling; Gavrilova, Oksana; Deng, Chu-Xia

    2011-01-01

    Insulin resistance is a major risk factor for type 2 diabetes mellitus. The protein encoded by the sirtuin 1 (Sirt1) gene, which is a mouse homolog of yeast Sir2, is implicated in the regulation of glucose metabolism and insulin sensitivity; however, the underlying mechanism remains elusive. Here, using mice with a liver-specific null mutation of Sirt1, we have identified a signaling pathway involving Sirt1, Rictor (a component of mTOR complex 2 [mTorc2]), Akt, and Foxo1 that regulates gluconeogenesis. We found that Sirt1 positively regulates transcription of the gene encoding Rictor, triggering a cascade of phosphorylation of Akt at S473 and Foxo1 at S253 and resulting in decreased transcription of the gluconeogenic genes glucose-6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (Pepck). Liver-specific Sirt1 deficiency caused hepatic glucose overproduction, chronic hyperglycemia, and increased ROS production. This oxidative stress disrupted mTorc2 and impaired mTorc2/Akt signaling in other insulin-sensitive organs, leading to insulin resistance that could be largely reversed with antioxidant treatment. These data delineate a pathway through which Sirt1 maintains insulin sensitivity and suggest that treatment with antioxidants might provide protection against progressive insulin resistance in older human populations. PMID:21965330

  5. RES-529: a PI3K/AKT/mTOR pathway inhibitor that dissociates the mTORC1 and mTORC2 complexes

    PubMed Central

    2016-01-01

    RES-529 (previously named Palomid 529, P529) is a phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway inhibitor that interferes with the pathway through both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) dissociation. This compound is currently being developed in oncology and ophthalmology. The oncology focus is for the treatment of glioblastoma, where it has received orphan designation by the US Food and Drug Administration, and prostate cancer. We present a review of the PI3K/AKT/mTOR pathway, its role in tumorigenesis, and the potential of RES-529 in cancer treatment. RES-529 inhibits mTORC1/mTORC2 activity in various cancer cell lines, as noted by decreased phosphorylation of substrates including ribosomal protein S6, 4E-BP1, and AKT, leading to cell growth inhibition and death, with activity generally in the range of 5–15 μmol/l. In animal tumor models where the PI3K/AKT/mTOR pathway is abnormally activated (i.e. glioblastoma, prostate cancer, and breast cancer), RES-529 reduces tumor growth by as much as 78%. RES-529 treatment is synergistic with radiation therapy, chemotherapy, and hormonal therapy in reducing tumor growth, potentially by preventing PI3K/AKT/mTOR pathway activation associated with these treatments. Furthermore, this compound has shown antiangiogenic activity in several animal models. mTORC1 and mTORC2 have redundant and distinct activities that contribute toward oncogenesis. Current inhibitors of this pathway have primarily targeted mTORC1, but have shown limited clinical efficacy. Inhibitors of mTORC1 and mTORC2 such as RES-529 may therefore have the potential to overcome the deficiencies found in targeting only mTORC1. PMID:26918392

  6. RES-529: a PI3K/AKT/mTOR pathway inhibitor that dissociates the mTORC1 and mTORC2 complexes.

    PubMed

    Weinberg, Mark A

    2016-07-01

    RES-529 (previously named Palomid 529, P529) is a phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway inhibitor that interferes with the pathway through both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) dissociation. This compound is currently being developed in oncology and ophthalmology. The oncology focus is for the treatment of glioblastoma, where it has received orphan designation by the US Food and Drug Administration, and prostate cancer. We present a review of the PI3K/AKT/mTOR pathway, its role in tumorigenesis, and the potential of RES-529 in cancer treatment. RES-529 inhibits mTORC1/mTORC2 activity in various cancer cell lines, as noted by decreased phosphorylation of substrates including ribosomal protein S6, 4E-BP1, and AKT, leading to cell growth inhibition and death, with activity generally in the range of 5-15 μmol/l. In animal tumor models where the PI3K/AKT/mTOR pathway is abnormally activated (i.e. glioblastoma, prostate cancer, and breast cancer), RES-529 reduces tumor growth by as much as 78%. RES-529 treatment is synergistic with radiation therapy, chemotherapy, and hormonal therapy in reducing tumor growth, potentially by preventing PI3K/AKT/mTOR pathway activation associated with these treatments. Furthermore, this compound has shown antiangiogenic activity in several animal models. mTORC1 and mTORC2 have redundant and distinct activities that contribute toward oncogenesis. Current inhibitors of this pathway have primarily targeted mTORC1, but have shown limited clinical efficacy. Inhibitors of mTORC1 and mTORC2 such as RES-529 may therefore have the potential to overcome the deficiencies found in targeting only mTORC1. PMID:26918392

  7. mTORC2-Akt signaling axis is implicated in myocardial compensation and fibrosis.

    PubMed

    Dany, M; Rimmani, H H; Matar, S A; Hajj Hussein, I

    2015-01-01

    mTOR signaling has long been implicated in the mechanisms of cardiomyocyte survival in response to volume or pressure overload. Several studies have focused on the significance of mTORC1 in cardiomyocyte survival, questioning the role of mTORC2. mTORC2–Akt signaling is an emerging axis implicated in cardiomyocyte compensation and thus heart failure. Upon being subjected to chronic stress, cardiomyocytes activate mTORC2–Akt signaling pathway to promote survival by activating the ubiquitin proteasome system, inducing the degradation of pro-apoptotic proteins, and altering the actin cytoskeleton. Given the importance of mTORC2 in cardiomyocyte survival, studies suggest that loss of mTORC2 signaling would result in loss of cardiomyocytes, fibrosis, and heart failure. This review serves to elaborate on how mTORC2-Akt signaling plays a role in cardiomyocyte growth and survival under stress and how the loss of the axis would result in fibrosis and heart failure. PMID:26753634

  8. In vivo evidence for mTORC2-mediated actin cytoskeleton rearrangement in neurons.

    PubMed

    Angliker, Nico; Rüegg, Markus A

    2013-01-01

    The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. While mTORC1 controls the signaling pathways important for cell growth, the physiological function of mTORC2 is only partially known. Here we comment on recent work on gene-targeted mice lacking mTORC2 in the cerebellum or the hippocampus that provided strong evidence that mTORC2 plays an important role in neuron morphology and synapse function. We discuss that this phenotype might be based on the perturbed regulation of the actin cytoskeleton and the lack of activation of several PKC isoforms. The fact that PKC isoforms and their targets have been implicated in neurological disease including spinocerebellar ataxia and that they have been shown to affect learning and memory, suggests that aberration of mTORC2 signaling might be involved in diseases of the brain. PMID:24721730

  9. ATP-site binding inhibitor effectively targets mTORC1 and mTORC2 complexes in glioblastoma.

    PubMed

    Neil, Jayson; Shannon, Craig; Mohan, Avinash; Laurent, Dimitri; Murali, Raj; Jhanwar-Uniyal, Meena

    2016-03-01

    The PI3K-AKT-mTOR signaling axis is central to the transformed phenotype of glioblastoma (GBM) cells, due to frequent loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10). The mechanistic target of rapamycin (mTOR) kinase is present in two cellular multi-protein complexes, mTORC1 and mTORC2, which have distinct subunit composition, substrates and mechanisms of action. Targeting the mTOR protein is a promising strategy for GBM therapy. However, neither of these complexes is fully inhibited by the allosteric inhibitor of mTOR, rapamycin or its analogs. Herein, we provide evidence that the combined inhibition of mTORC1/2, using the ATP-competitive binding inhibitor PP242, would effectively suppress GBM growth and dissemination as compared to an allosteric binding inhibitor of mTOR. GBM cells treated with PP242 demonstrated significantly decreased activation of mTORC1 and mTORC2, as shown by reduced phosphorylation of their substrate levels, p70 S6KThr389 and AKTSer473, respectively, in a dose-dependent manner. Furthermore, insulin induced activation of these kinases was abrogated by pretreatment with PP242 as compared with rapamycin. Unlike rapamycin, PP242 modestly activates extracellular regulated kinase (ERK1/2), as shown by expression of pERKThr202/Tyr204. Cell proliferation and S-phase entry of GBM cells was significantly suppressed by PP242, which was more pronounced compared to rapamycin treatment. Lastly, PP242 significantly suppressed the migration of GBM cells, which was associated with a change in cellular behavior rather than cytoskeleton loss. In conclusion, these results underscore the potential therapeutic use of the PP242, a novel ATP-competitive binding inhibitor of mTORC1/2 kinase, in suppression of GBM growth and dissemination. PMID:26719046

  10. Inhibition of mTORC2 Induces Cell-Cycle Arrest and Enhances the Cytotoxicity of Doxorubicin by Suppressing MDR1 Expression in HCC Cells.

    PubMed

    Chen, Bryan Wei; Chen, Wei; Liang, Hui; Liu, Hao; Liang, Chao; Zhi, Xiao; Hu, Li-Qiang; Yu, Xia-Zhen; Wei, Tao; Ma, Tao; Xue, Fei; Zheng, Lei; Zhao, Bin; Feng, Xin-Hua; Bai, Xue-Li; Liang, Ting-Bo

    2015-08-01

    mTOR is aberrantly activated in hepatocellular carcinoma (HCC) and plays pivotal roles in tumorigenesis and chemoresistance. Rapamycin has been reported to exert antitumor activity in HCC and sensitizes HCC cells to cytotoxic agents. However, due to feedback activation of AKT after mTOR complex 1 (mTORC1) inhibition, simultaneous targeting of mTORC1/2 may be more effective. In this study, we examined the interaction between the dual mTORC1/2 inhibitor OSI-027 and doxorubicin in vitro and in vivo. OSI-027 was found to reduce phosphorylation of both mTORC1 and mTORC2 substrates, including 4E-BP1, p70S6K, and AKT (Ser473), and inhibit HCC cell proliferation. Similar to OSI-027 treatment, knockdown of mTORC2 induced G0-G1 phase cell-cycle arrest. In contrast, rapamycin or knockdown of mTORC1 increased phosphorylation of AKT (Ser473), yet had little antiproliferative effect. Notably, OSI-027 synergized with doxorubicin for the antiproliferative efficacy in a manner dependent of MDR1 expression in HCC cells. The synergistic antitumor effect of OSI-027 and doxorubicin was also observed in a HCC xenograft mouse model. Moreover, AKT was required for OSI-027-induced cell-cycle arrest and downregulation of MDR1. Our findings provide a rationale for dual mTORC1/mTORC2 inhibitors, such as OSI-027, as monotherapy or in combination with cytotoxic agents to treat HCC. Mol Cancer Ther; 14(8); 1805-15. ©2015 AACR. PMID:26026051

  11. mTORC2 in Thymic Epithelial Cells Controls Thymopoiesis and T Cell Development.

    PubMed

    Wang, Hong-Xia; Cheng, Joyce S; Chu, Shuai; Qiu, Yu-Rong; Zhong, Xiao-Ping

    2016-07-01

    Thymic epithelial cells (TECs) play important roles in T cell generation. Mechanisms that control TEC development and function are still not well defined. The mammalian or mechanistic target of rapamycin complex (mTORC)2 signals to regulate cell survival, nutrient uptake, and metabolism. We report in the present study that mice with TEC-specific ablation of Rictor, a critical and unique adaptor molecule in mTORC2, display thymic atrophy, which accompanies decreased TEC numbers in the medulla. Moreover, generation of multiple T cell lineages, including conventional TCRαβ T cells, regulatory T cells, invariant NKT cells, and TCRγδ T cells, was reduced in TEC-specific Rictor-deficient mice. Our data demonstrate that mTORC2 in TECs is important for normal thymopoiesis and efficient T cell generation. PMID:27233961

  12. The Adenosine A2A Receptor Agonist, CGS-21680, Blocks Excessive Rearing, Acquisition of Wheel Running, and Increases Nucleus Accumbens CREB Phosphorylation in Chronically Food-Restricted Rats

    PubMed Central

    de Vaca, Soledad Cabeza; Kannan, Pavitra; Pan, Yan; Jiang, Nancy; Sun, Yanjie; Carr, Kenneth D.

    2007-01-01

    Adenosine A2A receptors are preferentially expressed in rat striatum, where they are concentrated in dendritic spines of striatopallidal medium spiny neurons and exist in a heteromeric complex with D2 dopamine (DA) receptors. Behavioral and biochemical studies indicate an antagonistic relationship between A2A and D2 receptors. Previous studies have demonstrated that food-restricted (FR) rats display behavioral and striatal cellular hypersensitivity to D1 and D2 DA receptor stimulation. These alterations may underlie adaptive, as well as maladaptive, behaviors characteristic of the FR rat. The present study examined whether FR rats are hypersensitive to the A2A receptor agonist, CGS-21680. In Experiment 1, spontaneous horizontal motor activity did not differ between FR and ad libitum fed (AL) rats, while vertical activity was greater in the former. Intracerebroventricular (i.c.v.) administration of CGS-21680 (0.25 and 1.0 nmol) decreased both types of motor activity in FR rats, and returned vertical activity levels to those observed in AL rats. In Experiment 2, FR rats given access to a running wheel for a brief period outside of the home cage rapidly acquired wheel running while AL rats did not. Pretreatment with CGS-21680 (1.0 nmol) blocked the acquisition of wheel running. When administered to FR subjects that had previously acquired wheel running, CGS-21680 suppressed the behavior. In Experiment 3, CGS-21680 (1.0 nmol) activated both ERK 1/2 and CREB in caudate-putamen with no difference between feeding groups. However, in nucleus accumbens (NAc), CGS-21680 failed to activate ERK 1/2 and selectively activated CREB in FR rats. These results indicate that FR subjects are hypersensitive to several effects of an adenosine A2A agonist, and suggest the involvement of an upregulated A2A receptor-linked signaling pathway in NAc. Medications targeting the A2A receptor may have utility in the treatment of maladaptive behaviors associated with FR, including substance abuse

  13. Chemical-Genetics of Rapamycin-Insensitive TORC2 in S. cerevisiae

    PubMed Central

    Kliegman, Joseph I.; Fiedler, Dorothea; Ryan, Colm J.; Xu, Yi-Fan; Su, Xiao-yang; Thomas, David; Caccese, Max C.; Cheng, Ada; Shales, Michael; Rabinowitz, Joshua D.; Krogan, Nevan J.; Shokat, Kevan M.

    2014-01-01

    Current approaches for identifying synergistic targets use cell culture models with combinations of clinically available drugs to see if the combined effect of the combination is better than predicted by their individual efficacy. New techniques are needed to systematically and rationally identify targets and pathways that have a high potential as synergistic targets. In this study, we create a tool to screen and identify molecular targets that may synergize with new inhibitors of TOR (Target of Rapamycin), a conserved protein that is a major integrator of cell proliferation signals in the nutrient-signaling pathway. While clinical results from TORC1 inhibition using rapamycin analogs (that only inhibit TORC1) have been disappointing, trials using inhibitors that also target TORC2 have been promising. To understand the molecular basis for this increased therapeutic efficacy and to discover secondary targets that may have potential in targeted combination therapy, we engineered TOR2 in S. cerevisiae to accept an orthogonal inhibitor in order to create the first chemical tool to selectively inhibit TORC2. We used this tool to create a Chemical Epistasis Mini-Array Profile, or ChE-MAP, by measuring interactions between the chemically inhibited TOR2 kinase and a diverse library of deletion mutants. The ChE-MAP identified known TOR components and distinguished between TORC1 (assessed using rapamycin) and TORC2 dependent functions. Results showed a novel TORC2-specific interaction with the pentose phosphate pathway (PPP). We used global metabolic profiling to show that that TORC2 inhibition led to decreases in metabolites specific to the PPP and confirmed that TOR2 was regulating this process using metabolic flux analysis. Regulation of the PPP is a previously unappreciated role for TORC2 that may suggest a role for the complex in balancing the high energy demand required for ribosome biogenesis. PMID:24360963

  14. mTORC1 and mTORC2 have largely distinct functions in Purkinje cells.

    PubMed

    Angliker, Nico; Burri, Michael; Zaichuk, Mariana; Fritschy, Jean-Marc; Rüegg, Markus A

    2015-10-01

    The mammalian target of rapamycin (mTOR) is a key regulator of cellular growth which associates with other proteins to form two multi-protein complexes called mTORC1 and mTORC2. Dysregulation of mTORC1 signalling in brain is implicated in neuropathological conditions such as autism spectrum or neurodegenerative disorders. Accordingly, allosteric mTOR inhibitors are currently in clinical trials for the treatment of such disorders. Here, we ablated either mTORC1 or mTORC2 conditionally in Purkinje cells of the mouse cerebellum to dissect their role in the development, function and survival of these neurons. We find that the two mouse models largely differ from each other by phenotype and cellular responses. Inactivation of mTORC2, but not of mTORC1, led to motor coordination deficits at an early age. This phenotype correlated with developmental deficits in climbing fibre elimination and impaired dendritic self-avoidance in mTORC2-deficient Purkinje cells. In contrast, inactivation of mTORC1, but not of mTORC2, affected social interest of the mice and caused a progressive loss of Purkinje cells due to apoptosis. This cell loss was paralleled by age-dependent motor deficits. Comparison of mTORC1-deficient Purkinje cells with those deficient for the mTORC1 inhibitor TSC1 revealed a striking overlap in Purkinje cell degeneration and death, which included neurofilamentopathy and reactive gliosis. Altogether, our study reveals distinct roles of mTORC1 and mTORC2 in Purkinje cells for mouse behaviour and the survival of neurons. Our study also highlights a convergence between the phenotypes of Purkinje cells lacking mTORC1 activity and those expressing constitutively active mTORC1 due to TSC1 deficiency. PMID:26296489

  15. Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation.

    PubMed

    Zheng, Bing; Mao, Jia-Hui; Li, Xiao-Qing; Qian, Lin; Zhu, Hua; Gu, Dong-Hua; Pan, Xiao-Dong

    2016-01-01

    Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation. PMID:27412013

  16. mTORC1 is necessary but mTORC2 and GSK3β are inhibitory for AKT3-induced axon regeneration in the central nervous system

    PubMed Central

    Miao, Linqing; Yang, Liu; Huang, Haoliang; Liang, Feisi; Ling, Chen; Hu, Yang

    2016-01-01

    Injured mature CNS axons do not regenerate in mammals. Deletion of PTEN, the negative regulator of PI3K, induces CNS axon regeneration through the activation of PI3K-mTOR signaling. We have conducted an extensive molecular dissection of the cross-regulating mechanisms in axon regeneration that involve the downstream effectors of PI3K, AKT and the two mTOR complexes (mTORC1 and mTORC2). We found that the predominant AKT isoform in CNS, AKT3, induces much more robust axon regeneration than AKT1 and that activation of mTORC1 and inhibition of GSK3β are two critical parallel pathways for AKT-induced axon regeneration. Surprisingly, phosphorylation of T308 and S473 of AKT play opposite roles in GSK3β phosphorylation and inhibition, by which mTORC2 and pAKT-S473 negatively regulate axon regeneration. Thus, our study revealed a complex neuron-intrinsic balancing mechanism involving AKT as the nodal point of PI3K, mTORC1/2 and GSK3β that coordinates both positive and negative cues to regulate adult CNS axon regeneration. DOI: http://dx.doi.org/10.7554/eLife.14908.001 PMID:27026523

  17. Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation

    PubMed Central

    Zheng, Bing; Mao, Jia-Hui; Li, Xiao-Qing; Qian, Lin; Zhu, Hua; Gu, Dong-hua; Pan, Xiao-dong

    2016-01-01

    Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation. PMID:27412013

  18. Rictor Phosphorylation on the THR-1135 Site Does Not Require Mammalian Target of Rapamycin Complex 2

    PubMed Central

    Boulbes, Delphine; Chen, Chien-Hung; Shaikenov, Tattym; Agarwal, Nitin K.; Peterson, Timothy R.; Addona, Terri A.; Keshishian, Hasmik; Carr, Steven A.; Magnuson, Mark A.; Sabatini, David M.; Sarbassov, Dos D.

    2010-01-01

    In animal cells growth factors coordinate cell proliferation and survival by regulating the PI3K/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K dependent signaling kinase complex defined as mTORC2 functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. Rictor phosphorylation is sensitive to inhibition of PI3K, mTOR, or expression of ILK. The substitution of wild-type rictor with its specific phospho-mutants in rictor null mouse embryonic fibroblasts did not alter the growth factor-dependent phosphorylation of Akt indicating that the rictor Thr-1135 phosphorylation is not critical in regulation of the mTORC2 kinase activity. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells suggesting that this modification might play a role in regulation not only mTORC2 but also the mTORC2-independent function of rictor. PMID:20501647

  19. A Critical Role for the mTORC2 Pathway in Lung Fibrosis

    PubMed Central

    Chang, Wenteh; Wei, Ke; Ho, Lawrence; Berry, Gerald J.; Jacobs, Susan S.; Chang, Cheryl H.; Rosen, Glenn D.

    2014-01-01

    A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases. PMID:25162417

  20. Oxytocin Regulates Stress-Induced Crf Gene Transcription through CREB-Regulated Transcription Coactivator 3

    PubMed Central

    Jurek, Benjamin; Slattery, David A.; Hiraoka, Yuichi; Liu, Ying; Nishimori, Katsuhiko; Aguilera, Greti; van den Burg, Erwin H.

    2015-01-01

    The major regulator of the neuroendocrine stress response in the brain is corticotropin releasing factor (CRF), whose transcription is controlled by CREB and its cofactors CRTC2/3 (TORC2/3). Phosphorylated CRTCs are sequestered in the cytoplasm, but rapidly dephosphorylated and translocated into the nucleus following a stressful stimulus. As the stress response is attenuated by oxytocin (OT), we tested whether OT interferes with CRTC translocation and, thereby, Crf expression. OT (1 nmol, i.c.v.) delayed the stress-induced increase of nuclear CRTC3 and Crf hnRNA levels in the paraventricular nucleus of male rats and mice, but did not affect either parameter in the absence of the stressor. The increase in Crf hnRNA levels at later time points was parallel to elevated nuclear CRTC2/3 levels. A direct effect of Thr4 Gly7-OT (TGOT) on CRTC3 translocation and Crf expression was found in rat primary hypothalamic neurons, amygdaloid (Ar-5), hypothalamic (H32), and human neuroblastoma (Be(2)M17) cell lines. CRTC3, but not CRCT2, knockdown using siRNA in Be(2)M17 cells prevented the effect of TGOT on Crf hnRNA levels. Chromatin-immunoprecipitation demonstrated that TGOT reduced CRTC3, but not CRTC2, binding to the Crf promoter after 10 min of forskolin stimulation. Together, the results indicate that OT modulates CRTC3 translocation, the binding of CRTC3 to the Crf promoter and, ultimately, transcription of the Crf gene. SIGNIFICANCE STATEMENT The neuropeptide oxytocin has been proposed to reduce hypothalamic-pituitary-adrenal (HPA) axis activation during stress. The underlying mechanisms are, however, elusive. In this study we show that activation of the oxytocin receptor in the paraventricular nucleus delays transcription of the gene encoding corticotropin releasing factor (Crf), the main regulator of the stress response. It does so by sequestering the coactivator of the transcription factor CREB, CRTC3, in the cytosol, resulting in reduced binding of CRTC3 to the Crf

  1. Regulation of CREB by moderate hypoxia in PC12 cells.

    PubMed

    Beitner-Johnson, D; Rust, R T; Hsieh, T; Millhorn, D E

    2000-01-01

    The mechanisms by which excitable cells adapt and respond to changes in O2 levels remain largely unknown. We have investigated the effect of hypoxia on the cyclic AMP response element binding protein (CREB) transcription factor. PC12 cells were exposed to moderate levels of hypoxia (5% O2) for various times between 20 min and 6 hr. We found that hypoxia rapidly and persistently induced ser133 phosphorylation of CREB. This effect was more robust than that produced by exposing PC12 cells to either forskolin, KCl, or NGF. This effect was not due to activation of any of the previously known CREB kinases, including PKA, CaMK, PKC, p70s6k, or MAPKAP kinase-2. Thus, hypoxia may induce activation of a novel CREB kinase. To test whether phosphorylation of CREB was associated with an activation of CRE-dependent gene expression, cells were transfected with wild type and mutated regions of the 5'-flanking region of the tyrosine hydroxylase (TH) gene fused to a CAT reporter gene. Mutation of the CRE element in a TH reporter gene reduced, but did not abolish, the effects of hypoxia on TH gene expression. However, hypoxia did not induce transactivation of a GAL4-luciferase reporter by a GAL4-CREB fusion protein. Thus, the mechanism by which hypoxia regulates CREB is distinct, and more complex, than that induced by forskolin, depolarization, or nerve growth factor. PMID:10849656

  2. mTORC2 coordinates pulmonary artery smooth muscle cell metabolism, proliferation and survival in pulmonary arterial hypertension

    PubMed Central

    Goncharov, Dmitry A.; Kudryashova, Tatiana V.; Ziai, Houman; Ihida-Stansbury, Kaori; DeLisser, Horace; Krymskaya, Vera P.; Tuder, Rubin M.; Kawut, Steven M.; Goncharova, Elena A.

    2014-01-01

    Background Enhanced proliferation, resistance to apoptosis and metabolic shift to glycolysis of pulmonary arterial vascular smooth muscle cells (PAVSMC) are key pathophysiological components of pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH). The role of distinct mTOR complexes mTORC1 (mTOR-raptor) and mTORC2 (mTOR-rictor) in PAVSMC proliferation and survival in PAH and their therapeutic relevance is unknown. Methods and Results Immunohistochemical and immunoblot analyses revealed that mTORC1 and mTORC2 pathways are markedly up-regulated in small remodeled PAs and isolated distal PAVSMC from IPAH subjects that have increased ATP levels, proliferation and survival that depend on glycolytic metabolism. siRNA- and pharmacological-based analysis showed that while both mTORC1 and mTORC2 contributing to proliferation, only mTORC2 is required for ATP generation and survival of IPAH PAVSMC. mTORC2 down-regulated energy sensor AMPK allowing activation of mTORC1-S6 and increased proliferation, and deficiency of pro-apoptotic protein Bim and IPAH PAVSMC survival. Nox4 protein levels were increased in IPAH PAVSMC that was necessary for mTORC2 activation, proliferation and survival. Nox4 levels and mTORC2 signaling were significantly up-regulated in small PAs from hypoxia-exposed rats at days 2-28 of hypoxia. Treatment with the mTOR kinase inhibitor PP242 at days 15-28 suppressed mTORC2, but not Nox4, induced SM-specific apoptosis in small PAs and reversed hypoxia-induced pulmonary vascular remodeling in rats. Conclusions These data provide a novel mechanistic link of Nox4-dependent activation of mTORC2 via energy sensor AMPK to increased proliferation and survival of PAVSMC in PAH suggesting a new potential pathway for the therapeutic interventions. PMID:24270265

  3. Insulin-Dependent Regulation of mTORC2-Akt-FoxO Suppresses TLR4 Signaling in Human Leukocytes: Relevance to Type 2 Diabetes.

    PubMed

    Zhang, Zhiyong; Amorosa, Louis F; Coyle, Susette M; Macor, Marie A; Birnbaum, Morris J; Lee, Leonard Y; Haimovich, Beatrice

    2016-08-01

    Leukocyte signaling in patients with systemic insulin resistance is largely unexplored. We recently discovered the presence of multiple Toll-like receptor 4 (TLR4) signaling intermediates in leukocytes from patients with type 2 diabetes or acute insulin resistance associated with cardiopulmonary bypass surgery. We extend this work to show that in addition to matrix metalloproteinase 9, hypoxia-inducible factor 1α, and cleaved AMPKα, patient leukocytes also express IRS-1 phosphorylated on Ser(312), Akt phosphorylated on Thr(308), and elevated TLR4 expression. Similar signaling intermediates were detected in leukocytes and neutrophils treated with lipopolysaccharide (LPS), a ligand of TLR4, in vitro. In contrast, insulin, but not LPS, induced mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt on Ser(473) and FoxO1/O3a on Thr(24/32) in leukocytes and neutrophils. Insulin suppressed LPS-induced responses in a dose- and time-dependent manner. AS1842856, a FoxO1 inhibitor, also suppressed TLR4 signaling. We propose that insulin is a homeostatic regulator of leukocyte responses to LPS/TLR4 and that the signaling intermediates expressed in leukocytes of patients with type 2 diabetes indicate TLR4 signaling dominance and deficient insulin signaling. The data suggest that insulin suppresses LPS/TLR4 signals in leukocytes through the mTORC2-Akt-FoxO signaling axis. Better understanding of leukocyte signaling in patients with type 2 diabetes may shed new light on disease causation and progression. PMID:27207509

  4. DNMT3b modulates melanoma growth by controlling levels of mTORC2 component RICTOR

    PubMed Central

    Micevic, Goran; Muthusamy, Viswanathan; Damsky, William; Theodosakis, Nicholas; Liu, Xiaoni; Meeth, Katrina; Wingrove, Emily; Santhanakrishnan, Manjula; Bosenberg, Marcus

    2016-01-01

    SUMMARY DNA methyltransferase DNMT3B is frequently overexpressed in tumor cells and plays important roles during the formation and progression of several cancer types. However, the specific signaling pathways controlled by DNMT3B in cancers, including melanoma, are poorly understood. Here, we report that DNMT3B plays a pro-tumorigenic role in human melanoma and that DNMT3B loss dramatically suppresses melanoma formation in the Braf/Pten mouse melanoma model. Loss of DNMT3B results in hypomethylation of the miR-196b promoter and increased miR-196b expression, which directly targets the mTORC2 component Rictor. Loss of RICTOR in turn prevents mTORC2 activation, which is critical for melanoma formation and growth. These findings establish Dnmt3b as a regulator of melanoma formation through its effect on mTORC2 signaling. Based on these results, DNMT3B is a potential therapeutic target in melanoma. PMID:26923591

  5. TORC2 signaling pathway guarantees genome stability in the face of DNA strand breaks.

    PubMed

    Shimada, Kenji; Filipuzzi, Ireos; Stahl, Michael; Helliwell, Stephen B; Studer, Christian; Hoepfner, Dominic; Seeber, Andrew; Loewith, Robbie; Movva, N Rao; Gasser, Susan M

    2013-09-26

    A chemicogenetic screen was performed in budding yeast mutants that have a weakened replication stress response. This identified an inhibitor of target of rapamycin (TOR) complexes 1 and 2 that selectively enhances the sensitivity of sgs1Δ cells to hydroxyurea and camptothecin. More importantly, the inhibitor has strong synthetic lethality in combination with either the break-inducing antibiotic Zeocin or ionizing radiation, independent of the strain background. Lethality correlates with a rapid fragmentation of chromosomes that occurs only when TORC2, but not TORC1, is repressed. Genetic inhibition of Tor2 kinase, or its downstream effector kinases Ypk1/Ypk2, conferred similar synergistic effects in the presence of Zeocin. Given that Ypk1/Ypk2 controls the actin cytoskeleton, we tested the effects of actin modulators latrunculin A and jasplakinolide. These phenocopy TORC2 inhibition on Zeocin, although modulation of calcineurin-sensitive transcription does not. These results implicate TORC2-mediated actin filament regulation in the survival of low levels of DNA damage. PMID:24035500

  6. 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

  7. mTORC2 Responds to Glutamine Catabolite Levels to Modulate the Hexosamine Biosynthesis Enzyme GFAT1.

    PubMed

    Moloughney, Joseph G; Kim, Peter K; Vega-Cotto, Nicole M; Wu, Chang-Chih; Zhang, Sisi; Adlam, Matthew; Lynch, Thomas; Chou, Po-Chien; Rabinowitz, Joshua D; Werlen, Guy; Jacinto, Estela

    2016-09-01

    Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and macromolecule biosynthesis. The signals that respond to nutrient fluctuations to maintain metabolic homeostasis remain poorly understood. Here, we found that mTORC2 is activated by nutrient deprivation due to decreasing glutamine catabolites. We elucidate how mTORC2 modulates a glutamine-requiring biosynthetic pathway, the hexosamine biosynthesis pathway (HBP) via regulation of expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of the HBP. GFAT1 expression is dependent on sufficient amounts of glutaminolysis catabolites particularly α-ketoglutarate, which are generated in an mTORC2-dependent manner. Additionally, mTORC2 is essential for proper expression and nuclear accumulation of the GFAT1 transcriptional regulator, Xbp1s. Thus, while mTORC1 senses amino acid abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and have implications for understanding insulin resistance and tumorigenesis. PMID:27570073

  8. AMPK-mediated up-regulation of mTORC2 and MCL-1 compromises the anti-cancer effects of aspirin

    PubMed Central

    Hua, Hui; Yin, Yancun; Wang, Jiao; Luo, Ting; Jiang, Yangfu

    2016-01-01

    AMP-activated protein kinase (AMPK) is an important energy sensor that may inhibit cell proliferation or promote cell survival during stresses. Besides cyclooxygenase, AMPK is another target of the nonsteroid anti-inflammatory agent aspirin. Preclinical and clinical investigations demonstrate that aspirin can inhibit several types of cancer such as colorectal adenomas and hepatocellular carcinoma (HCC). However, little is known about the cellular response to aspirin that may lead to aspirin resistance. Here, we show that aspirin induces the expression of MCL-1 in HepG2 and SW480 cells through AMPK-mTOR-Akt/ERK axis. Treatment of HepG2 and SW480 cells with aspirin leads to increased MCL-1 expression, Akt and ERK1/2 phosphorylation. Inhibition of Akt/MEK abrogates the induction of MCL-1 by aspirin. Aspirin activates AMPK, which in turn up-regulates mTORC2 activity, Akt, ERK1/2 phosphorylation and MCL-1 expression. MCL-1 knockdown sensitizes cancer cells to aspirin-induced apoptosis. Combination of aspirin and AMPK, Akt or MEK inhibitor results in more significant inhibition of cell proliferation and induction of apoptosis than single agent. Moreover, sorafenib blocks aspirin-induced MCL-1 up-regulation. Combination of aspirin and sorafenib leads to much more cell death and less cell proliferation than each drug alone. Treatment of HCC and colon cancer xenografts with both aspirin and sorafenib results in more significant tumor suppression than single agent. These data demonstrate that AMPK-mediated up-regulation of mTORC2 and MCL-1 may compromise the anticancer effects of aspirin. Combination of aspirin and sorafenib may be an effective regimen to treat HCC and colon cancer. PMID:26918349

  9. AMPK-mediated up-regulation of mTORC2 and MCL-1 compromises the anti-cancer effects of aspirin.

    PubMed

    Gao, Mei; Kong, Qingbin; Hua, Hui; Yin, Yancun; Wang, Jiao; Luo, Ting; Jiang, Yangfu

    2016-03-29

    AMP-activated protein kinase (AMPK) is an important energy sensor that may inhibit cell proliferation or promote cell survival during stresses. Besides cyclooxygenase, AMPK is another target of the nonsteroid anti-inflammatory agent aspirin. Preclinical and clinical investigations demonstrate that aspirin can inhibit several types of cancer such as colorectal adenomas and hepatocellular carcinoma (HCC). However, little is known about the cellular response to aspirin that may lead to aspirin resistance. Here, we show that aspirin induces the expression of MCL-1 in HepG2 and SW480 cells through AMPK-mTOR-Akt/ERK axis. Treatment of HepG2 and SW480 cells with aspirin leads to increased MCL-1 expression, Akt and ERK1/2 phosphorylation. Inhibition of Akt/MEK abrogates the induction of MCL-1 by aspirin. Aspirin activates AMPK, which in turn up-regulates mTORC2 activity, Akt, ERK1/2 phosphorylation and MCL-1 expression. MCL-1 knockdown sensitizes cancer cells to aspirin-induced apoptosis. Combination of aspirin and AMPK, Akt or MEK inhibitor results in more significant inhibition of cell proliferation and induction of apoptosis than single agent. Moreover, sorafenib blocks aspirin-induced MCL-1 up-regulation. Combination of aspirin and sorafenib leads to much more cell death and less cell proliferation than each drug alone. Treatment of HCC and colon cancer xenografts with both aspirin and sorafenib results in more significant tumor suppression than single agent. These data demonstrate that AMPK-mediated up-regulation of mTORC2 and MCL-1 may compromise the anticancer effects of aspirin. Combination of aspirin and sorafenib may be an effective regimen to treat HCC and colon cancer. PMID:26918349

  10. 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…

  11. microRNA-153 Targets mTORC2 Component Rictor to Inhibit Glioma Cells

    PubMed Central

    Cui, Yan; Zhao, Jizong; Yi, Lei; Jiang, Yugang

    2016-01-01

    Rictor upregulation and mTORC complex 2 (mTORC2) over-activation participate in glioma cell progression, yet the underling mechanisms are not known. We here identified microRNA-153 (miR-153) as a potential anti-Rictor miRNA, which was downregulated in multiple human glioma tissues and glioma cell lines (U87MG, T98G, U373MG and U251MG). miR-153 downregulation was correlated with Rictor (mRNA and protein) upregulation and p-Akt Ser473 (the mTORC2 indicator) over-activation in the glioma tissues and cells. Our in vitro evidences suggested that Rictor could be one primary target of miR-153 in glioma cells. Exogenous overexpression of miR-153 downregulated Rictor (mRNA and protein) and decreased p-Akt Ser473 in U87MG cells, leading to significant growth inhibition and apoptosis activation. Notably, U87MG cells with Rictor shRNA knockdown showed similar phenotypes of cells with miR-153 overexpression. More importantly, in Rictor-silenced U87MG cells, miR-153 expression failed to further affect cell growth nor apoptosis. In vivo, we showed that miR-153 overexpression dramatically inhibited U87MG tumor growth in nude mice. Together, these results suggest that miR-153 downregulation could be one important reason of Rictor upregulation and mTORC2 over-activation in glioma cells. Further, miR-153-induced anti-glioma cell activity is possibly via downregulating Rictor. PMID:27295037

  12. Rictor/mTORC2 loss in osteoblasts impairs bone mass and strength.

    PubMed

    Liu, Dong-Mei; Zhao, Lin; Liu, Ting-Ting; Jiao, Pei-Lin; Zhao, Dian-Dian; Shih, Mei-Shu; Tao, Bei; Sun, Li-Hao; Zhao, Hong-Yan; Liu, Jian-Min

    2016-09-01

    Mammalian target of rapamycin (mTOR) is a Ser/Thr kinase conserved through evolution that coordinates extra cellular signals associated with cell growth. Main functions of mTOR present in the form of two complexes, namely mTORC1 and mTORC2, which are distinct in their unique components, raptor and rictor. In the current study, using a Cre/loxp system, we found an anabolic effect of mTORC2 signaling on skeleton. Osteoblast differentiation was reduced, with down-regulation of mTORC2 signaling activity in primary cultures of osteoblasts that did not contain rictor. Mice with a specific deletion of rictor in mature osteoblasts showed a significant reduction in lean mass and bone mineral density by dual energy x-ray absorptiometry analysis. Micro-computed tomography, histomorphometric, and molecular biological analyses revealed a marked impairment of the cortical bone mass and microarchitecture, as well as minor changes in trabecular bone, of the Rictorob(-/-) mice. Cortical bone mass and thickness of the femoral mid-shaft were dramatically reduced, with unusual increases in porosity and marrow area in Rictorob(-/-) mice. Thinner trabeculae were found in the L4 vertebrae with relatively normal structural indices of trabecular numbers and separation. A lower rate of bone turnover was observed, as the consequence of the decreased individual osteoblast activity and bone resorption. Furthermore, these changes were associated with significantly decreased bone biomechanical properties. In conclusion, expression of rictor in osteoblasts is essential for the maintenance of normal bone remodeling and microarchitecture, especially for the maintenance of the cortical bone. PMID:27262777

  13. Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans.

    PubMed

    Soukas, Alexander A; Kane, Elizabeth A; Carr, Christopher E; Melo, Justine A; Ruvkun, Gary

    2009-02-15

    Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. We identified mutations in the Caenorhabditis elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span. PMID:19240135

  14. Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans

    PubMed Central

    Soukas, Alexander A.; Kane, Elizabeth A.; Carr, Christopher E.; Melo, Justine A.; Ruvkun, Gary

    2009-01-01

    Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. We identified mutations in the Caenorhabditis elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span. PMID:19240135

  15. Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

    PubMed

    Thompson, Scott M; Callstrom, Matthew R; Jondal, Danielle E; Butters, Kim A; Knudsen, Bruce E; Anderson, Jill L; Lien, Karen R; Sutor, Shari L; Lee, Ju-Seog; Thorgeirsson, Snorri S; Grande, Joseph P; Roberts, Lewis R; Woodrum, David A

    2016-01-01

    Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2-3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin. PMID:27611696

  16. CREB Inhibits AP-2α Expression to Regulate the Malignant Phenotype of Melanoma

    PubMed Central

    Zigler, Maya; Villares, Gabriel J.; Braeuer, Russell R.; Wang, Hua; Huang, Li; Bar-Eli, Menashe

    2010-01-01

    Background The loss of AP-2α and increased activity of cAMP-responsive element binding (CREB) protein are two hallmarks of malignant progression of cutaneous melanoma. However, the molecular mechanism responsible for the loss of AP-2α during melanoma progression remains unknown. Methodology/Principal Findings Herein, we demonstrate that both inhibition of PKA-dependent CREB phosphorylation, as well as silencing of CREB expression by shRNA, restored AP-2α protein expression in two metastatic melanoma cell lines. Moreover, rescue of CREB expression in CREB-silenced cell lines downregulates expression of AP-2α. Loss of AP-2α expression in metastatic melanoma occurs via a dual mechanism involving binding of CREB to the AP-2α promoter and CREB-induced overexpression of another oncogenic transcription factor, E2F-1. Upregulation of AP-2α expression following CREB silencing increases endogenous p21Waf1 and decreases MCAM/MUC18, both known to be downstream target genes of AP-2α involved in melanoma progression. Conclusions/Significance Since AP-2α regulates several genes associated with the metastatic potential of melanoma including c-KIT, VEGF, PAR-1, MCAM/MUC18, and p21Waf1, our data identified CREB as a major regulator of the malignant melanoma phenotype. PMID:20805990

  17. PRICKLE1 Contributes to Cancer Cell Dissemination through Its Interaction with mTORC2.

    PubMed

    Daulat, Avais M; Bertucci, François; Audebert, Stéphane; Sergé, Arnauld; Finetti, Pascal; Josselin, Emmanuelle; Castellano, Rémy; Birnbaum, Daniel; Angers, Stéphane; Borg, Jean-Paul

    2016-05-23

    Components of the evolutionarily conserved developmental planar cell polarity (PCP) pathway were recently described to play a prominent role in cancer cell dissemination. However, the molecular mechanisms by which PCP molecules drive the spread of cancer cells remain largely unknown. PRICKLE1 encodes a PCP protein bound to the promigratory serine/threonine kinase MINK1. We identify RICTOR, a member of the mTORC2 complex, as a PRICKLE1-binding partner and show that the integrity of the PRICKLE1-MINK1-RICTOR complex is required for activation of AKT, regulation of focal adhesions, and cancer cell migration. Disruption of the PRICKLE1-RICTOR interaction results in a strong impairment of breast cancer cell dissemination in xenograft assays. Finally, we show that upregulation of PRICKLE1 in basal breast cancers, a subtype characterized by high metastatic potential, is associated with poor metastasis-free survival. PMID:27184734

  18. Adipose tissue mTORC2 regulates ChREBP-driven de novo lipogenesis and hepatic glucose metabolism.

    PubMed

    Tang, Yuefeng; Wallace, Martina; Sanchez-Gurmaches, Joan; Hsiao, Wen-Yu; Li, Huawei; Lee, Peter L; Vernia, Santiago; Metallo, Christian M; Guertin, David A

    2016-01-01

    Adipose tissue de novo lipogenesis (DNL) positively influences insulin sensitivity, is reduced in obesity, and predicts insulin resistance. Therefore, elucidating mechanisms controlling adipose tissue DNL could lead to therapies for type 2 diabetes. Here, we report that mechanistic target of rapamycin complex 2 (mTORC2) functions in white adipose tissue (WAT) to control expression of the lipogenic transcription factor ChREBPβ. Conditionally deleting the essential mTORC2 subunit Rictor in mature adipocytes decreases ChREBPβ expression, which reduces DNL in WAT, and impairs hepatic insulin sensitivity. Mechanistically, Rictor/mTORC2 promotes ChREBPβ expression in part by controlling glucose uptake, but without impairing pan-AKT signalling. High-fat diet also rapidly decreases adipose tissue ChREBPβ expression and insulin sensitivity in wild-type mice, and does not further exacerbate insulin resistance in adipose tissue Rictor knockout mice, implicating adipose tissue DNL as an early target in diet-induced insulin resistance. These data suggest mTORC2 functions in WAT as part of an extra-hepatic nutrient-sensing mechanism to control glucose homeostasis. PMID:27098609

  19. Adipose tissue mTORC2 regulates ChREBP-driven de novo lipogenesis and hepatic glucose metabolism

    PubMed Central

    Tang, Yuefeng; Wallace, Martina; Sanchez-Gurmaches, Joan; Hsiao, Wen-Yu; Li, Huawei; Lee, Peter L.; Vernia, Santiago; Metallo, Christian M.; Guertin, David A.

    2016-01-01

    Adipose tissue de novo lipogenesis (DNL) positively influences insulin sensitivity, is reduced in obesity, and predicts insulin resistance. Therefore, elucidating mechanisms controlling adipose tissue DNL could lead to therapies for type 2 diabetes. Here, we report that mechanistic target of rapamycin complex 2 (mTORC2) functions in white adipose tissue (WAT) to control expression of the lipogenic transcription factor ChREBPβ. Conditionally deleting the essential mTORC2 subunit Rictor in mature adipocytes decreases ChREBPβ expression, which reduces DNL in WAT, and impairs hepatic insulin sensitivity. Mechanistically, Rictor/mTORC2 promotes ChREBPβ expression in part by controlling glucose uptake, but without impairing pan-AKT signalling. High-fat diet also rapidly decreases adipose tissue ChREBPβ expression and insulin sensitivity in wild-type mice, and does not further exacerbate insulin resistance in adipose tissue Rictor knockout mice, implicating adipose tissue DNL as an early target in diet-induced insulin resistance. These data suggest mTORC2 functions in WAT as part of an extra-hepatic nutrient-sensing mechanism to control glucose homeostasis. PMID:27098609

  20. mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue.

    PubMed

    Albert, Verena; Svensson, Kristoffer; Shimobayashi, Mitsugu; Colombi, Marco; Muñoz, Sergio; Jimenez, Veronica; Handschin, Christoph; Bosch, Fatima; Hall, Michael N

    2016-03-01

    Activation of non-shivering thermogenesis (NST) in brown adipose tissue (BAT) has been proposed as an anti-obesity treatment. Moreover, cold-induced glucose uptake could normalize blood glucose levels in insulin-resistant patients. It is therefore important to identify novel regulators of NST and cold-induced glucose uptake. Mammalian target of rapamycin complex 2 (mTORC2) mediates insulin-stimulated glucose uptake in metabolic tissues, but its role in NST is unknown. We show that mTORC2 is activated in brown adipocytes upon β-adrenergic stimulation. Furthermore, mice lacking mTORC2 specifically in adipose tissue (AdRiKO mice) are hypothermic, display increased sensitivity to cold, and show impaired cold-induced glucose uptake and glycolysis. Restoration of glucose uptake in BAT by overexpression of hexokinase II or activated Akt2 was sufficient to increase body temperature and improve cold tolerance in AdRiKO mice. Thus, mTORC2 in BAT mediates temperature homeostasis via regulation of cold-induced glucose uptake. Our findings demonstrate the importance of glucose metabolism in temperature regulation. PMID:26772600

  1. Structurally distinct modes of recognition of the KIX domain of CBP by Jun and CREB.

    PubMed

    Campbell, Kathleen M; Lumb, Kevin J

    2002-11-26

    Gene expression is coordinated in part by interactions between transcriptional activators and other transcription factors such as coactivators. The KIX domain of the coactivator and histone acetyltransferase CREB binding protein (CBP) binds numerous mammalian and viral transcriptional activators such as BRCA1, CREB, c-Jun, c-Myb, p53, papillomavirus E2, and HTLV-1 Tax. Formation of the CREB-CBP complex depends on phosphorylation of the KID region of CREB and involves induced folding of KID upon binding a hydrophobic groove of the KIX domain of CBP. Here we investigate the formation of the complex formed by human KIX and the N-terminal activation domain of human c-Jun. The c-Jun activation domain and KID do not share significant sequence similarity. Circular dichroism spectroscopy shows that the Jun N-terminal activation domain is intrinsically disordered in isolation and that KIX binding is independent of Jun phosphorylation. In contrast to the mode of binding exhibited by CREB, NMR chemical shift mapping indicates that the c-Jun activation domain binds to a distinctly different surface of KIX than used by CREB. Moreover, NMR and sedimentation equilibrium studies show that the activation domains of c-Jun and CREB can simultaneously bind the KIX domain of CBP. The results illustrate a new mode of binding and combinatorial recruitment via the KIX domain of CBP by multiple transcriptional activators. PMID:12437352

  2. The Rac1 Inhibitor NSC23766 Suppresses CREB Signaling by Targeting NMDA Receptor Function

    PubMed Central

    Hou, Hailong; Chávez, Andrés E.; Wang, Chih-Chieh; Yang, Hongtian; Gu, Hua; Siddoway, Benjamin A.; Hall, Benjamin J.; Castillo, Pablo E.

    2014-01-01

    NMDA receptor signaling plays a complex role in CREB activation and CREB-mediated gene transcription, depending on the subcellular location of NMDA receptors, as well as how strongly they are activated. However, it is not known whether Rac1, the prototype of Rac GTPase, plays a role in neuronal CREB activation induced by NMDA receptor signaling. Here, we report that NSC23766, a widely used specific Rac1 inhibitor, inhibits basal CREB phosphorylation at S133 (pCREB) and antagonizes changes in pCREB levels induced by NMDA bath application in rat cortical neurons. Unexpectedly, we found that NSC23766 affects the levels of neuronal pCREB in a Rac1-independent manner. Instead, our results indicate that NSC23766 can directly regulate NMDA receptors as indicated by their strong effects on both exogenous and synaptically evoked NMDA receptor-mediated currents in mouse and rat neurons, respectively. Our findings strongly suggest that Rac1 does not affect pCREB signaling in cortical neurons and reveal that NSC23766 could be a novel NMDA receptor antagonist. PMID:25319697

  3. Cyclin-dependent Kinase 1-dependent Phosphorylation of cAMP Response Element-binding Protein Decreases Chromatin Occupancy*

    PubMed Central

    Trinh, Anthony T.; Kim, Sang Hwa; Chang, Hae-yoon; Mastrocola, Adam S.; Tibbetts, Randal S.

    2013-01-01

    The cyclic AMP response element-binding protein (CREB) initiates transcriptional responses to a wide variety of stimuli. CREB activation involves its phosphorylation on Ser-133, which promotes interaction between the CREB kinase-inducible domain (KID) and the KID-interacting domain of the transcriptional coactivator, CREB-binding protein (CBP). The KID also contains a highly conserved phosphorylation cluster, termed the ATM/CK cluster, which is processively phosphorylated in response to DNA damage by the coordinated actions of ataxia-telangiectasia-mutated (ATM) and casein kinases (CKs) 1 and 2. The ATM/CK cluster phosphorylation attenuates CBP binding and CREB transcriptional activity. Paradoxically, it was recently reported that DNA damage activates CREB through homeodomain-interacting protein kinase 2-dependent phosphorylation of Ser-271 near the CREB bZIP DNA binding domain. In this study we sought to further clarify DNA damage-dependent CREB phosphorylation as well as to explore the possibility that the ATM/CK cluster and Ser-271 synergistically or antagonistically modulate CREB activity. We show that, rather than being induced by DNA damage, Ser-270 and Ser-271 of CREB cophosphorylated in a CDK1-dependent manner during G2/M phase. Functionally, we show that phosphorylation of CREB on Ser-270/Ser-271 during mitosis correlated with reduced CREB chromatin occupancy. Furthermore, CDK1-dependent phosphorylation of CREB in vitro inhibited its DNA binding activity. The combined results suggest that CDK1-dependent phosphorylation of CREB on Ser-270/Ser-271 facilitates its dissociation from chromatin during mitosis by reducing its intrinsic DNA binding potential. PMID:23814058

  4. mTORC2 REGULATES NEUTROPHIL CHEMOTAXIS IN A cAMP- AND RhoA-DEPENDENT FASHION

    PubMed Central

    Liu, Lunhua; Das, Satarupa; Losert, Wolfgang; Parent, Carole A.

    2010-01-01

    SUMMARY We studied the role of the target of rapamycin complex 2 (mTORC2) during neutrophil chemotaxis, a process that is mediated through the polarization of actin and myosin filament networks. We show that inhibition of mTORC2 activity, achieved via knock down (KD) of Rictor, severely inhibits neutrophil polarization and directed migration induced by chemoattractants, independently of Akt. Rictor KD also abolishes the ability of chemoattractants to induce cAMP production, a process mediated through the activation of the adenylyl cyclase 9 (AC9). Cells with either reduced or higher AC9 levels also exhibit specific and severe tail retraction defects that are mediated through RhoA. We further show that cAMP is excluded from extending pseudopods and remains restricted to the cell body of migrating neutrophils. We propose that the mTORC2-dependent regulation of MyoII occurs through a cAMP/RhoA signaling axis, independently of actin reorganization during neutrophil chemotaxis. PMID:21145500

  5. 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

  6. 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

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

    PubMed

    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 SCF(β-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. The miR-491-3p/mTORC2/FOXO1 regulatory loop modulates chemo-sensitivity in human tongue cancer

    PubMed Central

    Peng, Cong; Deng, Yingen; Yin, Jiang; Zhang, Zhijie; Li, Nan; Deng, Min; Liu, Xiaorong; Liu, Hao; Lu, Minying; Wang, Chengkun; Gu, Yixue; He, Zhimin

    2015-01-01

    We found that levels of miR-491-3p were decreased in multidrug-resistant tongue cancer (TC) cells. Induction of miR-491-3p expression sensitized TC cells to chemotherapy. In agreement, functional inhibition of miR-491-3p enhanced resistance of TC cells to chemotherapy. We found that miR-491-3p directly targeted mTORC2 component Rictor and inhibited mTORC2 activity, which was increased in resistant TC cells with high p-Akt(Ser473), p-SGK1(Ser422) and p-FOXO1(Thr24) levels. Inhibition of mTORC2 activity via either Rictor knockdown or mTOR inhibitor in turn sensitized TC cells to chemotherapy. In agreement, overexpression of Rictor increased the mTORC2 activity and induced resistance of TC cells to chemotherapy. As a feedback loop, mTORC2 downregulated miR-491-3p expression by inactivating FOXO1, which otherwise would transcriptionally induce miR-491-3p expression. Levels of miR-491–3 and Rictor or mTORC2 activity negatively correlated in TC tissues. Finally, low levels of miR-491-3p and highly expressed Rictor were associated with poor prognosis in tongue cancer patients. These data provide a rationale for targeted intervention on miR-491-3p/mTORC2 axis to enhance the efficacy of chemotherapy against tongue cancer. PMID:25749387

  9. A phase I dose‐escalation study to assess safety, tolerability, pharmacokinetics, and preliminary efficacy of the dual mTORC1/mTORC2 kinase inhibitor CC‐223 in patients with advanced solid tumors or multiple myeloma

    PubMed Central

    Bendell, Johanna C.; Kelley, Robin K.; Shih, Kent C.; Grabowsky, Jennifer A.; Bergsland, Emily; Jones, Suzanne; Martin, Thomas; Infante, Jeffrey R.; Mischel, Paul S.; Matsutani, Tomoo; Xu, Shuichan; Wong, Lilly; Liu, Yong; Wu, Xiaoling; Mortensen, Deborah S.; Chopra, Rajesh; Hege, Kristen

    2015-01-01

    BACKGROUND The mammalian target of rapamycin (mTOR) pathway is essential for tumor development, yet mTOR inhibitors have yielded modest results. This phase 1 study investigated the mTORC1/mTORC2 inhibitor CC‐223 in patients with advanced cancer. METHODS Patients with advanced solid tumors or multiple myeloma received an initial dose of 7.5‐60 mg of CC‐223, followed by oral daily dosing in 28‐day cycles until disease progression. The primary objective was to determine the safety, tolerability, nontolerated dosage, maximum tolerated dosage (MTD), and preliminary pharmacokinetic profile. Secondary objectives were to evaluate pharmacodynamic effects and to describe preliminary efficacy. RESULTS Twenty‐eight patients were enrolled and received ≥1 dose of CC‐223. The most common treatment‐related grade 3 adverse events were hyperglycemia, fatigue, and rash. Four patients had dose‐limiting toxicities, including hyperglycemia, rash, fatigue, and mucositis. Therefore, 45 mg/d was determined to be the MTD. The pharmacokinetics of CC‐223 demonstrated a mean terminal half‐life ranging from 4.86 to 5.64 hours and maximum observed plasma concentration ranging from 269 to 480 ng/mL in patients who received CC‐223 ≥45 mg/d. Phosphorylation of mTORC1/mTORC2 pathway biomarkers in blood cells was inhibited by CC‐223 ≥30 mg/d with an exposure‐response relationship. Best responses included 1 partial response (breast cancer; response duration 220 days; 30‐mg/d cohort), stable disease (8 patients across ≥15 mg/d cohorts; response duration range, 36‐168 days), and progressive disease (12 patients). The disease control rate was 32%. CONCLUSIONS CC‐223 was tolerable, with manageable toxicities. Preliminary antitumor activity, including tumor regression, and evidence of mTORC1/mTORC2 pathway inhibition were observed. Cancer 2015;121:3435–43. © 2015 American Cancer Society. PMID:26177599

  10. Hippocampal overexpression of mutant creb blocks long-term, but not short-term memory for a socially transmitted food preference.

    PubMed

    Brightwell, Jennifer J; Smith, Clayton A; Countryman, Renee A; Neve, Rachael L; Colombo, Paul J

    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 phosphorylation in the hippocampus of trained rats in comparisons with controls. In the current study, we tested the hypothesis that CREB function is necessary for long-term memory for STFP using herpes simplex viral (HSV) vector-mediated gene transfer. Rats received intrahippocampal infusions of HSV-mCREB (a mutant form of CREB, in which Ser133 has been replaced with Ala), HSV-LacZ, or saline, and were trained 3 d later. Rats were tested for food preference (demonstrated vs. novel foods) immediately (short-term test) and 11 d (long-term test) after training. Rats in all treatment groups showed a significant preference for the demonstrated food at the short-term memory test. At the long-term memory test, however, the percentage of demonstrated food eaten by mCREB-treated rats was significantly less than that eaten by the LacZ- or saline-treated rats. Quantitative Western blotting confirmed that mCREB-infused rats had significantly more hippocampal CREB protein than controls during training. The present results show that hippocampal CREB function is necessary for long-term, but not short-term memory for STFP. PMID:15687228

  11. The anxious amygdala: CREB signaling and predisposition to anxiety and alcoholism.

    PubMed

    Wand, Gary

    2005-10-01

    The amygdala is believed to play a key role in assigning emotional significance to specific sensory input, and conditions such as anxiety, autism, stress, and phobias are thought to be linked to its abnormal function. Growing evidence has also implicated the amygdala in mediation of the stress-dampening properties of alcohol. In this issue of the JCI, Pandey and colleagues identify a central amygdaloid signaling pathway involved in anxiety-like and alcohol-drinking behaviors in rats. They report that decreased phosphorylation of cAMP responsive element-binding protein (CREB) resulted in decreased neuropeptide Y (NPY) expression in the central amygdala of alcohol-preferring rats, causing high anxiety-like behavior. Alcohol intake by these animals was shown to increase PKA-dependent CREB phosphorylation and thereby NPY expression, subsequently ameliorating anxiety-like behavior. These provocative data suggest that a CREB-dependent neuromechanism underlies high anxiety-like and excessive alcohol-drinking behavior. PMID:16200206

  12. Fluoxetine Increases the Expression of NCAM140 and pCREB in Rat C6 Glioma Cells

    PubMed Central

    Choi, Mi Ran; Oh, Dong Hoon; Kim, Seok Hyeon; Jung, Kyoung Hwa; Das, Nando Dulal

    2012-01-01

    Objective Dysfunction of neural plasticity in the brain is known to alter neural networks, resulting in depression. To understand how fluoxetine regulates molecules involved in neural plasticity, the expression levels of NCAM, NCAM140, CREB and pCREB, in rat C6 glioma cells after fluoxetine treatment were examined. Methods C6 cells were cultured after 20 min or after 6, 24 or 72 h treatments with 10 µM fluoxetine. Immunocytochemistry was used to determine the effect of fluoxetine on the expression of NCAM. Western blot analysis was used to measure the expression levels of NCAM140 and CREB and the induction of pCREB after fluoxetine treatment. Results NCAM expression following 72-h fluoxetine treatment was significantly increased around cell membranes compared to control cells. Cells treated with fluoxetine for 6 and 72 h showed a significant increase in NCAM140 expression compared to cells treated for 20 min. The level of pCREB in the cells treated with fluoxetine for 72 h not only increased more than 60%, but was also significantly different when compared with the other treatment times. The 72-h fluoxetine treatment led to the increase of NCAM140 and the phosphorylation of CREB in C6 cells. Conclusion Our findings indicate that fluoxetine treatment regulates neuronal plasticity and neurite outgrowth by phosphorylating and activating CREB via the NCAM140 homophilic interaction-induced activation of the Ras-MAPK pathway. PMID:22707970

  13. 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

  14. FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2.

    PubMed Central

    Tan, Y; Rouse, J; Zhang, A; Cariati, S; Cohen, P; Comb, M J

    1996-01-01

    Fibroblast growth factor (FGF) activates a protein kinase cascade in SK-N-MC cells that regulates gene expression at a cyclic-AMP response element (CRE) by stimulating the transcriptional activity of CREB. The activation of CREB is prevented by a dominant negative mutant of Ras and triggered via the same site (Ser133) that becomes phosphorylated in response to cyclic AMP and Ca2+. However, the effect of FGF is not mediated by cyclic AMP-dependent protein kinase, TPA-sensitive isoforms of protein kinase-C, p70S6K or p90rsk (all of which phosphorylate CREB at Ser133 in vitro). Instead, we identify the FGF-stimulated CREB kinase as MAP kinase-activated protein (MAPKAP) kinase-2, an enzyme that lies immediately downstream of p38 MAP kinase, in a pathway that is also stimulated by cellular stresses. We show that MAPKAP kinase-2 phosphorylates CREB at Ser133 in vitro, that the FGF- or stress-induced activation of MAPKAP kinase-2 and phosphorylation of CREB and ATF-1 are prevented by similar concentrations of the specific p38 MAP kinase inhibitor SB 203580, and that MAPKAP kinase-2 is the only detectable SB 203580-sensitive CREB kinase in SK-N-MC cell extracts. We also show that transfection of RK/p38 MAP kinase in SK-N-MC cells, but not transfection of p44 MAP kinase, activates Gal4-CREB-dependent transcription via Ser133. These findings identify a new growth factor and stress-activated signaling pathway that regulates gene expression at the CRE. Images PMID:8887554

  15. Rapamycin protects livers from ischemia and reperfusion Injury via both autophagy induction and mTORC2-Akt activation

    PubMed Central

    Zhu, Jianjun; Lu, Tianfei; Yue, Shi; Shen, Xiuda; Gao, Feng; Busuttil, Ronald W.; Kupiec-Weglinski, Jerzy W.; Xia, Qiang; Zhai, Yuan

    2014-01-01

    Background Although Rapamycin (RPM) have been studied extensively in ischemia models, its functional mechanisms remains to be defined. Methods We determined how RPM impacted the pathogenesis of ischemia reperfusion injury (IRI) in a murine liver partial warm ischemia model, with emphasis on its regulation of hepatocyte death. Results RPM protected livers from IRI in the presence of fully developed liver inflammatory immune response. RPM enhanced liver autophagy induction at the reperfusion stage. Dual mTOR1/2 inhibitor Torin 1, despite its ability to induced autophagy, failed to protect livers from IRI. The treatment with RPM, but not Torin 1, resulted in the enhanced activation of the mTORC2-Akt signaling pathway activation in livers post reperfusion. Inactivation of Akt by Triciribine abolished liver protective effect of RPM. The differential cytoprotective effect of RPM and Torin 1 was confirmed in vitro in hepatocyte cultures. RPM, but not Trin 1, protected hepatocytes from stress and TNF-α induced cell death; and inhibition of either autophagy by chloroquine or Akt by Triciribine abolished RPM-mediated cytoprotection. Conclusion RPM protected livers from IRI via both autophagy and mTORC2-Akt activation mechanisms. PMID:25340604

  16. Membrane Tension Acts Through PLD2 and mTORC2 to Limit Actin Network Assembly During Neutrophil Migration

    PubMed Central

    Diz-Muñoz, Alba; Thurley, Kevin; Chintamen, Sana; Altschuler, Steven J.; Fletcher, Daniel A.; Weiner, Orion D.

    2016-01-01

    For efficient polarity and migration, cells need to regulate the magnitude and spatial distribution of actin assembly. This process is coordinated by reciprocal interactions between the actin cytoskeleton and mechanical forces. Actin polymerization-based protrusion increases tension in the plasma membrane, which in turn acts as a long-range inhibitor of actin assembly. These interactions form a negative feedback circuit that limits the magnitude of membrane tension in neutrophils and prevents expansion of the existing front and the formation of secondary fronts. It has been suggested that the plasma membrane directly inhibits actin assembly by serving as a physical barrier that opposes protrusion. Here we show that efficient control of actin polymerization-based protrusion requires an additional mechanosensory feedback cascade that indirectly links membrane tension with actin assembly. Specifically, elevated membrane tension acts through phospholipase D2 (PLD2) and the mammalian target of rapamycin complex 2 (mTORC2) to limit actin nucleation. In the absence of this pathway, neutrophils exhibit larger leading edges, higher membrane tension, and profoundly defective chemotaxis. Mathematical modeling suggests roles for both the direct (mechanical) and indirect (biochemical via PLD2 and mTORC2) feedback loops in organizing cell polarity and motility—the indirect loop is better suited to enable competition between fronts, whereas the direct loop helps spatially organize actin nucleation for efficient leading edge formation and cell movement. This circuit is essential for polarity, motility, and the control of membrane tension. PMID:27280401

  17. Membrane Tension Acts Through PLD2 and mTORC2 to Limit Actin Network Assembly During Neutrophil Migration.

    PubMed

    Diz-Muñoz, Alba; Thurley, Kevin; Chintamen, Sana; Altschuler, Steven J; Wu, Lani F; Fletcher, Daniel A; Weiner, Orion D

    2016-06-01

    For efficient polarity and migration, cells need to regulate the magnitude and spatial distribution of actin assembly. This process is coordinated by reciprocal interactions between the actin cytoskeleton and mechanical forces. Actin polymerization-based protrusion increases tension in the plasma membrane, which in turn acts as a long-range inhibitor of actin assembly. These interactions form a negative feedback circuit that limits the magnitude of membrane tension in neutrophils and prevents expansion of the existing front and the formation of secondary fronts. It has been suggested that the plasma membrane directly inhibits actin assembly by serving as a physical barrier that opposes protrusion. Here we show that efficient control of actin polymerization-based protrusion requires an additional mechanosensory feedback cascade that indirectly links membrane tension with actin assembly. Specifically, elevated membrane tension acts through phospholipase D2 (PLD2) and the mammalian target of rapamycin complex 2 (mTORC2) to limit actin nucleation. In the absence of this pathway, neutrophils exhibit larger leading edges, higher membrane tension, and profoundly defective chemotaxis. Mathematical modeling suggests roles for both the direct (mechanical) and indirect (biochemical via PLD2 and mTORC2) feedback loops in organizing cell polarity and motility-the indirect loop is better suited to enable competition between fronts, whereas the direct loop helps spatially organize actin nucleation for efficient leading edge formation and cell movement. This circuit is essential for polarity, motility, and the control of membrane tension. PMID:27280401

  18. The role of nitric oxide in the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein in the spinal cord after intradermal injection of capsaicin.

    PubMed

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

    2002-06-01

    We investigated the involvement of nitric oxide (NO) in the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein (CREB) in the spinal cord of rats during central sensitization after intradermal capsaicin injection. CREB and phosphorylated CREB (p-CREB) were measured by immunoblotting. The level of p-CREB increased by 20 minutes, peaked between 20 and 60 minutes after capsaicin injection, and started to decrease after 150 minutes. CREB itself did not show an obvious change after capsaicin injection. The p-CREB expression on the ipsilateral side of the spinal dorsal horn, but not on the contralateral side, increased significantly after capsaicin injection. The increase in p-CREB induced by capsaicin injection was partially blocked by pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, administered through a microdialysis fiber placed across the spinal cord. D-NAME, an inactive form of L-NAME, had no effect. CREB phosphorylation, not the level of CREB, was induced within 20 minutes by microdialysis administration of SIN-1, an NO donor. These results indicate that CREB phosphorylation in the spinal cord results from both endogenous and exogenous NO release and that p-CREB may play a role in central sensitization or in longer-term changes in gene expression induced by strong peripheral noxious stimulation. PMID:14622772

  19. mTORC2 deficiency in myeloid DC enhances their allogeneic Th1 and Th17 stimulatory ability after TLR4 ligation in vitro and in vivo

    PubMed Central

    Raïch-Regué, Dàlia; Rosborough, Brian R.; Watson, Alicia R.; McGeachy, Mandy J.; Turnquist, Hēth R.; Thomson, Angus W.

    2015-01-01

    The mammalian/mechanistic target of rapamycin (mTOR) is a key integrative kinase that functions in two independent complexes, mTOR complex (mTORC) 1 and mTORC2. In contrast to the well-defined role of mTORC1 in dendritic cells (DC), little is known about the function of mTORC2. Here, we demonstrate for the first time an enhanced ability of mTORC2-deficient myeloid DC to stimulate and polarize allogeneic T cells. We show that activated bone marrow-derived DC from conditional Rictor−/− mice exhibit lower co-inhibitory B7-H1 molecule expression independently of the stimulus and enhanced IL-6, TNFα, IL-12p70 and IL-23 production following TLR4 ligation. Accordingly, TLR4-activated Rictor−/− DC display augmented allogeneic T cell stimulatory ability, expanding IFN-γ+ and IL-17+, but not IL-10+ or CD4+Foxp3+ regulatory T cells in vitro. A similar DC profile was obtained by stimulating Dectin-1 (C-type lectin family member) on Rictor−/− DC. Using novel CD11c-specific Rictor−/− mice, we confirm the alloreactive Th1 and Th17 cell-polarizing ability of endogenous mTORC2-deficient DC after TLR4 ligation in vivo. Furthermore, we demonstrate that pro-inflammatory cytokines produced by Rictor−/− DC after LPS stimulation are key in promoting Th1/Th17 responses. These data establish that mTORC2 activity restrains conventional DC pro-inflammatory capacity and their ability to polarize T cells following TLR and non-TLR stimulation. Our findings provide new insight into the role of mTORC2 in regulating DC function and may have implications for emerging therapeutic strategies that target mTOR in cancer, infectious diseases, and transplantation. PMID:25840913

  20. PKA-CREB-BDNF signaling regulated long lasting antidepressant activities of Yueju but not ketamine

    PubMed Central

    Xue, Wenda; Wang, Wei; Gong, Tong; Zhang, Hailou; Tao, Weiwei; Xue, Lihong; Sun, Yan; Wang, Fushun; Chen, Gang

    2016-01-01

    Yueju confers antidepressant effects in a rapid and long-lasting manner, similar to ketamine. CREB (cAMP-response element binding protein) signaling is implicated in depression pathology and antidepressant responses. However, the role of CREB and associated brain derived neurotrophic factor (BDNF) signaling in rapid and long-lasting antidepressant effects remains unclear. Here, we demonstrated that ICR and Kunming strain mice conferred antidepressant responses lasting for 1 and 5 days, respectively, following a single dose of Yueju. One day post Yueju in Kunming but not ICR strain mice, expression of total and phosphorylated CREB, as well as the CREB signaling activator, PKA (protein kinase A) was up-regulated in the hippocampus. Although BDNF gene expression increased at 3 hours in both strains, it remained up-regulated at 1 day only in Kunming mice. Ketamine showed similar strain-dependent behavioral effects. However, blockade of PKA/CREB signaling blunted the antidepressant effects and reversed the up-regulation of BDNF gene expression by Yueju, but not ketamine. Conversely, blockade of mammalian target of rapamycin signaling led to opposite effects. Taken altogether, prolonged transcriptional up-regulation of hippocampal BDNF may account for the stain-dependent enduring antidepressant responses to Yueju and ketamine, but it was mediated via PKA/CREB pathway only for Yueju. PMID:27197752

  1. PKA-CREB-BDNF signaling regulated long lasting antidepressant activities of Yueju but not ketamine.

    PubMed

    Xue, Wenda; Wang, Wei; Gong, Tong; Zhang, Hailou; Tao, Weiwei; Xue, Lihong; Sun, Yan; Wang, Fushun; Chen, Gang

    2016-01-01

    Yueju confers antidepressant effects in a rapid and long-lasting manner, similar to ketamine. CREB (cAMP-response element binding protein) signaling is implicated in depression pathology and antidepressant responses. However, the role of CREB and associated brain derived neurotrophic factor (BDNF) signaling in rapid and long-lasting antidepressant effects remains unclear. Here, we demonstrated that ICR and Kunming strain mice conferred antidepressant responses lasting for 1 and 5 days, respectively, following a single dose of Yueju. One day post Yueju in Kunming but not ICR strain mice, expression of total and phosphorylated CREB, as well as the CREB signaling activator, PKA (protein kinase A) was up-regulated in the hippocampus. Although BDNF gene expression increased at 3 hours in both strains, it remained up-regulated at 1 day only in Kunming mice. Ketamine showed similar strain-dependent behavioral effects. However, blockade of PKA/CREB signaling blunted the antidepressant effects and reversed the up-regulation of BDNF gene expression by Yueju, but not ketamine. Conversely, blockade of mammalian target of rapamycin signaling led to opposite effects. Taken altogether, prolonged transcriptional up-regulation of hippocampal BDNF may account for the stain-dependent enduring antidepressant responses to Yueju and ketamine, but it was mediated via PKA/CREB pathway only for Yueju. PMID:27197752

  2. 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. PMID:27220266

  3. GRM7 regulates embryonic neurogenesis via CREB and YAP.

    PubMed

    Xia, Wenlong; Liu, YanLi; Jiao, Jianwei

    2015-05-12

    Metabotropic glutamate receptor 7 (GRM7) has recently been identified to be associated with brain developmental defects, such as attention deficit hyperactivity disorder (ADHD) and autism. However, the function of GRM7 during brain development remains largely unknown. Here, we used gain- and loss-of-function strategies to investigate the role of GRM7 in early cortical development. We demonstrate that Grm7 knockdown increases neural progenitor cell (NPC) proliferation, decreases terminal mitosis and neuronal differentiation, and leads to abnormal neuronal morphology. GRM7 regulates the phosphorylation of cyclic AMP response element-binding protein (CREB) and the expression of Yes-associated protein (YAP) by directly interacting with CaM, which subsequently regulates the expression of CyclinD1 and ultimately affects early cortical development. These defects in neurogenesis are ameliorated by Grm7 overexpression, Creb knockdown, or Yap knockdown. Thus, our findings indicate that GRM7 signaling via CREB and YAP is necessary for neurogenesis in the brain. PMID:25921811

  4. 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

  5. Structure–activity relationship studies of naphthol AS-E and its derivatives as anticancer agents by inhibiting CREB-mediated gene transcription

    PubMed Central

    Li, Bingbing X.; Yamanaka, Kinrin; Xiao, Xiangshu

    2012-01-01

    CREB (cyclic AMP-response element binding protein) is a downstream transcription factor of a multitude of signaling pathways emanating from receptor tyrosine kinases or G-protein coupled receptors. CREB is not activated until it is phosphorylated at Ser133 and its subsequent binding to CREB-binding protein (CBP) through kinase-inducible domain (KID) in CREB and KID-interacting (KIX) domain in CBP. Tumor tissues from various organs present higher level of expression and activation of CREB. Thus CREB has been proposed as a promising cancer drug target. We previously described naphthol AS-E (1a) as a small molecule inhibitor of CREB-mediated gene transcription in living cells. Here we report the structure–activity relationship (SAR) studies of 1a by modifying the appendant phenyl ring. All the compounds were evaluated for in vitro inhibition of KIX–KID interaction, cellular inhibition of CREB-mediated gene transcription and inhibition of proliferation of four cancer cell lines (A549, MCF-7, MDA-MB-231 and MDA-MB-468). SAR indicated that a small and electron-withdrawing group was preferred at the para-position for KIX–KID interaction inhibition. Compound 1a was selected for further biological characterization and it was found that 1a down-regulated the expression of endogenous CREB target genes. Expression of a constitutively active CREB mutant, VP16-CREB in MCF-7 cells rendered the cells resistant to 1a, suggesting that CREB was critical in mediating its anticancer activity. Furthermore, 1a was not toxic to normal human cells. Collectively, these data support that 1a represents a structural template for further development into potential cancer therapeutics with a novel mechanism of action. PMID:23102993

  6. Dramatic antitumor effects of the dual mTORC1 and mTORC2 inhibitor AZD2014 in hepatocellular carcinoma

    PubMed Central

    Liao, Hui; Huang, Yu; Guo, Botang; Liang, Bo; Liu, Xincheng; Ou, Huohui; Jiang, Chenglong; Li, Xianghong; Yang, Dinghua

    2015-01-01

    The mammalian target of rapamycin (mTOR) has emerged as a critical effector in cell growth, proliferation, survival, angiogenesis, and autophagy through direct interaction with mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2). The mTOR axis is aberrantly activated in about 50% of human hepatocellular carcinoma (HCC) cases and thus has become an attractive target for drug development in this disease. Allosteric inhibitors of mTORC1, rapamycin and its derivatives have been used to study in patients with HCC but have not shown significant clinical utility, likely because of the lack of inhibition of mTORC2. In the present study, we describe that AZD2014, a small molecular ATP-competitive inhibitor of mTOR, was a highly potent inhibitor of mTORC1 and mTORC2 in human HCC cells, which led to a more thorough inhibition of mTORC1 than rapamycin, and the inhibition of mTORC2 prevented the feedback activation of AKT signaling. Compared with rapamycin, AZD2014 resulted in more profound proliferation suppression, apoptosis, cell cycle arrest, and autophagy in HCC cells. Notably, we found blockage of both mTORC1 and mTORC2 by AZD2014 to be more efficacious than blockage of mTORC1 alone by rapamycin in inhibiting the migration, invasion and EMT progression of HCC cells. In conclusion, our current results highlight mechanistic differentiation between rapamycin and AZD2014 in targeting cancer cell proliferation, cell cycle, apoptosis, autophagy, migration, invasion and EMT progression, and provide support for further investigation of AZD2014 as an antitumor agent for the treatment of HCC in clinic. PMID:25628925

  7. mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity.

    PubMed

    Gleason, Catherine E; Frindt, Gustavo; Cheng, Chih-Jen; Ng, Michael; Kidwai, Atif; Rashmi, Priyanka; Lang, Florian; Baum, Michel; Palmer, Lawrence G; Pearce, David

    2015-01-01

    The epithelial Na+ channel (ENaC) is essential for Na+ homeostasis, and dysregulation of this channel underlies many forms of hypertension. Recent studies suggest that mTOR regulates phosphorylation and activation of serum/glucocorticoid regulated kinase 1 (SGK1), which is known to inhibit ENaC internalization and degradation; however, it is not clear whether mTOR contributes to the regulation of renal tubule ion transport. Here, we evaluated the effect of selective mTOR inhibitors on kidney tubule Na+ and K+ transport in WT and Sgk1-/- mice, as well as in isolated collecting tubules. We found that 2 structurally distinct competitive inhibitors (PP242 and AZD8055), both of which prevent all mTOR-dependent phosphorylation, including that of SGK1, caused substantial natriuresis, but not kaliuresis, in WT mice, which indicates that mTOR preferentially influences ENaC function. PP242 also substantially inhibited Na+ currents in isolated perfused cortical collecting tubules. Accordingly, patch clamp studies on cortical tubule apical membranes revealed that mTOR inhibition markedly reduces ENaC activity, but does not alter activity of K+ inwardly rectifying channels (ROMK channels). Together, these results demonstrate that mTOR regulates kidney tubule ion handling and suggest that mTOR regulates Na+ homeostasis through SGK1-dependent modulation of ENaC activity. PMID:25415435

  8. Optogenetic Inhibitor of the Transcription Factor CREB.

    PubMed

    Ali, Ahmed M; Reis, Jakeb M; Xia, Yan; Rashid, Asim J; Mercaldo, Valentina; Walters, Brandon J; Brechun, Katherine E; Borisenko, Vitali; Josselyn, Sheena A; Karanicolas, John; Woolley, G Andrew

    2015-11-19

    Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events. PMID:26590638

  9. NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

    SciTech Connect

    Li, Mei; Zhang, Dong-Qing; Wang, Xiang-Zhen; Xu, Tie-Jun

    2011-08-12

    Highlights: {yields} The NR2B component of the NMDARs is important for the NSPC proliferation. {yields} pCaMKIV and pCREB exist in NSPCs. {yields} The CaMKIV/CREB pathway mediates NSPC proliferation. -- Abstract: Accumulating evidence indicates the involvement of N-methyl-D-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.

  10. Docosahexaenoic acid consumption inhibits deoxynivalenol-induced CREB/ATF1 activation and IL-6 gene transcription in mouse macrophages.

    PubMed

    Jia, Qunshan; Zhou, Hui-Ren; Shi, Yuhui; Pestka, James J

    2006-02-01

    The mycotoxin deoxynivalenol (DON) induces IgA nephropathy in mice by upregulating IL-6 expression, which is suppressed by (n-3) PUFA consumption. The purpose of this study was to test the hypothesis that consumption of the (n-3) PUFA docosahexaenoic acid (DHA) interferes with DON-induced transcriptional and post-transcriptional upregulation of IL-6 mRNA in murine macrophages. DON evoked expression of IL-6 mRNA and IL-6 heterogenous nuclear RNA (hnRNA), an indicator of ongoing IL-6 transcription, in macrophages elicited from mice fed control AIN-93G diet for 4 wk, whereas expression of both RNA species was suppressed in macrophages from mice fed AIN-93G modified to contain 30 g DHA/kg diet for the same time period. DON enhanced IL-6 mRNA stability similarly in macrophages from control and DHA-fed mice suggesting that (n-3) PUFA effects were not post-transcriptional. DON upregulated binding activity of cAMP response element binding protein (CREB) and activator protein (AP-1) to their respective consensus sequences in nuclear extracts from control-fed mice, whereas both activities were suppressed in nuclear extracts from DHA-fed mice. DON induced phosphorylation of CREB at Ser-133 and ATF1 at Ser-63 as well as intranuclear binding of phospho-CREB/ATF1 to the cis element of the IL-6 promoter in control macrophages, whereas both activities were inhibited in macrophages from DHA-fed mice. DHA consumption blocked DON-induced phosphorylation of the CREB kinase AKT. Inhibition of AKT suppressed both CREB/ATF1 phosphorylation and IL-6 transcription. These data suggest that DHA consumption suppresses DON-induced IL-6 transcription in macrophages in part by interfering with AKT-dependent phosphorylation and subsequent binding of CREB/ATF1 to the IL-6 promoter. PMID:16424113

  11. Transducer of regulated CREB-binding proteins (TORCs) induce PGC-1α transcription and mitochondrial biogenesis in muscle cells

    PubMed Central

    Wu, Zhidan; Huang, Xueming; Feng, Yajun; Handschin, Christoph; Feng, Yan; Gullicksen, P. Scott; Bare, Olivia; Labow, Mark; Spiegelman, Bruce; Stevenson, Susan C.

    2006-01-01

    PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) is a master regulator of mitochondrial biogenesis and plays an important role in several other aspects of energy metabolism. To identify upstream regulators of PGC-1α gene transcription, 10,000 human full-length cDNAs were screened for induction of the PGC-1α promoter. A number of activators of PGC-1α transcription were found; the most potent activator was the transducer of regulated CREB (cAMP response element-binding protein) binding protein (TORC) 1, a coactivator of CREB. The other two members of the TORC family, TORC2 and TORC3, also strongly activated PGC-1α transcription. TORCs dramatically induced PGC-1α gene transcription through CREB. Forced expression of TORCs in primary muscle cells induced the endogenous mRNA of PGC-1α and its downstream target genes in the mitochondrial respiratory chain and TCA cycle. Importantly, these changes in gene expression resulted in increased mitochondrial oxidative capacity measured by cellular respiration and fatty acid oxidation. Finally, we demonstrated that the action of TORCs in promoting mitochondrial gene expression and function requires PGC-1α. Previous studies had indicated that TORCs function as a calcium- and cAMP-sensitive coincidence detector and mediate individual and synergistic effects of these two pathways. Our results, together with previous findings, strongly suggest that TORCs play a key role in linking these external signals to the transcriptional program of adaptive mitochondrial biogenesis by activating PGC-1α gene transcription. PMID:16980408

  12. Interferon γ (IFNγ) Signaling via Mechanistic Target of Rapamycin Complex 2 (mTORC2) and Regulatory Effects in the Generation of Type II Interferon Biological Responses.

    PubMed

    Kroczynska, Barbara; Rafidi, Robert L; Majchrzak-Kita, Beata; Kosciuczuk, Ewa M; Blyth, Gavin T; Jemielity, Jacek; Warminska, Zofia; Saleiro, Diana; Mehrotra, Swarna; Arslan, Ahmet Dirim; Fish, Eleanor N; Platanias, Leonidas C

    2016-01-29

    We provide evidence for a unique pathway engaged by the type II IFN receptor, involving mTORC2/AKT-mediated downstream regulation of mTORC1 and effectors. These events are required for formation of the eukaryotic translation initiation factor 4F complex (eIF4F) and initiation of mRNA translation of type II interferon-stimulated genes. Our studies establish that Rictor is essential for the generation of type II IFN-dependent antiviral and antiproliferative responses and that it controls the generation of type II IFN-suppressive effects on normal and malignant hematopoiesis. Together, our findings establish a central role for mTORC2 in IFNγ signaling and type II IFN responses. PMID:26645692

  13. Chronic mild stress inhibits BDNF protein expression and CREB activation in the dentate gyrus but not in the hippocampus proper.

    PubMed

    Grønli, Janne; Bramham, Clive; Murison, Robert; Kanhema, Tambudzai; Fiske, Eldbjørg; Bjorvatn, Bjørn; Ursin, Reidun; Portas, Chiara M

    2006-12-01

    Chronic stress is linked to development of depression and may trigger neurobiological changes underlying the disease. Downregulation of the secretory peptide brain-derived neurotrophic factor (BDNF) and the transcriptional regulator calcium/cyclic-AMP responsive binding protein (CREB) have been implicated in stress and depression-related pathology in animal studies. When animals are exposed to the chronic mild stress (CMS) protocol, multiple depression-like symptoms are observed. Here we investigated the effect of CMS on BDNF protein expression and CREB activation in the dentate gyrus and hippocampus proper. Rats exposed for 5 weeks to repeated, unpredictable, mild stressors showed reduced BDNF expression and inhibited phosphorylation of CREB (Ser-133) in the dentate gyrus (-25.0%+/-3.5% and -29.7+/-7.3%, respectively), whereas no significant effects were observed in the hippocampus proper. CMS-treated rats consumed less sucrose compared to control rats, indicating a state of anhedonia. Moreover, phospho-CREB levels in the dentate gyrus were positively correlated with the animals' sucrose intake at the end of the CMS protocol. These results couple chronic mild stress to a downregulation of CREB activity and BDNF protein expression specifically within the dentate gyrus and support the possibility that the BDNF-CREB system plays an important role in the response to environmental challenges. PMID:17204313

  14. Rescuing prefrontal cAMP-CREB pathway reverses working memory deficits during withdrawal from prolonged alcohol exposure.

    PubMed

    Dominguez, G; Dagnas, M; Decorte, L; Vandesquille, M; Belzung, C; Béracochéa, D; Mons, N

    2016-03-01

    Both human and animal studies indicate that alcohol withdrawal following chronic alcohol consumption (CAC) impairs many of the cognitive functions which rely on the prefrontal cortex (PFC). A candidate signaling cascade contributing to memory deficits during alcohol withdrawal is the protein kinase A (PKA)/cAMP-responsive element binding (CREB) cascade, although the role of PKA/CREB cascade in behavioral and molecular changes during sustained withdrawal period remains largely unknown. We demonstrated that 1 week (1W) or 6 weeks (6W) withdrawal after 6-month CAC impairs working memory (WM) in a T-maze spontaneous alternation task and reduces phosphorylated CREB (pCREB) in the PFC but not the dorsal CA1 region (dCA1) of the hippocampus compared with CAC and water conditions. In contrast, both CAC-unimpaired and withdrawn-impaired mice exhibited decreased pCREB in dCA1 as well as reduced histone H4 acetylation in PFC and dCA1, compared with water controls. Next, we showed that enhancing CREB activity through rolipram administration prior to testing improved WM performance in withdrawn mice but impaired WM function in water mice. In addition, WM improvement correlates positively with increased pCREB level selectively in the PFC of withdrawn mice. Results further indicate that direct infusion of the PKA activator (Sp-cAMPS) into the PFC significantly improves or impairs, respectively, WM performance in withdrawn and water animals. In contrast, Sp-cAMPS had no effect on WM when infused into the dCA1. Collectively, these results provide strong support that dysregulation of PKA/CREB-dependent processes in prefrontal neurons is a critical molecular signature underlying cognitive decline during alcohol withdrawal. PMID:25388276

  15. CREB in the Pathophysiology of Cancer: Implications for Targeting Transcription Factors for Cancer Therapy

    PubMed Central

    Sakamoto, Kathleen M.; Frank, David A.

    2010-01-01

    Transcription factors are key regulators of the pattern of gene expression in a cell and directly control central processes such as proliferation, survival, self-renewal, and invasion. Given this critical role, the function of transcription factors is normally regulated closely, often through transient phosphorylation. Although transcription factors are not often directly modified by mutations in cancer cells, they frequently become activated constitutively through mutations affecting “upstream” pathways. By continually driving the expression of key target genes, these oncogenic transcription factors play a central role in tumor pathogenesis. One such transcription factor is the cAMP-regulatory element-binding protein (CREB), which can be activated through phosphorylation by a number of kinases, including Akt, p90Rsk, protein kinase A, and calcium/calmodulin-dependent kinases and regulates genes whose deregulated expression promotes oncogenesis, including cyclins, Bcl-2 family members, and Egr-1. CREB is overexpressed and constitutively phosphorylated in a number of forms of human cancer, including acute myeloid leukemia (AML) and non – small cell lung cancer, and appears to play a direct role in disease pathogenesis and prognosis. Although transcription factors have not been a central focus of drug development, recent advances suggest that CREB and other such proteins may be worthwhile targets for cancer therapy. PMID:19351775

  16. 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. PMID:25792232

  17. GSK-3/CREB pathway involved in the gx-50's effect on Alzheimer's disease.

    PubMed

    Tang, Maoping; Shi, Shi; Guo, Yubing; Xu, Wangjie; Wang, Lianyun; Chen, Yi; Wang, Zhaoxia; Qiao, Zhongdong

    2014-06-01

    Aggregation of amyloid-beta (Aβ) fragments is one of the major pathological hallmarks of Alzheimer's disease (AD). Our previous study has demonstrated that a novel compound named N-[2-(3, 4-dimethoxyphenyl) ethyl]-3-phenyl-acrylamide (gx-50) can decrease the accumulation of Aβ oligomers in the cerebral cortex and improve the cognitive abilities in transgenic demented mice. To further study the mechanism of the neuroprotective effect of gx-50 against AD, we employed microarray to investigate the gene expression profile of the primary cultured neurons treated with gx-50 or/and Aβ. Microarray disclosed 351 genes associated with AD in the gx-50 plus Aβ treated group, out of the 22,523 probes. 217 of the 351 genes were significantly up-regulated, 134 of them were down-regulated. The 351 genes were mainly involved in neurotransmission, signal transduction, nervous system development, protein phosphorylation, transcription and apoptosis. By the Onto-pathway analysis, a network involved two molecules - GSK-3, CREB and another two closely linked proteins - AKT, BDNF was discovered. The GSK/CREB pathway was further studied at the gene and protein level both in vivo and in vitro. Western blot and immunohistochemistry analysis showed that the gx-50 elevated the AKT phosphorylation and inhibited its downstream protein - GSK-3's activity, then restored the CREB's transcriptional activity, and finally enhanced the expression of the CREB target gene - BDNF. In addition, the real-time PCR results displayed the same tendency. In conclusion, studies in this research indicated that the gx-50 may improve the cognitive ability of AD via the GSK-3/CREB pathway. PMID:24565641

  18. Regulation of genotoxic stress response by homeodomain-interacting protein kinase 2 through phosphorylation of cyclic AMP response element-binding protein at serine 271.

    PubMed

    Sakamoto, Kensuke; Huang, Bo-Wen; Iwasaki, Kenta; Hailemariam, Kiros; Ninomiya-Tsuji, Jun; Tsuji, Yoshiaki

    2010-08-15

    CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2-/- MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2-/- cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress. PMID:20573984

  19. Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

    PubMed

    Rawashdeh, Oliver; Jilg, Antje; Maronde, Erik; Fahrenkrug, Jan; Stehle, Jörg H

    2016-09-01

    Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6 kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus

  20. Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling.

    PubMed

    Yao, Jin; Wang, Xiao-Qun; Li, Yu-Jie; Shan, Kun; Yang, Hong; Wang, Yang-Ning-Zhi; Yao, Mu-Di; Liu, Chang; Li, Xiu-Miao; Shen, Yi; Liu, Jing-Yu; Cheng, Hong; Yuan, Jun; Zhang, Yang-Yang; Jiang, Qin; Yan, Biao

    2016-01-01

    The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non-coding RNAs (lncRNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up-regulated in the retinas, Müller cells, and primary retinal ganglion cells (RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Müller cell activation, and RGC survival in vivo and in vitro MALAT1-CREB binding maintains CREB phosphorylation by inhibiting PP2A-mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling. PMID:26964565

  1. Nitric oxide protects neuroblastoma cells from apoptosis induced by serum deprivation through cAMP-response element-binding protein (CREB) activation.

    PubMed

    Ciani, Elisabetta; Guidi, Sandra; Della Valle, Giuliano; Perini, Giovanni; Bartesaghi, Renata; Contestabile, Antonio

    2002-12-20

    The transcription factor cAMP-response element-binding protein (CREB) mediates survival in many cells, including neurons. Recently, death of cerebellar granule neurons due to nitric oxide (NO) deprivation was shown to be accompanied by down-regulation of CREB activity (). We now provide evidence that overproduction of endogenous NO or supplementation with exogenous NO renders SK-N-BE human neuroblastoma cells more resistant to apoptosis induced by serum deprivation. Parental cells underwent apoptosis after 24 h of serum deprivation, an outcome largely absent in clones overexpressing human neuronal nitric oxide synthase (nNOS). This protective effect was reversed by the inhibition of NOS itself or soluble guanylyl cyclase, pointing at cGMP as an intermediate effector of NO-mediated rescue. A slow-releasing NO donor protected parental cells to a significant extent, thus confirming the survival effect of NO. The impaired viability of serum-deprived parental cells was accompanied by a strong decrease of CREB phosphorylation and transcriptional activity, effects significantly attenuated in nNOS-overexpressing clones. To confirm the role of CREB in survival, the ectopic expression of CREB and/or protein kinase A largely counteracted serum deprivation-induced cell death of SK-N-BE cells, whereas transfection with a CREB negative mutant was ineffective. These experiments indicate that CREB activity is an important step for NO-mediated survival in neuronal cells. PMID:12368293

  2. 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

  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 Increases IGFBP-1 Phosphorylation Mediated by mTOR Inhibition.

    PubMed

    Damerill, Ian; Biggar, Kyle K; Abu Shehab, Majida; Li, Shawn Shun-Cheng; Jansson, Thomas; Gupta, Madhulika B

    2016-02-01

    In fetal growth restriction (FGR), fetal growth is limited by reduced nutrient and oxygen supply. Insulin-like growth factor I (IGF-I) is a key regulator of fetal growth and IGF binding protein -1(IGFBP-1) is the principal regulator of fetal IGF-I bioavailability. Phosphorylation enhances IGFBP-1's affinity for IGF-I. Hypoxia induces IGFBP-1 hyperphosphorylation, markedly decreasing IGF-I bioavailability. We recently reported that fetal liver IGFBP-1 hyperphosphorylation is associated with inhibition of the mechanistic target of rapamycin (mTOR) in a nonhuman primate model of FGR. Here, we test the hypothesis that IGFBP-1 hyperphosphorylation in response to hypoxia is mediated by mTOR inhibition. We inhibited mTOR either by rapamycin or small interfering RNA (siRNA) targeting raptor (mTOR complex [mTORC]1) and/or rictor (mTORC2) in HepG2 cells cultured under hypoxia (1% O2) or basal (20% O2) conditions. Conversely, we activated mTORC1 or mTORC1+mTORC2 by silencing endogenous mTOR inhibitors (tuberous sclerosis complex 2/DEP-domain-containing and mTOR-interacting protein). Immunoblot analysis demonstrated that both hypoxia and inhibition of mTORC1 and/or mTORC2 induced similar degrees of IGFBP-1 phosphorylation at Ser101/119/169 and reduced IGF-I receptor autophosphorylation. Activation of mTORC1+mTORC2 or mTORC1 alone prevented IGFBP-1 hyperphosphorylation in response to hypoxia. Multiple reaction monitoring-mass spectrometry showed that rapamycin and/or hypoxia increased phosphorylation also at Ser98 and at a novel site Ser174. In silico structural analysis indicated that Ser174 was in close proximity to the IGF-binding site. Together, we demonstrate that signaling through the mTORC1 or mTORC2 pathway is sufficient to induce IGFBP-1 hyperphosphorylation in response to hypoxia. This study provides novel understanding of the cellular mechanism that controls fetal IGFBP-1 phosphorylation in hypoxia, and we propose that mTOR inhibition constitutes a mechanistic link

  5. A first in man, dose-finding study of the mTORC1/mTORC2 inhibitor OSI-027 in patients with advanced solid malignancies

    PubMed Central

    Mateo, Joaquin; Olmos, David; Dumez, Herlinde; Poondru, Srinivasu; Samberg, Nancy L; Barr, Sharon; Van Tornout, Jan M; Jie, Fei; Sandhu, Shahneen; Tan, Daniel S; Moreno, Victor; LoRusso, Patricia M; Kaye, Stan B; Schöffski, Patrick

    2016-01-01

    Background: The kinase activity of mTOR involves 2 multiprotein complexes, (mTORC1-mTORC2). Targeting mTORC1 with rapalogues induces compensatory feedback loops resulting in AKT/ERK activation, which may be abrogated by mTORC2 inhibition. A first-in-human trial evaluating tolerability, pharmacokinetics and pharmacodynamics of the dual TORC1/TORC2 inhibitor OSI-027 was conducted. Methods: Dose escalation was pursued for three schedules of administration (three consecutive days per week (S1), once a week (S2) and daily dosing (S3)), until dose-limiting toxicities (DLT) were identified. Expansion cohorts with paired tumour biopsies were initiated based on tolerability and pharmacodynamics. Results: One hundred and twenty eight patients with advanced cancer were enrolled. DLT consisted predominantly of fatigue, renal function disturbances and cardiac events. OSI-027 exposure was dose proportional, with Tmax within 4 h and a half-life of ∼14 h. Expansion cohorts were initiated for S1 and S2, as MTD for S3 was overall considered suboptimal. Target modulation in peripheral blood mononuclear cells were observed from 30 mg, but in tumour biopsies 120 mg QD were needed, which was a non-tolerable dose due to renal toxicity. No RECIST responses were recorded, with stable disease >6 months in six (5%) patients. Conclusions: OSI-027 inhibits mTORC1/2 in patients with advanced tumour s in a dose-dependent manner but doses above the tolerable levels in S1 and S3 are required for a sustained biological effect in tumour biopsies. PMID:27002938

  6. Effect of mTORC1/mTORC2 inhibition on T cell function: potential role in graft-versus-host disease control.

    PubMed

    Herrero-Sánchez, Ma Carmen; Rodríguez-Serrano, Concepción; Almeida, Julia; San-Segundo, Laura; Inogés, Susana; Santos-Briz, Ángel; García-Briñón, Jesús; SanMiguel, Jesús F; Del Cañizo, Consuelo; Blanco, Belén

    2016-06-01

    The mechanistic target of rapamycin (mTOR) pathway is crucial for the activation and function of T cells, which play an essential role in the development of graft-versus-host disease (GvHD). Despite its partial ability to block mTOR pathway, the mTORC1 inhibitor rapamycin has shown encouraging results in the control of GvHD. Therefore, we considered that simultaneous targeting of both mTORC1 and mTORC2 complexes could exert a more potent inhibition of T cell activation and, thus, could have utility in GvHD control. To assess this assumption, we have used the dual mTORC1/mTORC2 inhibitors CC214-1 and CC214-2. In vitro studies confirmed the superior ability of CC214-1 versus rapamycin to block mTORC1 and mTORC2 activity and to reduce T cell proliferation. Both drugs induced a similar decrease in Th1/Th2 cytokine secretion, but CC214-1 was more efficient in inhibiting naïve T cell activation and the expression of T-cell activation markers. In addition, CC214-1 induced specific tolerance against alloantigens, while preserving anti-cytomegalovirus response. Finally, in a mouse model of GvHD, the administration of CC214-2 significantly improved mice survival and decreased GvHD-induced damages. In conclusion, the current study shows, for the first time, the immunosuppressive ability of CC214-1 on T lymphocytes and illustrates the role of CC214-2 in the allogeneic transplantation setting as a possible GvHD prophylaxis agent. PMID:26914848

  7. Vasoactive intestinal peptide stimulates melanogenesis in B16F10 mouse melanoma cells via CREB/MITF/tyrosinase signaling.

    PubMed

    Yuan, Xing-Hua; Yao, Cheng; Oh, Jang-Hee; Park, Chi-Hyun; Tian, Yu-Dan; Han, Mira; Kim, Ji Eun; Chung, Jin Ho; Jin, Zhe-Hu; Lee, Dong Hun

    2016-08-26

    Vasoactive intestinal peptide (VIP), one of the major skin neuropeptides, has been suggested to have active roles in the pathogenesis of inflammatory skin disorders such as atopic dermatitis and psoriasis, which can commonly cause post-inflammatory hyperpigmentation. However, the effect of VIP on melanogenesis remains unknown. In this study, we showed that the melanin contents, tyrosinase activity, and gene expression of tyrosinase and microphthalmia-associated transcription factor (MITF) were significantly increased by treatment with VIP in B16F10 mouse melanoma cells and the stimulatory melanogenic effect was further examined in human epidermal melanocytes (HEMns). In addition, phosphorylated levels of CRE-binding protein (CREB) and protein kinase A (PKA) were markedly increased after VIP treatment, but not p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), or Akt, indicating the possible PKA-CREB signaling pathway involved in VIP-induced melanogenesis. This result was further verified by the fact that VIP induced increased melanin synthesis, and protein levels of phosphorylated CREB, MITF, tyrosinase were significantly attenuated by H89 (a specific PKA inhibitor). These data suggest that VIP-induced upregulation of tyrosinase through the CREB-MITF signaling pathway plays an important role in finding new treatment strategy for skin inflammatory diseases related pigmentation disorders. PMID:27343558

  8. A microRNA program in the C. elegans hypodermis couples to intestinal mTORC2/PQM-1 signaling to modulate fat transport.

    PubMed

    Dowen, Robert H; Breen, Peter C; Tullius, Thomas; Conery, Annie L; Ruvkun, Gary

    2016-07-01

    Animals integrate metabolic, developmental, and environmental information before committing key resources to reproduction. In Caenorhabditis elegans, adult animals transport fat from intestinal cells to the germline to promote reproduction. We identified a microRNA (miRNA)-regulated developmental timing pathway that functions in the hypodermis to nonautonomously coordinate the mobilization of intestinal fat stores to the germline upon initiation of adulthood. This developmental timing pathway, which is controlled by the lin-4 and let-7 miRNAs, engages mTOR signaling in the intestine. The intestinal signaling component is specific to mTORC2 and functions in parallel to the insulin pathway to modulate the activity of the serum/glucocorticoid-regulated kinase (SGK-1). Surprisingly, SGK-1 functions independently of DAF-16/FoxO; instead, SGK-1 promotes the cytoplasmic localization of the PQM-1 transcription factor, which antagonizes intestinal fat mobilization at the transcriptional level when localized to the nucleus. These results revealed that a non-cell-autonomous developmental input regulates intestinal fat metabolism by engaging mTORC2 signaling to promote the intertissue transport of fat reserves from the soma to the germline. PMID:27401555

  9. Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett’s Esophageal Adenocarcinoma Cells

    PubMed Central

    Li, Dan; Cao, Weibiao

    2016-01-01

    The mechanisms whereby bile acid reflux may accelerate the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett’s patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA. PMID:27511066

  10. Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett's Esophageal Adenocarcinoma Cells.

    PubMed

    Li, Dan; Cao, Weibiao

    2016-01-01

    The mechanisms whereby bile acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett's patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA. PMID:27511066

  11. 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…

  12. Glucocorticoids Alter CRTC-CREB Signaling in Muscle Cells: Impact on PGC-1α Expression and Atrophy Markers

    PubMed Central

    Rahnert, Jill A.; Zheng, Bin; Hudson, Matthew B.; Woodworth-Hobbs, Myra E.; Price, S. Russ

    2016-01-01

    Muscle wasting associated with chronic diseases has been linked to decreased expression of PGC-1α and overexpression of PGC-1α counters muscle loss. CREB, in conjunction with the CREB-regulated transcription coactivator (CRTC2), is a positive modulator of PGC-1α transcription. We previously reported that PGC-1α expression is decreased in skeletal muscle of diabetic rats despite a high level of CREB phosphorylation (i.e., activation), suggesting that CRTC2-CREB signaling may be dysregulated. In this study, the relationship between CREB/CRTC signaling and PGC-1α expression was examined in L6 myotubes treated with dexamethasone (Dex, 48h) to induce atrophy. Dex decreased PGC-1α mRNA and protein as well as the levels of CRTC1 and CRTC2 in the nucleus. Dex also altered the nuclear levels of two known regulators of CRTC2 localization; the amount of calcinuerin catalytic A subunit (CnA) was decreased whereas SIK was increased. To assess PGC-1α transcription, muscle cells were transfected with a PGC-1α luciferase reporter plasmid (PGC-1α-Luc). Dex suppressed PGC-1α luciferase activity while both isobutylmethylxanthine (IBMX) and over-expression of CRTC1 or CRTC2 increased PGC-1α-Luc activity. Mutation of the CRE binding site from PGC-1α-Luc reporter attenuated the responses to both IBMX and the CRTC proteins. Consistent with the reporter gene results, overexpression of CRTC2 produced an increase in CRTC2 in the nucleus and in PGC-1α mRNA and PGC-1α protein. Overexpression of CRTC2 was not sufficient to prevent the decrease in PGC-1α mRNA or protein by Dex. In summary, these data suggest that attenuated CREB/CRTC signaling contributes to the decrease in PGC-1α expression during atrophy. PMID:27404111

  13. The dual mTORC1 and mTORC2 inhibitor AZD8055 inhibits head and neck squamous cell carcinoma cell growth in vivo and in vitro

    SciTech Connect

    Li, Qiang; Song, Xin-mao; Ji, Yang-yang; Jiang, Hui; Xu, Lin-gen

    2013-11-01

    Highlights: •AZD8055 induces significant cytotoxic effects in cultured HNSCC cells. •AZD8055 blocks mTORC1 and mTORC2 activation in cultured HNSCC cells. •JNK activation is required for AZD8055-induced HNSCC cell death. •AZD8055 inhibits Hep-2 cell growth in vivo, and was more efficient than rapamycin. -- Abstract: The serine/threonine kinase mammalian target of rapamycin (mTOR) promotes cell survival and proliferation, and is constitutively activated in head and neck squamous cell carcinoma (HNSCC). Thus mTOR is an important target for drug development in this disease. Here we tested the anti-tumor ability of AZD8055, the novel mTOR inhibitor, in HNSCC cells. AZD8055 induced dramatic cell death of HNSCC lines (Hep-2 and SCC-9) through autophagy. AZD8055 blocked both mTOR complex (mTORC) 1 and mTORC2 activation without affecting Erk in cultured HNSCC cells. Meanwhile, AZD8055 induced significant c-Jun N-terminal kinase (JNK) activation, which was also required for cancer cell death. JNK inhibition by its inhibitors (SP 600125 and JNK-IN-8), or by RNA interference (RNAi) alleviated AZD8055-induced cell death. Finally, AZD8055 markedly increased the survival of Hep-2 transplanted mice through a significant reduction of tumor growth, without apparent toxicity, and its anti-tumor ability was more potent than rapamycin. Meanwhile, AZD8055 administration activated JNK while blocking mTORC1/2 in Hep-2 tumor engrafts. Our current results strongly suggest that AZD8055 may be further investigated for HNSCC treatment in clinical trials.

  14. Rictor/mTORC2 regulates blood-testis barrier dynamics via its effects on gap junction communications and actin filament network

    PubMed Central

    Mok, Ka-Wai; Mruk, Dolores D.; Lee, Will M.; Cheng, C. Yan

    2013-01-01

    In the mammalian testis, coexisting tight junctions (TJs), basal ectoplasmic specializations, and gap junctions (GJs), together with desmosomes near the basement membrane, constitute the blood-testis barrier (BTB). The most notable feature of the BTB, however, is the extensive network of actin filament bundles, which makes it one of the tightest blood-tissue barriers. The BTB undergoes restructuring to facilitate the transit of preleptotene spermatocytes at stage VIII-IX of the epithelial cycle. Thus, the F-actin network at the BTB undergoes cyclic reorganization via a yet-to-be explored mechanism. Rictor, the key component of mTORC2 that is known to regulate actin cytoskeleton, was shown to express stage-specifically at the BTB in the seminiferous epithelium. Its expression was down-regulated at the BTB in stage VIII-IX tubules, coinciding with BTB restructuring at these stages. Using an in vivo model, a down-regulation of rictor at the BTB was also detected during adjudin-induced BTB disruption, illustrating rictor expression is positively correlated with the status of the BTB integrity. Indeed, the knockdown of rictor by RNAi was found to perturb the Sertoli cell TJ-barrier function in vitro and the BTB integrity in vivo. This loss of barrier function was accompanied by changes in F-actin organization at the Sertoli cell BTB in vitro and in vivo, associated with a loss of interaction between actin and α-catenin or ZO-1. Rictor knockdown by RNAi was also found to impede Sertoli cell-cell GJ communication, disrupting protein distribution (e.g., occludin, ZO-1) at the BTB, illustrating that rictor is a crucial BTB regulator.—Mok, K., Mruk, D. D., Lee, W. M., Cheng, C. Y. Rictor/mTORC2 regulates blood-testis barrier dynamics via its effects on gap junction communications and actin filament network. PMID:23288930

  15. Prohibitin-Mediated Lifespan and Mitochondrial Stress Implicate SGK-1, Insulin/IGF and mTORC2 in C. elegans

    PubMed Central

    Gatsi, Roxani; Schulze, Bettina; Rodríguez-Palero, María Jesús; Hernando-Rodríguez, Blanca; Baumeister, Ralf; Artal-Sanz, Marta

    2014-01-01

    Lifespan regulation by mitochondrial proteins has been well described, however, the mechanism of this regulation is not fully understood. Amongst the mitochondrial proteins profoundly affecting ageing are prohibitins (PHB-1 and PHB-2). Paradoxically, in C. elegans prohibitin depletion shortens the lifespan of wild type animals while dramatically extending that of metabolically compromised animals, such as daf-2-insulin-receptor mutants. Here we show that amongst the three kinases known to act downstream of daf-2, only loss of function of sgk-1 recapitulates the ageing phenotype observed in daf-2 mutants upon prohibitin depletion. Interestingly, signalling through SGK-1 receives input from an additional pathway, parallel to DAF-2, for the prohibitin-mediated lifespan phenotype. We investigated the effect of prohibitin depletion on the mitochondrial unfolded protein response (UPRmt). Remarkably, the lifespan extension upon prohibitin elimination, of both daf-2 and sgk-1 mutants, is accompanied by suppression of the UPRmt induced by lack of prohibitin. On the contrary, gain of function of SGK-1 results in further shortening of lifespan and a further increase of the UPRmt in prohibitin depleted animals. Moreover, SGK-1 interacts with RICT-1 for the regulation of the UPRmt in a parallel pathway to DAF-2. Interestingly, prohibitin depletion in rict-1 loss of function mutant animals also causes lifespan extension. Finally, we reveal an unprecedented role for mTORC2-SGK-1 in the regulation of mitochodrial homeostasis. Together, these results give further insight into the mechanism of lifespan regulation by mitochondrial function and reveal a cross-talk of mitochondria with two key pathways, Insulin/IGF and mTORC2, for the regulation of ageing and stress response. PMID:25265021

  16. The Bardet–Biedl syndrome-related protein CCDC28B modulates mTORC2 function and interacts with SIN1 to control cilia length independently of the mTOR complex

    PubMed Central

    Cardenas-Rodriguez, Magdalena; Irigoín, Florencia; Osborn, Daniel P.S.; Gascue, Cecilia; Katsanis, Nicholas; Beales, Philip L.; Badano, Jose L.

    2013-01-01

    CCDC28B encodes a coiled coil domain-containing protein involved in ciliogenesis that was originally identified as a second site modifier of the ciliopathy Bardet–Biedl syndrome. We have previously shown that the depletion of CCDC28B leads to shortened cilia; however, the mechanism underlying how this protein controls ciliary length is unknown. Here, we show that CCDC28B interacts with SIN1, a component of the mTOR complex 2 (mTORC2), and that this interaction is important both in the context of mTOR signaling and in a hitherto unknown, mTORC-independent role of SIN1 in cilia biology. We show that CCDC28B is a positive regulator of mTORC2, participating in its assembly/stability and modulating its activity, while not affecting mTORC1 function. Further, we show that Ccdc28b regulates cilia length in vivo, at least in part, through its interaction with Sin1. Importantly, depletion of Rictor, another core component of mTORC2, does not result in shortened cilia. Taken together, our findings implicate CCDC28B in the regulation of mTORC2, and uncover a novel function of SIN1 regulating cilia length that is likely independent of mTOR signaling. PMID:23727834

  17. 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.

  18. 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

  19. RKIP Regulates Neural Cell Apoptosis Induced by Exposure to Microwave Radiation Partly Through the MEK/ERK/CREB Pathway.

    PubMed

    Zuo, Hongyan; Lin, Tao; Wang, Dewen; Peng, Ruiyun; Wang, Shuiming; Gao, Yabing; Xu, Xinping; Zhao, Li; Wang, Shaoxia; Su, Zhentao

    2015-01-01

    In the present study, we investigated whether Raf-1 kinase inhibitory protein (RKIP) is important for neural cell apoptosis induced by microwave exposure and explored the role of MEK/ERK/CREB pathway regulated by RKIP in the apoptosis. Differentiated PC12 cells were exposed to continuous microwave radiation at 2.856 GHz for 5 min with average power density of 30 mW/cm(2). RKIP sense and anti-sense recombinant plasmids were constructed and transfected into PC12 cells, respectively. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and caspase-3 activity assay were used to detect cell apoptosis. The results showed that RKIP was downregulated after microwave exposure while the MEK/ERK/CREB signaling pathway was activated excessively. Moreover, the ratio of Bcl-2/Bax decreased, activity of caspase-3 increased, and thus apoptotic DNA fragmentation increased. RKIP overexpression significantly inhibited the phosphorylation of MEK, ERK, and CREB, while RKIP downregulation had the reverse effect. Furthermore, U0126 was found to antagonize the changes caused by RKIP downregulation after exposure to radiation. In conclusion, RKIP plays an important role in the neural cell apoptosis induced by microwave radiation, and the regulation of cell apoptosis by RKIP is partly through the MEK/ERK/CREB pathway. This suggests that RKIP may act as a key regulator of neuronal damage caused by microwave radiation. PMID:25108669

  20. Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Fa, Svetlana; Pogrmic-Majkic, Kristina; Andric, Nebojsa

    2016-01-01

    Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells. PMID:26494252

  1. Dipeptidyl peptidase-4 inhibition in diabetic rats leads to activation of the transcription factor CREB in β-cells.

    PubMed

    Pugazhenthi, Subbiah; Qin, LiMei; Bouchard, Ron

    2015-05-15

    Incretin therapies are effective in controlling blood glucose levels in type 2 diabetic patients by improving the survival and function of β-cells. They include dipeptidyl peptidase-4 (DPP-4) inhibitors and long-acting glucagon-like peptide-1 (GLP-1) analogs. We have previously reported that GLP-1 enhances the survival of cultured human islets by activation of the transcription factor CREB. To test the in vivo relevance of these findings, we examined the effects of alogliptin, a DPP-4 inhibitor, in Zucker Diabetic rats, a model for type 2 diabetes. The plasma levels of GLP-1 increased in alogliptin-treated diabetic rats leading to normoglycemia. Pancreatic islets of untreated diabetic rats were characterized by decreased immunostaining for insulin and PDX-1. Elevation of GLP-1 in treated diabetic rats resulted in the improved survival of β-cells. Dual immunofluorescent staining showed phosphorylation/activation of CREB in insulin-positive β-cells of islets. This led to increases in the levels of CREB targets including Bcl-2, an antiapoptotic mitochondrial protein, BIRC3, a caspase inhibitor and IRS-2, an adapter protein needed for insulin signaling. Findings from this study suggest potential activation of cytoprotective CREB by GLP-1 in pancreatic β-cells of diabetic patients undergoing incretin-based therapies. PMID:25720341

  2. Phosphorylation of the TOR ATP binding domain by AGC kinase constitutes a novel mode of TOR inhibition.

    PubMed

    Hálová, Lenka; Du, Wei; Kirkham, Sara; Smith, Duncan L; Petersen, Janni

    2013-11-25

    TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell division through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase-controlled phosphorylation to generate physiologically significant changes in TOR signaling. PMID:24247430

  3. Hypoxia induces phosphorylation of the cyclic AMP response element-binding protein by a novel signaling mechanism.

    PubMed

    Beitner-Johnson, D; Millhorn, D E

    1998-07-31

    To investigate signaling mechanisms by which hypoxia regulates gene expression, we examined the effect of hypoxia on the cyclic AMP response element-binding protein (CREB) in PC12 cells. Exposure to physiological levels of hypoxia (5% O2, approximately 50 mm Hg) rapidly induced a persistent phosphorylation of CREB on Ser133, an event that is required for CREB-mediated transcriptional activation. Hypoxia-induced phosphorylation of CREB was more robust than that induced by any other stimulus tested, including forskolin, depolarization, and osmotic stress. Furthermore, this effect was not mediated by any of the previously known signaling pathways that lead to phosphorylation of CREB, including protein kinase A, calcium/calmodulin-dependent protein kinase, protein kinase C, ribosomal S6 kinase-2, and mitogen-activated protein kinase-activated protein kinase-2. Hypoxic activation of a CRE-containing reporter (derived from the 5'-flanking region of the tyrosine hydroxylase gene) was attenuated markedly by mutation of the CRE. Thus, a physiological reduction in O2 levels induces a functional phosphorylation of CREB at Ser133 via a novel signaling pathway. PMID:9677418

  4. Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter

    PubMed Central

    Rogge, George A; Shen, Li-Ling; Kuhar, Michael J.

    2010-01-01

    Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action. PMID:20451507

  5. CREB Binds to Multiple Loci on Human Chromosome 22

    PubMed Central

    Euskirchen, Ghia; Royce, Thomas E.; Bertone, Paul; Martone, Rebecca; Rinn, John L.; Nelson, F. Kenneth; Sayward, Fred; Luscombe, Nicholas M.; Miller, Perry; Gerstein, Mark; Weissman, Sherman; Snyder, Michael

    2004-01-01

    The cyclic AMP-responsive element-binding protein (CREB) is an important transcription factor that can be activated by hormonal stimulation and regulates neuronal function and development. An unbiased, global analysis of where CREB binds has not been performed. We have mapped for the first time the binding distribution of CREB along an entire human chromosome. Chromatin immunoprecipitation of CREB-associated DNA and subsequent hybridization of the associated DNA to a genomic DNA microarray containing all of the nonrepetitive DNA of human chromosome 22 revealed 215 binding sites corresponding to 192 different loci and 100 annotated potential gene targets. We found binding near or within many genes involved in signal transduction and neuronal function. We also found that only a small fraction of CREB binding sites lay near well-defined 5′ ends of genes; the majority of sites were found elsewhere, including introns and unannotated regions. Several of the latter lay near novel unannotated transcriptionally active regions. Few CREB targets were found near full-length cyclic AMP response element sites; the majority contained shorter versions or close matches to this sequence. Several of the CREB targets were altered in their expression by treatment with forskolin; interestingly, both induced and repressed genes were found. Our results provide novel molecular insights into how CREB mediates its functions in humans. PMID:15082775

  6. 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.

  7. 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

  8. Phosphorylation-dependent targeting of cAMP response element binding protein to the ubiquitin/proteasome pathway in hypoxia

    PubMed Central

    Taylor, Cormac T.; Furuta, Glenn T.; Synnestvedt, Kristin; Colgan, Sean P.

    2000-01-01

    Hypoxia activates a number of gene products through degradation of the transcriptional coactivator cAMP response element binding protein (CREB). Other transcriptional regulators (e.g., β-catenin and NF-κB) are controlled through phosphorylation-targeted proteasomal degradation, and thus, we hypothesized a similar degradative pathway for CREB. Differential display analysis of mRNA derived from hypoxic epithelia revealed a specific and time-dependent repression of protein phosphatase 1 (PP1), a serine phosphatase important in CREB dephosphorylation. Subsequent studies identified a previously unappreciated proteasomal-targeting motif within the primary structure of CREB (DSVTDS), which functions as a substrate for PP1. Ambient hypoxia resulted in temporally sequential CREB serine phosphorylation, ubiquitination, and degradation (in vitro and in vivo). HIV-tat peptide-facilitated loading of intact epithelia with phosphopeptides corresponding to this proteasome targeting motif resulted in inhibition of CREB ubiquitination. Further studies revealed that PP1 inhibitors mimicked hypoxia-induced gene expression, whereas proteasome inhibitors reversed the hypoxic phenotype. Thus, hypoxia establishes conditions that target CREB to proteasomal degradation. These studies may provide unique insight into a general mechanism of transcriptional regulation by hypoxia. PMID:11035795

  9. 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.

    PubMed

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

    2013-10-18

    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 IP3/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. PMID:24064350

  10. NF-κB Regulates Spatial Memory Formation and Synaptic Plasticity through Protein Kinase A/CREB Signaling

    PubMed Central

    Kaltschmidt, Barbara; Ndiaye, Delphine; Korte, Martin; Pothion, Stéphanie; Arbibe, Laurence; Prüllage, Maria; Pfeiffer, Julia; Lindecke, Antje; Staiger, Volker; Israël, Alain; Kaltschmidt, Christian; Mémet, Sylvie

    2006-01-01

    Synaptic activity-dependent de novo gene transcription is crucial for long-lasting neuronal plasticity and long-term memory. In a forebrain neuronal conditional NF-κB-deficient mouse model, we demonstrate here that the transcription factor NF-κB regulates spatial memory formation, synaptic transmission, and plasticity. Gene profiling experiments and analysis of regulatory regions identified the α catalytic subunit of protein kinase A (PKA), an essential memory regulator, as a new NF-κB target gene. Consequently, NF-κB inhibition led to a decrease in forskolin-induced CREB phosphorylation. Collectively, these results disclose a novel hierarchical transcriptional network involving NF-κB, PKA, and CREB that leads to concerted nuclear transduction of synaptic signals in neurons, accounting for the critical function of NF-κB in learning and memory. PMID:16581769

  11. The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2.

    PubMed

    Chen, Bo; Xu, Ming; Zhang, Hui; Xu, Ming-zheng; Wang, Xu-jing; Tang, Qing-he; Tang, Jian-ying

    2015-10-01

    In the present study, we investigated the potential activity of OSI-027, a potent and selective mammalian target of rapamycin (mTOR) complex 1/2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells both in vitro and in vivo. We demonstrated that OSI-027 inhibited survival and growth of both primary and transformed (PANC-1 and MIA PaCa-2 lines) human pancreatic cancer cells. Meanwhile, OSI-027 induced caspase-dependent apoptotic death of the pancreatic cancer cells. On the other hand, caspase inhibitors alleviated cytotoxicity by OSI-027. At the molecular level, OSI-027 treatment blocked mTORC1 and mTORC2 activation simultaneously, without affecting ERK-mitogen-activated protein kinase activation. Importantly, OSI-027 activated cytoprotective autophagy in the above cancer cells. Whereas pharmacological blockage of autophagy or siRNA knockdown of Beclin-1 significantly enhanced the OSI-027-induced activity against pancreatic cancer cells. Specifically, a relatively low dose of OSI-027 sensitized gemcitabine-induced pancreatic cancer cell death in vitro. Further, administration of OSI-027 or together with gemcitabine dramatically inhibited PANC-1 xenograft growth in severe combined immunodeficiency mice, leading to significant mice survival improvement. In summary, the preclinical results of this study suggest that targeting mTORC1/2 synchronously by OSI-027 could be further investigated as a valuable treatment for pancreatic cancer. PMID:26284306

  12. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes. PMID:22948226

  13. The Steroid Hormone 20-Hydroxyecdysone Enhances Gene Transcription through the cAMP Response Element-binding Protein (CREB) Signaling Pathway.

    PubMed

    Jing, Yu-Pu; Wang, Di; Han, Xiao-Lin; Dong, Du-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2016-06-10

    Animal steroid hormones regulate gene transcription through genomic pathways by binding to nuclear receptors. These steroid hormones also rapidly increase intracellular calcium and cyclic adenosine monophosphate (cAMP) levels and activate the protein kinase C (PKC) and protein kinase A (PKA) nongenomic pathways. However, the function and mechanism of the nongenomic pathways of the steroid hormones are unclear, and the relationship between the PKC and PKA pathways is also unclear. We propose that the steroid hormone 20-hydroxyecdysone (20E) activates the PKA pathway to enhance 20E-induced gene transcription in the lepidopteran insect Helicoverpa armigera The expression of the catalytic subunit 1 of PKA (PKAC1) increased during metamorphosis, and PKAC1 knockdown blocked pupation and repressed 20E-responsive gene expression. 20E regulated PKAC1 phosphorylation at threonine 200 and nuclear translocation through an ecdysone-responsive G-protein-coupled receptor 2. PKAC1 induced cAMP response element-binding protein (CREB) phosphorylation at serine 143, which bound to the cAMP response element on DNA to enhance 20E-responsive gene transcription. Through ecdysone-responsive G-protein-coupled receptor 2, 20E increased cAMP levels, which induced CREB PKA phosphorylation and 20E-responsive gene expression. This study demonstrates that the PKA/CREB pathway tightly and critically regulates 20E-induced gene transcription as well as its relationship with the 20E-induced PKC pathway. PMID:27129227

  14. Prenatal choline supplementation advances hippocampal development and enhances MAPK and CREB activation.

    PubMed

    Mellott, Tiffany J; Williams, Christina L; Meck, Warren H; Blusztajn, Jan Krzysztof

    2004-03-01

    Choline is an essential nutrient for animals and humans. Previous studies showed that supplementing the maternal diet with choline during the second half of gestation in rats permanently enhances memory performance of the adult offspring. Here we show that prenatal choline supplementation causes a 3-day advancement in the ability of juvenile rats to use relational cues in a water maze task, indicating that the treatment accelerates hippocampal maturation. Moreover, phosphorylation and therefore activation of hippocampal mitogen-activated protein kinase (MAPK) and cAMP-response element binding protein (CREB) in response to stimulation by glutamate, N-methyl-D-aspartate, or depolarizing concentrations of K+ were increased by prenatal choline supplementation and reduced by prenatal choline deficiency. These data provide the first evidence that developmental plasticity of the hippocampal MAPK and CREB signaling pathways is controlled by the supply of a single essential nutrient, choline, during fetal development and point to these pathways as candidate mechanisms for the developmental and long-term cognitive enhancement induced by prenatal choline supplementation. PMID:14715695

  15. Phosphorylated testis-specific serine/threonine kinase 4 may phosphorylate Crem at Ser-117.

    PubMed

    Fu, Guolong; Wei, Youheng; Wang, Xiaoli; Yu, Long

    2016-06-01

    We aimed to investigate the internal existence status of testis-specific serine/threonine kinase 4 (Tssk4) and the interaction of Tssk4 and Cre-responsive element modulator (Crem). The internal existence status of Tssk4 in testis of mice was detected using western blotting and dephosphorylation method. The interaction of Tssk4 and Crem was analyzed by western blotting, immunohistochemistry, immunofluorescence, in vitro co-immunoprecipitation assays, and in vitro kinase assay. The results revealed that Tssk4 existed in testis both in phosphorylation and unphosphorylation status by a temporal manner with the development of testis. Immunofluorescence results showed that Tssk4 had identical distribution pattern with Crem in testis, which was utterly different to the localization of Cre-responsive element binding (Creb). In conclusion, our study demonstrated that phosphorylated Tssk4 might participate in testis genes expressions by phosphorylating Crem at Ser-117. PMID:26940607

  16. 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

  17. Electroacupuncture improves cognitive ability following cerebral ischemia reperfusion injury via CaM-CaMKIV-CREB signaling in the rat hippocampus

    PubMed Central

    Zhang, Yun; Lin, Ruhui; Tao, Jing; Wu, Yunan; Chen, Bin; Yu, Kunqiang; Chen, Jixiang; Li, Xiaojie; Chen, Li-Dian

    2016-01-01

    The aim of the present study was to investigate the effect of electroacupuncture (EA) on cognitive deficits, and the underlying mechanism following cerebral ischemia-reperfusion (I/R) via the calmodulin (CaM)-calmodulin-dependent protein kinase type IV (CaMKIV)-cyclic adenosine monophosphate response elements binding protein (CREB) intracellular signaling pathway in the hippocampus. In total, 45 adult female Sprague-Dawley rats were randomly divided into three groups, namely the sham group, the middle cerebral artery occlusion (MCAO) group and the MCAO + EA group. Rats in the MCAO and MCAO + EA groups were modeled for post-stroke cognitive impairment. EA was performed at the Baihui and Shenting acupuncture points for 30 min/day for one week in the MCAO + EA group. Behavioral testing was analyzed using a step-down apparatus, while 2,3,5-triphenyl tetrazolium chloride was used to detect the infarct volume and lesion size. In addition, CaM activity was assessed by cyclic nucleotide-dependent phosphodiesterase analysis, and the protein expression levels of CaM, CaMKIV, phosphorylated (p)-CaMKIV, CREB and p-CREB were analyzed by western blot analysis. The cerebral I/R injured rat model in the MCAO group was established successfully with regard to the infarct volume and neuronal lesion size, as compared with the sham group. EA was demonstrated to effectively improve the cognitive ability, as measured by the step-down apparatus test, and decrease the infarct volume when compared with the MCAO group (P<0.05). The step-down apparatus test for the EA-treated rats revealed improved learning and reduced memory impairment when compared with the MCAO group. Furthermore, CaM activity and CaM protein expression levels in the MCAO + EA group were lower compared with those in the MCAO group (P<0.05). By contrast, the protein expression levels of CaMKIV, p-CaMKIV, CREB and p-CREB were significantly reduced in the MCAO group when compared with the sham group (P<0.05), although the

  18. Sodium Phenylbutyrate Enhances Astrocytic Neurotrophin Synthesis via Protein Kinase C (PKC)-mediated Activation of cAMP-response Element-binding Protein (CREB)

    PubMed Central

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

    2013-01-01

    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 (Ser133) 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. PMID:23404502

  19. 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

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

    PubMed

    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

  1. Yifei Xuanfei Jiangzhuo formula, a Chinese herbal decoction, improves memory impairment through inhibiting apoptosis and enhancing PKA/CREB signal transduction in rats with cerebral ischemia/reperfusion

    PubMed Central

    WU, LIN; ZHAO, QING-SHAN; LI, TIAN-WEI; LI, HAI-YUAN; WANG, QING-BI; BI, XIN-YA; CAI, XIN-KUN; TANG, NONG

    2015-01-01

    Apoptosis and the dysfunction of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) signaling pathway have a key role in memory impairment in vascular dementia (VaD), a challenging clinical problem. Yifei Xuanfei Jiangzhuo formula (YXJF), a Chinese herbal decoction, has been used to treat VaD in clinical practice and has produced positive outcomes; however, convincing evidence is currently lacking. The present study aimed to investigate the effects of YXJF on memory impairment in rats with cerebral ischemia/reperfusion and to explore the underlying mechanism. YXJF ameliorated memory impairment in rats with cerebral ischemia/reperfusion, inhibited hippocampal apoptosis in a dose-dependent manner and attenuated increases in the protein expression of B-cell lymphoma 2 (Bcl-2)-associated X protein as well as c-Jun and a reduction in Bcl-2 protein expression in the hippocampal tissue of the rats. Furthermore, administration of YXJF significantly increased the protein expression of PKA C-α and CREB, and promoted CREB phosphorylation. The results indicated that YXJF improves memory impairment through inhibiting apoptosis and enhancing PKA/CREB signal transduction in rats with cerebral ischemia/reperfusion. PMID:26094797

  2. Reduced pCREB in Alzheimer's disease prefrontal cortex is reflected in peripheral blood mononuclear cells.

    PubMed

    Bartolotti, N; Bennett, D A; Lazarov, O

    2016-09-01

    Cyclic-AMP response element-binding protein (CREB) signaling has a critical role in the formation of memories. CREB signaling is dysfunctional in the brains of mouse models of Alzheimer's disease (AD), and evidence suggests that CREB signaling may be disrupted in human AD brains as well. Here, we show that both CREB and its activated form pCREB-Ser(133) (pCREB) are reduced in the prefrontal cortex of AD patients. Similarly, the transcription cofactors CREB-binding protein (CBP) and p300 are reduced in the prefrontal cortex of AD patients, indicating additional dysfunction of CREB signaling in AD. Importantly, we show that pCREB expression is reduced in peripheral blood mononuclear cells (PBMC) of AD subjects. In addition, pCREB levels in PBMC positively correlated with pCREB expression in the postmortem brain of persons with AD. These results suggest that pCREB expression in PBMC may be indicative of its expression in the brain, and thus offers the intriguing possibility of pCREB as a biomarker of cognitive function and disease progression in AD. PMID:27480489

  3. Reduced pCREB in Alzheimer's disease prefrontal cortex is reflected in peripheral blood mononuclear cells

    PubMed Central

    Bartolotti, N; Bennett, D A; Lazarov, O

    2016-01-01

    Cyclic-AMP response element-binding protein (CREB) signaling has a critical role in the formation of memories. CREB signaling is dysfunctional in the brains of mouse models of Alzheimer's disease (AD), and evidence suggests that CREB signaling may be disrupted in human AD brains as well. Here, we show that both CREB and its activated form pCREB-Ser133 (pCREB) are reduced in the prefrontal cortex of AD patients. Similarly, the transcription cofactors CREB-binding protein (CBP) and p300 are reduced in the prefrontal cortex of AD patients, indicating additional dysfunction of CREB signaling in AD. Importantly, we show that pCREB expression is reduced in peripheral blood mononuclear cells (PBMC) of AD subjects. In addition, pCREB levels in PBMC positively correlated with pCREB expression in the postmortem brain of persons with AD. These results suggest that pCREB expression in PBMC may be indicative of its expression in the brain, and thus offers the intriguing possibility of pCREB as a biomarker of cognitive function and disease progression in AD. PMID:27480489

  4. Environmental enrichment: effects on spatial memory and hippocampal CREB immunoreactivity.

    PubMed

    Williams, B M; Luo, Y; Ward, C; Redd, K; Gibson, R; Kuczaj, S A; McCoy, J G

    2001-07-01

    Environmental enrichment has been shown to improve performance in tests of spatial memory, induce neurogenesis in the hippocampus, enhance survival of newly formed granule cells, and inhibit spontaneous apoptosis. Although neuroplasticity of the mammalian brain declines with age, recent evidence suggests that the adult brain exhibits significant plasticity in response to environmental stimulation. The present study was designed to evaluate the effect of environmental enrichment on spatial memory and on immunoreactivity to cAMP response element binding protein (CREB) from the hippocampus. C57/BL/6 mice were trained in a Morris water maze after exposure to an enriched environment, either from 35 to 94 days or from 100 to 159 days of age. Hippocampal tissue from representative animals was later analyzed by Western blot for CREB immunoreactivity. Results indicate that environmental enrichment (particularly during the earlier period) improved performance on the Morris water maze and tended to increase immunoreactivity to CREB in the hippocampus. Social interaction by itself did not result in significant differences in navigational performance. Results with regard to social interaction and CREB immunoreactivity were mixed. Results are discussed in terms of evaluating the construct of enrichment, the correlation of CREB transcription and behavior change, and the importance of the developmental period for enrichment. PMID:11495671

  5. Hyperactivation of mTORC1 and mTORC2 by multiple oncogenic events causes addiction to eIF4E-dependent mRNA translation in T-cell leukemia.

    PubMed

    Schwarzer, A; Holtmann, H; Brugman, M; Meyer, J; Schauerte, C; Zuber, J; Steinemann, D; Schlegelberger, B; Li, Z; Baum, C

    2015-07-01

    High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). The activity of the master regulator of this pathway, PTEN, is often impaired in T-ALL. However, experimental evidence suggests that input from receptor tyrosine kinases (RTKs) is required for sustained mTOR activation, even in the absence of PTEN. We previously reported the expression of Neurotrophin receptor tyrosine kinases (TRKs) and their respective ligands in primary human leukemia samples. In the present study we aimed to dissect the downstream signaling cascades of TRK-induced T-ALL in a murine model and show that T-ALLs induced by deregulated receptor tyrosine kinase signaling acquire activating mutations in Notch1 and lose PTEN during clonal evolution. Some clones additionally lost one allele of the homeodomain transcription factor Cux1. All events independently led to a gradual hyperactivation of both mTORC1 and mTORC2 signaling. We dissected the role of the individual mTOR complexes by shRNA knockdown and found that the separate depletion of mTORC1 or mTORC2 reduced the growth of T-ALL blasts, but was not sufficient to induce apoptosis. In contrast, knockdown of the mTOR downstream effector eIF4E caused a striking cytotoxic effect, demonstrating a critical addiction to cap-dependent mRNA-translation. Although high mTORC2-AKT activation is commonly associated with drug-resistance, we demonstrate that T-ALL displaying a strong mTORC2-AKT activation were specifically susceptible to 4EGI-1, an inhibitor of the eIF4E-eIF4G interaction. To decipher the mechanism of 4EGI-1, we performed a genome-wide analysis of mRNAs that are translationally regulated by 4EGI-1 in T-ALL. 4EGI-1 effectively reduced the ribosomal occupancy of mRNAs that were strongly upregulated in T-ALL blasts compared with normal thymocytes including transcripts important for translation, mitochondria and cell cycle progression, such as cyclins and ribosomal proteins. These data

  6. ZIP4 Regulates Pancreatic Cancer Cell Growth by Activating IL-6/STAT3 Pathway via Zinc Finger Transcription Factor CREB

    PubMed Central

    Zhang, Yuqing; Bharadwaj, Uddalak; Logsdon, Craig D.; Chen, Changyi; Yao, Qizhi; Li, Min

    2010-01-01

    Purpose Recent studies indicate a strong correlation of zinc transporter ZIP4 and pancreatic cancer progression; however, the underlying mechanisms are unclear. We have recently found that ZIP4 is overexpressed in pancreatic cancer. In this study, we investigated the signaling pathway through which ZIP4 regulates pancreatic cancer growth. Experimental Design The expression of cyclin D1, IL-6, and STAT3 in pancreatic cancer xenografts and cells were examined by real time PCR, Bio-Plex cytokine assay, and Western blot, respectively. The activity of CREB is examined by a promoter activity assay. Results Cyclin D1 was significantly increased in the ZIP4 overexpressing MIA PaCa-2 cells (MIA-ZIP4)-injected orthotopic xenografts and was downregulated in the ZIP4 silenced ASPC-1 (ASPC-shZIP4) group. The phosphorylation of signal transducer and activator of transcription 3 (STAT3), an upstream activator of cyclin D1, was increased in MIA-ZIP4 cells, and decreased in ASPC-shZIP4 cells. IL-6, a known upstream activator for STAT3, was also found to be significantly increased in the MIA-ZIP4 cells and xenografts, and decreased in the ASPC-shZIP4 group. Overexpression of ZIP4 led to a 75% increase of IL-6 promoter activity, and caused increased phosphorylation of cAMP response element binding protein (CREB). Conclusions Our study suggest that ZIP4 overexpression causes increased IL-6 transcription via CREB, which in turn activates STAT3, and leads to increased cyclin D1 expression, resulting in increased cell proliferation and tumor progression in pancreatic cancer. These results elucidated a novel pathway in ZIP4-mediated pancreatic cancer growth, and suggest new therapeutic targets including ZIP4, IL-6, and STAT3 in pancreatic cancer treatment. PMID:20160059

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

    PubMed Central

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

    2016-01-01

    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. PMID:26830449

  8. SCM-198 Ameliorates Cognitive Deficits, Promotes Neuronal Survival and Enhances CREB/BDNF/TrkB Signaling without Affecting Aβ Burden in AβPP/PS1 Mice

    PubMed Central

    Hong, Zhen-Yi; Yu, Shuang-Shuang; Wang, Zhi-Jun; Zhu, Yi-Zhun

    2015-01-01

    SCM-198 is an alkaloid found only in Herba leonuri and it has been reported to possess considerable neuroprotective effects in animal models of ischemic stroke, Parkinson’s disease and Alzheimer’s disease (AD). In this study, we demonstrated for the first time that 3-month oral SCM-198 treatment could significantly improve both recognition and spatial memory, inhibit microgliosis and promote neuronal survival in amyloid-β protein precursor and presenilin-1(AβPP/PS1) double-transgenic mice without affecting amyloid-β (Aβ) burden. In addition, decreases in cAMP-response element-binding protein (CREB) phosphorylation, brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) phosphorylation were attenuated by SCM-198 both in vivo and in primary cortical neurons, which could be blocked by protein kinase A (PKA) inhibitors, suggesting the involvement of upstream PKA in enhancing the BDNF/TrkB/CREB signaling by SCM-198. Our results indicate that SCM-198, a drug that could promote neuronal survival and enhance BDNF/TrkB/CREB signaling, has beneficial effects on behavioral and biochemical alterations without affecting Aβ burden in AβPP/PS1 mice and might become a potential drug candidate for AD treatment in the future. PMID:26262618

  9. 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…

  10. CREB Binding Protein (CBP) Activation Is Required for Luteinizing Hormone Beta Expression and Normal Fertility in Mice

    PubMed Central

    Miller, Ryan S.; Wolfe, Andrew; He, Ling; Radovick, Sally

    2012-01-01

    Normal function of the hypothalamic-pituitary-gonadal axis is dependent on gonadotropin-releasing hormone (GNRH)-stimulated synthesis and secretion of luteinizing hormone (LH) from the pituitary gonadotroph. While the transcriptional coactivator CREB binding protein (CBP) is known to interact with Egr-1, the major mediator of GNRH action on the Lhb gene, the role of CBP in Lhb gene expression has yet to be characterized. We show that in the LβT2 gonadotroph cell line, overexpression of CBP augmented the response to GNRH and that knockdown of CBP eliminated GNRH responsiveness. While GNRH-mediated phosphorylation of CBP at Ser436 increased the interaction with Egr-1 on the Lhb promoter, loss of this phosphorylation site eliminated GNRH-mediated Lhb expression in LβT2 cells. In vivo, loss of CBP phosphorylation at Ser436 rendered female mice subfertile. S436A knock-in mice had disrupted estrous cyclicity and reduced responsiveness to GNRH. Our results show that GNRH-mediated phosphorylation of CBP at Ser436 is required for Egr-1 to activate Lhb expression and is a requirement for normal fertility in female mice. As CBP can be phosphorylated by other factors, such as insulin, our studies suggest that CBP may act as a key regulator of Lhb expression in the gonadotroph by integrating homeostatic information with GNRH signaling. PMID:22508984

  11. Intra-ventral tegmental area HIV-1 Tat1–86 attenuates nicotine-mediated locomotor sensitization and alters mesocorticolimbic ERK and CREB signaling in rats

    PubMed Central

    Zhu, Jun; Midde, Narasimha M.; Gomez, Adrian M.; Sun, Wei-Lun; Harrod, Steven B.

    2015-01-01

    Cigarette smoking prevalence in the HIV-positive individuals is profoundly higher than that in the HIV-negative individuals. We have demonstrated that HIV-1 transgenic rats exhibit attenuated nicotine-mediated locomotor activity, altered cAMP response element binding protein (CREB) and extracellular regulated kinase (ERK1/2) signaling in the mesocorticolimbic regions. This study investigated the role of HIV-1 transactivator of transcription (Tat) protein in the alterations of nicotine-mediated behavior and the signaling pathway observed in the HIV-1 transgenic rats. Rats received bilateral microinjection of recombinant Tat1–86 (25 μg/side) or vehicle directed at ventral tegmental area (VTA) followed by locomotor testing in response to 13 daily intravenous injections of nicotine (0.05 mg/kg, freebase, once/day) or saline. Further, we examined the phosphorylated levels of CREB (pCREB) and ERK1/2 (pERK1/2) in the prefrontal cortex (PFC), nucleus accumbens (NAc) and VTA. Tat diminished baseline activity in saline control rats, and attenuated nicotine-induced behavioral sensitization. Following repeated saline injection, the basal levels of pERK1 in the NAc and VTA and pERK2 in VTA were lower in the vehicle control group, relative to the Tat group. After repeated nicotine injection, pERK1 in NAc and VTA and pERK2 in VTA were increased in the vehicle group, but not in the Tat group. Moreover, repeated nicotine injections decreased pCREB in the PFC and VTA in the Tat group but not in the vehicle group. Thus, these findings indicate that the direct injection of Tat at the VTA may mediate CREB and ERK activity in response to nicotine-induced locomotor activity. PMID:26150803

  12. 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

  13. Expression Patterns of CREBs in Oocyte Growth and Maturation of Fish.

    PubMed

    Senthilkumaran, Balasubramanian; Sreenivasulu, Gunti; Wang, De-Shou; Sudhakumari, Cheni-Chery; Kobayashi, Tohru; Nagahama, Yoshitaka

    2015-01-01

    In fish, oocyte meiotic maturation is regulated by 17α, 20β-dihydroxy-progesterone through cAMP. To study the role of cAMP response element binding protein (CREB) in meiotic maturation, we cloned and characterized the expression pattern of CREBs from two fish models, the Nile tilapia and catfish. In the Nile tilapia three different CREBs were identified where in CREB1 was found in many tissues including gonads with abundant expression in testis. CREB2, few amino acids shorter than CREB1, was expressed in several tissues with abundant expression in ovary. In addition, a 3'UTR variant form, CREB3 was exclusively found in ovary. During natural 14-day ovarian cycle of the Nile tilapia, CREB1 expression was stable throughout vitellogenesis with a sharp decrease on the day of spawning. In contrast, CREB2 remain unchanged throughout the ovarian cycle, however elevated in 11-day full-grown immature ovarian follicle and after hCG-induction. Interestingly, CREB3 expression was induced three folds on the day of spawning as well as during hCG-induced oocyte maturation. Based on the synergistic expression pattern, CREB1 is likely to control oocyte growth, whereas CREB 2 and 3 contribute to oocyte maturation in tilapia and the latter seems to be critical. In catfish, a single form of CREB showed a maximum expression during spawning phase and hCG-induced maturation both in vivo and in vitro augmented CREB expression. These results suggest that spatial and temporal expression of CREBs seems to be important for final oocyte maturation and may also regulate oocyte growth in fish. PMID:26700177

  14. Expression Patterns of CREBs in Oocyte Growth and Maturation of Fish

    PubMed Central

    Wang, De-Shou; Sudhakumari, Cheni-Chery; Kobayashi, Tohru; Nagahama, Yoshitaka

    2015-01-01

    In fish, oocyte meiotic maturation is regulated by 17α, 20β-dihydroxy-progesterone through cAMP. To study the role of cAMP response element binding protein (CREB) in meiotic maturation, we cloned and characterized the expression pattern of CREBs from two fish models, the Nile tilapia and catfish. In the Nile tilapia three different CREBs were identified where in CREB1 was found in many tissues including gonads with abundant expression in testis. CREB2, few amino acids shorter than CREB1, was expressed in several tissues with abundant expression in ovary. In addition, a 3’UTR variant form, CREB3 was exclusively found in ovary. During natural 14-day ovarian cycle of the Nile tilapia, CREB1 expression was stable throughout vitellogenesis with a sharp decrease on the day of spawning. In contrast, CREB2 remain unchanged throughout the ovarian cycle, however elevated in 11-day full-grown immature ovarian follicle and after hCG-induction. Interestingly, CREB3 expression was induced three folds on the day of spawning as well as during hCG-induced oocyte maturation. Based on the synergistic expression pattern, CREB1 is likely to control oocyte growth, whereas CREB 2 and 3 contribute to oocyte maturation in tilapia and the latter seems to be critical. In catfish, a single form of CREB showed a maximum expression during spawning phase and hCG-induced maturation both in vivo and in vitro augmented CREB expression. These results suggest that spatial and temporal expression of CREBs seems to be important for final oocyte maturation and may also regulate oocyte growth in fish. PMID:26700177

  15. CREB (cAMP response element binding protein) and C/EBPalpha (CCAAT/enhancer binding protein) are required for the superstimulation of phosphoenolpyruvate carboxykinase gene transcription by adenoviral E1a and cAMP.

    PubMed Central

    Routes, J M; Colton, L A; Ryan, S; Klemm, D J

    2000-01-01

    In the present study, we observed superstimulated levels of cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter in cells infected with wild-type adenovirus expressing 12 S and 13 S E1a proteins, or in cells expressing 13 S E1a alone. cAMP-stimulated transcription was inhibited in cells expressing only 12 S E1a, but slightly elevated in cells expressing E1a proteins with mutations in conserved regions 1 or 2, leading us to conclude that the superstimulation was mediated by conserved region 3 of 13 S E1a. E1a failed to enhance cAMP-stimulated transcription from promoters containing mutations that abolish binding by cAMP response element binding protein (CREB) or CCAAT/enhancer binding proteins (C/EBPs). This result was supported by experiments in which expression of dominant-negative CREB and/or C/EBP proteins repressed E1a- and cAMP-stimulated transcription from the PEPCK gene promoter. In reconstitution experiments using a Gal4-responsive promoter, E1a enhanced cAMP-stimulated transcription when chimaeric Gal4-CREB and Gal4-C/EBPalpha were co-expressed. Phosphorylation of CREB on serine-133 was stimulated in cells treated with dibutyryl cAMP, whereas phosphorylation of C/EBPalpha was increased by E1a expression. Our data support a model in which cAMP agonists increase CREB activity and stimulate PEPCK gene transcription, a process that is enhanced by E1a through the phosphorylation of C/EBPalpha. PMID:11085926

  16. CREB-regulated transcription coactivator 1 enhances CREB-dependent gene expression in spinal cord to maintain the bone cancer pain in mice

    PubMed Central

    Liang, Ying; Liu, Yue; Hou, Bailing; Zhang, Wei; Liu, Ming; Sun, Yu-E; Gu, Xiaoping

    2016-01-01

    Background cAMP response element binding protein (CREB)-dependent gene expression plays an important role in central sensitization. CREB-regulated transcription coactivator 1 (CRTC1) dramatically increases CREB-mediated transcriptional activity. Brain-derived neurotrophic factor, N-methyl-d-aspartate receptor subunit 2B, and miRNA-212/132, which are highly CREB responsive, function downstream from CREB/CRTC1 to mediate activity-dependent synaptic plasticity and in turn loops back to amplify CREB/CRTC1 signaling. This study aimed to investigate the role of spinal CRTC1 in the maintenance of bone cancer pain using an RNA interference method. Results Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeNCrlVr mice to induce bone cancer pain. Western blotting was applied to examine the expression of spinal phospho-Ser133 CREB and CRTC1. We further investigated effects of repeated intrathecal administration with Adenoviruses expressing CRTC1-small interfering RNA (siRNA) on nociceptive behaviors and on the upregulation of CREB/CRTC1-target genes associated with bone cancer pain. Inoculation of osteosarcoma cells induced progressive mechanical allodynia and spontaneous pain, and resulted in upregulation of spinal p-CREB and CRTC1. Repeated intrathecal administration with Adenoviruses expressing CRTC1-siRNA attenuated bone cancer–evoked pain behaviors, and reduced CREB/CRTC1-target genes expression in spinal cord, including BDNF, NR2B, and miR-212/132. Conclusions Upregulation of CRTC1 enhancing CREB-dependent gene transcription in spinal cord may play an important role in bone cancer pain. Inhibition of spinal CRTC1 expression reduced bone cancer pain. Interruption to the positive feedback circuit between CREB/CRTC1 and its targets may contribute to the analgesic effects. These findings may provide further insight into the mechanisms and treatment of bone cancer pain. PMID:27060162

  17. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

    PubMed

    Kovács, Krisztián A; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J; Cardinaux, Jean-René

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. PMID:26247811

  18. 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…

  19. Small molecule screen for inhibitors of expression from canonical CREB response element-containing promoters

    PubMed Central

    Mitton, Bryan; Hsu, Katie; Dutta, Ritika; Tiu, Bruce C.; Cox, Nick; McLure, Kevin G.; Chae, Hee-Don; Smith, Mark; Eklund, Elizabeth A.; Solow-Cordero, David E.; Sakamoto, Kathleen M.

    2016-01-01

    The transcription factor CREB (cAMP Response Element Binding Protein) is an important determinant in the growth of Acute Myeloid Leukemia (AML) cells. CREB overexpression increases AML cell growth by driving the expression of key regulators of apoptosis and the cell cycle. Conversely, CREB knockdown inhibits proliferation and survival of AML cells but not normal hematopoietic cells. Thus, CREB represents a promising drug target for the treatment of AML, which carries a poor prognosis. In this study, we performed a high-throughput small molecule screen to identify compounds that disrupt CREB function in AML cells. We screened ∼114,000 candidate compounds from Stanford University's small molecule library, and identified 5 molecules that inhibit CREB function at micromolar concentrations, but are non-toxic to normal hematopoietic cells. This study suggests that targeting CREB function using small molecules could provide alternative approaches to treat AML. PMID:26840025

  20. Dysregulation of neurotrophic and inflammatory systems accompanied by decreased CREB signaling in ischemic rat retina.

    PubMed

    Guo, Xian Jun; Tian, Xue Song; Ruan, Zhi; Chen, Yu Ting; Wu, Lei; Gong, Qi; Wang, Wei; Zhang, Hai Yan

    2014-08-01

    Although permanent bilateral common carotid artery occlusion (2VO) has been demonstrated to induce retinal injury, there is still a lack of systematic research on the complex processing of retinal degeneration. In the present study, time-dependent (at three, 14, 60 days after 2VO surgery) changes of neurotrophic and inflammatory systems, as well as cAMP-responsive element binding protein (CREB) signaling, which has been previously reported to effectively regulate these two systems, were evaluated. First, a morphological study confirmed that 2VO surgery progressively induced severe inner retinal degeneration and down-regulation of synaptic proteins, PSD95 and synaptophysin. The mRNA or protein levels of neurotrophic factors (NGF, BDNF, NT-3 and GDNF) and their receptors (TrkA, TrkB and TrkC) showed marked and persistent down-regulation in the rat retina since three days after 2VO surgery, whereas the gene transcription levels of CNTF were increased and p75(NTR) mRNA levels remained unchanged. In contrast to inner retinal degeneration, retinal Müller cells displayed rapid and prolonged activation since three days after 2VO lesion, whereas the microglia cell number, and TNF-α and IL-1β levels showed a robust increase with a maximal effect at three days and returned to levels that were slightly over baseline at 14 and 60 days after 2VO lesion. Interestingly, the gene expression levels of iNOS significantly decreased in the rat retina at both three and 14 days after 2VO surgery. Finally, as we hypothesized, remarkable reduction of CREB and extracellular signal-regulated kinase (ERK) phosphorylation levels were observed in the rat retina at three days after 2VO surgery. Thus, for the first time, our study demonstrated that chronic ischemia induced long-term aberrant CREB signaling and time-dependent progressive dysregulation of neurotrophic and inflammatory systems in the retina, which may provide important clues for a better understanding of the pathogenesis of

  1. MAPK, CREB and zif268 are all required for the consolidation of recognition memory.

    PubMed

    Bozon, Bruno; Kelly, Aine; Josselyn, Sheena A; Silva, Alcino J; Davis, Sabrina; Laroche, Serge

    2003-04-29

    There has been nearly a century of interest in the idea that encoding and storage of information in the brain requires changes in the efficacy of synaptic connections between neurons that are activated during learning. Recent research into the molecular mechanisms of long-term potentiation (LTP) has brought about new knowledge that has provided valuable insights into the neural mechanisms of memory storage. The evidence indicates that rapid activation of the genetic machinery can be a key mechanism underlying the enduring modification of neural networks required for the stability of memories. In recent years, a wealth of experimental data has highlighted the importance of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signalling in the regulation of gene transcription in neurons. Here, we briefly review experiments that have shown MAPK/ERK, cAMP response element-binding protein (CREB) and the immediate early gene (IEG) zif268 are essential components of a signalling cascade required for the expression of late phase LTP and of certain forms of long-term memory. We also present experiments in which we have assessed the role of these three molecules in recognition memory. We show that pharmacological blockade of MAPK/ERK phosphorylation, functional inactivation of CREB in an inducible transgenic mouse and inactivation of zif268 in a mutant mouse result in a similar deficit in long-term recognition memory. In the continuing debate about the role of LTP mechanisms in memory, these findings provide an important complement to the suggestion that synaptic changes brought about by LTP and memory consolidation and storage share, at least in part, common underlying molecular mechanisms. PMID:12740127

  2. MAPK, CREB and zif268 are all required for the consolidation of recognition memory.

    PubMed Central

    Bozon, Bruno; Kelly, Aine; Josselyn, Sheena A; Silva, Alcino J; Davis, Sabrina; Laroche, Serge

    2003-01-01

    There has been nearly a century of interest in the idea that encoding and storage of information in the brain requires changes in the efficacy of synaptic connections between neurons that are activated during learning. Recent research into the molecular mechanisms of long-term potentiation (LTP) has brought about new knowledge that has provided valuable insights into the neural mechanisms of memory storage. The evidence indicates that rapid activation of the genetic machinery can be a key mechanism underlying the enduring modification of neural networks required for the stability of memories. In recent years, a wealth of experimental data has highlighted the importance of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signalling in the regulation of gene transcription in neurons. Here, we briefly review experiments that have shown MAPK/ERK, cAMP response element-binding protein (CREB) and the immediate early gene (IEG) zif268 are essential components of a signalling cascade required for the expression of late phase LTP and of certain forms of long-term memory. We also present experiments in which we have assessed the role of these three molecules in recognition memory. We show that pharmacological blockade of MAPK/ERK phosphorylation, functional inactivation of CREB in an inducible transgenic mouse and inactivation of zif268 in a mutant mouse result in a similar deficit in long-term recognition memory. In the continuing debate about the role of LTP mechanisms in memory, these findings provide an important complement to the suggestion that synaptic changes brought about by LTP and memory consolidation and storage share, at least in part, common underlying molecular mechanisms. PMID:12740127

  3. 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. PMID:25049196

  4. The role of CREB signaling in Alzheimer's disease and other cognitive disorders.

    PubMed

    Saura, Carlos A; Valero, Jorge

    2011-01-01

    Gene expression changes in the brain affect cognition during normal and pathological aging. Progress in understanding the cellular processes regulating gene expression networks in cognition is relevant to develop therapeutic interventions for age-related cognitive disorders. Synaptic efficacy mediating memory storage requires the activation of specific gene expression programs regulated, among others, by the transcription factor cAMP-response element binding protein (CREB). CREB signaling is essential for long-lasting changes in synaptic plasticity that mediates the conversion of short-term memory to long-term memory. CREB signaling has been recently involved in several brain pathological conditions including cognitive and neurodegenerative disorders. The β-amyloid (Aβ) peptide, which plays a crucial role in the pathogenesis of Alzheimer's disease, alters hippocampal-dependent synaptic plasticity and memory and mediates synapse loss through the CREB signaling pathway. The fact that altered CREB signaling has been implicated in other cognitive disorders including Huntington's disease and Rubinstein-Taybi and Coffin-Lowry syndromes suggests a crucial role of CREB signaling in cognitive dysfunction. In this review paper, we summarize recent findings indicating a role of CREB and its coactivators CREB binding protein and CREB-regulated transcription coactivator in cognition during normal and pathological aging. We also discuss the development of novel therapeutic strategies based on CREB targeting to ameliorate cognitive decline in aging and cognitive disorders. PMID:21476939

  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. 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. PMID:26352003

  7. GSK-3α Is a Novel Target of CREB and CREB-GSK-3α Signaling Participates in Cell Viability in Lung Cancer

    PubMed Central

    Herbst, Roy S.; Koo, Ja Seok

    2016-01-01

    Overexpression or activation of cyclic AMP-response element-binding protein (CREB) has been known to be involved in several human malignancies, including lung cancer. Genes regulated by CREB have been reported to suppress apoptosis, induce cell proliferation, inflammation, and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. Here, we identified GSK-3α as one of the CREB target genes which is critical for the viability of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3α and the direct binding of CREB on the promoter of GSK3A was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3α expression in lung cancer. Inhibition of GSK-3α suppressed cell viability, colony formation, and tumor growth. For the first time, we demonstrated that GSK-3α is regulated by CREB in lung cancer and is required for the cell viability. These findings implicate CREB-GSK-3α axis as a novel therapeutic target for lung cancer treatment. PMID:27049759

  8. Antidepressant- and anxiolytic-like effects of the phosphodiesterase-4 (PDE4) inhibitor rolipram on behavior depend on cyclic AMP-response element binding protein (CREB)-mediated neurogenesis in the hippocampus

    PubMed Central

    Li, Yun-Feng; Huang, Ying; Amsdell, Simon L.; Xiao, Lan; O'Donnell, James M.; Zhang, Han-Ting

    2009-01-01

    Inhibition of phosphodiesterase-4 (PDE4), an enzyme that catalyzes the hydrolysis of cyclic AMP (cAMP), increases phosphorylation of cAMP-response element binding protein (pCREB) and hippocampal neurogenesis, and produces antidepressant-like effects on behavior; however, causal links among these have not been established. In the present study, chronic administration of rolipram produced antidepressant- and anxiolytic-like effects on behavior in mice. It also increased cAMP and pCREB levels in the hippocampus and prefrontal cortex, but increased Sox2, a marker for mitotic progenitor cells, only in the hippocampus. Chronic rolipram treatment also increased hippocampal neurogenesis, as evidenced by increased bromodeoxyuridine (BrdU)-positive cells in the hippocampal dentate gyrus. Methylazoxymethanol (MAM), which is toxic to proliferating cells, reversed rolipram-induced increases in BrdU-positive cells and pCREB in the hippocampus and partially blocked its behavioral effects. Approximately 84% of BrdU-positive cells became newborn neurons, 93% of which co-expressed pCREB; these proportions were not altered by rolipram or MAM, either alone or in combination. Finally, three weeks following the end of MAM treatment, when neurogenesis was no longer inhibited, rolipram again increased hippocampal pCREB, with its antidepressant- and anxiolytic-like effects resumed. Overall, the present results suggest that rolipram produces its effects on behavior in a manner that at least partially depends on its neurogenic action in the hippocampus, targeting mitotic progenitor cells rather than newborn or mature neurons; cAMP/CREB signaling in hippocampal newborn neurons is critical for neurogenesis and contributes to the behavioral effects of rolipram. PMID:19516250

  9. 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

    2015-12-01

    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. PMID:26485503

  10. Akt- and CREB-Mediated Prostate Cancer Cell Proliferation Inhibition by Nexrutine, a Phellodendron amurense Extract1

    PubMed Central

    Garcia, Gretchen E; Nicole, Arevalo; Bhaskaran, Shylesh; Gupta, Ashima; Kyprianou, Natasha; Kumar, Addanki P

    2006-01-01

    Abstract Evidence from epidemiological studies suggests that plant-based diets can reduce the risk of prostate cancer. However, very little information is available concerning the use of botanicals in preventing prostate cancer. As a first step toward developing botanicals as prostate cancer preventives, we examined the effect of Nexrutine on human prostate cancer cells. Nexrutine is a herbal extract developed from Phellodendron amurense. Phellodendron extracts have been used traditionally in Chinese medicine for hundreds of years as an anti-diarrheal, astringent, and anti-inflammatory agent. The present study investigated its potential antitumor effect on human prostate cancer cells. Our results suggest that it inhibits tumor cell proliferation through apoptosis induction and inhibition of cell survival signaling. The results of the present study indicate that Nexrutine treatment 1) inhibits the proliferation of both androgen-responsive and androgen-independent human prostate cancer cells through induction of apoptosis; 2) reduces levels of pAkt, phosphorylated cAMP response-binding protein (pCREB), and CREB DNA-binding activity; and 3) induces apoptosis in prostate cancer cells stably overexpressing Bcl-2. Further Akt kinase activity was reduced in cells treated with Nexrutine, and ectopic expression of myristoylated Akt protected from Nexrutine induced inhibition of proliferation, implicating a role for Akt signaling. PMID:16820098

  11. Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes the vascular endothelial growth factor-stimulated MAPKs and downstream effectors AP-1 and CREB in mouse mesangial cells

    PubMed Central

    Tripathi, Satyabha; Pandey, Kailash N.

    2012-01-01

    Along with its natriuretic, diuretic, and vasodilatory properties, atrial natriuretic peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) exhibit an inhibitory effect on cell growth and proliferation. However, the signaling pathways mediating this inhibition are not well understood. The objective of this study was to determine the effect of ANP-NPRA system on mitogen-activated protein kinases (MAPKs) and the downstream proliferative transcription factors involving activating protein-1 (AP-1) and cAMP-response element binding protein (CREB) in agonist-stimulated mouse mesangial cells (MMCs). We found that ANP inhibited vascular endothelial growth factor (VEGF)-stimulated phosphorylation of MAPKs (Erk1, Erk2, JNK, and p38), to a greater extent in NPRA-transfected cells (50–60%) relative to vector-transfected cells (25–30%). The analyses of the phosphorylated transcription factors revealed that ANP inhibited VEGF-stimulated activation of CREB, and the AP-1 subunits (c-jun and c-fos). Gel shift assays demonstrated that ANP inhibited VEGF-stimulated AP-1 and CREB DNA-binding ability by 67 % and 62 %, respectively. The addition of the protein kinase G (PKG) inhibitor, KT-5823, restored the VEGF-stimulated activation of MAPKs, AP-1, and CREB, demonstrating the integral role of cGMP/PKG signaling in NPRA-mediated effects. Our results delineate the under lying mechanisms through which ANP-NPRA system exerts an inhibitory effect on MAPKs and down-stream effector molecules, AP-1 and CREB, critical for cell growth and proliferation. PMID:22610792

  12. 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

    2013-01-01

    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. PMID:24025424

  13. 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

    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

  14. Chronic administration of aripiprazole activates GSK3β-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats.

    PubMed

    Pan, Bo; Huang, Xu-Feng; Deng, Chao

    2016-01-01

    Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3β-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with haloperidol (a D2R antagonist) and bifeprunox (a potent D2R partial agonist). We found that the Akt-GSK3β pathway was activated by aripiprazole and bifeprunox in the prefrontal cortex; NMDA NR2A levels were reduced by aripiprazole and haloperidol. In the nucleus accumbens, all three drugs increased Akt-GSK3β signalling; in addition, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3, β-catenin and GABAA receptors, NMDA receptor subunits, as well as CREB1 phosphorylation levels. The results suggest that chronic oral administration of aripiprazole affects schizophrenia-related cellular signalling pathways and markers (including Akt-GSK3β signalling, Dvl-GSK3β-β-catenin signalling, GABAA receptor, NMDA receptor and CREB1) in a brain-region-dependent manner; the selective effects of aripiprazole on these signalling pathways might be associated with its unique clinical effects. PMID:27435909

  15. 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

  16. Chronic administration of aripiprazole activates GSK3β-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats

    PubMed Central

    Pan, Bo; Huang, Xu-Feng; Deng, Chao

    2016-01-01

    Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3β-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with haloperidol (a D2R antagonist) and bifeprunox (a potent D2R partial agonist). We found that the Akt-GSK3β pathway was activated by aripiprazole and bifeprunox in the prefrontal cortex; NMDA NR2A levels were reduced by aripiprazole and haloperidol. In the nucleus accumbens, all three drugs increased Akt-GSK3β signalling; in addition, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3, β-catenin and GABAA receptors, NMDA receptor subunits, as well as CREB1 phosphorylation levels. The results suggest that chronic oral administration of aripiprazole affects schizophrenia-related cellular signalling pathways and markers (including Akt-GSK3β signalling, Dvl-GSK3β-β-catenin signalling, GABAA receptor, NMDA receptor and CREB1) in a brain-region-dependent manner; the selective effects of aripiprazole on these signalling pathways might be associated with its unique clinical effects. PMID:27435909

  17. Reciprocal Phosphorylation of Yeast Glycerol-3-Phosphate Dehydrogenases in Adaptation to Distinct Types of Stress

    PubMed Central

    Lee, Yong Jae; Jeschke, Grace R.; Roelants, Françoise M.; Thorner, Jeremy

    2012-01-01

    Eukaryotic cells have evolved mechanisms for ensuring growth and survival in the face of stress caused by a fluctuating environment. Saccharomyces cerevisiae has two homologous glycerol-3-phosphate dehydrogenases, Gpd1 and Gpd2, that are required to endure various stresses, including hyperosmotic shock and hypoxia. These enzymes are only partially redundant, and their unique functions were attributed previously to differential transcriptional regulation and localization. We find that Gpd1 and Gpd2 are negatively regulated through phosphorylation by distinct kinases under reciprocal conditions. Gpd2 is phosphorylated by the AMP-activated protein kinase Snf1 to curtail glycerol production when nutrients are limiting. Gpd1, in contrast, is a target of TORC2-dependent kinases Ypk1 and Ypk2. Inactivation of Ypk1 by hyperosmotic shock results in dephosphorylation and activation of Gpd1, accelerating recovery through increased glycerol production. Gpd1 dephosphorylation acts synergistically with its transcriptional upregulation, enabling long-term growth at high osmolarity. Phosphorylation of Gpd1 and Gpd2 by distinct kinases thereby enables rapid adaptation to specific stress conditions. Introduction of phosphorylation motifs targeted by distinct kinases provides a general mechanism for functional specialization of duplicated genes during evolution. PMID:22988299

  18. GABA-CREB signalling regulates maturation and survival of newly generated neurons in the adult hippocampus

    PubMed Central

    Jagasia, Ravi; Steib, Kathrin; Englberger, Elisabeth; Herold, Sabine; Faus-Kessler, Theresa; Saxe, Michael; Gage, Fred H.; Song, Hongjun; Lie, D. Chichung

    2009-01-01

    Survival and integration of new neurons in the hippocampal circuit are rate-limiting steps in adult hippocampal neurogenesis. Neuronal network activity is a major regulator of these processes, yet little is known about the respective downstream signalling pathways. Here, we investigate the role of CREB signalling in adult hippocampal neurogenesis. CREB is activated in new granule neurons during a distinct developmental period. Loss of CREB function in a cell-autonomous fashion impairs dendritic development, decreases the expression of the neurogenic transcription factor NeuroD and of the neuronal microtubule associated protein, DCX, and compromises the survival of newborn neurons. In addition, GABA-mediated excitation regulates CREB activation at early developmental stages. Importantly, developmental defects following loss of GABA-mediated excitation can be compensated by enhanced CREB signalling. These results indicate that CREB signalling is a central pathway in adult hippocampal neurogenesis, regulating the development and survival of new hippocampal neurons downstream of GABA-mediated excitation. PMID:19553437

  19. WSKY, a traditional Chinese decoction, rescues cognitive impairment associated with NMDA receptor antagonism by enhancing BDNF/ERK/CREB signaling.

    PubMed

    Guo, Xin; Chen, Zheng-Hua; Wang, Hui-Ling; Liu, Zhong-Chun; Wang, Xiao-Ping; Zhou, Ben-Hong; Yang, Can; Zhang, Xue-Ping; Xiao, Ling; Shu, Chang; Chen, Jian-Xin; Wang, Gao-Hua

    2015-04-01

    Warm‑supplementing kidney yang (WSKY) is an herbal prescription that has been used in Traditional Chinese Medicine for the treatment of psychiatric conditions. A previous study by our group found that WSKY significantly improved cognitive function of schizophrenia patients. In the present study, the effects of WSKY on cognitive function and their underlying mechanisms were investigated. WSKY was administered to an MK‑801‑induced rat model of chronic schizophrenia for 14 days. Memory performance was assessed using the Morris water maze (MWM) test. The expression of brain‑derived neurotrophic factor (BDNF), activation of cAMP response element binding protein (pCREB/CREB) and activation of extracellular signal‑regulated kinase (pERK/ERK) in the hippocampus was detected using western blot analysis. In the acquisition phase of the MWM test, the escape latency was significantly increased in the MK‑801‑treated group compared with the normal control group (P<0.01). Treatment with WSKY for 14 days at doses of 100 or 250 mg/kg rescued this cognitive impairment (P<0.05). In the probe test, 250 mg/kg WSKY treatment increased the time spent in the target quadrant (P<0.05) and number of platform crossings (P<0.01). Western blot analysis demonstrated that the levels of BDNF expression in the hippocampus of rats without behavioral tests were elevated following 14 days of WSKY treatment, and the effect of WSKY treatment on hippocampal BDNF expression was presented in an inverted U‑shaped dose‑response pattern. The pERK1/2 in the hippocampus was significantly enhanced following 100 mg/kg (P<0.01) and 250 mg/kg (P<0.01) WSKY treatment, while only 250 mg/kg WSKY increased the phosphorylation of CREB (P<0.01). The results of the present study indicated that WSKY enhances cognitive performance via the upregulation of BDNF/ERK/CREB signaling, and that WSKY has potential therapeutic implications for cognitive impairment of schizophrenia. PMID:25503442

  20. CREB regulates memory allocation in the insular cortex.

    PubMed

    Sano, Yoshitake; Shobe, Justin L; Zhou, Miou; Huang, Shan; Shuman, Tristan; Cai, Denise J; Golshani, Peyman; Kamata, Masakazu; Silva, Alcino J

    2014-12-01

    The molecular and cellular mechanisms of memory storage have attracted a great deal of attention. By comparison, little is known about memory allocation, the process that determines which specific neurons in a neural network will store a given memory. Previous studies demonstrated that memory allocation is not random in the amygdala; these studies showed that amygdala neurons with higher levels of the cyclic-AMP-response-element-binding protein (CREB) are more likely to be recruited into encoding and storing fear memory. To determine whether specific mechanisms also regulate memory allocation in other brain regions and whether CREB also has a role in this process, we studied insular cortical memory representations for conditioned taste aversion (CTA). In this task, an animal learns to associate a taste (conditioned stimulus [CS]) with the experience of malaise (such as that induced by LiCl; unconditioned stimulus [US]). The insular cortex is required for CTA memory formation and retrieval. CTA learning activates a subpopulation of neurons in this structure, and the insular cortex and the basolateral amygdala (BLA) interact during CTA formation. Here, we used a combination of approaches, including viral vector transfections of insular cortex, arc fluorescence in situ hybridization (FISH), and designer receptors exclusively activated by designer drugs (DREADD) system, to show that CREB levels determine which insular cortical neurons go on to encode a given conditioned taste memory. PMID:25454591

  1. Distinct effect of CacyBP/SIP on the ERK1/2-CREB-BDNF pathway in undifferentiated and differentiated neuroblastoma NB2a cells.

    PubMed

    Rosińska, Sara; Leśniak, Wiesława; Filipek, Anna

    2016-07-01

    CacyBP/SIP, a protein expressed to high extent in the brain, has been shown to act as ERK1/2 phosphatase in vitro and in cultured cells. It has been demonstrated recently that CacyBP/SIP can modulate the activity of some transcription factors in neurons and glioma cells. In the present work we have examined the effect of CacyBP/SIP overexpression and silencing on the phosphorylation/activity of ERK1/2 (pERK1/2) and CREB (pCREB) and on the level of BDNF mRNA in differentiated and undifferentiated neuroblastoma NB2a cells. We have shown that in undifferentiated cells the amount of pERK1/2 decreased upon CacyBP/SIP overexpression. Further studies have shown that the activity of CREB and the level of BDNF mRNA, downstream effectors of the ERK1/2 signaling pathway, also depended on the CacyBP/SIP level and strictly matched the level of pERK1/2. Interestingly, in differentiated NB2a cells, overexpression of CacyBP/SIP appeared to have a distinct effect on the pERK1/2 level from that observed in undifferentiated cells. Subsequent studies have revealed that distinct function of CacyBP/SIP in undifferentiated and differentiated NB2a cells might be due to changes in its posttranslational modifications and protein ligands. Altogether, our studies suggest that CacyBP/SIP is involved in the ERK1/2-CREB-BDNF pathway and that it might regulate this pathway depending on the stage of NB2a cell differentiation. PMID:27180052

  2. Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms

    PubMed Central

    Antony, N.; McDougall, A. R.; Mantamadiotis, T.; Cole, T. J.; Bird, A. D.

    2016-01-01

    During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1−/− mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1−/− mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1−/− mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development. PMID:27150575

  3. Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms.

    PubMed

    Antony, N; McDougall, A R; Mantamadiotis, T; Cole, T J; Bird, A D

    2016-01-01

    During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1(-/-) mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1(-/-) mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1(-/-) mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development. PMID:27150575

  4. Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity

    PubMed Central

    Akhmedov, Dmitry; Rajendran, Kavitha; Mendoza-Rodriguez, Maria G.

    2016-01-01

    The cAMP response element binding protein (CREB) is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo. PMID:27336479

  5. CREB regulates spine density of lateral amygdala neurons: implications for memory allocation

    PubMed Central

    Sargin, Derya; Mercaldo, Valentina; Yiu, Adelaide P.; Higgs, Gemma; Han, Jin-Hee; Frankland, Paul W.; Josselyn, Sheena A.

    2013-01-01

    Neurons may compete against one another for integration into a memory trace. Specifically, neurons in the lateral nucleus of the amygdala with relatively higher levels of cAMP Responsive Element Binding Protein (CREB) seem to be preferentially allocated to a fear memory trace, while neurons with relatively decreased CREB function seem to be excluded from a fear memory trace. CREB is a ubiquitous transcription factor that modulates many diverse cellular processes, raising the question as to which of these CREB-mediated processes underlie memory allocation. CREB is implicated in modulating dendritic spine number and morphology. As dendritic spines are intimately involved in memory formation, we investigated whether manipulations of CREB function alter spine number or morphology of neurons at the time of fear conditioning. We used viral vectors to manipulate CREB function in the lateral amygdala (LA) principal neurons in mice maintained in their homecages. At the time that fear conditioning normally occurs, we observed that neurons with high levels of CREB had more dendritic spines, while neurons with low CREB function had relatively fewer spines compared to control neurons. These results suggest that the modulation of spine density provides a potential mechanism for preferential allocation of a subset of neurons to the memory trace. PMID:24391565

  6. Dl-3-n-butylphthalide-induced upregulation of antioxidant defense is involved in the enhancement of cross talk between CREB and Nrf2 in an Alzheimer's disease mouse model.

    PubMed

    Wang, Chun-Yan; Wang, Zhan-You; Xie, Jing-Wei; Wang, Tao; Wang, Xu; Xu, Ye; Cai, Jian-Hui

    2016-02-01

    Synapse impairment in the Alzheimer's disease (AD) brain is an early event leading to cognitive dysfunction. Most oxidative stress localizes to the synapse, and synapse loss is the basis of cognitive decline in AD. Dl-3-n-butylphthalide (Dl-NBP), a small molecule compound has been shown to ameliorate oxidative stress. We evaluated the effects of a 5-month oral delivery with Dl-NBP on oxidative stress and cognitive function in APP/PS1 transgenic mice. Dl-NBP treatment reduced oxidative stress in the APP/PS1 mouse brain and alleviated learning and memory deficits. Dl-NBP supplementation meliorated synaptic plasticity, diminished soluble amyloid beta and amyloid beta oligomer in the APP/PS1 mouse brain. Dl-NBP administration caused an increase of cyclic adenosine monophosphate-response element binding protein (CREB)-binding protein (CBP)-associated Ser133-phosphorylated CREB (p-CREB) protein. Chromatin immunoprecipitation analysis revealed that Dl-NBP increased the recruitment of CBP to the promoters of best-characterized genes downstream of nuclear factor erythroid 2-related factor 2, nicotinamide adenine dinucleotide phosphate (NADPH) quinone oxidoreductase 1, and γ-glutamylcysteine synthetase modifier subunit. We demonstrate that the Dl-NBP-triggered upregulation of antioxidant defenses is involved in the enhancement of cross talk between CREB and nuclear factor erythroid 2-related factor 2 via CBP. Our results suggest that Dl-NBP may be a useful agent for the treatment of AD. PMID:26827641

  7. 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. PMID:26147081

  8. Chronic enhancement of CREB activity in the hippocampus interferes with the retrieval of spatial information

    PubMed Central

    Viosca, Jose; Malleret, Gaël; Bourtchouladze, Rusiko; Benito, Eva; Vronskava, Svetlana; Kandel, Eric R.; Barco, Angel

    2009-01-01

    The activation of cAMP-responsive element-binding protein (CREB)-dependent gene expression is thought to be critical for the formation of different types of long-term memory. To explore the consequences of chronic enhancement of CREB function on spatial memory in mammals, we examined spatial navigation in bitransgenic mice that express in a regulated and restricted manner a constitutively active form of CREB, VP16-CREB, in forebrain neurons. We found that chronic enhancement of CREB activity delayed the acquisition of an allocentric strategy to solve the hidden platform task. The ability to turn on and off transgene expression allowed us to dissect the role of CREB in dissociable memory processes. In mice in which transgene expression was turned on during memory acquisition, turning off the transgene re-established the access to the memory trace, whereas in mice in which transgene expression was turned off during acquisition, turning on the transgene impaired memory expression in a reversible manner, indicating that CREB enhancement specifically interfered with the retrieval of spatial information. The defects on spatial navigation in mice with chronic enhancement of CREB function were not corrected by conditions that increased further CREB-dependent activation of hippocampal memory systems, such as housing in an enriched environment. These results along with previous findings in CREB-deficient mutants indicate that the relationship of CREB-mediated plasticity to spatial memory is an inverted-U function, and that optimal learning in the water maze requires accurate regulation of this pathway. PMID:19237642

  9. BDNF–ERK–CREB signalling mediates the role of miR-132 in the regulation of the effects of oleanolic acid in male mice

    PubMed Central

    Yi, Li-Tao; Li, Jing; Liu, Bin-Bin; Luo, Liu; Liu, Qing; Geng, Di

    2014-01-01

    Background Although previous study has demonstrated that brain-derived neurotrophic factor (BDNF) is involved in the antidepressant-like effect of oleanolic acid, there is little information regarding the details of the molecular mechanism involved in this effect. Methods We used a chronic unpredictable mild stress (CUMS) model to test the antidepressant-like effect of oleanolic acid on depressant-like behaviour, miR-132 expression and synaptic protein expression in the male mouse hippocampus. Furthermore, we explored the possible signalling pathways associated with miR-132 expression that mediate the effect of oleanolic acid on neuronal proliferation. Results The results demonstrated that a 3-week treatment with oleanolic acid ameliorated CUMS-induced anhedonic and anxiogenic behaviours. Furthermore, we found that oleanolic acid led to the BDNF-related phosphorylation and activation of extracellular signal-regulated kinases (ERK) and cyclic adenosine monophosphate response element binding protein (CREB), which was associated with the upregulation of miR-132 and hippocampal neuronal proliferation. Moreover, experiments with an miR-132 antagomir revealed that targeting miR-132 led to inhibition of neuronal proliferation and the postsynaptic density protein 95, but did not affect presynaptic protein synapsin I. Limitations Several other stimuli can also induce CREB phosphorylation in the hippocampus. Thus, regulation of miR-132 may not be restricted to neurotrophic signalling. Conclusion Our results show that oleanolic acid induces the upregulation of miR-132, which serves as an important regulator of neurotrophic actions, mainly through the activation of the hippocampal BDNF–ERK–CREB signalling pathways. PMID:25079084

  10. p21-activated kinase 4 critically regulates melanogenesis via activation of the CREB/MITF and β-catenin/MITF pathways.

    PubMed

    Yun, Cheong-Yong; You, Soon-Tae; Kim, Jin-Hwa; Chung, Jin H; Han, Sang-Bae; Shin, Eun-Young; Kim, Eung-Gook

    2015-05-01

    p21-activated kinase 4 (PAK4) regulates a wide range of cellular events, including cytoskeletal remodeling, cell growth, and survival. Our previous study identified PAK4 as a key regulator of cAMP-response element-binding protein (CREB) that acts upstream of microphthalmia-associated transcription factor (MITF), a master transcription factor in melanogenesis. We therefore investigated the role of PAK4 in melanogenesis. Melanocytes express both PAK2 and PAK4 isoforms, but only RNA interference knockdown of PAK4 significantly influenced α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in B16 melanoma cells. Consistent with this result, PAK4 inhibition by PF3758309, a potent ATP-competitive inhibitor of PAKs, suppressed not only α-MSH-induced melanogenesis in B16 melanoma and human epithelial melanocyte cells but also UVB-induced melanogenesis in the skin of melanin-possessing hairless mice (HRM-2) in a dose-dependent manner. Inhibition of PAK4 over several days markedly decreased the levels of CREB, MITF, and tyrosinase in both HRM-2 mice and B16 melanoma cells. Moreover, PAK4 knockdown and inhibition suppressed α-MSH-stimulated β-catenin phosphorylation at serine 675 (S675) but enhanced phosphorylation at S33/37, an indicator for ubiquitination-dependent proteolysis. Together, our results provide evidence that PAK4 promotes α-MSH/UVB-induced melanogenesis via the CREB and Wnt/β-catenin signaling pathways and suggest that PAK4 may be a potential therapeutic target in pigmentation disorders. PMID:25560280

  11. 6-Hydroxydopamine lesions of rat substantia nigra up-regulate dopamine-induced phosphorylation of the cAMP-response element-binding protein in striatal neurons.

    PubMed Central

    Cole, D G; Kobierski, L A; Konradi, C; Hyman, S E

    1994-01-01

    Destruction of the substantia nigra produces striatal D1 dopamine receptor supersensitivity without increasing receptor number or affinity, thus implicating postreceptor mechanisms. The nature of these mechanisms is unknown. Increased striatal c-fos expression ipsilateral to a unilateral lesion of the substantia nigra in rats treated with appropriate dopamine agonists provides a cellular marker of D1 receptor supersensitivity. D1 receptors are positively linked to adenylate cyclase and therefore to cAMP-dependent protein kinase. Because expression of the c-fos gene in response to cAMP- and Ca2+/calmodulin-regulated protein kinases depends on phosphorylation of cAMP-response element-binding protein (CREB) at Ser-133, we examined CREB phosphorylation after dopaminergic stimulation in cultured striatal neurons and in the striatum of rats after unilateral 6-hydroxydopamine ablation of the substantia nigra. Using an antiserum specific for CREB phosphorylated at Ser-133, we found that dopamine increases CREB phosphorylation in cultured striatal neurons. This effect was blocked by a D1 antagonist. L-Dopa produced marked CREB phosphorylation in striatal neurons in rats ipsilateral, but not contralateral, to a 6-hydroxydopamine lesion. This response was blocked by a D1 antagonist, but not a D2 antagonist, and was reproduced by a D1 agonist, but not a D2 agonist. These findings are consistent with the hypothesis that D1 receptor supersensitivity is associated with upregulated activity of cAMP-dependent or Ca2+/calmodulin-dependent protein kinases, or both, following dopamine denervation of striatal neurons. Images PMID:7937819

  12. Resveratrol protects CA1 neurons against focal cerebral ischemic reperfusion-induced damage via the ERK-CREB signaling pathway in rats.

    PubMed

    Li, Zhen; Fang, Fang; Wang, Yuanyin; Wang, Liecheng

    2016-01-01

    Ischemic stroke is a primary cause of mortality and disability in the aged population. Resveratrol (Res), a natural polyphenol enriched in plants, presents diverse biological activities, e.g., antiinflammatory and anti-oxidation effects. Here, we evaluated whether Res pretreatment influenced focal cerebral ischemia-induced cognitive impairment, and we explored the underlying mechanisms in rats. The results showed that a single administration of Res (30mg/kg, i.p.) at 1 or 4h, but not at 24h before focal cerebral ischemia exerted significant neuroprotective effects, including a reduction in hippocampal CA1 neuronal death and spatial cognition deficits caused by ischemia. The neuroprotective effects of Res were suppressed by pretreatment with MK801, an NMDA receptor blocker, or U0126, an extracellular signal regulated kinase 1/2 (ERK1/2) kinase inhibitor. A western blot analysis revealed that Res treatment at 1h before ischemia significantly increased ERK1/2 phosphorylation and cyclic-AMP response element binding protein (CREB) phosphorylation in the CA1 region of the hippocampus, which can be prevented with U0126 pretreatment. The results showed that the NMDA receptor-mediated ERK-CREB signaling pathway might participates in Res-induced neuroprotection in rats with focal cerebral ischemia. PMID:27143440

  13. Regulation of proliferation and histone acetylation in embryonic neural precursors by CREB/CREM signaling

    PubMed Central

    Parlato, Rosanna; Mandl, Claudia; Hölzl-Wenig, Gabriele; Liss, Birgit; Tucker, Kerry L; Ciccolini, Francesca

    2014-01-01

    The transcription factor CREB (cAMP-response element binding protein) regulates differentiation, migration, survival and activity-dependent gene expression in the developing and mature nervous system. However, its specific role in the proliferation of embryonic neural progenitors is still not completely understood. Here we investigated how CREB regulates proliferation of mouse embryonic neural progenitors by a conditional mutant lacking Creb gene in neural progenitors. In parallel, we explored possible compensatory effects by the genetic ablation of another member of the same gene family, the cAMP-responsive element modulator (Crem). We show that CREB loss differentially impaired the proliferation, clonogenic potential and self-renewal of precursors derived from the ganglionic eminence (GE), in comparison to those derived from the cortex. This phenotype was associated with a specific reduction of histone acetylation in the GE of CREB mutant mice, and this reduction was rescued in vivo by inhibition of histone deacetylation. These observations indicate that the impaired proliferation could be caused by a reduced acetyltransferase activity in Creb conditional knock-out mice. These findings support a crucial role of CREB in controlling embryonic neurogenesis and propose a novel mechanism by which CREB regulates embryonic neural development.

  14. 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…

  15. Chronic Enhancement of CREB Activity in the Hippocampus Interferes with the Retrieval of Spatial Information

    ERIC Educational Resources Information Center

    Viosca, Jose; Malleret, Gael; Bourtchouladze, Rusiko; Benito, Eva; Vronskava, Svetlana; Kandel, Eric R.; Barco, Angel

    2009-01-01

    The activation of cAMP-responsive element-binding protein (CREB)-dependent gene expression is thought to be critical for the formation of different types of long-term memory. To explore the consequences of chronic enhancement of CREB function on spatial memory in mammals, we examined spatial navigation in bitransgenic mice that express in a…

  16. Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation

    PubMed Central

    Tobias, Irene S.; Kaulich, Manuel; Kim, Peter K.; Simon, Nitya; Jacinto, Estela; Dowdy, Steven F.; King, Charles C.; Newton, Alexandra C.

    2016-01-01

    Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ. PMID:26635352

  17. Identification of compounds that potentiate CREB signaling as possible enhancers of long-term memory

    PubMed Central

    Xia, Menghang; Huang, Ruili; Guo, Vicky; Southall, Noel; Cho, Ming-Hsuang; Inglese, James; Austin, Christopher P.; Nirenberg, Marshall

    2009-01-01

    Many studies have implicated the cAMP Response Element Binding (CREB) protein signaling pathway in long-term memory. To identify small molecule enhancers of CREB activation of gene expression, we screened ≈73,000 compounds, each at 7–15 concentrations in a quantitative high-throughput screening (qHTS) format, for activity in cells by assaying CREB mediated β-lactamase reporter gene expression. We identified 1,800 compounds that potentiated CREB mediated gene expression, with potencies as low as 16 nM, comprising 96 structural series. Mechanisms of action were systematically determined, and compounds that affect phosphodiesterase 4, protein kinase A, and cAMP production were identified, as well as compounds that affect CREB signaling via apparently unidentified mechanisms. qHTS folowed by interrogation of pathway targets is an efficient paradigm for lead generation for chemical genomics and drug development. PMID:19196967

  18. Computational analysis of human and mouse CREB3L4 Protein

    PubMed Central

    Velpula, Kiran Kumar; Rehman, Azeem Abdul; Chigurupati, Soumya; Sanam, Ramadevi; Inampudi, Krishna Kishore; Akila, Chandra Sekhar

    2012-01-01

    CREB3L4 is a member of the CREB/ATF transcription factor family, characterized by their regulation of gene expression through the cAMP-responsive element. Previous studies identified this protein in mice and humans. Whereas CREB3L4 in mice (referred to as Tisp40) is found in the testes and functions in spermatogenesis, human CREB3L4 is primarily detected in the prostate and has been implicated in cancer. We conducted computational analyses to compare the structural homology between murine Tisp40α human CREB3L4. Our results reveal that the primary and secondary structures of the two proteins contain high similarity. Additionally, predicted helical transmembrane structure reveals that the proteins likely have similar structure and function. This study offers preliminary findings that support the translation of mouse Tisp40α findings into human models, based on structural homology. PMID:22829733

  19. Computational analysis of human and mouse CREB3L4 Protein.

    PubMed

    Velpula, Kiran Kumar; Rehman, Azeem Abdul; Chigurupati, Soumya; Sanam, Ramadevi; Inampudi, Krishna Kishore; Akila, Chandra Sekhar

    2012-01-01

    CREB3L4 is a member of the CREB/ATF transcription factor family, characterized by their regulation of gene expression through the cAMP-responsive element. Previous studies identified this protein in mice and humans. Whereas CREB3L4 in mice (referred to as Tisp40) is found in the testes and functions in spermatogenesis, human CREB3L4 is primarily detected in the prostate and has been implicated in cancer. We conducted computational analyses to compare the structural homology between murine Tisp40α human CREB3L4. Our results reveal that the primary and secondary structures of the two proteins contain high similarity. Additionally, predicted helical transmembrane structure reveals that the proteins likely have similar structure and function. This study offers preliminary findings that support the translation of mouse Tisp40α findings into human models, based on structural homology. PMID:22829733

  20. CREB Activation Induces Adipogenesis in 3T3-L1 Cells

    PubMed Central

    Reusch, Jane E. B.; Colton, Lilliester A.; Klemm, Dwight J.

    2000-01-01

    Obesity is the result of numerous, interacting behavioral, physiological, and biochemical factors. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding and/or large increases in body fat composition. The generation of new adipocytes is controlled by several “adipocyte-specific” transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed only following the induction of adipogenesis. The transcription factor(s) that are involved in initiating adipocyte differentiation have not been identified. Here we demonstrate that the transcription factor, CREB, is constitutively expressed in preadipocytes and throughout the differentiation process and that CREB is stimulated by conventional differentiation-inducing agents such as insulin, dexamethasone, and dibutyryl cAMP. Stably transfected 3T3-L1 preadipocytes were generated in which we could induce the expression of either a constitutively active CREB (VP16-CREB) or a dominant-negative CREB (KCREB). Inducible expression of VP16-CREB alone was sufficient to initiate adipogenesis as determined by triacylglycerol storage, cell morphology, and the expression of two adipocyte marker genes, peroxisome proliferator activated receptor gamma 2, and fatty acid binding protein. Alternatively, KCREB alone blocked adipogenesis in cells treated with conventional differentiation-inducing agents. These data indicate that activation of CREB was necessary and sufficient to induce adipogenesis. Finally, CREB was shown to bind to putative CRE sequences in the promoters of several adipocyte-specific genes. These data firmly establish CREB as a primary regulator of adipogenesis and suggest that CREB may play similar roles in other cells and tissues. PMID:10629058

  1. Upregulation of CREB-mediated transcription enhances both short- and long-term memory.

    PubMed

    Suzuki, Akinobu; Fukushima, Hotaka; Mukawa, Takuya; Toyoda, Hiroki; Wu, Long-Jun; Zhao, Ming-Gao; Xu, Hui; Shang, Yuze; Endoh, Kengo; Iwamoto, Taku; Mamiya, Nori; Okano, Emiko; Hasegawa, Shunsuke; Mercaldo, Valentina; Zhang, Yue; Maeda, Ryouta; Ohta, Miho; Josselyn, Sheena A; Zhuo, Min; Kida, Satoshi

    2011-06-15

    Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression. PMID:21677163

  2. Fission yeast TORC1 regulates phosphorylation of ribosomal S6 proteins in response to nutrients and its activity is inhibited by rapamycin

    PubMed Central

    Nakashima, Akio; Sato, Tatsuhiro; Tamanoi, Fuyuhiko

    2010-01-01

    Cellular activities are regulated by environmental stimuli through protein phosphorylation. Target of rapamycin (TOR), a serine/threonine kinase, plays pivotal roles in cell proliferation and cell growth in response to nutrient status. In Schizosaccharomyces pombe, TORC1, which contains Tor2, plays crucial roles in nutrient response. Here we find a nitrogen-regulated phosphoprotein, p27, in S. pombe using the phospho-Akt substrate antibody. Response of p27 phosphorylation to nitrogen availability is mediated by TORC1 and the TSC-Rhb1 signaling, but not by TORC2 or other nutrient stress-related pathways. Database and biochemical analyses indicate that p27 is identical to ribosomal protein S6 (Rps6). Ser235 and Ser236 in Rps6 are necessary for Rps6 phosphorylation by TORC1. These Rps6 phosphorylations are dispensable for cell viability. Rps6 phosphorylation by TORC1 also responds to availability of glucose and is inhibited by osmotic and oxidative stresses. Rapamycin inhibits the ability of TORC1 to phosphorylate Rps6, owing to interaction of the rapamycin-FKBP12 complex with the FRB domain in Tor2. Rapamycin also leads to a decrease in cell size in a TORC1-dependent manner. Our findings demonstrate that the nutrient-responsive and rapamycin-sensitive TORC1-S6 signaling exists in S. pombe, and that this pathway plays a role in cell size control. PMID:20144990

  3. Regulation of Blood–Testis Barrier (BTB) Dynamics during Spermatogenesis via the “Yin” and “Yang” Effects of Mammalian Target of Rapamycin Complex 1 (mTORC1) and mTORC2

    PubMed Central

    Mok, Ka Wai; Mruk, Dolores D.; Cheng, C. Yan

    2014-01-01

    In mammalian testes, haploid spermatozoa are formed from diploid spermatogonia during spermatogenesis, which is a complicated cellular process. While these cellular events were reported in the 1960s and 1970s, the underlying molecular mechanism(s) that regulates these events remained unexplored until the past ~10 years. For instance, adhesion proteins were shown to be integrated components at the Sertoli cell–cell interface and/or the Sertoli–spermatid interface in the late 1980s. But only until recently, studies have demonstrated that some of the adhesion proteins serve as the platform for signal transduction that regulates cell adhesion. In this chapter, a brief summary and critical discussion are provided on the latest findings regarding these cell-adhesion proteins in the testis and their relationship to spermatogenesis. Moreover, antagonistic effects of two mammalian target of rapamycin (mTOR) complexes, known as mTORC1 and mTORC2, on cell-adhesion function in the testis are discussed. Finally, a hypothetic model is presented to depict how these two mTOR-signaling complexes having the “yin” and “yang” antagonistic effects on the Sertoli cell tight junction (TJ)-permeability barrier can maintain the blood–testis barrier (BTB) integrity during the epithelial cycle while preleptotene spermatocytes are crossing the BTB. PMID:23317821

  4. Region-specific tolerance to cocaine-regulated cAMP-dependent protein phosphorylation following chronic self-administration.

    PubMed

    Edwards, Scott; Graham, Danielle L; Bachtell, Ryan K; Self, David W

    2007-04-01

    Chronic cocaine self-administration can produce either tolerance or sensitization to certain cocaine-regulated behaviours, but whether differential alterations develop in the biochemical response to cocaine is less clear. We measured cocaine-induced phosphorylation of multiple cAMP-dependent and -independent protein substrates in mesolimbic dopamine terminal regions following chronic self-administration. Changes in self-administering rats were compared to changes produced by passive yoked injection to identify reinforcement-related regulation, whereas acute and chronic yoked groups were compared to identify the development tolerance or sensitization in the biochemical response to cocaine. Microwave-fixed brain tissue was collected immediately following 4 h of intravenous cocaine administration, and subjected to Western blot analysis of phosphorylated and total protein substrates. Chronic cocaine produced region- and substrate-specific tolerance to cAMP-dependent protein phosphorylation, including GluR1(S845) phosphorylation in striatal and amygdala subregions and NR1(S897) phosphorylation in the CA1 subregion of the hippocampus. Tolerance also developed to cAMP-independent GluR1(S831) phosphorylation in the prefrontal cortex. In contrast, sensitization to presynaptic regulation of synapsin(S9) phosphorylation developed in the hippocampal CA3 subregion while cAMP-dependent tyrosine hydroxylase(S40) phosphorylation decreased in striatal dopamine terminals. Cocaine-induced ERK and CREB(S133) phosphorylation were dissociated in many brain regions and failed to develop either tolerance or sensitization with chronic administration. Positive reinforcement-related correlations between cocaine intake and protein phosphorylation were found only in self-administering animals, while negative dose-related correlations were found primarily with yoked administration. These regional- and substrate-specific adaptations in cocaine-induced protein phosphorylation are discussed in

  5. 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. PMID:27412469

  6. Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus

    PubMed Central

    Liu, Pengda; Begley, Michael; Michowski, Wojciech; Inuzuka, Hiroyuki; Ginzberg, Miriam; Gao, Daming; Tsou, Peiling; Gan, Wenjian; Papa, Antonella; Kim, Byeong Mo; Wan, Lixin; Singh, Amrik; Zhai, Bo; Yuan, Min; Wang, Zhiwei; Gygi, Steven P.; Lee, Tae Ho; Lu, Kun-Ping; Toker, Alex; Pandolfi, Pier Paolo; Asara, John M.; Kirschner, Marc W.; Sicinski, Piotr; Cantley, Lewis; Wei, Wenyi

    2014-01-01

    Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers1–3, and is closely associated with poor prognosis and chemo- or radio-therapeutic resistance4. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark7. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer. PMID:24670654

  7. Local translation and retrograde axonal transport of CREB regulates IL-6-induced nociceptive plasticity

    PubMed Central

    2014-01-01

    Transcriptional regulation of genes by cyclic AMP response element binding protein (CREB) is essential for the maintenance of long-term memory. Moreover, retrograde axonal trafficking of CREB in response to nerve growth factor (NGF) is critical for the survival of developing primary sensory neurons. We have previously demonstrated that hindpaw injection of interleukin-6 (IL-6) induces mechanical hypersensitivity and hyperalgesic priming that is prevented by the local injection of protein synthesis inhibitors. However, proteins that are locally synthesized that might lead to this effect have not been identified. We hypothesized that retrograde axonal trafficking of nascently synthesized CREB might link local, activity-dependent translation to nociceptive plasticity. To test this hypothesis, we determined if IL-6 enhances the expression of CREB and if it subsequently undergoes retrograde axonal transport. IL-6 treatment of sensory neurons in vitro caused an increase in CREB protein and in vivo treatment evoked an increase in CREB in the sciatic nerve consistent with retrograde transport. Importantly, co-injection of IL-6 with the methionine analogue azido-homoalanine (AHA), to assess nascently synthesized proteins, revealed an increase in CREB containing AHA in the sciatic nerve 2 hrs post injection, indicating retrograde transport of nascently synthesized CREB. Behaviorally, blockade of retrograde transport by disruption of microtubules or inhibition of dynein or intrathecal injection of cAMP response element (CRE) consensus sequence DNA oligonucleotides, which act as decoys for CREB DNA binding, prevented the development of IL-6-induced mechanical hypersensitivity and hyperalgesic priming. Consistent with previous studies in inflammatory models, intraplantar IL-6 enhanced the expression of BDNF in dorsal root ganglion (DRG). This effect was blocked by inhibition of retrograde axonal transport and by intrathecal CRE oligonucleotides. Collectively, these findings

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

    PubMed

    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

    2013-04-01

    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

  9. Member of the CREB/ATF protein family, but not CREB alpha plays an active role in BLV tax trans activation in vivo.

    PubMed Central

    Kiss-Toth, E; Paca-uccaralertkun, S; Unk, I; Boros, I

    1993-01-01

    The trans activator protein of Bovine Leukaemia Virus (tax) increases the rate of transcription from the virus promoter through 21 bp sequences located in three tandem copies in the virus LTR. Based on data obtained by three different experimental approaches we concluded that the central CRE-like motif found in each of the BLV 21 bp repeats plays an important and indispensable role in tax mediated trans activation. These include (i) in vivo analysis of the function of mutant 21 bp sequences in transient transfection, (ii) gel mobility shift assay to show that CREB binds to BLV 21 bp repeats in vitro and (iii) the demonstration that the production of antisense CREB mRNA inhibits tax trans activation. Further studies with different deletion mutant CREB proteins suggest that although CREB alpha can interact with factors involved in BLV trans activation, it does not promote transcription initiation; consequently some other member/s of the CREB/ATF family must be involved. Images PMID:8396235

  10. Long-Term Dietary Alpha-Linolenic Acid Supplement Alleviates Cognitive Impairment Correlate with Activating Hippocampal CREB Signaling in Natural Aging Rats.

    PubMed

    Gao, Hui; Yan, Peipei; Zhang, Shun; Huang, Hao; Huang, Fenghong; Sun, Taoping; Deng, Qianchun; Huang, Qingde; Chen, Sijing; Ye, Keqiang; Xu, Jiqu; Liu, Liegang

    2016-09-01

    Alpha-linolenic acid (ALA) is a major precursor of the essential n-3 polyunsaturated fatty acid (PUFA), whose deficiency alters the structure and function of membranes and induces cerebral dysfunctions. The major purpose of this study was to investigate the protective effect of prolonged ALA intake on cognitive function during natural aging. Female Sprague-Dawley rats aged 6 months were chronically treated with ALA and/or lard per day for 12 months. Regular diet-treated rats, both young and old (4 and 18 months old, respectively) served as controls. Rats fed on regular diet during aging showed memory deficits in Morris water maze, which were further exacerbated by lard intake. However, supplementation with ALA for 12 months dose-dependently improved the performance in spatial working memory tasks. Memory performance correlated well with the activation of cAMP response element-binding protein (CREB) and increases in both levels of brain-derived neurotrophic factor (BDNF) and its specific receptor tyrosine kinase B (TrkB) phosphorylation in the hippocampus. Further study identified that hippocampal extracellular signal-related kinase (ERK) and Akt rather than calcium calmodulin kinase IV (CaMKIV) and protein kinase A (PKA), the upstream signalings of CREB, were also activated by ALA supplement. Moreover, memory improvement was accompanied with alterations of hippocampal synaptic structure and number, suggestive of enhancement in synaptic plasticity. Together, these results suggest that long-term dietary intake of ALA enhances CREB/BDNF/TrkB pathway through the activation of ERK and Akt signalings in hippocampus, which contributes to its ameliorative effects on cognitive deficits in natural aging. PMID:26328539

  11. 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

    2015-12-01

    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. PMID:26408985

  12. NLK phosphorylates Raptor to mediate stress-induced mTORC1 inhibition

    PubMed Central

    Yuan, Hai-Xin; Wang, Zhen; Yu, Fa-Xing; Li, Fulong; Russell, Ryan C.; Jewell, Jenna L.; Guan, Kun-Liang

    2015-01-01

    The mechanistic target of rapamycin (mTOR) is a central cell growth controller and forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates a wide range of upstream signals, both positive and negative, to regulate cell growth. Although mTORC1 activation by positive signals, such as growth factors and nutrients, has been extensively investigated, the mechanism of mTORC1 regulation by stress signals is less understood. In this study, we identified the Nemo-like kinase (NLK) as an mTORC1 regulator in mediating the osmotic and oxidative stress signals. NLK inhibits mTORC1 lysosomal localization and thereby suppresses mTORC1 activation. Mechanistically, NLK phosphorylates Raptor on S863 to disrupt its interaction with the Rag GTPase, which is important for mTORC1 lysosomal recruitment. Cells with Nlk deletion or knock-in of the Raptor S863 phosphorylation mutants are defective in the rapid mTORC1 inhibition upon osmotic stress. Our study reveals a function of NLK in stress-induced mTORC1 modulation and the underlying biochemical mechanism of NLK in mTORC1 inhibition in stress response. PMID:26588989

  13. UVB Stimulates the Expression of Endothelin B Receptor in Human Melanocytes via a Sequential Activation of the p38/MSK1/CREB/MITF Pathway Which Can Be Interrupted by a French Maritime Pine Bark Extract through a Direct Inactivation of MSK1

    PubMed Central

    Tagashira, Hideki; Miyamoto, Aki; Kitamura, Sei-ichi; Tsubata, Masahito; Yamaguchi, Kazuya; Takagaki, Kinya; Imokawa, Genji

    2015-01-01

    Melanogenesis is the physiological process by which melanin is synthesized to protect the skin from UV damage. While paracrine interactions between keratinocytes and melanocytes are crucial for regulating epidermal pigmentation, the endothelin (EDN)-endothelin B-receptor (EDNRB) interaction is one of the key linkages. In this study, we found that a single exposure of normal human melanocytes (NHMs) with UVB stimulates the expression of EDNRB and its upstream transcription factor microphthalmia-associated transcription factor (MITF) at the transcriptional and translational levels. That stimulation can be abrogated by post-irradiation treatment with a French maritime pine bark extract (PBE). UVB stimulated the phosphorylation of p38 and c-jun N-terminal kinase (JNK), but not ERK, followed by the increased phosphorylation of MSK1 and CREB. The post-irradiation treatment with PBE did not affect the increased phosphorylation of p38 and JNK, but distinctly abrogated the phosphorylation of MSK1 and CREB. Post-irradiation treatment with the MSK1 inhibitor H89 significantly down-regulated the increased gene expression of MITF and EDNRB in UVB-exposed NHMs. Our findings indicate for the first time that the increased expression of MITF that leads to the up-regulation of melanocyte-specific proteins in UVB-exposed NHMs is mediated via activation of the p38/MSK1/CREB pathway but not the ERK/RSK/CREB pathway. The mode of action by PBE demonstrates that interrupting MSK1 activation is a new target for antioxidants including PBE which can serve as anti-pigmenting agents in a reactive oxygen species-depletion-independent manner. PMID:26030901

  14. UVB Stimulates the Expression of Endothelin B Receptor in Human Melanocytes via a Sequential Activation of the p38/MSK1/CREB/MITF Pathway Which Can Be Interrupted by a French Maritime Pine Bark Extract through a Direct Inactivation of MSK1.

    PubMed

    Tagashira, Hideki; Miyamoto, Aki; Kitamura, Sei-Ichi; Tsubata, Masahito; Yamaguchi, Kazuya; Takagaki, Kinya; Imokawa, Genji

    2015-01-01

    Melanogenesis is the physiological process by which melanin is synthesized to protect the skin from UV damage. While paracrine interactions between keratinocytes and melanocytes are crucial for regulating epidermal pigmentation, the endothelin (EDN)-endothelin B-receptor (EDNRB) interaction is one of the key linkages. In this study, we found that a single exposure of normal human melanocytes (NHMs) with UVB stimulates the expression of EDNRB and its upstream transcription factor microphthalmia-associated transcription factor (MITF) at the transcriptional and translational levels. That stimulation can be abrogated by post-irradiation treatment with a French maritime pine bark extract (PBE). UVB stimulated the phosphorylation of p38 and c-jun N-terminal kinase (JNK), but not ERK, followed by the increased phosphorylation of MSK1 and CREB. The post-irradiation treatment with PBE did not affect the increased phosphorylation of p38 and JNK, but distinctly abrogated the phosphorylation of MSK1 and CREB. Post-irradiation treatment with the MSK1 inhibitor H89 significantly down-regulated the increased gene expression of MITF and EDNRB in UVB-exposed NHMs. Our findings indicate for the first time that the increased expression of MITF that leads to the up-regulation of melanocyte-specific proteins in UVB-exposed NHMs is mediated via activation of the p38/MSK1/CREB pathway but not the ERK/RSK/CREB pathway. The mode of action by PBE demonstrates that interrupting MSK1 activation is a new target for antioxidants including PBE which can serve as anti-pigmenting agents in a reactive oxygen species-depletion-independent manner. PMID:26030901

  15. Transgenic songbirds with suppressed or enhanced activity of CREB transcription factor.

    PubMed

    Abe, Kentaro; Matsui, Sumiko; Watanabe, Dai

    2015-06-16

    Songbirds postnatally develop their skill to utter and to perceive a vocal signal for communication. How genetic and environmental influences act in concert to regulate the development of such skill is not fully understood. Here, we report the phenotype of transgenic songbirds with altered intrinsic activity of cAMP response element-binding protein (CREB) transcription factor. By viral vector-mediated modification of genomic DNA, we established germ line-transmitted lines of zebra finches, which exhibited enhanced or suppressed activity of CREB. Although intrinsically acquired vocalizations or their hearing ability were not affected, the transgenic birds showed reduced vocal learning quality of their own songs and impaired audio-memory formation against conspecific songs. These results thus demonstrate that appropriate activity of CREB is necessary for the postnatal acquisition of learned behavior in songbirds, and the CREB transgenic birds offer a unique opportunity to separately manipulate both genetic and environmental factors that impinge on the postnatal song learning. PMID:26048905

  16. Deficits in Memory Tasks of Mice with CREB Mutations Depend on Gene Dosage

    PubMed Central

    Gass, Peter; Wolfer, David P.; Balschun, Detlef; Rudolph, Dorothea; Frey, Uwe; Lipp, Hans-Peter; Schütz, Günther

    1998-01-01

    Studies in Aplysia, Drosophila, and mice have shown that the transcription factor CREB is involved in formation and retention of long-term memory. To analyze the impact of differential CREB levels on learning and memory, we varied the gene dosage of CREB in two strains of mutant mice: (1) CREBαΔ mice, in which the α and Δ isoforms are disrupted, but a third isoform β is strongly up-regulated; (2) CREBcomp, a compound strain with one αΔ allele and one CREBnull allele in which all CREB isoforms are disrupted. To minimize genetic background effects, CREB mutations were backcrossed into a C57BL/6 and a FVB/N strain, respectively, and studies were performed in F1 hybrids from these lines. CREBcomp but not CREBαΔ F1 hybrids were impaired in water maze learning and fear conditioning, demonstrating a CREB gene dosage effect. However, analysis of the platform searching strategies in the water maze task suggested that CREBcomp mutants are impaired in behavioral flexibility rather than in spatial memory. In contrast to previous experiments using CREBαΔ mice with different genetic background, the F1 hybrid CREBαΔ and CREBcomp mice did not show deficits in a social transmission of food preference task nor in dentate gyrus and CA1 LTP as recorded from slice preparations. These data indicate that the hybrid vigor typical for F1 hybrids may compensate for a reduction in CREB levels in some tests. On the other hand, the persistence of clear behavioral deficits as shown by the F1 hybrid CREBcomp mice in water maze and fear conditioning indicates a robust and repeatable phenomenon that will permit further functional analysis of CREB. PMID:10454354

  17. Nuclear TRAF3 is a negative regulator of CREB in B cells.

    PubMed

    Mambetsariev, Nurbek; Lin, Wai W; Stunz, Laura L; Hanson, Brett M; Hildebrand, Joanne M; Bishop, Gail A

    2016-01-26

    The adaptor protein TNF receptor-associated factor 3 (TRAF3) regulates signaling through B-lymphocyte receptors, including CD40, BAFF receptor, and Toll-like receptors, and also plays a critical role inhibiting B-cell homoeostatic survival. Consistent with these findings, loss-of-function human TRAF3 mutations are common in B-cell cancers, particularly multiple myeloma and B-cell lymphoma. B cells of B-cell-specific TRAF3(-/-) mice (B-Traf3(-/-)) display remarkably enhanced survival compared with littermate control (WT) B cells. The mechanism for this abnormal homeostatic survival is poorly understood, a key knowledge gap in selecting optimal treatments for human B-cell cancers with TRAF3 deficiency. We show here for the first time to our knowledge that TRAF3 is a resident nuclear protein that associates with the transcriptional regulator cAMP response element binding protein (CREB) in both mouse and human B cells. The TRAF-C domain of TRAF3 was necessary and sufficient to localize TRAF3 to the nucleus via a functional nuclear localization signal. CREB protein was elevated in TRAF3(-/-) B cells, without change in mRNA, but with a decrease in CREB ubiquitination. CREB-mediated transcriptional activity was increased in TRAF3-deficient B cells. Consistent with these findings, Mcl-1, an antiapoptotic target of CREB-mediated transcription, was increased in the absence of TRAF3 and enhanced Mcl-1 was suppressed with CREB inhibition. TRAF3-deficient B cells were also preferentially sensitive to survival inhibition with pharmacologic CREB inhibitor. Our results identify a new mechanism by which nuclear TRAF3 regulates B-cell survival via inhibition of CREB stability, information highly relevant to the role of TRAF3 in B-cell malignancies. PMID:26755589

  18. Importin-mediated retrograde transport of CREB2 from distal processes to the nucleus in neurons

    PubMed Central

    Lai, Kwok-On; Zhao, Yali; Ch'ng, Toh Hean; Martin, Kelsey C.

    2008-01-01

    Signals received at distal synapses of neurons must be conveyed to the nucleus to initiate the changes in transcription that underlie long-lasting synaptic plasticity. The presence of importin nuclear transporters and of select transcription factors at synapses raises the possibility that importins directly transport transcription factors from synapse to nucleus to modulate gene expression. Here, we show that cyclic AMP response element binding protein 2 (CREB2)/activating transcription factor 4 (ATF4), a transcriptional repressor that modulates long-term synaptic plasticity and memory, localizes to distal dendrites of rodent hippocampal neurons and neurites of Aplysia sensory neurons (SNs) and binds to specific importin α isoforms. Binding of CREB2 to importin α is required for its transport from distal dendrites to the soma and for its translocation into the nucleus. CREB2 accumulates in the nucleus during long-term depression (LTD) but not long-term potentiation of rodent hippocampal synapses, and during LTD but not long-term facilitation (LTF) of Aplysia sensory-motor synapses. Time-lapse microscopy of CREB2 tagged with a photoconvertible fluorescent protein further reveals retrograde transport of CREB2 from distal neurites to the nucleus of Aplysia SN during phenylalanine-methionine-arginine-phenylalanine-amide (FMRFamide)-induced LTD. Together, our findings indicate that CREB2 is a novel cargo of importin α that translocates from distal synaptic sites to the nucleus after stimuli that induce LTD of neuronal synapses. PMID:18957537

  19. Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1

    PubMed Central

    Denard, Bray; Lee, Ching; Ye, Jin

    2012-01-01

    Doxorubicin is used extensively for chemotherapy of diverse types of cancer, yet the mechanism through which it inhibits proliferation of cancer cells remains unclear. Here we report that doxorubicin stimulates de novo synthesis of ceramide, which in turn activates CREB3L1, a transcription factor synthesized as a membrane-bound precursor. Doxorubicin stimulates proteolytic cleavage of CREB3L1 by Site-1 Protease and Site-2 Protease, allowing the NH2-terminal domain of CREB3L1 to enter the nucleus where it activates transcription of genes encoding inhibitors of the cell cycle, including p21. Knockdown of CREB3L1 mRNA in human hepatoma Huh7 cells and immortalized human fibroblast SV589 cells conferred increased resistance to doxorubicin, whereas overexpression of CREB3L1 in human breast cancer MCF-7 cells markedly enhanced the sensitivity of these cells to doxorubicin. These results suggest that measurement of CREB3L1 expression may be a useful biomarker in identifying cancer cells sensitive to doxorubicin. DOI: http://dx.doi.org/10.7554/eLife.00090.001 PMID:23256041

  20. Mitochondrial Cyclic AMP Response Element-binding Protein (CREB) Mediates Mitochondrial Gene Expression and Neuronal Survival*S

    PubMed Central

    Lee, Junghee; Kim, Chun-Hyung; Simon, David K.; Aminova, Lyaylya R.; Andreyev, Alexander Y.; Kushnareva, Yulia E.; Murphy, Anne N.; Lonze, Bonnie E.; Kim, Kwang-Soo; Ginty, David D.; Ferrante, Robert J.; Ryu, Hoon; Ratan, Rajiv R.

    2008-01-01

    Cyclic AMP response element-binding protein (CREB) is a widely expressed transcription factor whose role in neuronal protection is now well established. Here we report that CREB is present in the mitochondrial matrix of neurons and that it binds directly to cyclic AMP response elements (CREs) found within the mitochondrial genome. Disruption of CREB activity in the mitochondria decreases the expression of a subset of mitochondrial genes, including the ND5 subunit of complex I, down-regulates complex I-dependent mitochondrial respiration, and increases susceptibility to 3-nitropropionic acid, a mitochondrial toxin that induces a clinical and pathological phenotype similar to Huntington disease. These results demonstrate that regulation of mitochondrial gene expression by mitochondrial CREB, in part, underlies the protective effects of CREB and raise the possibility that decreased mitochondrial CREB activity contributes to the mitochondrial dysfunction and neuronal loss associated with neurodegenerative disorders. PMID:16207717

  1. Activation of CREB in the nucleus accumbens shell produces anhedonia and resistance to extinction of fear in rats.

    PubMed

    Muschamp, John W; Van't Veer, Ashlee; Parsegian, Aram; Gallo, Miranda S; Chen, Melissa; Neve, Rachael L; Meloni, Edward G; Carlezon, William A

    2011-02-23

    Stress triggers psychiatric conditions including depressive and anxiety disorders. The mechanisms by which stress produces persistent changes in behavior are not fully understood. Here we show in rats that stress (footshock) activates the transcription factor cAMP response element binding protein (CREB) within the nucleus accumbens shell (NAS), a brain area involved in encoding reward and aversion. To examine the behavioral significance of altered CREB function in the NAS, we used viral vectors to elevate or disrupt CREB in this region. Elevated CREB produced increases in intracranial self-stimulation thresholds, a depressive-like sign reflecting anhedonia (decreased sensitivity to reward), whereas disruption of CREB function by expression of a dominant-negative CREB had the opposite effect. To determine whether neuroadaptations that produce anhedonia subsequently affect vulnerability to stress-induced behavioral adaptations, we subjected rats with altered CREB function in the NAS to fear conditioning. Although neither elevation nor disruption of CREB function altered the development of conditioned fear, elevation of CREB impaired extinction of conditioned fear. To mimic downstream effects of CREB activation on expression of the opioid peptide dynorphin, we microinjected the κ-opioid receptor (KOR) agonist U50,488 directly into the NAS. KOR stimulation produced anhedonia but had no effect on expression or extinction of conditioned fear. These findings demonstrate that activation of CREB in the NAS produces multiple behavioral signs (anhedonia, impaired extinction) characteristic of experience-dependent psychiatric conditions such as posttraumatic stress disorder. Although CREB activation is a common trigger, expression of these individual signs appears to involve divergent downstream mechanisms. PMID:21414930

  2. Phosphorylation and RLK signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant genomes encode hundreds of receptor-like kinases (RLKs) with an organization of functional domains similar to that of animal receptor kinases. Ligand-dependent phosphorylation has now been demonstrated for several plant RLKs and identification of specific phosphorylation sites followed by thei...

  3. 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

  4. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside confers neuroprotection in cell and animal models of ischemic stroke through calpain1/PKA/CREB-mediated induction of neuronal glucose transporter 3

    SciTech Connect

    Yu, Shu; Cheng, Qiong; Li, Lu; Liu, Mei; Yang, Yumin; Ding, Fei

    2014-06-15

    Salidroside is proven to be a neuroprotective agent of natural origin, and its analog, 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside (named SalA-4 g), has been synthesized in our lab. In this study, we showed that SalA-4 g promoted neuronal survival and inhibited neuronal apoptosis in primary hippocampal neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to ischemia by transient middle cerebral artery occlusion (MCAO), respectively, and that SalA-4 g was more neuroprotective than salidroside. We further found that SalA-4 g elevated glucose uptake in OGD-injured primary hippocampal neurons and increased the expression and recruitment of glucose transporter 3 (GLUT3) in ischemic brain. Signaling analysis revealed that SalA-4 g triggered the phosphorylation of CREB, and increased the expression of PKA RII in primary hippocampal neurons exposed to OGD injury, while inhibition of PKA/CREB by H-89 alleviated the elevation in glucose uptake and GLUT3 expression, and blocked the protective effects of SalA-4 g. Moreover, SalA-4 g was noted to inhibit intracellular Ca{sup 2+} influx and calpain1 activation in OGD-injured primary hippocampal neurons. Our results suggest that SalA-4 g neuroprotection might be mediated by increased glucose uptake and elevated GLUT3 expression through calpain1/PKA/CREB pathway. - Highlights: • A salidroside (Sal) analog (SalA-4 g) is prepared to be more neuroprotective than Sal. • SalA-4 g protected hippocampal neurons from oxygen and glucose deprivation insult. • SalA-4 g reduced ischemic injury after transient middle cerebral artery occlusion in rats. • Neuroprotection of SalA-4 g was mediated by GLUT3 level via calpain/PKA/CREB pathway.

  5. 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

  6. CREB1 gene polymorphisms combined with environmental risk factors increase susceptibility to major depressive disorder (MDD)

    PubMed Central

    Wang, Peng; Yang, Yanjie; Yang, Xiuxian; Qiu, Xiaohui; Qiao, Zhengxue; Wang, Lin; Zhu, Xiongzhao; Sui, Hong; Ma, Jingsong

    2015-01-01

    Major depressive disorder (MDD) is one of the most severe psychiatric disorders. The objective of this study was to explore the effects of CREB1 gene polymorphisms on risk of developing MDD and the joint effects of gene-environment interactions. Genotyping was performed by Taqman allelic discrimination assay among 586 patients and 586 healthy controls. A significant impact on rs6740584 genotype distribution was found for childhood trauma (P = 0.015). We did not find an association of CREB1 polymorphisms with MDD susceptibility. However, we found a significantly increased risk associated with the interactions of CREB1 polymorphisms and drinking (OR = 11.67, 95% CI = 2.52-54.18; OR = 11.52, 95% CI = 2.55-51.95 for rs11904814; OR = 4.18, 95% CI = 1.87-9.38; OR = 5.02, 95% CI = 2.27-11.14 for rs6740584; OR = 7.58, 95% CI = 2.05-27.98; OR = 7.59, 95% CI = 2.12-27.14 for rs2553206; OR = 8.37, 95% CI = 3.02-23.23; OR = 7.84, 95% CI = 2.93-20.98 for rs2551941). We also noted that CREB polymorphisms combined with family harmony and childhood trauma conferred increased susceptibility for MDD. In conclusion, polymorphisms in the CREB gene may not be independently associated with MDD risk, but they are likely to confer increased susceptibility by interacting with environmental risk factors in the Chinese population. PMID:25755794

  7. Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

    PubMed

    Hunter, A J; Morris, T A; Jin, B; Saint, C P; Kelly, J M

    2013-09-01

    Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes. PMID:23835170

  8. CREB1 Genotype Modulates Adaptive Reward-Based Decisions in Humans.

    PubMed

    Wolf, Claudia; Mohr, Holger; Diekhof, Esther K; Vieker, Henning; Goya-Maldonado, Roberto; Trost, Sarah; Krämer, Bernd; Keil, Maria; Binder, Elisabeth B; Gruber, Oliver

    2016-07-01

    Cyclic AMP response element-binding protein (CREB) contributes to adaptation of mesocorticolimbic networks by modulating activity-regulated transcription and plasticity in neurons. Activity or expression changes of CREB in the nucleus accumbens (NAc) and orbital frontal cortex (OFC) interact with behavioral changes during reward-motivated learning. However, these findings from animal models have not been evaluated in humans. We tested whether CREB1 genotypes affect reward-motivated decisions and related brain activation, using BOLD fMRI in 224 young and healthy participants. More specifically, participants needed to adapt their decision to either pursue or resist immediate rewards to optimize the reward outcome. We found significant CREB1 genotype effects on choices to pursue increases of the reward outcome and on BOLD signal in the NAc, OFC, insula cortex, cingulate gyrus, hippocampus, amygdala, and precuneus during these decisions in comparison with those decisions avoiding total reward loss. Our results suggest that CREB1 genotype effects in these regions could contribute to individual differences in reward- and associative memory-based decision-making. PMID:26045569

  9. Deletion of creB in Aspergillus oryzae Increases Secreted Hydrolytic Enzyme Activity

    PubMed Central

    Hunter, A. J.; Morris, T. A.; Jin, B.; Saint, C. P.

    2013-01-01

    Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes. PMID:23835170

  10. Mining Conditional Phosphorylation Motifs.

    PubMed

    Liu, Xiaoqing; Wu, Jun; Gong, Haipeng; Deng, Shengchun; He, Zengyou

    2014-01-01

    Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage. Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge. net/projects/cmotif/. PMID:26356863

  11. Silencing cAMP-response element-binding protein (CREB) identifies CYR61 as a tumor suppressor gene in melanoma.

    PubMed

    Dobroff, Andrey S; Wang, Hua; Melnikova, Vladislava O; Villares, Gabriel J; Zigler, Maya; Huang, Li; Bar-Eli, Menashe

    2009-09-18

    Metastatic progression of melanoma is associated with overexpression and activity of cAMP-response element-binding protein (CREB). However, the mechanism by which CREB contributes to tumor progression and metastasis remains unclear. Here, we demonstrate that stably silencing CREB expression in two human metastatic melanoma cell lines, A375SM and C8161-c9, suppresses tumor growth and experimental metastasis. Analysis of cDNA microarrays revealed that CREB silencing leads to increased expression of cysteine-rich protein 61 (CCN1/CYR61) known to mediate adhesion, chemostasis, survival, and angiogenesis. Promoter analysis and chromatin immunoprecipitation assays demonstrated that CREB acts as a negative regulator of CCN1/CYR61 transcription by directly binding to its promoter. Re-expression of CREB in CREB-silenced cells rescued the low CCN1/CYR61 expression phenotype. CCN1/CYR61 overexpression resulted in reduced tumor growth and metastasis and inhibited the activity of matrix metalloproteinase-2. Furthermore, its overexpression decreased melanoma cell motility and invasion through Matrigel, which was abrogated by silencing CCN1/CYR61 in low metastatic melanoma cells. Moreover, a significant decrease in angiogenesis as well as an increase in apoptosis was seen in tumors overexpressing CCN1/CYR61. Our results demonstrate that CREB promotes melanoma growth and metastasis by down-regulating CCN1/CYR61 expression, which acts as a suppressor of melanoma cell motility, invasion and angiogenesis. PMID:19632997

  12. Silencing cAMP-response Element-binding Protein (CREB) Identifies CYR61 as a Tumor Suppressor Gene in Melanoma*

    PubMed Central

    Dobroff, Andrey S.; Wang, Hua; Melnikova, Vladislava O.; Villares, Gabriel J.; Zigler, Maya; Huang, Li; Bar-Eli, Menashe

    2009-01-01

    Metastatic progression of melanoma is associated with overexpression and activity of cAMP-response element-binding protein (CREB). However, the mechanism by which CREB contributes to tumor progression and metastasis remains unclear. Here, we demonstrate that stably silencing CREB expression in two human metastatic melanoma cell lines, A375SM and C8161-c9, suppresses tumor growth and experimental metastasis. Analysis of cDNA microarrays revealed that CREB silencing leads to increased expression of cysteine-rich protein 61 (CCN1/CYR61) known to mediate adhesion, chemostasis, survival, and angiogenesis. Promoter analysis and chromatin immunoprecipitation assays demonstrated that CREB acts as a negative regulator of CCN1/CYR61 transcription by directly binding to its promoter. Re-expression of CREB in CREB-silenced cells rescued the low CCN1/CYR61 expression phenotype. CCN1/CYR61 overexpression resulted in reduced tumor growth and metastasis and inhibited the activity of matrix metalloproteinase-2. Furthermore, its overexpression decreased melanoma cell motility and invasion through Matrigel, which was abrogated by silencing CCN1/CYR61 in low metastatic melanoma cells. Moreover, a significant decrease in angiogenesis as well as an increase in apoptosis was seen in tumors overexpressing CCN1/CYR61. Our results demonstrate that CREB promotes melanoma growth and metastasis by down-regulating CCN1/CYR61 expression, which acts as a suppressor of melanoma cell motility, invasion and angiogenesis. PMID:19632997

  13. CREB-H: a novel mammalian transcription factor belonging to the CREB/ATF family and functioning via the box-B element with a liver-specific expression.

    PubMed

    Omori, Y; Imai, J; Watanabe, M; Komatsu, T; Suzuki, Y; Kataoka, K; Watanabe, S; Tanigami, A; Sugano, S

    2001-05-15

    The expression of liver-specific genes is regulated by unequivocally allocated transcription factors via proper responsible elements within their promoters. We identified a novel transcription factor, CREB-H, and found that its expression was restricted in the liver among 16 human tissues tested. A region of CREB-H exhibited significant homology to the basic leucine zipper (b-Zip) domain of members of the CREB/ATF family: mammalian LZIP and Drosophila BBF-2 that binds to box-B, a Drosophila enhancer modulating the fat-body-specific gene expression. CREB-H contained a hydrophobic region representing a putative transmembrane domain, like LZIP. Constructing a variety of CREB-H fusion proteins with the GAL4 DNA-binding domain disclosed that CREB-H functioned as a transcriptional activator and its N-terminal 149 amino acids accounted for the activation ability. Gel mobility sift assays revealed that CREB-H did not bind to the C/EBP, AP-1 and NF-kappaB elements but specifically bound to CRE and the box-B element. Luciferase reporter assays demonstrated that like BBF-2, CREB-H activated transcription via the box-B element and that a deletion of the putative transmembrane domain increased the activation of reporter expression significantly. Furthermore, a fusion protein of GFP and full-length CREB-H was localized in reticular structures surrounding the nucleus, whereas a fusion protein of GFP and a deletion mutant lacking the putative transmembrane domain was mainly in the nucleus. These findings suggest that CREB-H plays an important role in transcriptional regulation of genes specifically expressed in the liver, and that the putative transmembrane domain may be associated with modulation of its function as the transcriptional activator. PMID:11353085

  14. Recurrent EWSR1-CREB3L1 gene fusions in sclerosing epithelioid fibrosarcoma.

    PubMed

    Arbajian, Elsa; Puls, Florian; Magnusson, Linda; Thway, Khin; Fisher, Cyril; Sumathi, Vaiyapuri P; Tayebwa, Johnbosco; Nord, Karolin H; Kindblom, Lars-Gunnar; Mertens, Fredrik

    2014-06-01

    Sclerosing epithelioid fibrosarcoma (SEF) and low-grade fibromyxoid sarcoma (LGFMS) are 2 distinct types of sarcoma, with a subset of cases showing overlapping morphologic and immunohistochemical features. LGFMS is characterized by expression of the MUC4 protein, and about 90% of cases display a distinctive FUS-CREB3L2 gene fusion. In addition, SEF is often MUC4 positive, but is genetically less well studied. Fluorescence in situ hybridization (FISH) studies have shown involvement of the FUS gene in the majority of so-called hybrid LGFMS/SEF and in 10% to 25% of sarcomas with pure SEF morphology. In this study, we investigated a series of 10 primary tumors showing pure SEF morphology, 4 cases of LGFMS that at local or distant relapse showed predominant SEF morphology, and 1 primary hybrid LGFMS/SEF. All but 1 case showed diffuse expression for MUC4. Using FISH, reverse transcription polymerase chain reaction, and/or mRNA sequencing in selected cases, we found recurrent EWSR1-CREB3L1 fusion transcripts by reverse transcription polymerase chain reaction in 3/10 pure SEF cases and splits and deletions of the EWSR1 and/or CREB3L1 genes by FISH in 6 additional cases. All 5 cases of LGFMS with progression to SEF morphology or hybrid features had FUS-CREB3L2 fusion transcripts. Our results indicate that EWSR1 and CREB3L1 rearrangements are predominant over FUS and CREB3L2 rearrangements in pure SEF, highlighting that SEF and LGFMS are different tumor types, with different impacts on patient outcome. PMID:24441665

  15. The CREB Transcription Factor Controls Transcriptional Activity of the Human RIC8B Gene.

    PubMed

    Maureira, Alejandro; Sánchez, Rodolfo; Valenzuela, Nicole; Torrejón, Marcela; Hinrichs, María V; Olate, Juan; Gutiérrez, José L

    2016-08-01

    Proper regulation of gene expression is essential for normal development, cellular growth, and differentiation. Differential expression profiles of mRNA coding for vertebrate Ric-8B during embryo and adult stages have been observed. In addition, Ric-8B is expressed in few cerebral nuclei subareas. These facts point to a dynamic control of RIC8B gene expression. In order to understand the transcriptional regulation of this gene, we searched for cis-elements in the sequence of the human RIC8B promoter region, identifying binding sites for the basic/leucine zipper (bZip) CREB transcription factor family (CRE sites) and C/EBP transcription factor family (C/EBP sites). CRE sites were found clustered near the transcription start site, while the C/EBP sites were found clustered at around 300 bp upstream the CRE sites. Here, we demonstrate the ability of CREB1 and C/EBPβ to bind their respective elements identified in the RIC8B promoter. Comparative protein-DNA interaction analyses revealed only the proximal elements as high affinity sites for CREB1 and only the distal elements as high affinity sites for C/EBPβ. Chromatin immunoprecipitation analyses, carried out using a human neuroblastoma cell line, confirmed the preferential association of CREB to the proximal region of the RIC8B promoter. By performing luciferase reporter assays, we found the CRE sites as the most relevant elements for its transcriptional activity. Taken together, these data show the existence of functional CREB and C/EBP binding sites in the human RIC8B gene promoter, a particular distribution of these sites and demonstrate a relevant role of CREB in stimulating transcriptional activity of this gene. J. Cell. Biochem. 117: 1797-1805, 2016. © 2016 Wiley Periodicals, Inc. PMID:26729411

  16. CREB1 modulates the influence of childhood sexual abuse on adult's anger traits.

    PubMed

    Hasler, R; Perroud, N; Baud, P; Olié, E; Guillaume, S; Malafosse, A; Courtet, P

    2012-08-01

    Childhood maltreatment and genes underlie vulnerability to suicidal behaviours (SB), possibly by affecting the constitution of endophenotypes such as anger traits. The CREB protein has been implicated in antidepressant response, suicide and mood disorders in general. The aim of this study was to investigate if CREB1 gene is associated with SB and/or anger-related traits and if these associations are modulated by childhood maltreatment. Five hundred and thirty-four male suicide attempters and 357 male non-suicide attempters were genotyped for several polymorphisms within CREB1 gene. Four hundred and thirty-seven (156 non-suicide attempters and 281 suicide attempters) completed the State-Trait Anger Expression Inventory (STAXI) and 288 (265 suicide attempters and 23 controls) fulfilled the Childhood Trauma Questionnaire (CTQ). In total, 72 males had experienced childhood sexual abuse. Our results did not show any significant association between CREB1 and suicide behaviour. We found a significant interaction showing that CREB1 rs4675690 polymorphism modulated the effect of childhood sexual abuse on adulthood anger-out levels (P = 0.003). Sexually abused subjects carrying the CC genotype showed higher anger-out scores than T allele carriers, whereas no difference was observed in non-sexually abused subjects. CREB1 rs4675690 polymorphism modulates the association between childhood sexual abuse and adulthood anger-trait level. This is, to our knowledge, the first study to show such an interaction and to highlight the main effect of this gene on modulating the effect of child abuse on psychopathologies and warrant further investigation on this topic. PMID:22574704

  17. The α and Δ Isoforms of CREB1 Are Required to Maintain Normal Pulmonary Vascular Resistance

    PubMed Central

    Sands, Michelle; Banahan, Mark; Frohlich, Stephen; Rowan, Simon C.; Neary, Roisín; Ryan, Donal; McLoughlin, Paul

    2013-01-01

    Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the β isoform intact (CREBαΔ mice). Here we report that expression of CREB regulated genes was altered in the lungs of CREBαΔ mice. CREBαΔ mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREBαΔ and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREBαΔ. CREBαΔ mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases. PMID:24349008

  18. The cAMP response element binding protein, CREB, is a potent inhibitor of diverse transcriptional activators.

    PubMed Central

    Lemaigre, F P; Ace, C I; Green, M R

    1993-01-01

    Cyclic AMP response element binding protein (CREB) activates transcription of cAMP response element (CRE)-containing promoters following an elevation of intracellular cAMP. Here we show that CREB and the highly related protein ATF-1 are also potent transcription inhibitors. Strikingly, CREB inhibits transcription of multiple activators, whose DNA-binding domains and activation regions are unrelated to one another. Inhibition requires that the CREB dimerization and DNA-binding domains are intact. However, inhibition is not dependent upon the presence of a CRE in the promoter, and does not involve heterodimer formation between CREB and the activator. The ability of an activator protein to inhibit transcription in such a promiscuous fashion has not been previously reported. Images PMID:8332500

  19. Learning Strategy Selection in the Water Maze and Hippocampal CREB Phosphorylation Differ in Two Inbred Strains of Mice

    ERIC Educational Resources Information Center

    Sung, Jin-Young; Goo, June-Seo; Lee, Dong-Eun; Jin, Da-Qing; Bizon, Jennifer L.; Gallagher, Michela; Han, Jung-Soo

    2008-01-01

    Learning strategy selection was assessed in two different inbred strains of mice, C57BL/6 and DBA/2, which are used for developing genetically modified mouse models. Male mice received a training protocol in a water maze using alternating blocks of visible and hidden platform trials, during which mice escaped to a single location. After training,…

  20. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    PubMed Central

    Nichols, Grant S.; DeBello, William M.

    2015-01-01

    Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX), the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults. PMID:25789177

  1. Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12 cells.

    PubMed

    Beitner-Johnson, D; Rust, R T; Hsieh, T C; Millhorn, D E

    2001-01-01

    Akt is a serine/threonine kinase that has been shown to play a central role in promoting cell survival and opposing apoptosis. We evaluated the effect of hypoxia on Akt in rat pheochromocytoma (PC12) cells. PC12 cells were exposed to varying levels of hypoxia, including 21%, 15%, 10%, 5%, and 1% O(2). Hypoxia dramatically increased phosphorylation of Akt (Ser(473)). This effect peaked after 6 h exposure to hypoxia, but persisted strongly for up to 24 h. Phosphorylation of Akt was paralleled with a progressive increase in phosphorylation of glycogen synthase kinase-3 (GSK-3), one of its downstream substrates. The effect of hypoxia on phosphorylation of Akt was completely blocked by pretreatment of the cells with wortmannin (100 nM), indicating that this effect is mediated by phosphatidylinositol 3-kinase (P13K). In contrast, whereas hypoxia also strongly induced phosphorylation of the transcription factors CREB and EPAS1, these effects persisted in the presence of wortmannin. Thus, hypoxia regulates both P13K-dependent and P13K-independent signaling pathways. Furthermore, activation of the P13K and Akt signaling pathways may be one mechanism by which cells adapt and survive under conditions of hypoxia. PMID:11257444

  2. Intra-Amygdala Injections of CREB Antisense Impair Inhibitory Avoidance Memory: Role of Norepinephrine and Acetylcholine

    ERIC Educational Resources Information Center

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2008-01-01

    Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…

  3. Rapid Forgetting of Social Transmission of Food Preferences in Aged Rats: Relationship to Hippocampal CREB Activation

    ERIC Educational Resources Information Center

    Countryman, Renee A.; Gold, Paul E.

    2007-01-01

    A major characteristic of age-related changes in memory in rodents is an increase in the rate of forgetting of new information, even when tests given soon after training reveal intact memory. Interference with CREB functions similarly results in rapid decay of memory. Using quantitative immunocytochemistry, the present experiment examined the…

  4. Impairment of object recognition memory by maternal bisphenol A exposure is associated with inhibition of Akt and ERK/CREB/BDNF pathway in the male offspring hippocampus.

    PubMed

    Wang, Chong; Li, Zhihui; Han, Haijun; Luo, Guangying; Zhou, Bingrui; Wang, Shaolin; Wang, Jundong

    2016-02-01

    Bisphenol A (BPA) is a commonly used endocrine-disrupting chemical used as a component of polycarbonates plastics that has potential adverse effects on human health. Exposure to BPA during development has been implicated in memory deficits, but the mechanism of action underlying the effect is not fully understood. In this study, we investigated the effect of maternal exposure to BPA on object recognition memory and the expressions of proteins important for memory, especially focusing on the ERK/CREB/BDNF pathway. Pregnant Sprague-Dawley female rats were orally treated with either vehicle or BPA (0.05, 0.5, 5 or 50 mg/kg BW/day) during days 9-20 of gestation. Male offspring were tested on postnatal day 21 with the object recognition task. Recognition memory was assessed using the object recognition index (index=the time spent exploring the novel object/(the time spent exploring the novel object+the time spent exploring the familiar object)). In the test session performed 90 min after the training session, BPA-exposed male offspring not only spent more time in exploring the familiar object at the highest dose than the control, but also displayed a significantly decreased the object recognition index at the doses of 0.5, 5 and 50 mg/kg BW/day. During the test session performed 24h after the training session, BPA-treated males did not change the time spent exploring the familiar object, but had a decreased object recognition index at 5 and 50 mg/kg BW/day, when compared to control group. These findings indicate that object recognition memory was susceptible to maternal BPA exposure. Western blot analysis of hippocampi from BPA-treated male offspring revealed a decrease in Akt, phospho-Akt, p44/42 MAPK and phospho-p44/42 MAPK protein levels, compared to controls. In addition, BPA significantly inhibited the levels of phosphorylation of CREB and BDNF in the hippocampus. Our results show that maternal BPA exposure may full impair object recognition memory, and that

  5. Combination of early and delayed ischemic postconditioning enhances brain-derived neurotrophic factor production by upregulating the ERK-CREB pathway in rats with focal ischemia

    PubMed Central

    WU, HUI; YANG, SHAO-FENG; DAI, JIONG; QIU, YONG-MING; MIAO, YI-FENG; ZHANG, XIAO-HUA

    2015-01-01

    Ischemic postconditioning, including early and delayed ischemic postconditioning, has been recognized as a simple and promising strategy in the treatment of stroke. However, the effects of the combination of early and delayed ischemic postconditioning, and the mechanisms underlying these effects, remain unclear. The aim of the present study was to determine whether the combination of early and delayed ischemic postconditioning offers greater protection against stroke, and enhances the production of brain-derived neurotrophic factor (BDNF). A combination of early and delayed ischemic postconditioning was established by repeated, transient occlusion and reperfusion of the ipsilateral common carotid artery in a rat model of middle cerebral artery occlusion. Infarct size, motor function, cerebral blood flow and brain edema were then evaluated, in order to confirm the effects of combinative ischemic postconditioning. TUNEL staining was used to analyze the rate of apoptosis of cells in the penumbral area. BDNF, extracellular signal-regulated kinases 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) expression was detected using immunofluorescence staining and western blot analysis. The results of the present study indicated that the combination of early and delayed ischemic postconditioning further reduced the infarct volume, stabilized cerebral blood disturbance and attenuated neuronal apoptosis, compared with either alone. However, combinative postconditioning exerted the same effect on neurological function and brain edema, compared with early or delayed ischemic postconditioning alone. Further investigation indicated that combinative ischemic postconditioning increased the expression of BDNF, and a significantly higher number of BDNF-positive cells was observed in neurons and astrocytes from the combined group than in the early or delayed groups. Combinative ischemic postconditioning also induced the phosphorylation of ERK1/2 and CREB in the cortex

  6. Mogrol Derived from Siraitia grosvenorii Mogrosides Suppresses 3T3-L1 Adipocyte Differentiation by Reducing cAMP-Response Element-Binding Protein Phosphorylation and Increasing AMP-Activated Protein Kinase Phosphorylation.

    PubMed

    Harada, Naoki; Ishihara, Mikako; Horiuchi, Hiroko; Ito, Yuta; Tabata, Hiromitsu; Suzuki, Yasushi A; Nakano, Yoshihisa; Yamaji, Ryoichi; Inui, Hiroshi

    2016-01-01

    This study investigated the effects of mogrol, an aglycone of mogrosides from Siraitia grosvenorii, on adipogenesis in 3T3-L1 preadipocytes. Mogrol, but not mogrosides, suppressed triglyceride accumulation by affecting early (days 0-2) and late (days 4-8), but not middle (days 2-4), differentiation stages. At the late stage, mogrol increased AMP-activated protein kinase (AMPK) phosphorylation and reduced glycerol-3-phosphate dehydrogenase activity. At the early stage, mogrol promoted AMPK phosphorylation, inhibited the induction of CCAAT/enhancer-binding protein β (C/EBPβ; a master regulator of adipogenesis), and reduced 3T3-L1 cell contents (e.g., clonal expansion). In addition, mogrol, but not the AMPK activator AICAR, suppressed the phosphorylation and activity of the cAMP response element-binding protein (CREB), which regulates C/EBPβ expression. These results indicated that mogrol suppressed adipogenesis by reducing CREB activation in the initial stage of cell differentiation and by activating AMPK signaling in both the early and late stages of this process. PMID:27583359

  7. Neuritogenic Monoglyceride Derived from the Constituent of a Marine Fish for Activating the PI3K/ERK/CREB Signalling Pathways in PC12 Cells

    PubMed Central

    Yang, Wei; Luo, Yan; Tang, Ruiqi; Zhang, Hui; Ye, Ying; Xiang, Lan; Qi, Jianhua

    2013-01-01

    A neuritogenic monoglyceride, 1-O-(myristoyl) glycerol (MG), was isolated from the head of Ilisha elongate using a PC12 cell bioassay system, and its chemical structure was elucidated using spectroscopic methods. MG significantly induced 42% of the neurite outgrowth of PC12 cells at a concentration of 10 μM. To study the structure-activity relationships of MG, a series of monoglycerides was designed and synthesised. Bioassay results indicated that the alkyl chain length plays a key role in the neuritogenic activity of the monoglycerides. The groups that link the propane-1,2-diol and alkyl chain were also investigated. An ester linkage, rather than an amido one, was found to be optimal for neuritogenic activity. Therefore, 1-O-(stearoyl) glycerol (SG), which induces 57% of the neurite outgrowth of PC12 cells at 10 μM, was determined to be a lead compound for neuritogenic activity. We then investigated the mechanism of action of neurite outgrowth induced by SG on PC12 cells using protein specific inhibitors and Western blot analysis. The mitogen-activated kinase/ERK kinase (MEK) inhibitor U0126 and the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 significantly decreased neurite outgrowth. At the same time, SG increased phosphorylation of CREB in protein level. Thus, SG-induced neuritogenic activity depends on the activation of the extracellular-regulated protein kinase (ERK), cAMP responsive element-binding protein (CREB) and PI3K signalling pathways in PC12 cells. PMID:24351811

  8. Struvite and prebiotic phosphorylation.

    NASA Technical Reports Server (NTRS)

    Handschuh, G. J.; Orgel, L. E.

    1973-01-01

    Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

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

    PubMed

    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

  10. Targeting of p300/CREB binding protein coactivators by simian virus 40 is mediated through p53.

    PubMed

    Borger, Darrell R; DeCaprio, James A

    2006-05-01

    The primary transforming functions of simian virus 40 large T antigen (SV40 LT) are conferred primarily through the binding and inactivation of p53 and the retinoblastoma family members. Normal p53 function requires an association with the CREB binding protein (CBP)/p300 coactivators, and a ternary complex containing SV40 LT, p53, and CBP/p300 has been identified previously. In this report, we have evaluated a secondary function of p53 bound to the SV40 LT complex in mediating the binding of human CBP/p300. We demonstrate that p53 associated with SV40 LT was posttranslationally modified in a manner consistent with the binding of CBP/p300. Furthermore, expression of SV40 LT induced the proportion of p53 phosphorylated on S15. An essential function for p53 in bridging the interaction between SV40 LT and CBP/p300 was identified through the reconstitution of the SV40 LT-CBP/p300 complex upon p53 reexpression in p53-null cells. In addition, the SV40 LT-CBP/p300 complex was disrupted through RNA interference-mediated depletion of endogenous p53. We also demonstrate that SV40 LT was acetylated in a p300- and p53-dependent manner, at least in part through the CH3 domain of p300. Therefore, the binding of p53 serves to modify SV40 LT by targeting CBP and p300 binding to direct the acetylation of SV40 LT. PMID:16611888

  11. Targeting of p300/CREB Binding Protein Coactivators by Simian Virus 40 Is Mediated through p53

    PubMed Central

    Borger, Darrell R.; DeCaprio, James A.

    2006-01-01

    The primary transforming functions of simian virus 40 large T antigen (SV40 LT) are conferred primarily through the binding and inactivation of p53 and the retinoblastoma family members. Normal p53 function requires an association with the CREB binding protein (CBP)/p300 coactivators, and a ternary complex containing SV40 LT, p53, and CBP/p300 has been identified previously. In this report, we have evaluated a secondary function of p53 bound to the SV40 LT complex in mediating the binding of human CBP/p300. We demonstrate that p53 associated with SV40 LT was posttranslationally modified in a manner consistent with the binding of CBP/p300. Furthermore, expression of SV40 LT induced the proportion of p53 phosphorylated on S15. An essential function for p53 in bridging the interaction between SV40 LT and CBP/p300 was identified through the reconstitution of the SV40 LT-CBP/p300 complex upon p53 reexpression in p53-null cells. In addition, the SV40 LT-CBP/p300 complex was disrupted through RNA interference-mediated depletion of endogenous p53. We also demonstrate that SV40 LT was acetylated in a p300- and p53-dependent manner, at least in part through the CH3 domain of p300. Therefore, the binding of p53 serves to modify SV40 LT by targeting CBP and p300 binding to direct the acetylation of SV40 LT. PMID:16611888

  12. Identification of a Potent Inhibitor of CREB-Mediated Gene Transcription with Efficacious in Vivo Anticancer Activity

    PubMed Central

    2015-01-01

    Recent studies have shown that nuclear transcription factor cyclic adenosine monophosphate response element binding protein (CREB) is overexpressed in many different types of cancers. Therefore, CREB has been pursued as a novel cancer therapeutic target. Naphthol AS-E and its closely related derivatives have been shown to inhibit CREB-mediated gene transcription and cancer cell growth. Previously, we identified naphthamide 3a as a different chemotype to inhibit CREB’s transcription activity. In a continuing effort to discover more potent CREB inhibitors, a series of structural congeners of 3a was designed and synthesized. Biological evaluations of these compounds uncovered compound 3i (666-15) as a potent and selective inhibitor of CREB-mediated gene transcription (IC50 = 0.081 ± 0.04 μM). 666-15 also potently inhibited cancer cell growth without harming normal cells. In an in vivo MDA-MB-468 xenograft model, 666-15 completely suppressed the tumor growth without overt toxicity. These results further support the potential of CREB as a valuable cancer drug target. PMID:26023867

  13. Flow-induced protein kinase A-CREB pathway acts via BMP signaling to promote HSC emergence.

    PubMed

    Kim, Peter Geon; Nakano, Haruko; Das, Partha P; Chen, Michael J; Rowe, R Grant; Chou, Stephanie S; Ross, Samantha J; Sakamoto, Kathleen M; Zon, Leonard I; Schlaeger, Thorsten M; Orkin, Stuart H; Nakano, Atsushi; Daley, George Q

    2015-05-01

    Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)-cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition. PMID:25870201

  14. Quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside suppresses melanin synthesis by augmenting p38 MAPK and CREB signaling pathways and subsequent cAMP down-regulation in murine melanoma cells

    PubMed Central

    Jung, Hyun Gug; Kim, Han Hyuk; Paul, Souren; Jang, Jae Yoon; Cho, Yong Hun; Kim, Hyeon Jeong; Yu, Jae Myo; Lee, Eun Su; An, Bong Jeun; Kang, Sun Chul; Bang, Byung Ho

    2015-01-01

    In this study, the effect of purified quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosid (QCGG) on melanogenesis was investigated. QCGG was isolated from the calyx of a traditional Korean medicinal herb, Persimmon (Diospyros kaki). The hypopigmentation effects of QCGG were determined by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay, and Western blotting of α-MSH-stimulated B16F10 mouse melanoma cells. Our results showed that QCGG inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner as well as significantly reduced the expression of melanogenic proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1, tyrosinase-related protein-2, and tyrosinase. Moreover, QCGG inhibited intracellular cAMP levels, cAMP response element-binding protein (CREB), and p38 MAPK expression in α-MSH-stimulated B16F10 cells. Taken together, the suppressive effects of QCGG on melanogenesis may involve down-regulation of MITF and its downstream signaling pathway via phosphorylation of p38 MAPK and CREB along with reduced cAMP levels. These results indicate that QCGG reduced melanin synthesis by reducing expression of tyrosine and tyrosine-related proteins via extracellular signal-related protein kinase (ERK) activation, followed by down-regulation of CREB, p38, and MITF. PMID:26586997

  15. Quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside suppresses melanin synthesis by augmenting p38 MAPK and CREB signaling pathways and subsequent cAMP down-regulation in murine melanoma cells.

    PubMed

    Jung, Hyun Gug; Kim, Han Hyuk; Paul, Souren; Jang, Jae Yoon; Cho, Yong Hun; Kim, Hyeon Jeong; Yu, Jae Myo; Lee, Eun Su; An, Bong Jeun; Kang, Sun Chul; Bang, Byung Ho

    2015-11-01

    In this study, the effect of purified quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosid (QCGG) on melanogenesis was investigated. QCGG was isolated from the calyx of a traditional Korean medicinal herb, Persimmon (Diospyros kaki). The hypopigmentation effects of QCGG were determined by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay, and Western blotting of α-MSH-stimulated B16F10 mouse melanoma cells. Our results showed that QCGG inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner as well as significantly reduced the expression of melanogenic proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1, tyrosinase-related protein-2, and tyrosinase. Moreover, QCGG inhibited intracellular cAMP levels, cAMP response element-binding protein (CREB), and p38 MAPK expression in α-MSH-stimulated B16F10 cells. Taken together, the suppressive effects of QCGG on melanogenesis may involve down-regulation of MITF and its downstream signaling pathway via phosphorylation of p38 MAPK and CREB along with reduced cAMP levels. These results indicate that QCGG reduced melanin synthesis by reducing expression of tyrosine and tyrosine-related proteins via extracellular signal-related protein kinase (ERK) activation, followed by down-regulation of CREB, p38, and MITF. PMID:26586997

  16. Synaptic plasticity and phosphorylation

    PubMed Central

    Lee, Hey-Kyoung

    2009-01-01

    A number of neuronal functions, including synaptic plasticity, depend on proper regulation of synaptic proteins, many of which can be rapidly regulated by phosphorylation. Neuronal activity controls the function of these synaptic proteins by exquisitely regulating the balance of various protein kinase and protein phosphatase activity. Recent understanding of synaptic plasticity mechanisms underscores important roles that these synaptic phosphoproteins play in regulating both pre- and post-synaptic functions. This review will focus on key postsynaptic phosphoproteins that have been implicated to play a role in synaptic plasticity. PMID:16904750

  17. Roles of Nucleus Accumbens CREB and Dynorphin in Dysregulation of Motivation

    PubMed Central

    Muschamp, John W.; Carlezon, William A.

    2013-01-01

    Psychostimulants such as amphetamine and cocaine are believed to produce dependence by causing rapid, supraphysiological elevations in synaptic dopamine (DA) within the nucleus accumbens (NAc) (Volkow et al. 2009, Neuropharmacology 56: 3–8). These changes in forebrain DA transmission are similar to those evoked by natural reinforcers (Louilot et al. 1991, Brain Res 553: 313–317; Roitman et al. 2004, J Neurosci 24: 1265–1271), but are of greater magnitude and longer duration. Repeated drug exposure causes compensatory neuroadaptations in neurons of the NAc, some of which may modulate excess DA in a homeostatic fashion. One such adaptation is the activation of the transcription factor CREB (cAMP response element-binding protein) within neurons of the NAc. Although elevated levels of transcriptionally active CREB appear to attenuate DA transmission by increasing expression of the endogenous κ opioid receptor (KOR) ligand dynorphin, increased dynorphin transmission may ultimately have undesirable effects that contribute to drug withdrawal states as well as comorbid psychiatric illnesses such as depression. This state may prompt a return to drug use to mitigate the adverse effects of withdrawal. This article summarizes our current understanding of how CREB and dynorphin contribute to the dysregulation of motivation and describes novel therapeutic strategies that derive from preclinical research in this area. PMID:23293139

  18. Senescent-induced dysregulation of cAMP/CREB signaling and correlations with cognitive decline.

    PubMed

    Hansen, Rolf T; Zhang, Han-Ting

    2013-06-21

    It is well known that alongside senescence there is a gradual decline in cognitive ability, most noticeably certain kinds of memory such as working, episodic, spatial, and long term memory. However, until recently, not much has been known regarding the specific mechanisms responsible for the decline in cognitive ability with age. Over the past decades, researchers have become more interested in cAMP signaling, and its downstream transcription factor cAMP response element binding protein (CREB) in the context of senescence. However, there is still a lack of understanding on what ultimately causes the cognitive deficits observed with senescence. This review will focus on the changes in intracellular signaling in the brain, more specifically, alterations in cAMP/CREB signaling in aging. In addition, the downstream effects of altered cAMP signaling on cognitive ability with age will be further discussed. Overall, understanding the senescent-related changes that occur in cAMP/CREB signaling could be important for the development of novel drug targets for both healthy aging, and pathological aging such as Alzheimer's disease. PMID:23623816

  19. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

    PubMed

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J

    2015-01-01

    G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype. PMID:26344213

  20. Uncouplers of oxidative phosphorylation.

    PubMed

    Terada, H

    1990-07-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  1. Uncouplers of oxidative phosphorylation.

    PubMed Central

    Terada, H

    1990-01-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  2. Trafficking of the bZIP transmembrane transcription factor CREB-H into alternate pathways of ERAD and stress-regulated intramembrane proteolysis.

    PubMed

    Bailey, Daniel; Barreca, Cristina; O'Hare, Peter

    2007-12-01

    CREB-H is an ATF6-related, transmembrane transcription factor that, in response to endoplasmic reticulum (ER)-associated stress, is cleaved by Golgi proteases and transported to the nucleus to effect appropriate adaptive responses. We characterize the ER processing and turnover of CREB-H with results which have important implications for ER stress regulation and signalling. We show that CREB-H is glycosylated and demonstrate that both the ER and nuclear forms of CREB-H have short half-lives. We also show that CREB-H is subject to cycles of retrotranslocation, deglycosylation and degradation through the ER-associated degradation (ERAD) pathway. Proteasome inhibition resulted in accumulation of a cytosolic intermediate but additionally, in contrast to inhibition of glycosylation, promoted specific cleavage of CREB-H and nuclear transport of the N-terminal-truncated product. Our data indicate that under normal conditions CREB-H is transported back from the ER to the cytosol, where it is subject to ERAD, but under conditions that repress proteasome function or promote load CREB-H is diverted from this pathway instead undergoing cleavage and nuclear transport. Finally, we identify a cytoplasmic determinant involved in CREB-H ER retention, deletion of which results in constitutive Golgi transport and corresponding cleavage. We present a model where cellular stresses may be sensed at different levels by different members of the basic and leucine zipper domain transmembrane proteins. PMID:17875199

  3. OASIS/CREB3L1 is epigenetically silenced in human bladder cancer facilitating tumor cell spreading and migration in vitro

    PubMed Central

    Rose, Michael; Schubert, Claudia; Dierichs, Laura; Gaisa, Nadine T; Heer, Matthias; Heidenreich, Axel; Knüchel, Ruth; Dahl, Edgar

    2014-01-01

    CREB3L1 has been recently proposed as a novel metastasis suppressor gene in breast cancer. Our current study highlights CREB3L1 expression, regulation, and function in bladder cancer. We demonstrate a significant downregulation of CREB3L1 mRNA expression (n = 64) in primary bladder cancer tissues caused by tumor-specific CREB3L1 promoter hypermethylation (n = 51). Based on pyrosequencing CREB3L1 methylation was shown to be potentially associated with a more aggressive phenotype of bladder cancer. These findings were verified by an independent public data set containing data from 184 bladder tumors. In addition, immunohistochemical evaluation showed that CREB3L1 protein expression is decreased in bladder cancer tissues as well. Interestingly, protein loss is predominately observed in the nuclei of aggressive tumor cells. Based on in vitro models we clearly show that CREB3L1 re-expression mediates suppression of tumor cell migration and colony growth of high grade and invasive bladder cancer cells. The candidate tumor suppressor and TGF-β signaling inhibitor HTRA3 was furthermore identified as putative target gene of CREB3L1 in both invasive J82 bladder cells and primary bladder tumors. Hence, our data provide for the first time evidence that the transcription factor CREB3L1 may have an important role as a putative tumor suppressor in bladder cancer. PMID:25625847

  4. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  5. Phosphorylation site prediction in plants.

    PubMed

    Yao, Qiuming; Schulze, Waltraud X; Xu, Dong

    2015-01-01

    Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community. PMID:25930706

  6. Protein phosphorylation in stomatal movement.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  7. CREB and CREB-binding proteins play an important role in the IE2 86-kilodalton protein-mediated transactivation of the human cytomegalovirus 2.2-kilobase RNA promoter.

    PubMed Central

    Schwartz, R; Helmich, B; Spector, D H

    1996-01-01

    The human cytomegalovirus (HCMV) immediate-early region 2 86-kDa protein (IE2 86) is the major transactivator of the promoter for the 2.2-kb class of early RNAs (open reading frame UL 112-113). Previously, we reported that a DNA segment on this promoter between nucleotides (nt) -113 and -59 was critical for activation by IE2 86 in vivo and could be bound by IE2 86 in vitro (R. Schwartz, M. H. Sommer, A. Scully, and D. H. Spector, J. Virol. 68:5613-5622, 1994). With a set of site-specific mutations within nt -84 to -61, we have localized the essential cis-acting sequences to nt -72 to -61, which contain an ATF/CREB-binding site. The IE2 86-binding site between nt -113 and -85 is not essential for activation of the promoter by IE2 86 in transient-expression assays, but its presence can enhance the level of activation mediated through the sequences located between nt -84 and -59. Electrophoretic mobility shift assays with a segment containing nt -84 to -59 and nuclear extracts from human cells permissive for the HCMV infection revealed a complex band pattern. However, by supershift analysis with specific antibodies, we were able to identify CREB as the major ATF/CREB family member in the protein-DNA complexes. Further evidence that CREB is a target for IE2 86-mediated induction, is provided by the finding that IE2 86 activates the somatostatin promoter to high levels. Although the binding of IE2 86 to nonphosphorylated full-length CREB or deltaCREB is minimal, IE2 86 does form complexes with p300 and the CREB-binding protein (CBP), which in turn bind to CREB and can serve as adaptor proteins for CREB function. In addition, the in vivo functional relevance of the interaction between IE2 86 and CBP is indicated by the ability of IE2 86 to enhance transcriptional activation mediated by a GAL4-CBP fusion protein brought to a promoter by GAL4-binding sites. PMID:8794339

  8. Interferon Regulatory Factor 3 and CREB-Binding Protein/p300 Are Subunits of Double-Stranded RNA-Activated Transcription Factor DRAF1

    PubMed Central

    Weaver, Brian K.; Kumar, K. Prasanna; Reich, Nancy C.

    1998-01-01

    Cells respond to viral infection or double-stranded RNA with the transcriptional induction of a subset of alpha/beta interferon-stimulated genes by a pathway distinct from the interferon signal pathway. The transcriptional induction is mediated through a DNA sequence containing the alpha/beta interferon-stimulated response element (ISRE). We previously identified a novel transcription factor, designated double-stranded RNA-activated factor 1 (DRAF1), that recognizes this response element. The DNA-binding specificity of DRAF1 correlates with transcriptional induction, thereby distinguishing it as a positive regulator of alpha/beta interferon-stimulated genes. Two of the components of DRAF1 have now been identified as interferon regulatory factor 3 (IRF-3) and the transcriptional coactivator CREB-binding protein (CBP)/p300. We demonstrate that IRF-3 preexists in the cytoplasm of uninfected cells and translocates to the nucleus following viral infection. Translocation of IRF-3 is accompanied by an increase in serine and threonine phosphorylation. Coimmunoprecipitation analyses of endogenous proteins demonstrate an association of IRF-3 with the transcriptional coactivators CBP and p300 only subsequent to infection. In addition, antibodies to the IRF-3, CBP, and p300 molecules react with DRAF1 bound to the ISRE target site of induced genes. The cellular response that leads to DRAF1 activation and specific gene expression may serve to increase host survival during viral infection. PMID:9488451

  9. Insulin-Like Growth Factor 1 Mediates Negative Feedback to Somatotroph GH Expression via POU1F1/CREB Binding Protein Interactions

    PubMed Central

    Pine-Twaddell, Elyse; Sima, Daniela I.; Miller, Ryan S.; He, Ling; Wondisford, Fredric; Radovick, Sally

    2012-01-01

    Circulating insulin-like growth factor 1 (IGF-1) has been shown to act as a negative feedback regulator of growth hormone (GH) gene expression; however, the mechanism of this negative feedback is poorly understood. Activation and regulation of GH gene expression require the binding of the transcription factor POU1F1 to the GH promoter along with cyclic AMP (cAMP) response element binding protein (CREB) binding protein (CBP). We investigate the role of CBP as a target of IGF-1 somatotroph regulation using the MtT/S somatotroph cell line. IGF-1 significantly inhibits basal GH mRNA levels but not POU1F1 levels. Chromatin immunoprecipitation assays demonstrate inhibition of CBP binding to the GH promoter after IGF-1 treatment. We hypothesized that IGF-1 receptor (IGF-1R) signaling disrupts the POU1F1/CBP complex to inhibit gene expression. In support, the use of a mutant CBP (S436A) construct, which lacks a critical phosphorylation site, leads to the loss of IGF-1 inhibition. The studies of CBP (S436A) knock-in mice show elevated serum GH levels, a greater response to GH releasing hormone (GHRH) stimulation along with lower weight gain, and decreased body fat. Our data confirm the inhibitory effects of IGF-1 on GH expression at the level of the promoter and provide evidence of CBP's role as a target of IGF-1R signaling. PMID:22890843

  10. Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1.

    PubMed

    Zhang, Qingzhan; Shi, Kaichuang; Yoo, Dongwan

    2016-02-01

    Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression. PMID:26773386

  11. Phosphorylated. beta. -dicarbonyl compounds

    SciTech Connect

    Liorber, B.G.; Tarzivolova, T.A.; Pavlov, V.A.; Zykova, T.V.; Kisilev, V.V.; Tumasheva, N.A.; Slizkii, A.Yu.; Shagvaleev, F.S.

    1987-08-20

    The reaction of trialkyl phosphites with alkyl malonyl chlorides leads to alkyl 3-dialkoxyphosphoryl-3-oxopropionates, which exist in the stable E-enol form. Depending on the basicities of the bases, the reactions of alkyl 3-dialkoxyphosphoryl-3-oxopropionates with nitrogen bases proceed with retention of the C-P bond and the formation of phosphorylated azomethine derivatives or with cleavage of the C-P bond and the liberation of nitrogen-containing derivatives of malonic acid. The /sup 1/H, /sup 13/C, and /sup 13/P NMR spectra were recorded with a Bruker WP-80 NMR spectrometer. The chemical shifts of the protons and carbon atoms are presented relative to tetramethylsilane (TMS). The chemical shifts of the /sup 31/P nuclei were determined relative to H/sub 3/PO/sub 4/.

  12. 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. PMID:26210066

  13. Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence

    PubMed Central

    Kim, Peter Geon; Nakano, Haruko; Das, Partha P.; Chen, Michael J.; Rowe, R. Grant; Chou, Stephanie S.; Ross, Samantha J.; Sakamoto, Kathleen M.; Zon, Leonard I.; Schlaeger, Thorsten M.; Orkin, Stuart H.; Nakano, Atsushi

    2015-01-01

    Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta–gonad–mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)–cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA–CREB and BMP pathways in isolated AGM VE-cadherin+ cells from mid-gestation embryos, we demonstrate that PKA–CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA–CREB–BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition. PMID:25870201

  14. 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

  15. The Role of Protein Phosphorylation in the Gustatory Cortex and Amygdala During Taste Learning

    PubMed Central

    Adaikkan, Chinnakkaruppan

    2012-01-01

    Protein phosphorylation and dephosphorylation form a major post-translation mechanism that enables a given cell to respond to ever-changing internal and external environments. Neurons, similarly to any other cells, use protein phosphorylation/dephosphorylation to maintain an internal homeostasis, but they also use it for updating the state of synaptic and intrinsic properties, following activation by neurotransmitters and growth factors. In the present review we focus on the roles of several families of kinases, phosphatases, and other synaptic-plasticity-related proteins, which activate membrane receptors and various intracellular signals to promote transcription, translation and protein degradation, and to regulate the appropriate cellular proteomes required for taste memory acquisition, consolidation and maintenance. Attention is especially focused on the protein phosphorylation state in two forebrain areas that are necessary for taste-memory learning and retrieval: the insular cortex and the amygdala. The various temporal phases of taste learning require the activation of appropriate waves of biochemical signals. These include: extracellular signal regulated kinase I and II (ERKI/II) signal transduction pathways; Ca2+-dependent pathways; tyrosine kinase/phosphatase-dependent pathways; brain-derived neurotrophicfactor (BDNF)-dependent pathways; cAMP-responsive element bindingprotein (CREB); and translation-regulation factors, such as initiation and elongation factors, and the mammalian target of rapamycin (mTOR). Interestingly, coding of hedonic and aversive taste information in the forebrain requires activation of different signal transduction pathways. PMID:22792024

  16. Overlapping ETS and CRE Motifs ((G/C)CGGAAGTGACGTCA) preferentially bound by GABPα and CREB proteins.

    PubMed

    Chatterjee, Raghunath; Zhao, Jianfei; He, Ximiao; Shlyakhtenko, Andrey; Mann, Ishminder; Waterfall, Joshua J; Meltzer, Paul; Sathyanarayana, B K; FitzGerald, Peter C; Vinson, Charles

    2012-10-01

    Previously, we identified 8-bps long DNA sequences (8-mers) that localize in human proximal promoters and grouped them into known transcription factor binding sites (TFBS). We now examine split 8-mers consisting of two 4-mers separated by 1-bp to 30-bps (X(4)-N(1-30)-X(4)) to identify pairs of TFBS that localize in proximal promoters at a precise distance. These include two overlapping TFBS: the ETS⇔ETS motif ((C/G)CCGGAAGCGGAA) and the ETS⇔CRE motif ((C/G)CGGAAGTGACGTCAC). The nucleotides in bold are part of both TFBS. Molecular modeling shows that the ETS⇔CRE motif can be bound simultaneously by both the ETS and the B-ZIP domains without protein-protein clashes. The electrophoretic mobility shift assay (EMSA) shows that the ETS protein GABPα and the B-ZIP protein CREB preferentially bind to the ETS⇔CRE motif only when the two TFBS overlap precisely. In contrast, the ETS domain of ETV5 and CREB interfere with each other for binding the ETS⇔CRE. The 11-mer (CGGAAGTGACG), the conserved part of the ETS⇔CRE motif, occurs 226 times in the human genome and 83% are in known regulatory regions. In vivo GABPα and CREB ChIP-seq peaks identified the ETS⇔CRE as the most enriched motif occurring in promoters of genes involved in mRNA processing, cellular catabolic processes, and stress response, suggesting that a specific class of genes is regulated by this composite motif. PMID:23050235

  17. Multiple mRNA isoforms of the transcription activator protein CREB: generation by alternative splicing and specific expression in primary spermatocytes.

    PubMed Central

    Ruppert, S; Cole, T J; Boshart, M; Schmid, E; Schütz, G

    1992-01-01

    We have characterized cDNA clones representing mouse CREB (cyclic AMP responsive element binding protein) mRNA isoforms. These include CREB delta and CREB alpha, of which the rat and human homologues have been previously identified. Both encode proteins with CRE-binding activity and identical transactivation potential. The additional CREB mRNA isoforms potentially encode CREB related proteins. From the structural organization of the mouse CREB gene we conclude that the multiple transcripts are generated by alternative splicing. Furthermore we show that specific CREB mRNA isoforms are expressed at a high level in the adult testis. Expression of these isoforms is induced after commencement of spermatogenesis. In situ hybridization suggests that this expression occurs predominantly in the primary spermatocytes. Comparison of the CREB gene with the recently isolated CREM (cAMP responsive element modulator) cDNAs illustrates that the two genes have arisen by gene duplication and have diverged to encode transcriptional activators and repressors of the cAMP signal transduction pathway. Images PMID:1532935

  18. A case of Rubinstein-Taybi Syndrome with a CREB-binding protein gene mutation.

    PubMed

    Kim, Se Hee; Lim, Byung Chan; Chae, Jong Hee; Kim, Ki Joong; Hwang, Yong Seung

    2010-06-01

    Rubinstein-Taybi syndrome (RTS) is a congenital disorder characterized by typical facial features, broad thumbs and toes, with mental retardation. Additionally, tumors, keloids and various congenital anomalies including congenital heart defects have been reported in RTS patients. In about 50% of the patients, mutations in the CREB binding protein (CREBBP) have been found, which are understood to be associated with cell growth and proliferation. Here, we describe a typical RTS patient with Arnold-Chiari malformation. A mutation in the CREBBP gene, c.4944_4945insC, was identified by mutational analysis. PMID:21189944

  19. Aripiprazole Increases the PKA Signalling and Expression of the GABAA Receptor and CREB1 in the Nucleus Accumbens of Rats.

    PubMed

    Pan, Bo; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

    2016-05-01

    The GABAA receptor is implicated in the pathophysiology of schizophrenia and regulated by PKA signalling. Current antipsychotics bind with D2-like receptors, but not the GABAA receptor. The cAMP-responsive element-binding protein 1 (CREB1) is also associated with PKA signalling and may be related to the positive symptoms of schizophrenia. This study investigated the effects of antipsychotics in modulating D2-mediated PKA signalling and its downstream GABAA receptors and CREB1. Rats were treated orally with aripiprazole (0.75 mg/kg, ter in die (t.i.d.)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for 1 week. The levels of PKA-Cα and p-PKA in the prefrontal cortex (PFC), nucleus accumbens (NAc) and caudate putamen (CPu) were detected by Western blots. The mRNA levels of Gabrb1, Gabrb2, Gabrb3 and Creb1, and their protein expression were measured by qRT-PCR and Western blots, respectively. Aripiprazole elevated the levels of p-PKA and the ratio of p-PKA/PKA in the NAc, but not the PFC and CPu. Correlated with this elevated PKA signalling, aripiprazole elevated the mRNA and protein expression of the GABAA (β-1) receptor and CREB1 in the NAc. While haloperidol elevated the levels of p-PKA and the ratio of p-PKA/PKA in both NAc and CPu, it only tended to increase the expression of the GABAA (β-1) receptor and CREB1 in the NAc, but not the CPu. Bifeprunox had no effects on PKA signalling in these brain regions. These results suggest that aripiprazole has selective effects on upregulating the GABAA (β-1) receptor and CREB1 in the NAc, probably via activating PKA signalling. PMID:26894264

  20. CREB5 computational regulation network construction and analysis between frontal cortex of HIV encephalitis (HIVE) and HIVE-control patients.

    PubMed

    Wang, Lin; Huang, Juxiang; Jiang, Minghu

    2011-07-01

    CREB5 computational regulation network construction and analysis of frontal cortex of HIV encephalitis (HIVE) is very useful to identify novel markers and potential targets for prognosis and therapy. By integration of gene regulatory network infer and the database for annotation, visualization and integrated discovery we identified and constructed significant molecule CREB5 regulation network from 12 frontal cortex of HIVE-control patients and 16 HIVE in the same GEO Dataset GDS1726. Our result verified CREB5 biological regulation module in the upstream of frontal cortex of HIVE-control patients (MAPKAPK3 activation; DGKG, LY96, TNFRSF11B inhibition) and downstream (ATP6V0E1, CFB, DGKG, MX1, TGFBR3 activation; LGALS3BP, RASGRP3, RDX, STAT1 inhibition), whereas in the upstream of frontal cortex of HIVE (BST2, CFB, LCAT, TNFRSF11B activation; CFHR1, LY96 inhibition) and downstream (GAS1, LCAT, LGALS3BP, NFAT5, VEZF1, ZNF652 activation; DGKG, IFITM1, LY96, TNFRSF11B inhibition). Importantly, we datamined that CREB5 regulation cluster of HIVE was involved in inflammatory response, proteolysis, biological adhesion, and negative regulation of biological process (only in HIVE terms) without positive regulation of cellular process, phosphotransferase, kinase, post-translational protein modification, ATP binding, transmembrane protein, calcium ion binding, acetylation, and hydrolase activity (only in HIVE-control patients terms), the condition was vital to inflammation and cognition impairment of HIVE. Our result demonstrated that common terms in both HIVE-control patients and HIVE included biological regulation, phosphoprotein, metabolic process, zinc, biosynthetic process, organelle, signal transduction, defense response, membrane, secreted, signal peptide, and glycoprotein, and these terms were more relative to inflammation and cognition impairment, therefore we deduced the stronger CREB5 regulation network in HIVE consistent with our number computation. It would be

  1. Phosphorylation of paxillin confers cisplatin resistance in non-small cell lung cancer via activating ERK-mediated Bcl-2 expression.

    PubMed

    Wu, D-W; Wu, T-C; Wu, J-Y; Cheng, Y-W; Chen, Y-C; Lee, M-C; Chen, C-Y; Lee, H

    2014-08-28

    Paxillin (PXN) is required for receptor tyrosine kinase-mediated ERK activation, and the activation of the Raf/MEK/ERK cascade has been linked with Bcl-2 expression. We hypothesized that phosphorylation of PXN by the EGFR/Src pathway might contribute to cisplatin resistance via increased Bcl-2 expression. We show that cisplatin resistance was dependent on PXN expression, as evidenced by PXN overexpression in TL-13 and TL-10 cells and PXN knockdown in H23 and CL1-5 cells. Specific inhibitors of signaling pathways indicated that the phosphorylation of PXN at Y118 and Y31 via the Src pathway was responsible for cisplatin resistance. We further demonstrated that ERK activation was also dependent on this PXN phosphorylation. Bcl-2 transcription was upregulated by phosphorylated PXN-mediated ERK activation via increased binding of phosphorylated CREB to the Bcl-2 promoter. A subsequent increase in Bcl-2 levels by a PXN/ERK axis was responsible for the resistance to cisplatin. Animal models further confirmed the findings of in vitro cells indicating that xenograft tumors induced by TL-13-overexpressing cells were successfully suppressed by cisplatin combined with Src or ERK inhibitor compared with treatment of cisplatin, Src inhibitor or ERK inhibitor alone. A positive correlation of phosphorylated PXN with phosphorylated ERK and Bcl-2 was observed in lung tumors from NSCLC patients. Patients with tumors positive for PXN, phosphorylated PXN, phosphorylated ERK and Bcl-2 more commonly showed a poorer response to cisplatin-based chemotherapy than did patients with negative tumors. Collectively, PXN phosphorylation might contribute to cisplatin resistance via activating ERK-mediated Bcl-2 transcription. Therefore, we suggest that Src or ERK inhibitor might be helpful to improve the sensitivity for cisplatin-based chemotherapy in NSCLC patients with PXN-positive tumors. PMID:24096476

  2. Transcription Factor CREB3L1 Regulates Endoplasmic Reticulum Stress Response Genes in the Osmotically Challenged Rat Hypothalamus

    PubMed Central

    Greenwood, Mingkwan; Greenwood, Michael Paul; Paton, Julian F. R.; Murphy, David

    2015-01-01

    Arginine vasopressin (AVP) is synthesised in magnocellular neurons (MCNs) of supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. In response to the hyperosmotic stressors of dehydration (complete fluid deprivation, DH) or salt loading (drinking 2% salt solution, SL), AVP synthesis increases in MCNs, which over-burdens the protein folding machinery in the endoplasmic reticulum (ER). ER stress and the unfolded protein response (UPR) are signaling pathways that improve ER function in response to the accumulation of misfold/unfold protein. We asked whether an ER stress response was activated in the SON and PVN of DH and SL rats. We observed increased mRNA expression for the immunoglobulin heavy chain binding protein (BiP), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), and cAMP responsive element binding protein 3 like 1 (Creb3l1) in both SON and PVN of DH and SL rats. Although we found no changes in the splicing pattern of X box-binding protein 1 (Xbp1), an increase in the level of the unspliced form of Xbp1 (Xbp1U) was observed in DH and SL rats. CREB3L1, a novel ER stress inducer, has been shown to be activated by ER stress to regulate the expression of target genes. We have previously shown that CREB3L1 is a transcriptional regulator of the AVP gene; however, a role for CREB3L1 in the response to ER stress has yet to be investigated in MCNs. Here, we used lentiviral vectors to introduce a dominant negative form of CREB3L1 (CREB3L1DN) in the rat SON. Expression of CREB3L1DN in the SON decreased Chop and Xbp1U mRNA levels, but not BiP and Atf4 transcript expression. CREB3L1 is thus implicated as a transcriptional mediator of the ER stress response in the osmotically stimulated SON. PMID:25915053

  3. Transcription Factor CREB3L1 Regulates Endoplasmic Reticulum Stress Response Genes in the Osmotically Challenged Rat Hypothalamus.

    PubMed

    Greenwood, Mingkwan; Greenwood, Michael Paul; Paton, Julian F R; Murphy, David

    2015-01-01

    Arginine vasopressin (AVP) is synthesised in magnocellular neurons (MCNs) of supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. In response to the hyperosmotic stressors of dehydration (complete fluid deprivation, DH) or salt loading (drinking 2% salt solution, SL), AVP synthesis increases in MCNs, which over-burdens the protein folding machinery in the endoplasmic reticulum (ER). ER stress and the unfolded protein response (UPR) are signaling pathways that improve ER function in response to the accumulation of misfold/unfold protein. We asked whether an ER stress response was activated in the SON and PVN of DH and SL rats. We observed increased mRNA expression for the immunoglobulin heavy chain binding protein (BiP), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), and cAMP responsive element binding protein 3 like 1 (Creb3l1) in both SON and PVN of DH and SL rats. Although we found no changes in the splicing pattern of X box-binding protein 1 (Xbp1), an increase in the level of the unspliced form of Xbp1 (Xbp1U) was observed in DH and SL rats. CREB3L1, a novel ER stress inducer, has been shown to be activated by ER stress to regulate the expression of target genes. We have previously shown that CREB3L1 is a transcriptional regulator of the AVP gene; however, a role for CREB3L1 in the response to ER stress has yet to be investigated in MCNs. Here, we used lentiviral vectors to introduce a dominant negative form of CREB3L1 (CREB3L1DN) in the rat SON. Expression of CREB3L1DN in the SON decreased Chop and Xbp1U mRNA levels, but not BiP and Atf4 transcript expression. CREB3L1 is thus implicated as a transcriptional mediator of the ER stress response in the osmotically stimulated SON. PMID:25915053

  4. Inducible forebrain-specific ablation of the transcription factor Creb during adulthood induces anxiety but no spatial/contextual learning deficits

    PubMed Central

    Vogt, Miriam A.; Inta, Dragos; Luoni, Alessia; Elkin, Hasan; Pfeiffer, Natascha; Riva, Marco A.; Gass, Peter

    2014-01-01

    The cyclic AMP (cAMP)-response element binding protein (CREB) is an activity-dependent transcription factor playing a role in synaptic plasticity, learning and memory, and emotional behavior. However, the impact of Creb ablation on rodent behavior is vague as e.g., memory performance of different Creb mutant mice depends on the specific type of mutation per se but additionally on the background and learning protocol differences. Here we present the first targeted ablation of CREB induced during adulthood selectively in principal forebrain neurons in a pure background strain of C57BL/6 mice. All hippocampal principal neurons exhibited lack of CREB expression. Mutant mice showed a severe anxiety phenotype in the openfield and novel object exploration test as well as in the Dark-Light Box Test, but unaltered hippocampus-dependent long-term memory in the Morris water maze and in context dependent fear conditioning. On the molecular level, CREB ablation led to CREM up regulation in the hippocampus and frontal cortex which may at least in part compensate for the loss of CREB. BDNF, a postulated CREB target gene, was down regulated in the frontal lobe but not in the hippocampus; neurogenesis remained unaltered. Our data indicate that in the adult mouse forebrain the late onset of CREB ablation can, in case of memory functionality, be compensated for and is not essential for memory consolidation and retrieval during adulthood. In contrast, the presence of CREB protein during adulthood seems to be pivotal for the regulation of emotional behavior. PMID:25505876

  5. Oxidative and Photosynthetic Phosphorylation Mechanisms

    ERIC Educational Resources Information Center

    Wang, Jui H.

    1970-01-01

    Proposes a molecular mechanism for the coupling of phosphorylation to electron transport in both mitochondria and chloroplasts. Justifies the proposed reaction schemes in terms of thermodynamics and biochemical data. Suggests how areobic respiration could have evolved. (EB)

  6. Properties of phosphorylated thymidylate synthase.

    PubMed

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  7. CREB-BDNF Pathway Influences Alcohol Cue-Elicited Hyperactivation in Drinkers

    PubMed Central

    Chen, Jiayu; Hutchison, Kent E.; Calhoun, Vince D.; Claus, Eric; Turner, Jessica A.; Sui, Jing; Liu, Jingyu

    2016-01-01

    Alcohol dependence (AD) is suggested to have polygenic risk factors and also exhibits neurological complications, strongly encouraging a translational study to explore the associations between aggregates of genetic variants and brain function alterations related to alcohol use. In this study, we used a semiblind multivariate approach, parallel independent component analysis with multiple references (pICA-MR) to investigate relationships of genome-wide single nucleotide polymorphisms (SNPs) with alcohol cue elicited brain activations in 326 healthy drinkers. The genetic component derived from the CREB-BDNF pathway reference was significantly associated (r = −0.36, p = 2.98×10−11) with an imaging component reflecting hyperactivation in precuneus, superior parietal lobule, and posterior cingulate for drinkers with more severe AD scores. The highlighted brain regions participate in many cognitive processes and have been robustly implicated in craving-related studies. The genetic factor highlighted the CREB and BDNF references, as well as other genes including GRM5, GRM7, GRID1, GRIN2A, PRKCA and PRKCB. Ingenuity Pathway Analysis indicated that the genetic component was enriched in synaptic plasticity, GABA and protein kinase A signaling. In summary, our findings suggest genetic variations in various neural plasticity and signaling pathways partially explain the variance of precuneus reactivity to alcohol cue which appears to be associated with AD severity. PMID:25939814

  8. Creb coactivators direct anabolic responses and enhance performance of skeletal muscle

    PubMed Central

    Bruno, Nelson E; Kelly, Kimberly A; Hawkins, Richard; Bramah-Lawani, Mariam; Amelio, Antonio L; Nwachukwu, Jerome C; Nettles, Kendall W; Conkright, Michael D

    2014-01-01

    During the stress response to intense exercise, the sympathetic nervous system (SNS) induces rapid catabolism of energy reserves through the release of catecholamines and subsequent activation of protein kinase A (PKA). Paradoxically, chronic administration of sympathomimetic drugs (β-agonists) leads to anabolic adaptations in skeletal muscle, suggesting that sympathetic outflow also regulates myofiber remodeling. Here, we show that β-agonists or catecholamines released during intense exercise induce Creb-mediated transcriptional programs through activation of its obligate coactivators Crtc2 and Crtc3. In contrast to the catabolic activity normally associated with SNS function, activation of the Crtc/Creb transcriptional complex by conditional overexpression of Crtc2 in the skeletal muscle of transgenic mice fostered an anabolic state of energy and protein balance. Crtc2-overexpressing mice have increased myofiber cross-sectional area, greater intramuscular triglycerides and glycogen content. Moreover, maximal exercise capacity was enhanced after induction of Crtc2 expression in transgenic mice. Collectively these findings demonstrate that the SNS-adrenergic signaling cascade coordinates a transient catabolic stress response during high-intensity exercise, which is followed by transcriptional reprogramming that directs anabolic changes for recovery and that augments subsequent exercise performance. PMID:24674967

  9. Broad-Scale Phosphoprotein Profiling of Beta Adrenergic Receptor (β-AR) Signaling Reveals Novel Phosphorylation and Dephosphorylation Events

    PubMed Central

    Chruscinski, Andrzej J.; Singh, Harvir; Chan, Steven M.; Utz, Paul J.

    2013-01-01

    β-adrenergic receptors (β-ARs) are model G-protein coupled receptors that mediate signal transduction in the sympathetic nervous system. Despite the widespread clinical use of agents that target β-ARs, the signaling pathways that operate downstream of β-AR stimulation have not yet been completely elucidated. Here, we utilized a lysate microarray approach to obtain a broad-scale perspective of phosphoprotein signaling downstream of β-AR. We monitored the time course of phosphorylation states of 54 proteins after β-AR activation mouse embryonic fibroblast (MEF) cells. In response to stimulation with the non-selective β-AR agonist isoproterenol, we observed previously described phosphorylation events such as ERK1/2(T202/Y204) and CREB(S133), but also novel phosphorylation events such as Cdc2(Y15) and Pyk2(Y402). All of these events were mediated through cAMP and PKA as they were reproduced by stimulation with the adenylyl cyclase activator forskolin and were blocked by treatment with H89, a PKA inhibitor. In addition, we also observed a number of novel isoproterenol-induced protein dephosphorylation events in target substrates of the PI3K/AKT pathway: GSK3β(S9), 4E-BP1(S65), and p70s6k(T389). These dephosphorylations were dependent on cAMP, but were independent of PKA and correlated with reduced PI3K/AKT activity. Isoproterenol stimulation also led to a cAMP-dependent dephosphorylation of PP1α(T320), a modification known to correlate with enhanced activity of this phosphatase. Dephosphorylation of PP1α coincided with the secondary decline in phosphorylation of some PKA-phosphorylated substrates, suggesting that PP1α may act in a feedback loop to return these phosphorylations to baseline. In summary, lysate microarrays are a powerful tool to profile phosphoprotein signaling and have provided a broad-scale perspective of how β-AR signaling can regulate key pathways involved in cell growth and metabolism. PMID:24340001

  10. Rescue of Impaired Long-Term Facilitation at Sensorimotor Synapses of Aplysia following siRNA Knockdown of CREB1

    PubMed Central

    Zhou, Lian; Zhang, Yili; Liu, Rong-Yu; Smolen, Paul; Cleary, Leonard J.

    2015-01-01

    Memory impairment is often associated with disrupted regulation of gene induction. For example, deficits in cAMP response element-binding protein (CREB) binding protein (CBP; an essential cofactor for activation of transcription by CREB) impair long-term synaptic plasticity and memory. Previously, we showed that small interfering RNA (siRNA)-induced knockdown of CBP in individual sensory neurons significantly reduced levels of CBP and impaired 5-HT-induced long-term facilitation (LTF) in sensorimotor cocultures from Aplysia. Moreover, computational simulations of the biochemical cascades underlying LTF successfully predicted training protocols that restored LTF following CBP knockdown. We examined whether simulations could also predict a training protocol that restores LTF impaired by siRNA-induced knockdown of the transcription factor CREB1. Simulations based on a previously described model predicted rescue protocols that were specific to CREB1 knockdown. Empirical studies demonstrated that one of these rescue protocols partially restored impaired LTF. In addition, the effectiveness of the rescue protocol was enhanced by pretreatment with rolipram, a selective cAMP phosphodiesterase inhibitor. These results provide further evidence that computational methods can help rescue disruptions in signaling cascades underlying memory formation. Moreover, the study demonstrates that the effectiveness of computationally designed training protocols can be enhanced with complementary pharmacological approaches. PMID:25632137

  11. The Expression of Porcine Prdx6 Gene Is Up-Regulated by C/EBPβ and CREB

    PubMed Central

    Wu, Xinyu; Ji, Panlong; Zhang, Liang; Bu, Guowei; Gu, Hao; Wang, Xiaojing; Xiong, Yuanzhu; Zuo, Bo

    2015-01-01

    Peroxiredoxin6 (Prdx6) is one of the peroxiredoxin (Prdxs) family members that play an important role in maintaining cell homeostasis. Our previous studies demonstrated that Prdx6 was significantly associated with pig meat quality, especially meat tenderness. However, the transcriptional regulation of porcine Prdx6 remains unclear. In this study, we determined the transcription start site (TSS) of porcine Prdx6 gene by 5′ rapid-amplification of cDNA ends (5′ RACE). Several regulatory elements including CCAAT/enhancer-binding proteinβ (C/EBPβ), Myogenic Differentiation (MyoD), cAMP response element binding protein (CREB), stimulating protein1 (Sp1) and heat shock factor (HSF) binding sites were found by computational analyses together with luciferase reporter system. Overexpression and RNA interference experiments showed that C/EBPβ or CREB could up-regulate the expression of porcine Prdx6 gene at both mRNA and protein level. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP) confirmed that C/EBPβ and CREB could interact with Prdx6 promoter. Immuoprecipitation results also showed that C/EBPβ could interact with Prdx6 in vivo. Taken together, our findings identified C/EBPβ and CREB as the important regulators of porcine Prdx6 gene expression, and offered clues for further investigation of Prdx6 gene function. PMID:26659441

  12. Corticotropin Releasing Hormone and Urocortin 3 Stimulate Vascular Endothelial Growth Factor Expression through the cAMP/CREB Pathway.

    PubMed

    Rhee, Sang Hoon; Ma, Elise L; Lee, Yunna; Taché, Yvette; Pothoulakis, Charalabos; Im, Eunok

    2015-10-23

    Colonic epithelium is the first line of defense against various pathological offenses in the gut. Previous studies have shown that the peptides of the corticotropin-releasing hormone (CRH) family modulate vascular endothelial growth factor (VEGF)-A production in other cells. Here we sought to investigate whether CRH and urocortin (Ucn) 3 regulate VEGF-A secretion in colonocytes through CRH receptors and to elucidate the underlying mechanism of action. CRH and Ucn 3 significantly increased the expression levels of VEGF-A mRNA and protein through CRH receptor 1 and 2, respectively, in human colonic epithelial cells and primary mouse intestinal epithelial cells. Underlying mechanisms involve activation of adenylyl cyclase with subsequent increase of intracellular cAMP level and increased DNA binding activity of transcription factor CREB on VEGF-A promoter region. Finally, genetic deficiency of CREB decreased intestinal inflammation and VEGF-A expression in a dextran sodium sulfate-induced colitis model. These results show that activation of CRH receptors by CRH ligands stimulates VEGF-A expression in intestinal epithelial cells through the cAMP/CREB pathway. Since VEGF-A boosts inflammatory responses through angiogenesis, these data suggest that CREB may be a key effector of CRH and Ucn 3-dependent inflammatory angiogenesis. PMID:26350463

  13. BzpF is a CREB-like transcription factor that regulates spore maturation and stability in Dictyostelium.

    PubMed

    Huang, Eryong; Talukder, Shaheynoor; Hughes, Timothy R; Curk, Tomaz; Zupan, Blaz; Shaulsky, Gad; Katoh-Kurasawa, Mariko

    2011-10-01

    The cAMP response element-binding protein (CREB) is a highly conserved transcription factor that integrates signaling through the cAMP-dependent protein kinase A (PKA) in many eukaryotes. PKA plays a critical role in Dictyostelium development but no CREB homologue has been identified in this system. Here we show that Dictyostelium utilizes a CREB-like protein, BzpF, to integrate PKA signaling during late development. bzpF(-) mutants produce compromised spores, which are extremely unstable and germination defective. Previously, we have found that BzpF binds the canonical CRE motif in vitro. In this paper, we determined the DNA binding specificity of BzpF using protein binding microarray (PBM) and showed that the motif with the highest specificity is a CRE-like sequence. BzpF is necessary to activate the transcription of at least 15 PKA-regulated, late-developmental target genes whose promoters contain BzpF binding motifs. BzpF is sufficient to activate two of these genes. The comparison of RNA sequencing data between wild type and bzpF(-) mutant revealed that the mutant fails to express 205 genes, many of which encode cellulose-binding and sugar-binding proteins. We propose that BzpF is a CREB-like transcription factor that regulates spore maturation and stability in a PKA-related manner. PMID:21810415

  14. Retinoic acid induces caspase-8 transcription via phospho-CREB and increases apoptotic responses to death stimuli in neuroblastoma cells

    PubMed Central

    Jiang, Manrong; Zhu, Kejin; Grenet, Jose; Lahti, Jill M.

    2008-01-01

    Caspase-8 is frequently deleted or silenced in neuroblastoma and other solid tumor such as medulloblastoma and small cell lung carcinoma. Caspase-8 expression can be re-established in neuroblastoma cell lines by treatment with demethylating agents or with IFN-γ Here we show that four different retinoic acid (RA) derivatives also increase caspase-8 protein expression in neuroblastoma, medulloblastoma and small cell lung carcinoma cell lines. This increase in protein expression is mirrored by an increase in RNA expression in NB cells. However, the promoter region of the caspase-8 gene was not responsible for the induction of caspase-8 expression. Rather, we identified another intronic region containing a CREB binding site that was required for maximal induction of caspase-8 via RA. DNA-protein interaction assays revealed increased phospho-CREB binding to this response element in RA-treated NB cells. Furthermore, both mutation of the CREB binding site completely blocked caspase-8 induction in the luciferase reporter system assay and transfection of dominant-negative form of CREB repressed the up-regulation of caspase-8 by RA. Importantly, RA-released cells maintained caspase-8 expression for at least 2–5 days and were more sensitive to doxorubicin and TNFα. Thus, RA treatment in conjunction with TNFα and/or subsets of cytotoxic agents may have therapeutic benefits. PMID:18342014

  15. Rescue of impaired long-term facilitation at sensorimotor synapses of Aplysia following siRNA knockdown of CREB1.

    PubMed

    Zhou, Lian; Zhang, Yili; Liu, Rong-Yu; Smolen, Paul; Cleary, Leonard J; Byrne, John H

    2015-01-28

    Memory impairment is often associated with disrupted regulation of gene induction. For example, deficits in cAMP response element-binding protein (CREB) binding protein (CBP; an essential cofactor for activation of transcription by CREB) impair long-term synaptic plasticity and memory. Previously, we showed that small interfering RNA (siRNA)-induced knockdown of CBP in individual sensory neurons significantly reduced levels of CBP and impaired 5-HT-induced long-term facilitation (LTF) in sensorimotor cocultures from Aplysia. Moreover, computational simulations of the biochemical cascades underlying LTF successfully predicted training protocols that restored LTF following CBP knockdown. We examined whether simulations could also predict a training protocol that restores LTF impaired by siRNA-induced knockdown of the transcription factor CREB1. Simulations based on a previously described model predicted rescue protocols that were specific to CREB1 knockdown. Empirical studies demonstrated that one of these rescue protocols partially restored impaired LTF. In addition, the effectiveness of the rescue protocol was enhanced by pretreatment with rolipram, a selective cAMP phosphodiesterase inhibitor. These results provide further evidence that computational methods can help rescue disruptions in signaling cascades underlying memory formation. Moreover, the study demonstrates that the effectiveness of computationally designed training protocols can be enhanced with complementary pharmacological approaches. PMID:25632137

  16. cAMP-dependent Protein Kinase and c-Jun N-terminal Kinase Mediate Stathmin Phosphorylation for the Maintenance of Interphase Microtubules during Osmotic Stress*

    PubMed Central

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

    2014-01-01

    Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress. PMID:24302736

  17. Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

    PubMed

    Baginsky, Sacha

    2016-06-01

    The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation. PMID:26969742

  18. Functional interaction between the human cytomegalovirus 86-kilodalton IE2 protein and the cellular transcription factor CREB.

    PubMed Central

    Lang, D; Gebert, S; Arlt, H; Stamminger, T

    1995-01-01

    The 86-kDa IE2 protein (IE86) of human cytomegalovirus (HCMV) has been described as a promiscuous transactivator of viral, as well as cellular, gene expression. Investigation of the mechanism used by IE86 to activate gene expression from the early UL112/113 promoter of HCMV revealed the existence of three binding sites for IE86 located between nucleotides -290 and -120 relative to the transcriptional start site (H. Arlt, D. Lang, S. Gebert, and T. Stamminger, J. Virol. 68:4117-4125, 1994). As shown previously, deletion of these target sites resulted in a reduction of IE86-mediated transactivation by approximately 70%. The remaining promoter, however, could still be stimulated about 40-fold, indicating the presence of an additional responsive element within these sequences. Here, we provide evidence that a binding site for the cellular transcription factor CREB can also act as a target for IE86 transactivation. By DNase I protection analysis, a binding sequence for CREB could be detected between nucleotides -78 and -56 within the respective promoter region. After in vitro mutagenesis of this CREB-binding site within the context of the entire UL112/113 promoter, a marked reduction in transactivation levels was evident. Moreover, when individual CREB-binding sites were positioned upstream of a minimal, TATA box-containing UL112/113 promoter, they were able to confer strong IE86 responsiveness, whereas a mutated sequence did not exert any effect. In far Western blot and pull-down experiments, a direct interaction of IE86 with the cellular transcription factor CREB could be observed. The in vivo relevance of this in vitro interaction was confirmed by using various GAL4 fusion proteins in the presence or absence of IE86 which revealed a strong activation only in the presence of both a GAL4-CREB fusion and IE86. This shows that at least one specific member of the ATF/CREB family of transcription factors is involved in mediating transactivation by the HCMV IE86 protein

  19. Interphase phosphorylation of lamin A.

    PubMed

    Kochin, Vitaly; Shimi, Takeshi; Torvaldson, Elin; Adam, Stephen A; Goldman, Anne; Pack, Chan-Gi; Melo-Cardenas, Johanna; Imanishi, Susumu Y; Goldman, Robert D; Eriksson, John E

    2014-06-15

    Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells. PMID:24741066

  20. Preliminary investigation of the influence of CREB1 gene polymorphisms on cognitive dysfunction in Chinese patients with major depression.

    PubMed

    Guo, Junhui; Liu, Zhongchun; Dai, Hong; Zhu, Zhixian; Wang, Huiling; Yang, Can; Xiao, Ling; Huang, Yonglan; Wang, Gaohua

    2014-01-01

    Research has increasingly focused on the role of the cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein in learning and memory, particularly its role in cognitive disorders and neurodegeneration, such as Huntington's disease, Alzheimer's disease, Rubinstein-Taybi syndrome, and Coffin-Lowry syndrome. The cognitive dysfunction of patients with major depressive disorder (MDD), which is widely recognized, is not completely in accordance with depressive severity, and the dysfunction persists upon clinical remission in some patients. However, few studies have focused on the role of CREB on cognitive function in patients with MDD. This study aimed to investigate the influence of CREB1 polymorphism on cognitive function in patients with MDD. The current study comprised 113 patients with MDD. The severity of depression was measured using the 17-item Hamilton Depression Rating Scale, and cognitive function was assessed using the Stroop Neuropsychological Screening Test, verbal fluency test, and tests of immediate logical memory and visual reproduction. All subjects were genotyped with regard to CREB1 polymorphisms (rs10932201, rs2551645, rs2254137, rs6740584, and rs2551640). For the verbal fluency test, the results showed significant differences for all single-nucleotide polymorphism genotypic groups. For the Stroop color-word task, a significant difference was found only for rs6740584. No significant differences were found for the Stroop color task, the immediate logical memory test or the visual reproduction test. In conclusion, there was an effect of CREB1 polymorphism on selective attention and retrieval of long-term memory, but not on immediate memory. PMID:23844928

  1. An essential GT motif in the lamin A promoter mediates activation by CREB-binding protein

    SciTech Connect

    Janaki Ramaiah, M.; Parnaik, Veena K. . E-mail: veenap@ccmb.res.in

    2006-09-29

    Lamin A is an important component of nuclear architecture in mammalian cells. Mutations in the human lamin A gene lead to highly degenerative disorders that affect specific tissues. In studies directed towards understanding the mode of regulation of the lamin A promoter, we have identified an essential GT motif at -55 position by reporter gene assays and mutational analysis. Binding of this sequence to Sp transcription factors has been observed in electrophoretic mobility shift assays and by chromatin immunoprecipitation studies. Further functional analysis by co-expression of recombinant proteins and ChIP assays has shown an important regulatory role for CREB-binding protein in promoter activation, which is mediated by the GT motif.

  2. Transcriptional Profiling of a Selective CREB Binding Protein Bromodomain Inhibitor Highlights Therapeutic Opportunities.

    PubMed

    Chekler, Eugene L Piatnitski; Pellegrino, Jessica A; Lanz, Thomas A; Denny, R Aldrin; Flick, Andrew C; Coe, Jotham; Langille, Jonathan; Basak, Arindrajit; Liu, Shenping; Stock, Ingrid A; Sahasrabudhe, Parag; Bonin, Paul D; Lee, Kevin; Pletcher, Mathew T; Jones, Lyn H

    2015-12-17

    Bromodomains are involved in transcriptional regulation through the recognition of acetyl lysine modifications on diverse proteins. Selective pharmacological modulators of bromodomains are lacking, although the largely hydrophobic nature of the pocket makes these modules attractive targets for small-molecule inhibitors. This work describes the structure-based design of a highly selective inhibitor of the CREB binding protein (CBP) bromodomain and its use in cell-based transcriptional profiling experiments. The inhibitor downregulated a number of inflammatory genes in macrophages that were not affected by a selective BET bromodomain inhibitor. In addition, the CBP bromodomain inhibitor modulated the mRNA level of the regulator of G-protein signaling 4 (RGS4) gene in neurons, suggesting a potential therapeutic opportunity for CBP inhibitors in the treatment of neurological disorders. PMID:26670081

  3. Notch1 Regulates Hippocampal Plasticity Through Interaction with the Reelin Pathway, Glutamatergic Transmission and CREB Signaling

    PubMed Central

    Brai, Emanuele; Marathe, Swananda; Astori, Simone; Fredj, Naila Ben; Perry, Elisabeth; Lamy, Christophe; Scotti, Alessandra; Alberi, Lavinia

    2015-01-01

    Notch signaling plays a crucial role in adult brain function such as synaptic plasticity, memory and olfaction. Several reports suggest an involvement of this pathway in neurodegenerative dementia. Yet, to date, the mechanism underlying Notch activity in mature neurons remains unresolved. In this work, we investigate how Notch regulates synaptic potentiation and contributes to the establishment of memory in mice. We observe that Notch1 is a postsynaptic receptor with functional interactions with the Reelin receptor, apolipoprotein E receptor 2 (ApoER2) and the ionotropic receptor, N-methyl-D-aspartate receptor (NMDAR). Targeted loss of Notch1 in the hippocampal CA fields affects Reelin signaling by influencing Dab1 expression and impairs the synaptic potentiation achieved through Reelin stimulation. Further analysis indicates that loss of Notch1 affects the expression and composition of the NMDAR but not AMPAR. Glutamatergic signaling is further compromised through downregulation of CamKII and its secondary and tertiary messengers resulting in reduced cAMP response element-binding (CREB) signaling. Our results identify Notch1 as an important regulator of mechanisms involved in synaptic plasticity and memory formation. These findings emphasize the possible involvement of this signaling receptor in dementia. Highlights In this paper, we propose a mechanism for Notch1-dependent plasticity that likely underlies the function of Notch1 in memory formation: Notch1 interacts with another important developmental pathway, the Reelin cascade. Notch1 regulates both NMDAR expression and composition. Notch1 influences a cascade of cellular events culminating in CREB activation. PMID:26635527

  4. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    SciTech Connect

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  5. Autophagy proteins regulate ERK phosphorylation

    PubMed Central

    Martinez-Lopez, Nuria; Athonvarangkul, Diana; Mishall, Priti; Sahu, Srabani; Singh, Rajat

    2013-01-01

    Autophagy is a conserved pathway that maintains cellular quality control. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including proliferation. Multiple signalling cascades, including ERK, have been shown to regulate autophagy, however whether autophagy proteins (ATG) regulate cell signalling is unknown. Here we show that growth factor exposure increases the interaction of ERK cascade components with ATG proteins in the cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes. ERK2 interacts with ATG proteins via its substrate-binding domains. Deleting Atg7 or Atg5 or blocking LC3 lipidation or ATG5–ATG12 conjugation decreases ERK phosphorylation. Conversely, increasing LC3-II availability by silencing the cysteine protease ATG4B or acute trehalose exposure increases ERK phosphorylation. Decreased ERK phosphorylation in Atg5−/− cells does not occur from overactive phosphatases. Our findings thus reveal an unconventional function of ATG proteins as cellular scaffolds in the regulation of ERK phosphorylation. PMID:24240988

  6. CREBRF is a potent tumor suppressor of glioblastoma by blocking hypoxia-induced autophagy via the CREB3/ATG5 pathway.

    PubMed

    Xue, Hao; Zhang, Jinsen; Guo, Xing; Wang, Jian; Li, Jiangbing; Gao, Xiao; Guo, Xiaofan; Li, Tong; Xu, Shugang; Zhang, Ping; Liu, Qinglin; Li, Gang

    2016-08-01

    Hypoxia induces protective autophagy in advanced glioblastoma cells, and targeting this process may improve the outcome for glioblastoma patients. Recent studies have suggested that the autophagic process is upregulated in glioblastoma cells in response to extensive hypoxia. Here, we describe a novel tumor suppressor in glioblastoma cells, whereby hypoxia downregulated CREBRF expression and acts as a potent inhibitor of autophagy in glioblastoma cells via the CREB3/ATG5 pathway. Our results demonstrate that CREBRF expression negatively correlates with autophagic and HIF-1α levels in different grade gliomas. Given that CREBRF is a negative regulator of CREB3, CREB3 knockdown also repressed hypoxia-induced autophagy in glioblastoma cells in vitro. Collectively, our findings provide new insight into the molecular mechanisms underlying hypoxia-induced glioblastoma cell autophagy and indicate that the hypoxia/CREBRF/CREB3/ATG5 pathway plays a central role in malignant glioma progression. PMID:27278737

  7. Epigenetic modification of miR-10a regulates renal damage by targeting CREB1 in type 2 diabetes mellitus.

    PubMed

    Shan, Qun; Zheng, Guihong; Zhu, Aihua; Cao, Li; Lu, Jun; Wu, Dongmei; Zhang, ZiFeng; Fan, Shaohua; Sun, Chunhui; Hu, Bin; Zheng, Yuanlin

    2016-09-01

    Emerging evidence has shown that microRNA-mediated gene expression modulation plays a crucial role in the pathogenesis of type 2 diabetes mellitus, but the novel miRNAs involved in type 2 diabetes and its functional regulatory mechanisms still need to be determined. In this study, we assessed the role of miR-10a in extracellular matrix accumulation in the kidney of diabetic mellitus induced by combining administration of chronic high fat diet (HFD) and low dosage of streptozotocin (STZ, 35mg/kg). Here, we found that HFD/STZ administration decreased the level of microRNA (miR-10a) expression in ICR strain mice. Overexpression of miR-10a alleviated the increased ratio of urine albumin-to-creatinine (ACR) ratio of HFD/STZ mice. In contrast, knockdown of miR-10a increased the ratio of kidney ACR in naïve mice. Furthermore, cAMP response element binding protein 1 (CREB1) was validated as a target of miR-10a in vitro and in vivo. CREB1 and its downstream fibronectin (FN, extracellular matrix) were increased in HFD/STZ-treated mice, which was reversed by kidney miR-10a overexpression. The content of CREB1 and FN was increased by miR-10a knockdown in kidney of naïve mice. Furthermore, histone deacetylase 3 (HDAC3) was revealed to be increased in kidney of HFD/STZ mice, accompanied with the augmentation of ACR ratio and FN level. Knockdown of HDAC3 with siRNA significantly caused the increase of miR-10a, resulting in the decrease in CREB1 and FN expression in kidney of HFD/STZ mice. Contrarily, HDAC3 overexpression mediated by lentivirus decreased miR-10a content, and enhanced ACR value, CREB1 and FN formation in naïve mice. Collectively, these results elucidate that HDAC3/miR-10a/CREB1 serves as a new mechanism underlying kidney injury, providing potential therapeutic targets in type 2 diabetes. PMID:27292126

  8. Gene network inference and biochemical assessment delineates GPCR pathways and CREB targets in small intestinal neuroendocrine neoplasia.

    PubMed

    Drozdov, Ignat; Svejda, Bernhard; Gustafsson, Bjorn I; Mane, Shrikant; Pfragner, Roswitha; Kidd, Mark; Modlin, Irvin M

    2011-01-01

    Small intestinal (SI) neuroendocrine tumors (NET) are increasing in incidence, however little is known about their biology. High throughput techniques such as inference of gene regulatory networks from microarray experiments can objectively define signaling machinery in this disease. Genome-wide co-expression analysis was used to infer gene relevance network in SI-NETs. The network was confirmed to be non-random, scale-free, and highly modular. Functional analysis of gene co-expression modules revealed processes including 'Nervous system development', 'Immune response', and 'Cell-cycle'. Importantly, gene network topology and differential expression analysis identified over-expression of the GPCR signaling regulators, the cAMP synthetase, ADCY2, and the protein kinase A, PRKAR1A. Seven CREB response element (CRE) transcripts associated with proliferation and secretion: BEX1, BICD1, CHGB, CPE, GABRB3, SCG2 and SCG3 as well as ADCY2 and PRKAR1A were measured in an independent SI dataset (n = 10 NETs; n = 8 normal preparations). All were up-regulated (p<0.035) with the exception of SCG3 which was not differently expressed. Forskolin (a direct cAMP activator, 10(-5) M) significantly stimulated transcription of pCREB and 3/7 CREB targets, isoproterenol (a selective ß-adrenergic receptor agonist and cAMP activator, 10(-5) M) stimulated pCREB and 4/7 targets while BIM-53061 (a dopamine D(2) and Serotonin [5-HT(2)] receptor agonist, 10(-6) M) stimulated 100% of targets as well as pCREB; CRE transcription correlated with the levels of cAMP accumulation and PKA activity; BIM-53061 stimulated the highest levels of cAMP and PKA (2.8-fold and 2.5-fold vs. 1.8-2-fold for isoproterenol and forskolin). Gene network inference and graph topology analysis in SI NETs suggests that SI NETs express neural GPCRs that activate different CRE targets associated with proliferation and secretion. In vitro studies, in a model NET cell system, confirmed that transcriptional effects are

  9. Phosphorylation in halobacterial signal transduction.

    PubMed Central

    Rudolph, J; Tolliday, N; Schmitt, C; Schuster, S C; Oesterhelt, D

    1995-01-01

    Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli. Images PMID:7556066

  10. Integrative analysis identifies targetable CREB1/FoxA1 transcriptional co-regulation as a predictor of prostate cancer recurrence.

    PubMed

    Sunkel, Benjamin; Wu, Dayong; Chen, Zhong; Wang, Chiou-Miin; Liu, Xiangtao; Ye, Zhenqing; Horning, Aaron M; Liu, Joseph; Mahalingam, Devalingam; Lopez-Nicora, Horacio; Lin, Chun-Lin; Goodfellow, Paul J; Clinton, Steven K; Jin, Victor X; Chen, Chun-Liang; Huang, Tim H-M; Wang, Qianben

    2016-05-19

    Identifying prostate cancer-driving transcription factors (TFs) in addition to the androgen receptor promises to improve our ability to effectively diagnose and treat this disease. We employed an integrative genomics analysis of master TFs CREB1 and FoxA1 in androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC) cell lines, primary prostate cancer tissues and circulating tumor cells (CTCs) to investigate their role in defining prostate cancer gene expression profiles. Combining genome-wide binding site and gene expression profiles we define CREB1 as a critical driver of pro-survival, cell cycle and metabolic transcription programs. We show that CREB1 and FoxA1 co-localize and mutually influence each other's binding to define disease-driving transcription profiles associated with advanced prostate cancer. Gene expression analysis in human prostate cancer samples found that CREB1/FoxA1 target gene panels predict prostate cancer recurrence. Finally, we showed that this signaling pathway is sensitive to compounds that inhibit the transcription co-regulatory factor MED1. These findings not only reveal a novel, global transcriptional co-regulatory function of CREB1 and FoxA1, but also suggest CREB1/FoxA1 signaling is a targetable driver of prostate cancer progression and serves as a biomarker of poor clinical outcomes. PMID:26743006

  11. Integrative analysis identifies targetable CREB1/FoxA1 transcriptional co-regulation as a predictor of prostate cancer recurrence

    PubMed Central

    Sunkel, Benjamin; Wu, Dayong; Chen, Zhong; Wang, Chiou-Miin; Liu, Xiangtao; Ye, Zhenqing; Horning, Aaron M.; Liu, Joseph; Mahalingam, Devalingam; Lopez-Nicora, Horacio; Lin, Chun-Lin; Goodfellow, Paul J.; Clinton, Steven K.; Jin, Victor X.; Chen, Chun-Liang; Huang, Tim H.-M.; Wang, Qianben

    2016-01-01

    Identifying prostate cancer-driving transcription factors (TFs) in addition to the androgen receptor promises to improve our ability to effectively diagnose and treat this disease. We employed an integrative genomics analysis of master TFs CREB1 and FoxA1 in androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC) cell lines, primary prostate cancer tissues and circulating tumor cells (CTCs) to investigate their role in defining prostate cancer gene expression profiles. Combining genome-wide binding site and gene expression profiles we define CREB1 as a critical driver of pro-survival, cell cycle and metabolic transcription programs. We show that CREB1 and FoxA1 co-localize and mutually influence each other's binding to define disease-driving transcription profiles associated with advanced prostate cancer. Gene expression analysis in human prostate cancer samples found that CREB1/FoxA1 target gene panels predict prostate cancer recurrence. Finally, we showed that this signaling pathway is sensitive to compounds that inhibit the transcription co-regulatory factor MED1. These findings not only reveal a novel, global transcriptional co-regulatory function of CREB1 and FoxA1, but also suggest CREB1/FoxA1 signaling is a targetable driver of prostate cancer progression and serves as a biomarker of poor clinical outcomes. PMID:26743006

  12. CREB Negatively Regulates IGF2R Gene Expression and Downstream Pathways to Inhibit Hypoxia-Induced H9c2 Cardiomyoblast Cell Death

    PubMed Central

    Chen, Wei-Kung; Kuo, Wei-Wen; Hsieh, Dennis Jine-Yuan; Chang, Hsin-Nung; Pai, Pei-Ying; Lin, Kuan-Ho; Pan, Lung-Fa; Ho, Tsung-Jung; Viswanadha, Vijaya Padma; Huang, Chih-Yang

    2015-01-01

    During hypoxia, gene expression is altered by various transcription factors. Insulin-like growth factor-II (IGF2) is known to be induced by hypoxia, which binds to IGF2 receptor IGF2R that acts like a G protein-coupled receptor, might cause pathological hypertrophy or activation of the mitochondria-mediated apoptosis pathway. Cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB) is central to second messenger-regulated transcription and plays a critical role in the cardiomyocyte survival pathway. In this study, we found that IGF2R level was enhanced in H9c2 cardiomyoblasts exposed to hypoxia in a time-dependent manner but was down-regulated by CREB expression. The over-expression of CREB in H9c2 cardiomyoblasts suppressed the induction of hypoxia-induced IGF2R expression levels and reduced cell apoptosis. Gel shift assay results further indicated that CREB binds to the promoter sequence of IGF2R. With a luciferase assay method, we further observed that CREB represses IGF2R promoter activity. These results suggest that CREB plays an important role in the inhibition of IGF2R expression by binding to the IGF2R promoter and further suppresses H9c2 cardiomyoblast cell apoptosis induced by IGF2R signaling under hypoxic conditions. PMID:26610485

  13. Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system

    PubMed Central

    Nakagawa, Yoshimi; Oikawa, Fusaka; Mizuno, Seiya; Ohno, Hiroshi; Yagishita, Yuka; Satoh, Aoi; Osaki, Yoshinori; Takei, Kenta; Kikuchi, Takuya; Han, Song-iee; Matsuzaka, Takashi; Iwasaki, Hitoshi; Kobayashi, Kazuto; Yatoh, Shigeru; Yahagi, Naoya; Isaka, Masaaki; Suzuki, Hiroaki; Sone, Hirohito; Takahashi, Satoru; Yamada, Nobuhiro; Shimano, Hitoshi

    2016-01-01

    cAMP responsive element binding protein 3-like 3 (CREB3L3), a transcription factor expressed in the liver and small intestine, governs fasting-response energy homeostasis. Tissue-specific CREB3L3 knockout mice have not been generated till date. To our knowledge, this is the first study using the one-step CRISPR/Cas9 system to generate CREB3L3 floxed mice and subsequently obtain liver- and small intestine-specific Creb3l3 knockout (LKO and IKO, respectively) mice. While LKO mice as well as global KO mice developed hypertriglyceridemia, LKO mice exhibited hypercholesterolemia in contrast to hypocholesterolemia in global KO mice. LKO mice demonstrated up-regulation of hepatic Srebf2 and its corresponding target genes. No phenotypic differences were observed between IKO and floxed mice. Severe liver injury was observed in LKO mice fed a methionine-choline deficient diet, a model for non-alcoholic steatohepatitis. These results provide new evidence regarding the hepatic CREB3L3 role in plasma triglyceride metabolism and hepatic and intestinal CREB3L3 contributions to cholesterol metabolism. PMID:27291420

  14. Regulation of cAMP response element binding protein (CREB) binding in the mammalian clock pacemaker by light but not a circadian clock.

    PubMed

    Kako, K; Banasik, M; Lee, K; Ishida, N

    1997-02-01

    Mammalian circadian rhythms are considered to be regulated by a clock pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The molecular mechanism of entrainment and oscillation of circadian rhythm are not well understood but photic induction of immediate-early gene (IEG) expression in the SCN is thought to play a role. Here we show that under 12 h light:12 h dark (LD) condition, the cAMP response element binding protein (CREB) binding to cAMP responsive promoter element (CRE) of NMDAR1/zeta1 promoter region in the SCN is higher during the light than the dark by electro-mobility shift assay (EMSA). When animals are placed in constant dark, CREB DNA binding activity in the SCN is low and does not vary with circadian time when compared with cortex nuclear extract as a control. Most significantly, photic induction of CREB binding activity in the SCN occurs at all circadian times tested, indicating that CREB DNA binding in the SCN is not gated by the endogenous clock. These results implicate the role of CREB in photic neuronal signaling in the SCN and suggest that CREB DNA binding activities may not be regulated by a circadian clock. PMID:9030696

  15. Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system.

    PubMed

    Nakagawa, Yoshimi; Oikawa, Fusaka; Mizuno, Seiya; Ohno, Hiroshi; Yagishita, Yuka; Satoh, Aoi; Osaki, Yoshinori; Takei, Kenta; Kikuchi, Takuya; Han, Song-Iee; Matsuzaka, Takashi; Iwasaki, Hitoshi; Kobayashi, Kazuto; Yatoh, Shigeru; Yahagi, Naoya; Isaka, Masaaki; Suzuki, Hiroaki; Sone, Hirohito; Takahashi, Satoru; Yamada, Nobuhiro; Shimano, Hitoshi

    2016-01-01

    cAMP responsive element binding protein 3-like 3 (CREB3L3), a transcription factor expressed in the liver and small intestine, governs fasting-response energy homeostasis. Tissue-specific CREB3L3 knockout mice have not been generated till date. To our knowledge, this is the first study using the one-step CRISPR/Cas9 system to generate CREB3L3 floxed mice and subsequently obtain liver- and small intestine-specific Creb3l3 knockout (LKO and IKO, respectively) mice. While LKO mice as well as global KO mice developed hypertriglyceridemia, LKO mice exhibited hypercholesterolemia in contrast to hypocholesterolemia in global KO mice. LKO mice demonstrated up-regulation of hepatic Srebf2 and its corresponding target genes. No phenotypic differences were observed between IKO and floxed mice. Severe liver injury was observed in LKO mice fed a methionine-choline deficient diet, a model for non-alcoholic steatohepatitis. These results provide new evidence regarding the hepatic CREB3L3 role in plasma triglyceride metabolism and hepatic and intestinal CREB3L3 contributions to cholesterol metabolism. PMID:27291420

  16. Leptin augments recruitment of IRF-1 and CREB to thrombospondin-1 gene promoter in vascular smooth muscle cells in vitro.

    PubMed

    Sahu, Soumyadip; Ganguly, Rituparna; Raman, Priya

    2016-08-01

    We previously reported that high pathophysiological concentrations of leptin, the adipocyte-secreted peptide, upregulate the expression of a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in vascular smooth muscle cells. Moreover, this regulation was found to occur at the level of transcription; however, the underlying molecular mechanisms remain unknown. The goal of the present study was to investigate the specific transcriptional mechanisms that mediate upregulation of TSP-1 expression by leptin. Primary human aortic smooth muscle cell cultures were transiently transfected with different TSP-1 gene (THBS1) promoter-linked luciferase reporter constructs, and luciferase activity in response to leptin (100 ng/ml) was assessed. We identified a long THBS1 promoter (-1270/+750) fragment with specific leptin response elements that are required for increased TSP-1 transcription by leptin. Promoter analyses, protein/DNA array and gel shift assays demonstrated activation and association of transcription factors, interferon regulatory factor-1 (IRF-1) and cAMP response element-binding protein (CREB), to the distal fragment of the THBS1 promoter in response to leptin. Supershift, chromatin immunoprecipitation, and coimmunoprecipitation assays revealed formation of a single complex between IRF-1 and CREB in response to leptin; importantly, recruitment of this complex to the THBS1 promoter mediated leptin-induced TSP-1 transcription. Finally, binding sequence decoy oligomer and site-directed mutagenesis revealed that regulatory elements for both IRF-1 (-1019 to -1016) and CREB (-1198 to -1195), specific to the distal THBS1 promoter, were required for leptin-induced TSP-1 transcription. Taken together, these findings demonstrate that leptin promotes a cooperative association between IRF-1 and CREB on the THBS1 promoter driving TSP-1 transcription in vascular smooth muscle cells. PMID:27281481

  17. Interleukin-17A Acts to Maintain Neuropathic Pain Through Activation of CaMKII/CREB Signaling in Spinal Neurons.

    PubMed

    Yao, Cheng-Ye; Weng, Ze-Lin; Zhang, Jian-Cheng; Feng, Tao; Lin, Yun; Yao, Shanglong

    2016-08-01

    Immunity and neuroinflammation play major roles in neuropathic pain. Spinal interleukin (IL)-17A, as a mediator connecting innate and adaptive immunity, has been shown to be an important cytokine in neuroinflammation and acute neuropathic pain. However, the effects and underlying mechanisms of spinal IL-17A in the maintenance of neuropathic pain remain unknown. This study was designed to investigate whether spinal IL-17A acted to maintain neuropathic pain and to elucidate the underlying mechanisms in IL-17A knockout or wild-type (WT) mice following L4 spinal nerve ligation (L4 SNL). WT mice were treated with anti-IL-17A neutralized monoclonal antibody (mAb) or recombinant IL-17A (rIL-17A). We showed that IL-17A levels were significantly increased 1, 3, 7, and 14 days after SNL in spinal cord. Double immunofluorescence staining showed that astrocytes were the major cellular source of spinal IL-17A. IL-17A knockout or anti-IL-17A mAb treatment significantly ameliorated hyperalgesia 7 days after SNL, which was associated with a significant reduction of p-CaMKII and p-CREB levels in spinal cord, whereas rIL-17A treatment conferred the opposite effects. Furthermore, we showed that blocking CaMKII with KN93 significantly reduced SNL- or rIL-17A-induced hyperalgesia and p-CREB expression. Our in vitro data showed that KN93 also significantly inhibited rIL-17A-induced CREB activation in primary cultured spinal neurons. Taken together, our study indicates that astrocytic IL-17A plays important roles in the maintenance of neuropathic pain through CaMKII/CREB signaling pathway in spinal cord, and thus targeting IL-17A may offer an attractive strategy for the treatment of chronic persistent neuropathic pain. PMID:26166359

  18. CRTC2 Is a Coactivator of GR and Couples GR and CREB in the Regulation of Hepatic Gluconeogenesis.

    PubMed

    Hill, Micah J; Suzuki, Shigeru; Segars, James H; Kino, Tomoshige

    2016-01-01

    Glucocorticoid hormones play essential roles in the regulation of gluconeogenesis in the liver, an adaptive response that is required for the maintenance of circulating glucose levels during fasting. Glucocorticoids do this by cooperating with glucagon, which is secreted from pancreatic islets to activate the cAMP-signaling pathway in hepatocytes. The cAMP-response element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) is a coactivator known to be specific to CREB and plays a central role in the glucagon-mediated activation of gluconeogenesis in the early phase of fasting. We show here that CRTC2 also functions as a coactivator for the glucocorticoid receptor (GR). CRTC2 strongly enhances GR-induced transcriptional activity of glucocorticoid-responsive genes. CRTC2 physically interacts with the ligand-binding domain of the GR through a region spanning amino acids 561-693. Further, CRTC2 is required for the glucocorticoid-associated cooperative mRNA expression of the glucose-6-phosphatase, a rate-limiting enzyme for hepatic gluconeogenesis, by facilitating the attraction of GR and itself to its promoter region already occupied by CREB. CRTC2 is required for the maintenance of blood glucose levels during fasting in mice by enhancing the GR transcriptional activity on both the G6p and phosphoenolpyruvate carboxykinase (Pepck) genes. Finally, CRTC2 modulates the transcriptional activity of the progesterone receptor, indicating that it may influence the transcriptional activity of other steroid/nuclear receptors. Taken together, these results reveal that CRTC2 plays an essential role in the regulation of hepatic gluconeogenesis through coordinated regulation of the glucocorticoid/GR- and glucagon/CREB-signaling pathways on the key genes G6P and PEPCK. PMID:26652733

  19. Cellular regulation by protein phosphorylation.

    PubMed

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert. PMID:23058924

  20. cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth

    PubMed Central

    Chen, Zirong; Lin, Shuibin; Cao, Chunxia; Gimbrone, Nicholas T.; Yang, Rongqiang; Fu, Dongtao A.; Carper, Miranda B.; Haura, Eric B.; Schabath, Matthew B.; Cress, W. Douglas; Kaye, Frederic J.

    2016-01-01

    The LKB1 tumor suppressor gene is frequently mutated and inactivated in non–small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and influences therapeutic responses in preclinical studies; however, specific targeted therapies for lung cancer with LKB1 inactivation are currently unavailable. Here, we have identified a long noncoding RNA (lncRNA) signature that is associated with the loss of LKB1 function. We discovered that LINC00473 is consistently the most highly induced gene in LKB1-inactivated human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression correlated with poor prognosis, and sustained LINC00473 expression was required for the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent cyclic AMP–responsive element–binding protein (CREB)/CREB-regulated transcription coactivator (CRTC) activation. We determined that LINC00473 is a nuclear lncRNA and interacts with NONO, a component of the cAMP signaling pathway, thereby facilitating CRTC/CREB-mediated transcription. Collectively, our study demonstrates that LINC00473 expression potentially serves as a robust biomarker for tumor LKB1 functional status that can be integrated into clinical trials for patient selection and treatment evaluation, and implicates LINC00473 as a therapeutic target for LKB1-inactivated NSCLC. PMID:27140397

  1. Status epilepticus stimulates NDEL1 expression via the CREB/CRE pathway in the adult mouse brain.

    PubMed

    Choi, Yun-Sik; Lee, Boyoung; Hansen, Katelin F; Aten, Sydney; Horning, Paul; Wheaton, Kelin L; Impey, Soren; Hoyt, Kari R; Obrietan, Karl

    2016-09-01

    Nuclear distribution element-like 1 (NDEL1/NUDEL) is a mammalian homolog of the Aspergillus nidulans nuclear distribution molecule NudE. NDEL1 plays a critical role in neuronal migration, neurite outgrowth and neuronal positioning during brain development; however within the adult central nervous system, limited information is available regarding NDEL1 expression and functions. Here, the goal was to examine inducible NDEL1 expression in the adult mouse forebrain. Immunolabeling revealed NDEL1 within the forebrain, including the cortex and hippocampus, as well as the midbrain and hypothalamus. Expression was principally localized to perikarya. Using a combination of immunolabeling and RNA seq profiling, we detected a marked and long-lasting upregulation of NDEL1 expression within the hippocampus following a pilocarpine-evoked repetitive seizure paradigm. Chromatin immunoprecipitation (ChIP) analysis identified a cAMP response element-binding protein (CREB) binding site within the CpG island proximal to the NDEL1 gene, and in vivo transgenic repression of CREB led to a marked downregulation of seizure-evoked NDEL1 expression. Together these data indicate that NDEL1 is inducibly expressed in the adult nervous system, and that signaling via the CREB/CRE transcriptional pathway is likely involved. The role of NDEL1 in neuronal migration and neurite outgrowth during development raises the interesting prospect that inducible NDEL1 in the mature nervous system could contribute to the well-characterized structural and functional plasticity resulting from repetitive seizure activity. PMID:27298008

  2. DISC1 regulates expression of the neurotrophin VGF through the PI3K/AKT/CREB pathway.

    PubMed

    Rodríguez-Seoane, Carmen; Ramos, Adriana; Korth, Carsten; Requena, Jesús R

    2015-11-01

    Disrupted in schizophrenia (DISC1) is a risk factor for chronic mental disease. In a previous proteomic study, we reported that knocking down DISC1 results in a sharp decrease in the levels of the neuropeptide precursor VGF (non-acronymic) and leads to reduced activation of cAMP response element-binding protein (CREB) and protein kinase B (AKT) in neurons. The main objective of this study is to complete the characterization of the route, or routes, involving AKT and CREB through which DISC1 modulates the expression of VGF. For that we explored known players upstream of AKT and the DISC1 binding partners glycogen synthase kinase-3 beta and Phosphodiesterase-4, which might in turn reach out to CREB in murine neuron primary culture. We found that DISC1 modulates the activation of Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Furthermore, pharmacological inhibition of PI3K resulted in decreased expression of VGF. All this suggests that the PI3K/AKT pathway plays a role in mediating the effects of DISC1 silencing on VGF expression. Given the important roles of VGF in mental disease, and its drugability, the DISC1-VGF connection might prove to be important for efforts to develop new therapies for these diseases. PMID:26212236

  3. cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth.

    PubMed

    Chen, Zirong; Li, Jian-Liang; Lin, Shuibin; Cao, Chunxia; Gimbrone, Nicholas T; Yang, Rongqiang; Fu, Dongtao A; Carper, Miranda B; Haura, Eric B; Schabath, Matthew B; Lu, Jianrong; Amelio, Antonio L; Cress, W Douglas; Kaye, Frederic J; Wu, Lizi

    2016-06-01

    The LKB1 tumor suppressor gene is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and influences therapeutic responses in preclinical studies; however, specific targeted therapies for lung cancer with LKB1 inactivation are currently unavailable. Here, we have identified a long noncoding RNA (lncRNA) signature that is associated with the loss of LKB1 function. We discovered that LINC00473 is consistently the most highly induced gene in LKB1-inactivated human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression correlated with poor prognosis, and sustained LINC00473 expression was required for the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent cyclic AMP-responsive element-binding protein (CREB)/CREB-regulated transcription coactivator (CRTC) activation. We determined that LINC00473 is a nuclear lncRNA and interacts with NONO, a component of the cAMP signaling pathway, thereby facilitating CRTC/CREB-mediated transcription. Collectively, our study demonstrates that LINC00473 expression potentially serves as a robust biomarker for tumor LKB1 functional status that can be integrated into clinical trials for patient selection and treatment evaluation, and implicates LINC00473 as a therapeutic target for LKB1-inactivated NSCLC. PMID:27140397

  4. Appetitive Cue-Evoked ERK Signaling in the Nucleus Accumbens Requires NMDA and D1 Dopamine Receptor Activation and Regulates CREB Phosphorylation

    ERIC Educational Resources Information Center

    Kirschmann, Erin K. Z.; Mauna, Jocelyn C.; Willis, Cory M.; Foster, Rebecca L.; Chipman, Amanda M.; Thiels, Edda

    2014-01-01

    Conditioned stimuli (CS) can modulate reward-seeking behavior. This modulatory effect can be maladaptive and has been implicated in excessive reward seeking and relapse to drug addiction. We previously demonstrated that exposure to an appetitive CS causes an increase in the activation of extracellular signal-regulated kinase (ERK) and cyclic-AMP…

  5. Mycobacterium tuberculosis supports protein tyrosine phosphorylation

    PubMed Central

    Kusebauch, Ulrike; Ortega, Corrie; Ollodart, Anja; Rogers, Richard S.; Sherman, David R.; Moritz, Robert L.; Grundner, Christoph

    2014-01-01

    Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ∼1.4 million lives every year. PMID:24927537

  6. Leptin recruits Creb-regulated transcriptional coactivator 1 to improve hyperglycemia in insulin-deficient diabetes

    PubMed Central

    Kim, Geun Hyang; Szabo, Andras; King, Emily M.; Ayala, Jennifer; Ayala, Julio E.; Altarejos, Judith Y.

    2014-01-01

    Objective Leptin alleviates hyperglycemia in rodent models of Type 1 diabetes by activating leptin receptors within the central nervous system. Here we delineate whether non-canonical leptin signaling through the Creb-regulated transcriptional coactivator 1 (Crtc1) contributes to leptin-dependent improvements in diabetic glucose metabolism. Methods We employed mice with a targeted genetic disruption of Crtc1, tracer dilution techniques and neuroanatomical studies to interrogate whether Crtc1 enables leptin to improve glucose metabolism in streptozotocin-induced (STZ) diabetes. Results Here we show that leptin improves diabetic glucose metabolism through Crtc1-dependent and independent mechanisms. We find that leptin reduces diabetic hyperglycemia, hepatic gluconeogenic gene expression and selectively increases glucose disposal to brown adipose tissue and heart, in STZ-diabetic Crtc1WT mice but not Crtc1+/− mice. By contrast, leptin decreases circulating glucagon levels in both STZ-diabetic Crtc1WT and Crtc1+/− mice. We also demonstrate that leptin promotes Crtc1 nuclear translocation in pro-opiomelanocortin (Pomc) and non-Pomc neurons within the hypothalamic arcuate nucleus (ARC). Accordingly, leptin's ability to induce Pomc gene expression in the ARC is blunted in STZ-diabetic Crtc1+/− mice. Conclusions Our study reveals that Crtc1 functions as a conduit for leptin's glucoregulatory actions in insulin-dependent diabetes. This study also highlights a new role for Crtc1 in modulating peripheral glucose metabolism. PMID:25737949

  7. JAB1/CSN5 inhibits the activity of Luman/CREB3 by promoting its degradation

    PubMed Central

    DenBoer, Lisa M.; Iyer, Aarti; McCluggage, Adam R.R.; Li, Yu; Martyn, Amanda C.; Lu, Ray

    2016-01-01

    Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER)-bound transcription factor that has been implicated in the ER stress response. In this study, we used the region of Luman containing the basic DNA-binding domain as bait in a yeast two-hybrid screen and identified the Jun activation domain-binding protein 1 (JAB1) or the COP9 signalosome complex unit 5 (CSN5) as an interacting protein. We confirmed their direct binding by glutathione S-transferase pull-down assays, and verified the existence of such interaction in the cellular environment by mammalian two-hybrid and co-immunoprecipitation assays. Deletion mapping studies revealed that the MPN domain in JAB1 was essential and sufficient for the binding. JAB1 also colocalized with Luman in transfected cells. More interestingly, the nuclear form of Luman was shown to promote the translocation of JAB1 into the nucleus. We found that overexpression of JAB1 shortened the half-life of Luman by 67%, and repressed its transactivation function on GAL4 and unfolded protein response element (UPRE)-containing promoters. We therefore propose that JAB1 is a novel binding partner of Luman, which negatively regulates the activity of Luman by promoting its degradation. PMID:23583719

  8. Sp1 and CREB regulate basal transcription of the human SNF2L gene

    SciTech Connect

    Xia Yu; Jiang Baichun; Zou Yongxin; Gao Guimin; Shang Linshan; Chen Bingxi; Liu Qiji; Gong Yaoqin

    2008-04-04

    Imitation Switch (ISWI) is a member of the SWI2/SNF2 superfamily of ATP-dependent chromatin remodelers, which are involved in multiple nuclear functions, including transcriptional regulation, replication, and chromatin assembly. Mammalian genomes encode two ISWI orthologs, SNF2H and SNF2L. In order to clarify the molecular mechanisms governing the expression of human SNF2L gene, we functionally examined the transcriptional regulation of human SNF2L promoter. Reporter gene assays demonstrated that the minimal SNF2L promoter was located between positions -152 to -86 relative to the transcription start site. In this region we have identified a cAMP-response element (CRE) located at -99 to -92 and a Sp1-binding site at -145 to -135 that play a critical role in regulating basal activity of human SNF2L gene, which were proven by deletion and mutation of specific binding sites, EMSA, and down-regulating Sp1 and CREB via RNAi. This study provides the first insight into the mechanisms that control basal expression of human SNF2L gene.

  9. CREB-binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway.

    PubMed

    Tang, Zhipeng; Yu, Wendan; Zhang, Changlin; Zhao, Shilei; Yu, Zhenlong; Xiao, Xiangsheng; Tang, Ranran; Xuan, Yang; Yang, Wenjing; Hao, Jiaojiao; Xu, Tingting; Zhang, Qianyi; Huang, Wenlin; Deng, Wuguo; Guo, Wei

    2016-02-01

    CBP (CREB-binding protein) is a transcriptional co-activator which possesses HAT (histone acetyltransferases) activity and participates in many biological processes, including embryonic development, growth control and homeostasis. However, its roles and the underlying mechanisms in the regulation of carcinogenesis and tumor development remain largely unknown. Here we investigated the molecular mechanisms and potential targets of CBP involved in tumor growth and survival in lung cancer cells. Elevated expression of CBP was detected in lung cancer cells and tumor tissues compared to the normal lung cells and tissues. Knockdown of CBP by siRNA or inhibition of its HAT activity using specific chemical inhibitor effectively suppressed cell proliferation, migration and colony formation and induced apoptosis in lung cancer cells by inhibiting MAPK and activating cytochrome C/caspase-dependent signaling pathways. Co-immunoprecipitation and immunofluorescence analyses revealed the co-localization and interaction between CBP and CPSF4 (cleavage and polyadenylation specific factor 4) proteins in lung cancer cells. Knockdown of CPSF4 inhibited hTERT transcription and cell growth induced by CBP, and vice versa, demonstrating the synergetic effect of CBP and CPSF4 in the regulation of lung cancer cell growth and survival. Moreover, we found that high expression of both CBP and CPSF4 predicted a poor prognosis in the patients with lung adenocarcinomas. Collectively, our results indicate that CBP regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathways. PMID:26628108

  10. Withaferin A abolishes the stem cell factor-stimulated pigmentation of human epidermal equivalents by interrupting the auto-phosphorylation of c-KIT in human melanocytes.

    PubMed

    Terazawa, Shuko; Nakajima, Hiroaki; Fukasawa, Katsunori; Imokawa, Genji

    2015-01-01

    We characterized the mechanism(s) underlying the abrogating effect of withaferin A (WFA) on the stem cell factor (SCF)-stimulated pigmentation of human epidermal equivalents (HEEs). Increased gene and protein expression levels of tyrosinase, tyrosinase-related protein1, dopachrome tautomerase, PMEL17, c-KIT and their targeted transcription factor, microphthalmia-associated transcription factor (MITF) were significantly reversed at days 7 and 10, respectively, by treatment with WFA. In WFA-treated normal human melanocytes (NHMs), there was a marked deficiency in the SCF-stimulated series of phosphorylations of c-KIT, Shc, Raf-1, MEK, ERK, MITF and CREB. Treatment with dithiothreitol (DTT) distinctly abolished the suppressive effect of WFA on the SCF-stimulated phosphorylation of c-KIT in NHMs. On the other hand, even after incubation at 4 °C for 2 h with 5 nM SCF, followed by the removal of unbound SCF by washing and then raising the temperature to 37 °C to start the signaling reaction, c-KIT was distinctly phosphorylated to a similar extent by incubation for 15 min with SCF only or with SCF + WFA. These findings indicate that WFA attenuates the SCF-induced activation of c-KIT in NHMs by interrupting the auto-phosphorylation of c-KIT through DTT-suppressible Michael addition thioalkylation reactions without interrupting the binding of SCF to the c-KIT receptor. PMID:25376854

  11. β-Hydroxybutyric sodium salt inhibition of growth hormone and prolactin secretion via the cAMP/PKA/CREB and AMPK signaling pathways in dairy cow anterior pituitary cells.

    PubMed

    Fu, Shou-Peng; Wang, Wei; Liu, Bing-Run; Yang, Huan-Min; Ji, Hong; Yang, Zhan-Qing; Guo, Bin; Liu, Ju-Xiong; Wang, Jian-Fa

    2015-01-01

    β-hydroxybutyric acid (BHBA) regulates the synthesis and secretion of growth hormone (GH) and prolactin (PRL), but its mechanism is unknown. In this study, we detected the effects of BHBA on the activities of G protein signaling pathways, AMPK-α activity, GH, and PRL gene transcription, and GH and PRL secretion in dairy cow anterior pituitary cells (DCAPCs). The results showed that BHBA decreased intracellular cAMP levels and a subsequent reduction in protein kinase A (PKA) activity. Inhibition of PKA activity reduced cAMP response element-binding protein (CREB) phosphorylation, thereby inhibiting GH and PRL transcription and secretion. The effects of BHBA were attenuated by a specific Gαi inhibitor, pertussis toxin (PTX). In addition, intracellular BHBA uptake mediated by monocarboxylate transporter 1 (MCT1) could trigger AMPK signaling and result in the decrease in GH and PRL mRNA translation in DCAPCs cultured under low-glucose and non-glucose condition when compared with the high-glucose group. This study identifies a biochemical mechanism for the regulatory action of BHBA on GH and PRL gene transcription, translation, and secretion in DCAPCs, which may be one of the factors that regulate pituitary function during the transition period in dairy cows. PMID:25690038

  12. Modulation of type I interferon induction by porcine reproductive and respiratory syndrome virus and degradation of CREB-binding protein by non-structural protein 1 in MARC-145 and HeLa cells

    SciTech Connect

    Kim, Oekyung; Sun Yan; Lai, Frances W.; Song Cheng; Yoo, Dongwan

    2010-07-05

    Porcine reproductive and respiratory syndrome (PRRS) is an emerged disease of swine characterized by negligible response of type I IFNs and viral persistence. We show that the PRRSV non-structural protein 1 (Nsp1) is the viral component responsible for modulation of IFN response. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. Nsp1 did not block phosphorylation and nuclear translocation of IRF3 but inhibited IRF3 association with CREB-binding protein (CBP) in the nucleus. While IRF3 was stable, CBP was degraded, and CBP degradation was proteasome-dependent, suggesting that CBP degradation is not due to the protease activity of Nsp1 but an intermediary is involved. Our data suggest that the Nsp1-mediated CBP degradation inhibits the recruitment of CBP for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation.

  13. Transcriptional dysregulation in Down syndrome: predictions for altered protein complex stoichiometries and post-translational modifications, and consequences for learning/behavior genes ELK, CREB, and the estrogen and glucocorticoid receptors.

    PubMed

    Gardiner, Katheleen

    2006-05-01

    The phenotype of Down syndrome, trisomy of chromosome 21, is hypothesized to be produced by the increased expression due to gene dosage of normal chromosome 21 genes. Chromosome 21 encodes a number of proteins that, based on experimental evidence or domain composition, are classed as transcription factors or their co-regulators. Other chromosome 21 proteins contribute to post-translational modification of transcription factors, including their phosphorylation, dephosphorylation and sumoylation. Several of these chromosome 21 proteins and the pathways in which they function have overlapping transcription factor specificities. Thus, altered stoichiometry in complexes and altered levels of activation of individual transcription factors may contribute to the Down syndrome phenotype by perturbation of downstream gene expression. Here we review recent data on four chromosome 21 proteins: NRIP1, GABPA, DYRK1A and SUMO3. We discuss the implications for activation of ELK, CREB, C/EBP alpha, beta estrogen and glucocorticoid receptors, and for expression of BDNF. Each of these proteins is relevant to learning, behavior and/or development and therefore perturbation of their activation may contribute to the Down syndrome phenotype. PMID:16502135

  14. Pyrroloquinoline Quinone Stimulates Mitochondrial Biogenesis through cAMP Response Element-binding Protein Phosphorylation and Increased PGC-1α Expression*

    PubMed Central

    Chowanadisai, Winyoo; Bauerly, Kathryn A.; Tchaparian, Eskouhie; Wong, Alice; Cortopassi, Gino A.; Rucker, Robert B.

    2010-01-01

    Bioactive compounds reported to stimulate mitochondrial biogenesis are linked to many health benefits such increased longevity, improved energy utilization, and protection from reactive oxygen species. Previously studies have shown that mice and rats fed diets lacking in pyrroloquinoline quinone (PQQ) have reduced mitochondrial content. Therefore, we hypothesized that PQQ can induce mitochondrial biogenesis in mouse hepatocytes. Exposure of mouse Hepa1–6 cells to 10–30 μm PQQ for 24–48 h resulted in increased citrate synthase and cytochrome c oxidase activity, Mitotracker staining, mitochondrial DNA content, and cellular oxygen respiration. The induction of this process occurred through the activation of cAMP response element-binding protein (CREB) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a pathway known to regulate mitochondrial biogenesis. PQQ exposure stimulated phosphorylation of CREB at serine 133, activated the promoter of PGC-1α, and increased PGC-1α mRNA and protein expression. PQQ did not stimulate mitochondrial biogenesis after small interfering RNA-mediated reduction in either PGC-1α or CREB expression. Consistent with activation of the PGC-1α pathway, PQQ increased nuclear respiratory factor activation (NRF-1 and NRF-2) and Tfam, TFB1M, and TFB2M mRNA expression. Moreover, PQQ protected cells from mitochondrial inhibition by rotenone, 3-nitropropionic acid, antimycin A, and sodium azide. The ability of PQQ to stimulate mitochondrial biogenesis accounts in part for action of this compound and suggests that PQQ may be beneficial in diseases associated with mitochondrial dysfunction. PMID:19861415

  15. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    NASA Astrophysics Data System (ADS)

    Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Vezir Kahraman, Memet

    2014-08-01

    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased.

  16. Single nucleotide polymorphism near CREB1, rs7591784, is associated with pretreatment methamphetamine use frequency and outcome of outpatient treatment for methamphetamine use disorder.

    PubMed

    Heinzerling, Keith G; Demirdjian, Levon; Wu, Yingnian; Shoptaw, Steven

    2016-03-01

    Although stimulant dependence is highly heritable, few studies have examined genetic influences on methamphetamine dependence. We performed a candidate gene study of 52 SNPs and pretreatment methamphetamine use frequency among 263 methamphetamine dependent Hispanic and Non-Hispanic White participants of several methamphetamine outpatient clinical trials in Los Angeles. One SNP, rs7591784 was significantly associated with pretreatment methamphetamine use frequency following Bonferroni correction (p < 0.001) in males but not females. We then examined rs7591784 and methamphetamine urine drug screen results during 12 weeks of outpatient treatment among males with treatment outcome data available (N = 94) and found rs7591784 was significantly associated with methamphetamine use during treatment controlling for pretreatment methamphetamine use. rs7591784 is near CREB1 and in a linkage disequilibrium block with rs2952768, previously shown to influence CREB1 expression. The CREB signaling pathway is involved in gene expression changes related to chronic use of multiple drugs of abuse including methamphetamine and these results suggest that variability in CREB signaling may influence pretreatment frequency of methamphetamine use as well as outcomes of outpatient treatment. Medications targeting the CREB pathway, including phosphodiesterase inhibitors, warrant investigation as pharmacotherapies for methamphetamine use disorders. PMID:26736037

  17. CREB antisense oligodeoxynucleotide administration into the dorsal hippocampal CA3 region impairs long- but not short-term spatial memory in mice

    PubMed Central

    Florian, Cédrick; Mons, Nicole; Roullet, Pascal

    2006-01-01

    The transcription factor cAMP response-element binding protein (CREB) has a pivotal role in hippocampal synaptic plasticity and hippocampus-dependent long-term memory. We recently demonstrated that the dorsal hippocampal CA3 region is involved in memory consolidation of spatial information tested on a Morris water maze in mice. To test whether activation of CREB in the CA3 region is required for memory consolidation of spatial information, bilaterally cannulated mice were infused 18 h before the beginning of the behavioral training with antisense or control sense CREB oligodeoxynucleotides (ODNs) or buffer. Mice were then subjected to massed training in a spatial version of the water maze and tested for retention 0 or 24 h after the last training session. We showed that CREB antisense ODN-infusion in the CA3 region impaired long-term memory when tested 24 h later but had no effect on spatial acquisition or short-term memory tested immediately after behavioral training. These findings provide evidence that the regionally restricted activation of CREB in the dorsal hippocampal CA3 region is critical for the long-term memory consolidation phase of spatial learning but not for short-term memory. PMID:16882863

  18. Starch phosphorylation: insights and perspectives.

    PubMed

    Mahlow, Sebastian; Orzechowski, Sławomir; Fettke, Joerg

    2016-07-01

    During starch metabolism, the phosphorylation of glucosyl residues of starch, to be more precise of amylopectin, is a repeatedly observed process. This phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD) and the phosphoglucan, water dikinase (PWD). The starch-related dikinases utilize ATP as dual phosphate donor transferring the terminal γ-phosphate group to water and the β-phosphate group selectively to either C6 position or C3 position of a glucosyl residue within amylopectin. By the collaborative action of both enzymes, the initiation of a transition of α-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation. Consequently, mutants lacking either GWD or PWD reveal a starch excess phenotype as well as growth retardation. In this review, we focus on the increased knowledge collected over the last years related to enzymatic properties, the precise definition of the substrates, the physiological implications, and discuss ongoing questions. PMID:27147464

  19. Histone tyrosine phosphorylation comes of age

    PubMed Central

    Singh, Rakesh Kumar

    2011-01-01

    Histones were discovered over a century ago and have since been found to be the most extensively post-translationally modified proteins, although tyrosine phosphorylation of histones had remained elusive until recently. The year 2009 proved to be a landmark year for histone tyrosine (Y) phosphorylation as five research groups independently discovered this modification. Three groups describe phosphorylation of Y142 in the variant histone H2A.X, where it may be involved in the cellular decision making process to either undergo DNA repair or apoptosis in response to DNA damage. Further, one group suggests that phosphorylation of histone H3 on Y99 is crucial for its regulated proteolysis in yeast, while another found that Y41 phosphorylation modulates chromatin architecture and oncogenesis in mammalian cells. These pioneering studies provide the initial conceptual framework for further analyses of the diverse roles of tyrosine phosphorylation on different histones, with far reaching implications for human health and disease. PMID:20935492

  20. Prebiotic phosphorylation of nucleosides in formamide

    NASA Technical Reports Server (NTRS)

    Schoffstall, A. M.

    1976-01-01

    Results are presented for an experimental study intended to assess phosphorylation under neither aqueous nor dry thermal conditions. Instead, phosphorylations were attempted in possible nonaqueous prebiotic solvents. Formamide appeared to be the most obvious candidate for phosphorylation studies. Three main classes of phosphorylated products were formed in formamide solution: adenosine monophosphates, cyclic adenosine phosphate, and adenosine diphosphates. Experiments were designed to investigate the extent of phosphorylation of nucleosides in formamide, the relative amounts of nucleoside monophosphate, diphosphates and cyclic phosphate formed and the relative effectiveness of different sources of phosphate as phosphorylating agents in formamide. Reaction variables were temperature, nature of the phosphate or condensed phosphate, nucleoside, concentration of reactants and possible effects of additives. Product identification was based on qualitative and quantitative thin layer chromatography.

  1. [Sugar phosphorylation activities in acetogenic bacteria].

    PubMed

    Jiang, W; Patterson, J A

    1999-12-01

    Seven acetogenic bacteria (Acetitomaculum ruminis, Acetobacterium woodii, Eubacterium limosum as well as isolates A2, A4, A10 and H3HH) were tested for PEP- and ATP-dependent phosphorylation of glucose and 2-deoxyglucose. Although all organisms had detectable phosphorylation activity, substantial variation existed in the rates of both PEP- and ATP-dependent phosphorylation. Isolate Alo had the highest rate of PEP-dependent phosphorylation of 11.62 nmol.L-1.mg-1.min-1. Isolate A10, H3HH as well as E. limosum most likely have a glucose phosphotransferase system(PTS). In contrast, A ruminis, A. woodii and isolate A2, A4 had PEP-dependent glucose phosphorylation rates very similar to control rates, suggesting the lack of PTS activity. The rates of ATP-dependent glucose phosphorylation were higher than PEP-dependent phosphorylation in all organisms surveyed. However, substantial variation existed in the rates of ATP-dependent glucose phosphorylation. The glucose PTS of isolates A10 and H3HH were induced by the presence of extracellular glucose. Moreover, the specific activity of the glucose PTS of both isolates increased as cultures progressed from the early log to late log phase of growth. ATP- and PEP-dependent maltose and sucrose phosphorylation was detected in isolates A10 and H3HH. Although activity was detected in both isolates(A10 and H3HH), the rate of activity varied considerably, depending on the sugar and organism tested. PMID:12555560

  2. The CREB-binding protein inhibitor ICG-001 suppresses pancreatic cancer growth.

    PubMed

    Arensman, Michael D; Telesca, Donatello; Lay, Anna R; Kershaw, Kathleen M; Wu, Nanping; Donahue, Timothy R; Dawson, David W

    2014-10-01

    Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer due in part to a lack of highly robust cytotoxic or molecular-based therapies. Recent studies investigating ligand-mediated Wnt/β-catenin signaling have highlighted its importance in pancreatic cancer initiation and progression, as well as its potential as a therapeutic target in PDAC. The small-molecule ICG-001 binds cAMP-responsive element binding (CREB)-binding protein (CBP) to disrupt its interaction with β-catenin and inhibit CBP function as a coactivator of Wnt/β-catenin-mediated transcription. Given its ability to inhibit Wnt/β-catenin-mediated transcription in vitro and in vivo, as well as its efficacy in preclinical models of colorectal cancer and other Wnt-driven diseases, we examined ICG-001 and its potential role as a therapeutic in PDAC. ICG-001 alone significantly inhibited anchorage-dependent and -independent growth of multiple PDAC lines, and augmented in vitro growth inhibition when used in combination with gemcitabine. ICG-001 had only variable modest effects on PDAC apoptosis and instead mediated PDAC growth inhibition primarily through robust induction of G₁ cell-cycle arrest. These effects, however, seemed decoupled from its inhibition of Wnt/β-catenin-mediated transcription. DNA microarrays performed on PDAC cells in the context of ICG-001 treatment revealed ICG-001 altered the expression of several genes with well-established roles in DNA replication and cell-cycle progression, including direct actions on SKP2 and CDKN1A. ICG-001 also significantly prolonged survival in an in vivo orthotopic xenograft model of PDAC, indicating ICG-001 or derived compounds that disrupt CBP activity are potentially useful small-molecule therapeutics for pancreatic cancer. PMID:25082960

  3. Transcriptional regulation of miR-15b by c-Rel and CREB in Japanese encephalitis virus infection

    PubMed Central

    Zhu, Bibo; Ye, Jing; Ashraf, Usama; Li, Yunchuan; Chen, Huanchun; Song, Yunfeng; Cao, Shengbo

    2016-01-01

    MicroRNAs (miRNAs) have been well known to play diverse roles in viral infection at the level of posttranscriptional repression. However, much less is understood about the mechanism by which miRNAs are regulated during viral infection. It is likely that both host and virus contain factors to modulate miRNA expression. Here we report the up-regulation of microRNA-15b (miR-15b) in vitro upon infection with Japanese encephalitis virus (JEV). Analysis of miR-15b precursor, pri-miR-15b and pre-miR-15b, suggest that the regulation occurs transcriptionally. Further, we identified the transcriptional regulatory region of miR-15b that contains consensus binding motif for NF-κB subunit c-Rel and cAMP-response element binding protein (CREB), which are known as transcription factor to regulate gene expression. By promoter fusion and mutational analyses, we demonstrated that c-Rel and CREB bind directly to the promoter elements of miR-15b, which are responsible for miR-15b transcription in response to JEV infection. Finally, we showed that pharmacological inhibition of ERK and NF-κB signaling pathway blocked induction of miR-15b in JEV infection, suggesting important roles of ERK and NF-κB pathway in the regulation of miR-15b gene. Therefore, our observations indicate that induced expression of miR-15b is modulated by c-Rel and CREB in response to JEV infection. PMID:26931521

  4. Transcriptional regulation of miR-15b by c-Rel and CREB in Japanese encephalitis virus infection.

    PubMed

    Zhu, Bibo; Ye, Jing; Ashraf, Usama; Li, Yunchuan; Chen, Huanchun; Song, Yunfeng; Cao, Shengbo

    2016-01-01

    MicroRNAs (miRNAs) have been well known to play diverse roles in viral infection at the level of posttranscriptional repression. However, much less is understood about the mechanism by which miRNAs are regulated during viral infection. It is likely that both host and virus contain factors to modulate miRNA expression. Here we report the up-regulation of microRNA-15b (miR-15b) in vitro upon infection with Japanese encephalitis virus (JEV). Analysis of miR-15b precursor, pri-miR-15b and pre-miR-15b, suggest that the regulation occurs transcriptionally. Further, we identified the transcriptional regulatory region of miR-15b that contains consensus binding motif for NF-κB subunit c-Rel and cAMP-response element binding protein (CREB), which are known as transcription factor to regulate gene expression. By promoter fusion and mutational analyses, we demonstrated that c-Rel and CREB bind directly to the promoter elements of miR-15b, which are responsible for miR-15b transcription in response to JEV infection. Finally, we showed that pharmacological inhibition of ERK and NF-κB signaling pathway blocked induction of miR-15b in JEV infection, suggesting important roles of ERK and NF-κB pathway in the regulation of miR-15b gene. Therefore, our observations indicate that induced expression of miR-15b is modulated by c-Rel and CREB in response to JEV infection. PMID:26931521

  5. Changes in CREB and deltaFosB are associated with the behavioural sensitization induced by methylenedioxypyrovalerone.

    PubMed

    Buenrostro-Jáuregui, Mario; Ciudad-Roberts, Andres; Moreno, Josep; Muñoz-Villegas, Patricia; López-Arnau, Raúl; Pubill, David; Escubedo, Elena; Camarasa, Jorge

    2016-07-01

    Methylenedioxypyrovalerone (MDPV) is a synthetic cathinone which has recently emerged as a designer drug of abuse. The objective of this study was to investigate the locomotor sensitization induced by MDPV in adolescent mice, and associated neuroplastic changes in the nucleus accumbens and striatum through deltaFosB and CREB expression. Behavioural testing consisted of three phases: Phase I: conditioning regimen with MDPV (0.3 mg/kg/day for five days) or saline; Phase II: resting (11 days); Phase III: challenged with MDPV (0.3 mg/kg), cocaine (10 mg/kg) or saline on day 16 for both groups. Mice repeatedly exposed to MDPV increased locomotor activity by 165-200% following acute MDPV or cocaine administration after an 11-day resting period, showing a MDPV-induced sensitization to itself and to cocaine. An explanation for this phenomenon could be the common mechanism of action between these two psychostimulants. Furthermore, the MDPV challenge resulted in higher levels of phospho-CREB in MDPV-conditioned mice compared with MDPV-naive mice, probably due to an up-regulation of the cAMP pathway. Likewise, MDPV exposure induced a persistent increase in the striatal expression of deltaFosB; the priming dose of MDPV also produced a significant increase in the accumbal expression of this transcription factor. This study constitutes the first evidence that an exposure to a low dose of MDPV during adolescence induces behavioural sensitization and provides a neurobiological basis for a relationship between MDPV and cocaine. We hypothesize that, similar to cocaine, both CREB and deltaFosB play a role in the induction of this behavioural sensitization. PMID:27147595

  6. Different effects of prenatal stress on ERK2/CREB/Bcl-2 expression in the hippocampus and the prefrontal cortex of adult offspring rats.

    PubMed

    Zhu, Zeen; Sun, Hongli; Gong, Xiaojie; Li, Hui

    2016-05-25

    It has become increasingly evident that prenatal stress and its psychological and physiological concomitants are associated with the pathophysiology of mood disorders. However, the mechanisms underlying the prenatal stress-induced offspring's anxiety disorders remain unknown. We recently reported that prenatal stress enhanced anxiety-like behavior in adult offspring rat, and involved N-methyl-D-aspartate receptor subunits, including NR1 and NR2A. In the present research, using the same prenatal stress model, we measured the ERK2/CREB/Bcl-2 mRNA levels by real-time PCR. Our findings indicated that prenatal stress decreased ERK2 and CREB mRNA levels in the hippocampus and the prefrontal cortex and Bcl-2 mRNA levels in the hippocampus of offspring rat. The results showed that the abnormal ERK2, CREB, and Bcl-2 mRNA levels may be involved in the anxiety-like behavior of adult rats with prenatal stress. PMID:27096215

  7. Aluminium chloride impairs long-term memory and downregulates cAMP-PKA-CREB signalling in rats.

    PubMed

    Zhang, Lifeng; Jin, Cuihong; Lu, Xiaobo; Yang, Jinghua; Wu, Shengwen; Liu, Qiufang; Chen, Rong; Bai, Chunyu; Zhang, Di; Zheng, Linlin; Du, Yanqiu; Cai, Yuan

    2014-09-01

    Epidemiological investigations have indicated that aluminium (Al) is an important environmental neurotoxicant that may be involved in the aetiology of the cognitive dysfunction associated with neurodegenerative diseases. Additionally, exposure to Al is known to cause neurobehavioural abnormalities in animals. Previous studies demonstrated that Al impaired early-phase long-term potentiation (E-LTP) in vivo and in vitro. Our previous research revealed that Al could impair long-term memory via the impairment of late-phase long-term potentiation (L-LTP) in vivo. However, the exact mechanism by which Al impairs long-term memory has been poorly studied thus far. This study was designed not only to observe the effects of subchronic Al treatment on long-term memory and hippocampal ultrastructure but also to explore a possible underlying mechanism (involving the cAMP-PKA-CREB signalling pathway) in the hippocampus of rats.. Pregnant Wistar rats were assigned to four groups. Neonatal rats were exposed to Al by parental lactation for 3 weeks and then fed with distilled water containing 0, 0.2%, 0.4% or 0.6% Al chloride (AlCl3) for 3 postnatal months. The levels of Al in the blood and hippocampus were quantified by atomic absorption spectrophotometry. The shuttle-box test was performed to detect long-term memory. The hippocampus was collected for ultrastructure observation, and the level of cAMP-PKA-CREB signalling was examined. The results showed that the Al concentrations in the blood and hippocampus of Al-treated rats were higher than those of the control rats. Al may impair the long-term memory of rats. Hippocampal cAMP, cPKA, pCREB, BDNF and c-jun expression decreased significantly, and the neuronal and synaptic ultrastructure exhibited pathological changes after Al treatment. These results indicated that Al may induce long-term memory damage in rats by inhibiting cAMP-PKA-CREB signalling and altering the synaptic and neuronal ultrastructure in the hippocampus. PMID

  8. Phosphorylation of human link proteins

    SciTech Connect

    Oester, D.A.; Caterson, B.; Schwartz, E.R.

    1986-06-13

    Three link proteins of 48, 44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain /sup 32/P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO/sub 4//mole link protein.

  9. Phosphorylation of the translation initiation factor eIF2α at serine 51 determines the cell fate decisions of Akt in response to oxidative stress.

    PubMed

    Rajesh, K; Krishnamoorthy, J; Kazimierczak, U; Tenkerian, C; Papadakis, A I; Wang, S; Huang, S; Koromilas, A E

    2015-01-01

    Phosphorylation of the α subunit of the translation initiation factor eIF2 at serine 51 (eIF2αP) is a master regulator of cell adaptation to various forms of stress with implications in antitumor treatments with chemotherapeutic drugs. Herein, we demonstrate that genetic loss of the eIF2α kinases PERK and GCN2 or impaired eIF2αP by genetic means renders immortalized mouse fibroblasts as well as human tumor cells increasingly susceptible to death by oxidative stress. We also show that eIF2αP facilitates Akt activation in cells subjected to oxidative insults. However, whereas Akt activation has a pro-survival role in eIF2αP-proficient cells, the lesser amount of activated Akt in eIF2αP-deficient cells promotes death. At the molecular level, we demonstrate that eIF2αP acts through an ATF4-independent mechanism to control Akt activity via the regulation of mTORC1. Specifically, eIF2αP downregulates mTORC1 activity, which in turn relieves the feedback inhibition of PI3K resulting in the upregulation of the mTORC2-Akt arm. Inhibition of mTORC1 by rapamycin restores Akt activity in eIF2αP-deficient cells but renders them highly susceptible to Akt-mediated death by oxidative stress. Our data demonstrate that eIF2αP acts as a molecular switch that dictates either cell survival or death by activated Akt in response to oxidative stress. Hence, we propose that inactivation of eIF2αP may be a suitable approach to unleash the killing power of Akt in tumor cells treated with pro-oxidant drugs. PMID:25590801

  10. Functional Impact of Corticotropin-Releasing Factor Exposure on Tau Phosphorylation and Axon Transport

    PubMed Central

    Le, Michelle H.; Weissmiller, April M.; Monte, Louise; Lin, Po Han; Hexom, Tia C.; Natera, Orlangie; Wu, Chengbiao; Rissman, Robert A.

    2016-01-01

    Stress exposure or increased levels of corticotropin-releasing factor (CRF) induce hippocampal tau phosphorylation (tau-P) in rodent models, a process that is dependent on the type-1 CRF receptor (CRFR1). Although these preclinical studies on stress-induced tau-P provide mechanistic insight for epidemiological work that identifies stress as a risk factor for Alzheimer’s disease (AD), the actual impact of stress-induced tau-P on neuronal function remains unclear. To determine the functional consequences of stress-induced tau-P, we developed a novel mouse neuronal cell culture system to explore the impact of acute (0.5hr) and chronic (2hr) CRF treatment on tau-P and integral cell processes such as axon transport. Consistent with in vivo reports, we found that chronic CRF treatment increased tau-P levels and caused globular accumulations of phosphorylated tau in dendritic and axonal processes. Furthermore, while both acute and chronic CRF treatment led to significant reduction in CREB activation and axon transport of brain-derived neurotrophic factor (BDNF), this was not the case with mitochondrial transport. Acute CRF treatment caused increased mitochondrial velocity and distance traveled in neurons, while chronic CRF treatment modestly decreased mitochondrial velocity and greatly increased distance traveled. These results suggest that transport of cellular energetics may take priority over growth factors during stress. Tau-P was required for these changes, as co-treatment of CRF with a GSK kinase inhibitor prevented CRF-induced tau-P and all axon transport changes. Collectively, our results provide mechanistic insight into the consequences of stress peptide-induced tau-P and provide an explanation for how chronic stress via CRF may lead to neuronal vulnerability in AD. PMID:26790099

  11. Beta-adrenergic stimulation of cFOS via protein kinase A is mediated by cAMP regulatory element binding protein (CREB)-dependent and tissue-specific CREB-independent mechanisms in corticotrope cells.

    PubMed

    Boutillier, A L; Barthel, F; Roberts, J L; Loeffler, J P

    1992-11-25

    Catecholamines stimulate proopiomelanocortin (POMC) gene expression in corticotrope cells, but the molecular mechanisms of these effects are not known. While beta-adrenergic receptors stimulate the protein kinase A (PKA) system, the POMC promoter does not have classical cAMP-response elements (CREs). Therefore, we investigated the induction of the c-fos protooncogen, previously shown to increase POMC transcription in AtT20 cells. In this corticotrope-derived cell line, we show that activation of beta-receptors with isoprenaline (Iso) induces a transient rise in c-fos mRNA levels. Gel mobility shift assays with a labeled AP1 consensus sequence (TGACTCA) showed induction of specific binding activity after Iso treatment. Cotransfection experiments with dominant inhibitory PKA mutants and reporter genes containing c-fos promoter sequences showed that c-fos induction by Iso is entirely dependent on a functional PKA activity. Furthermore, we show that beta-receptor induction of c-fos in corticotrophs is mediated by at least two distinct cAMP-responsive sequences. cAMP regulatory element binding (CREB)-dependent induction is observed on the CRE located at -60 bp on the c-fos promoter. A region located in the vicinity of the dyad symetry element (-290) is also found to mediate tissue-specific cAMP induction. Transcriptional activation by this site, although sensitive to PKA antagonism, is not blocked by CREB mutants. PMID:1331087

  12. Phosphorylation of the multidrug resistance associated glycoprotein

    SciTech Connect

    Mellado, W.; Horwitz, S.B.

    1987-11-03

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistant phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl/sub 2/ was enhanced a minimum of 2-fold by 10 ..mu..M cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by (..gamma..-/sup 32/P)ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.

  13. Phosphorylation of the multidrug resistance associated glycoprotein.

    PubMed

    Mellado, W; Horwitz, S B

    1987-11-01

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance. PMID:3427052

  14. CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by hepatocyte growth factor

    SciTech Connect

    Li, Xin-Yu; Zhan, Xiao-Rong; Liu, Xiao-Min; Wang, Xiao-Chen

    2011-01-14

    Research highlights: {yields} CREB is a regulatory target for the protein kinase Akt/PKB in pancreatic duct cells. {yields} Activation of the PI3K/AKT/CREB pathway plays a critical role in the HGF-mediated differentiation of pancreatic duct cells in vivo. {yields} CREB was causally linked to the expression of transcription factors during PDEC differentiation induced by HGF. -- Abstract: We have previously reported that the PI3K/Akt signaling pathway is involved in hepatocyte growth factor (HGF)-induced differentiation of adult rat pancreatic ductal epithelial cells (PDECs) into islet {beta}-cells in vitro. The transcription factor CREB is one of the downstream key effectors of the PI3K/Akt signaling pathway. Recent studies showing that CREB is required for the survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the HGF-dependent Ser/Thr kinase Akt/PKB in the differentiation of pancreatic duct cell into islet {beta}-cells. In this study, we first attempted to examine whether HGF modulates the Akt-dependent activation of target gene CREB and then investigated whether CREB activity affects the differentiation of HGF-induced PDECs. Finally, we studied the role of CREB in modulating the expression of transcription factors in PDECs during the differentiation of HGF-induced PDECs. Our results demonstrated that CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by HGF.

  15. Cisplatin stimulates protein tyrosine phosphorylation in macrophages.

    PubMed

    Kumar, R; Shrivastava, A; Sodhi, A

    1995-03-01

    Cisplatin [cis-dichlorodiamine platinum (II)], a potent anti-tumor compound, stimulates immune responses by activating monocyte-macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event that mediates cellular responses, we examined whether cisplatin alters tyrosine phosphorylation in macrophages. We found that cisplatin increased tyrosine phosphorylation of several proteins in peritoneal macrophages and in P388D1 and IC-21 macrophage cell lines. Treatment of macrophages with tyrosine kinase inhibitors, genestein and lavendustin A, inhibited cisplatin-stimulated protein tyrosine phosphorylation in macrophages. Macrophages treated with cisplatin also exhibit increased fluorescence with anti-phosphotyrosine-FITC antibody. These data indicate that protein tyrosine phosphorylation plays a role in cisplatin-induced activation of macrophages. PMID:7539662

  16. Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene mcu

    PubMed Central

    Shanmughapriya, Santhanam; Rajan, Sudarsan; Hoffman, Nicholas E.; Zhang, Xueqian; Guo, Shuchi; Kolesar, Jill E.; Hines, Kevin J.; Ragheb, Jonathan; Jog, Neelakshi R.; Caricchio, Roberto; Baba, Yoshihiro; Zhou, Yandong; Kaufman, Brett; Cheung, Joseph Y.; Kurosaki, Tomohiro; Gill, Donald L.; Madesh, Muniswamy

    2016-01-01

    Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function and, when cytosolic Ca2+ concentrations become too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+-permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+-regulated transcription factor CREB directly bound the mcu promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulate mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+-dependent activation of CREB controls the Ca2+-uptake capability of mitochondria and hence regulates mitochondrial metabolism. PMID:25737585

  17. Macrophage-derived BAFF induces AID expression through the p38MAPK/CREB and JNK/AP-1 pathways.

    PubMed

    Kim, Hyun-A; Seo, Goo-Young; Kim, Pyeung-Hyeun

    2011-03-01

    BAFF is expressed primarily by macrophages and DCs. BAFF stimulates the differentiation and survival of B cells and induces Ig production. We have demonstrated previously that murine macrophages treated with TGF-β1 or IFN-γ express membrane-bound and soluble forms of BAFF. The ability of these two forms of BAFF to induce expression of AID, which plays a critical role in Ig CSR in B cells, was investigated. Both forms of BAFF, derived from macrophages activated by IFN-γ or TGF-β1, can increase AID expression. Subsequent analysis of BAFF signaling suggested that BAFF induces AID through BCMA, a BAFF-receptor, and p38MAPK and CREB act as intermediates in AID expression. In addition, JNK and AP-1 have similar activities. Our findings suggest that macrophage-derived BAFF stimulates B cells to express AID through BCMA and at least two different pathways, including the p38MAPK/CREB and the JNK/AP-1 pathways. PMID:21169521

  18. Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU.

    PubMed

    Shanmughapriya, Santhanam; Rajan, Sudarsan; Hoffman, Nicholas E; Zhang, Xueqian; Guo, Shuchi; Kolesar, Jill E; Hines, Kevin J; Ragheb, Jonathan; Jog, Neelakshi R; Caricchio, Roberto; Baba, Yoshihiro; Zhou, Yandong; Kaufman, Brett A; Cheung, Joseph Y; Kurosaki, Tomohiro; Gill, Donald L; Madesh, Muniswamy

    2015-03-01

    Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+ -permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+ -regulated transcription factor CREB (cyclic adenosine monophosphate response element-binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+ -dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism. PMID:25737585

  19. A negative feedback control of transforming growth factor-beta signaling by glycogen synthase kinase 3-mediated Smad3 linker phosphorylation at Ser-204.

    PubMed

    Millet, Caroline; Yamashita, Motozo; Heller, Mary; Yu, Li-Rong; Veenstra, Timothy D; Zhang, Ying E

    2009-07-24

    Through the action of its membrane-bound type I receptor, transforming growth factor-beta (TGF-beta) elicits a wide range of cellular responses that regulate cell proliferation, differentiation, and apo ptosis. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-beta and its related factors. Here, we show that TGF-beta induces phosphorylation at three sites in the Smad3 linker region in addition to the two C-terminal residues, and glycogen synthase kinase 3 is responsible for phosphorylation at one of these sites, namely Ser-204. Alanine substitution at Ser-204 and/or the neighboring Ser-208, the priming site for glycogen synthase kinase 3 in vivo activity, strengthened the affinity of Smad3 to CREB-binding protein, suggesting that linker phosphorylation may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of the Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-beta. PMID:19458083

  20. Virus-Dependent Phosphorylation of the IRF-3 Transcription Factor Regulates Nuclear Translocation, Transactivation Potential, and Proteasome-Mediated Degradation

    PubMed Central

    Lin, Rongtuan; Heylbroeck, Christophe; Pitha, Paula M.; Hiscott, John

    1998-01-01

    The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-α/β) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of IRF-3. A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation. Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome inhibitors. Interestingly, virus infection resulted in the association of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-396 and

  1. Oxidative phosphorylation and lacunar stroke

    PubMed Central

    Anderson, Christopher D.; Hurford, Robert; Bevan, Steve; Markus, Hugh S.

    2016-01-01

    Objective: We investigated whether oxidative phosphorylation (OXPHOS) abnormalities were associated with lacunar stroke, hypothesizing that these would be more strongly associated in patients with multiple lacunar infarcts and leukoaraiosis (LA). Methods: In 1,012 MRI-confirmed lacunar stroke cases and 964 age-matched controls recruited from general practice surgeries, we investigated associations between common genetic variants within the OXPHOS pathway and lacunar stroke using a permutation-based enrichment approach. Cases were phenotyped using MRI into those with multiple infarcts or LA (MLI/LA) and those with isolated lacunar infarcts (ILI) based on the number of subcortical infarcts and degree of LA, using the Fazekas grading. Using gene-level association statistics, we tested for enrichment of genes in the OXPHOS pathway with all lacunar stroke and the 2 subtypes. Results: There was a specific association with strong evidence of enrichment in the top 1% of genes in the MLI/LA (subtype p = 0.0017) but not in the ILI subtype (p = 1). Genes in the top percentile for the all lacunar stroke analysis were not significantly enriched (p = 0.07). Conclusions: Our results implicate the OXPHOS pathway in the pathogenesis of lacunar stroke, and show the association is specific to patients with the MLI/LA subtype. They show that MRI-based subtyping of lacunar stroke can provide insights into disease pathophysiology, and imply that different radiologic subtypes of lacunar stroke subtypes have distinct underlying pathophysiologic processes. PMID:26674331

  2. In the Beginning, There Was Protein Phosphorylation

    PubMed Central

    Kyriakis, John M.

    2014-01-01

    The importance of reversible protein phosphorylation to cellular regulation cannot be overstated. In eukaryotic cells, protein kinase/phosphatase signaling pathways regulate a staggering number of cellular processes, including cell proliferation, cell death (apoptosis, necroptosis, necrosis), metabolism (at both the cellular and organismal levels), behavior and neurological function, development, and pathogen resistance. Although protein phosphorylation as a mode of eukaryotic cell regulation is familiar to most biochemists, many are less familiar with protein kinase/phosphatase signaling networks that function in prokaryotes. In this thematic minireview series, we present four minireviews that cover the important field of prokaryotic protein phosphorylation. PMID:24554697

  3. Activation of PTHrP-cAMP-CREB1 signaling following p53 loss is essential for osteosarcoma initiation and maintenance.

    PubMed

    Walia, Mannu K; Ho, Patricia Mw; Taylor, Scott; Ng, Alvin Jm; Gupte, Ankita; Chalk, Alistair M; Zannettino, Andrew Cw; Martin, T John; Walkley, Carl R

    2016-01-01

    Mutations in the P53 pathway are a hallmark of human cancer. The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53. In contrast to P53 point mutations in other cancer, complete loss of P53 is a frequent event in osteosarcoma (OS), the most common cancer of bone. The consequences of p53 loss for osteoblastic cells and OS development are poorly understood. Here we use murine OS models to demonstrate that elevated Pthlh (Pthrp), cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS. Normal osteoblasts survive depletion of both PTHrP and CREB1. In contrast, p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1. Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS. PMID:27070462

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

    PubMed

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

    1995-11-25

    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. PMID:8524640

  5. Interaction of the human T-lymphotropic virus type 1 Tax dimer with CREB and the viral 21-base-pair repeat.

    PubMed Central

    Tie, F; Adya, N; Greene, W C; Giam, C Z

    1996-01-01

    Human T-lymphotropic virus type 1 Tax interacts specifically with the cellular transcription factor CREB and the viral 21-bp repeat element to form a Tax-CREB-DNA ternary complex which mediates activation of viral mRNA transcription. Analyses of Tax and Tax mutants indicate that, like CREB, Tax incorporates into the ternary complex as a dimer. The ability of Tax to form a dimer is necessary for its interaction with CREB and the 21-bp element. Analyses of several Tax mutants with amino acid substitutions spanning residues 123 to 204 indicate that intersubunit Tax dimerization correlates with its ability to assemble into the ternary complex and activate transcription. Tax also enhances the DNA binding activities of specific bZip domains in vitro. The ability of Tax to enhance DNA binding of bZip proteins can be explained in part by Tax dimerization. This activity alone is not sufficient for transactivation. A dual amino acid substitution mutant of Tax, M47 (L319R, L320S), completely abrogated for activation of the human T-lymphotropic virus type 1 long terminal repeat as a result of a defect in the transactivation domain, continues to stimulate binding of bZip proteins to DNA. PMID:8970957

  6. CREB Antisense Oligodeoxynucleotide Administration into the Dorsal Hippocampal CA3 Region Impairs Long- but Not Short-Term Spatial Memory in Mice

    ERIC Educational Resources Information Center

    Florian, Cedrick; Mons, Nicole; Roullet, Pascal

    2006-01-01

    The transcription factor cAMP response-element binding protein (CREB) has a pivotal role in hippocampal synaptic plasticity and hippocampus-dependent long-term memory. We recently demonstrated that the dorsal hippocampal CA3 region is involved in memory consolidation of spatial information tested on a Morris water maze in mice. To test whether…

  7. PFOS Disturbs BDNF-ERK-CREB Signalling in Association with Increased MicroRNA-22 in SH-SY5Y Cells

    PubMed Central

    Li, Wu; He, Qing-zhi; Wu, Cheng-qiu; Pan, Xiao-yuan; Wang, Jing; Tan, Yan; Shan, Xiao-yun; Zeng, Huai-cai

    2015-01-01

    Perfluorooctane sulfonate (PFOS), a ubiquitous environmental pollutant, is neurotoxic to mammalian species. However, the underlying mechanism of its neurotoxicity was unclear. We hypothesized that PFOS suppresses BDNF expression to produce its neurotoxic effects by inhibiting the ERK-CREB pathway. SH-SY5Y human neuroblastoma cells were exposed to various concentrations of PFOS to examine the role of the BDNF-ERK-CREB signalling pathway in PFOS-induced apoptosis and cytotoxicity. Furthermore, to ascertain the mechanism by which PFOS reduces BDNF signalling, we examined the expression levels of miR-16 and miR-22, which potentially regulate BDNF mRNA translation at the posttranscriptional level. Results indicated that PFOS significantly decreased cell viability and induced apoptosis in SH-SY5Y cells. In addition, BDNF and pERK protein levels decreased after PFOS treatment; however, pCREB protein levels were significantly elevated in PFOS treated groups. TrkB protein expression increased in the 10 μM and 50 μM PFOS groups and significantly decreased in the 100 μM PFOS group. Our results demonstrated that PFOS exposure decreased miR-16 expression and increased miR-22 expression, which may represent a possible mechanism by which PFOS decreases BDNF protein levels. PFOS may inhibit BDNF-ERK-CREB signalling by increasing miR-22 levels, which may, in part, explain the mechanism of PFOS neurotoxicity. PMID:26649298

  8. An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DR alpha promoter and activation by SV40 T-antigen.

    PubMed Central

    Cox, P M; Goding, C R

    1992-01-01

    Constitutive expression of major histocompatibility complex class II (MHC II) antigens normally occurs in B-lymphocytes and antigen presenting cells of the monocyte/macrophage lineage. However, many malignant tumours and transformed cells express these proteins aberrantly. We demonstrate here that the MHC II DR alpha promoter is constitutively active both in the SV40 large T antigen transformed cell line, COS, and in CV1 cells from which they are derived. As an approach to understanding the molecular mechanisms underlying aberrant DR alpha expression we have examined the cis- and trans-acting requirements for DR alpha transcription in these cell types. Electrophoretic mobility shift assays showed that the region immediately 3' to the X-box was bound by a member of the ATF/CREB family of transcription factors. Using deletions and point mutations in the DR alpha promoter we demonstrate that, in contrast to B-cells, the octamer motif and conserved X- and Y-boxes make only a minor contribution to promoter function while single point mutations in the ATF/CREB motif reduced transcription up to 20-fold. In addition, we show that the DR alpha promoter is activated by SV40 large T-antigen and that activation requires an intact ATF/CREB motif. Similar data were obtained using B16 melanoma cells. These results suggest that the ATF/CREB motif may be a target for transcription deregulation in several transformed cell types. Images PMID:1329030

  9. Transgenic Mice Expressing a Truncated Form of CREB-Binding Protein (CBP) Exhibit Deficits in Hippocampal Synaptic Plasticity and Memory Storage

    ERIC Educational Resources Information Center

    Wood, Marcelo A.; Kaplan, Michael P.; Park, Alice; Blanchard, Edward J.; Oliveira, Ana M. M.; Lombardi, Thomas L.; Abel, Ted

    2005-01-01

    Deletions, translocations, or point mutations in the CREB-binding protein (CBP) gene have been associated with Rubinstein-Taybi Syndrome; a human developmental disorder characterized by retarded growth and reduced mental function. To examine the role of CBP in memory, transgenic mice were generated in which the CaMKII[alpha] promoter drives…

  10. 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

  11. Activation of PTHrP-cAMP-CREB1 signaling following p53 loss is essential for osteosarcoma initiation and maintenance

    PubMed Central

    Walia, Mannu K; Ho, Patricia MW; Taylor, Scott; Ng, Alvin JM; Gupte, Ankita; Chalk, Alistair M; Zannettino, Andrew CW; Martin, T John; Walkley, Carl R

    2016-01-01

    Mutations in the P53 pathway are a hallmark of human cancer. The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53. In contrast to P53 point mutations in other cancer, complete loss of P53 is a frequent event in osteosarcoma (OS), the most common cancer of bone. The consequences of p53 loss for osteoblastic cells and OS development are poorly understood. Here we use murine OS models to demonstrate that elevated Pthlh (Pthrp), cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS. Normal osteoblasts survive depletion of both PTHrP and CREB1. In contrast, p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1. Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS. DOI: http://dx.doi.org/10.7554/eLife.13446.001 PMID:27070462

  12. The Chemical Biology of Protein Phosphorylation

    PubMed Central

    Tarrant, Mary Katherine; Cole, Philip A.

    2011-01-01

    The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain. PMID:19489734

  13. CREB, ATF, and AP-1 transcription factors regulate IFN-gamma secretion by human T cells in response to mycobacterial antigen.

    PubMed

    Samten, Buka; Townsend, James C; Weis, Steven E; Bhoumik, Anindita; Klucar, Peter; Shams, Homayoun; Barnes, Peter F

    2008-08-01

    IFN-gamma production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-gamma, -73 to -48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-gamma in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-gamma upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-gamma proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-gamma and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-gamma proximal promoter and that they up-regulate IFN-gamma transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation. PMID:18641343

  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. Time-dependent co-relation of BDNF and CREB mRNAs in adult rat brains following acute psychological stress in the communication box paradigm.

    PubMed

    Li, Gongying; Wang, Yanmei; Yan, Min; Ma, Hongxia; Gao, Yanjie; Li, Zexuan; Li, Changqi; Tian, Hongjun; Zhuo, Chuanjun

    2016-06-15

    Psychological stress affects human health, and chronic stress leads to life-threatening diseases, such as depression and post-traumatic stress disorder. Psychological stress coping mechanisms involve the brain-derived neurotrophic factor (BDNF) and downstream cAMP response element binding protein (CREB), which are targets of the adverse effects of stress paradigms. Fourty-seven adult male Sprague-Dawley rats were divided into control, physical stress and six psychological stress groups which were assayed at 0h, 0.5h, 1h, 2h, 6h and 24h after communication box (CB) stress induction. Behavioral assessment using open field and elevated plus maze tests determined that CB stress significantly increased anxiety. After CB stress, the alternation of mRNA levels of BDNF and CREB were assessed at different time points by in situ hybridization. The mRNA levels of BDNF and CREB were significantly decreased, then gradually recovered over 24h to maximum levels in the hippocampus (CA1 region), prefrontal cortex (PFC), central amygdaloid nuclei (AG), shell of accumbens nucleus (NAC), periaqueductal gray (PAG) and ventral tegmental area, except for the ventral tegmental area (VTA). Moreover, mRNA levels of BDNF and CREB were positively correlated in all examined brain regions, except for the VTA region at 0 and 24h after CB stress induction. These findings suggest that BDNF and CREB may belong to the same pathway and be involved in psychological stress response mechanisms, and protect the organism from stress induced, aversive processes leading to disease. PMID:27132084

  16. Curcumin modulates dopaminergic receptor, CREB and phospholipase c gene expression in the cerebral cortex and cerebellum of streptozotocin induced diabetic rats

    PubMed Central

    2010-01-01

    Curcumin, an active principle component in rhizome of Curcuma longa, has proved its merit for diabetes through its anti-oxidative and anti-inflammatory properties. This study aims at evaluating the effect of curcumin in modulating the altered dopaminergic receptors, CREB and phospholipase C in the cerebral cortex and cerebellum of STZ induced diabetic rats. Radioreceptor binding assays and gene expression was done in the cerebral cortex and cerebellum of male Wistar rats using specific ligands and probes. Total dopaminergic receptor binding parameter, Bmax showed an increase in cerebral cortex and decrease in the cerebellum of diabetic rats. Gene expression studies using real time PCR showed an increased expression of dopamine D1 and D2 receptor in the cerebral cortex of diabetic rats. In cerebellum dopamine D1 receptor was down regulated and D2 receptor showed an up regulation. Transcription factor CREB and phospholipase C showed a significant down regulation in cerebral cortex and cerebellum of diabetic rats. We report that curcumin supplementation reduces diabetes induced alteration of dopamine D1, D2 receptors, transcription factor CREB and phospholipase C to near control. Our results indicate that curcumin has a potential to regulate diabetes induced malfunctions of dopaminergic signalling, CREB and Phospholipase C expression in cerebral cortex and cerebellum and thereby improving the cognitive and emotional functions associated with these regions. Furthermore, in line with these studies an interaction between curcumin and dopaminergic receptors, CREB and phospholipase C is suggested, which attenuates the cortical and cerebellar dysfunction in diabetes. These results suggest that curcumin holds promise as an agent to prevent or treat CNS complications in diabetes. PMID:20513244

  17. The coactivator dTAF(II)110/hTAF(II)135 is sufficient to recruit a polymerase complex and activate basal transcription mediated by CREB.

    PubMed

    Felinski, E A; Quinn, P G

    2001-11-01

    A specific TATA binding protein-associated factor (TAF), dTAF(II)110/hTAF(II)135, interacts with cAMP response element binding protein (CREB) through its constitutive activation domain (CAD), which recruits a polymerase complex and activates transcription. The simplest explanation is that the TAF is a coactivator, but several studies have questioned this role of TAFs. Using a reverse two-hybrid analysis in yeast, we previously mapped the interaction between dTAF(II)110 (amino acid 1-308) and CREB to conserved hydrophobic amino acid residues in the CAD. That mapping was possible only because CREB fails to activate transcription in yeast, where all TAFs are conserved, except for the TAF recognizing CREB. To test whether CREB fails to activate transcription in yeast because it lacks a coactivator, we fused dTAF(II)110 (amino acid 1-308) to the TATA binding protein domain of the yeast scaffolding TAF, yTAF(II)130. Transformation of yeast with this hybrid TAF conferred activation by the CAD, indicating that interaction with yTFIID is sufficient to recruit a polymerase complex and activate transcription. The hybrid TAF did not mediate activation by VP16 or vitamin D receptor, each of which interacts with TFIIB, but not with dTAF(II)110 (amino acid 1-308). Enhancement of transcription activation by dTAF(II)110 in mammalian cells required interaction with both the CAD and TFIID and was inhibited by mutation of core hydrophobic residues in the CAD. These data demonstrate that dTAF(II)110/hTAF(II)135 acts as a coactivator to recruit TFIID and polymerase and that this mechanism of activation is conserved in eukaryotes. PMID:11687654

  18. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  19. Phosphorylation of human skeletal muscle myosin

    SciTech Connect

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-03-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30/sup 0/C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with (/sup 30/P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ.

  20. Protein phosphorylation in neurodegeneration: friend or foe?

    PubMed Central

    Tenreiro, Sandra; Eckermann, Katrin; Outeiro, Tiago F.

    2014-01-01

    Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system. PMID:24860424

  1. Protein phosphorylation during Plasmodium berghei gametogenesis.

    PubMed

    Alonso-Morales, Alberto; González-López, Lorena; Cázares-Raga, Febe Elena; Cortés-Martínez, Leticia; Torres-Monzón, Jorge Aurelio; Gallegos-Pérez, José Luis; Rodríguez, Mario Henry; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

    2015-09-01

    Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies. PMID:26008612

  2. Fibronectin phosphorylation by ecto-protein kinase

    SciTech Connect

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru )

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  3. Long-term dynamics of multisite phosphorylation.

    PubMed

    Rubinstein, Boris Y; Mattingly, Henry H; Berezhkovskii, Alexander M; Shvartsman, Stanislav Y

    2016-07-15

    Multisite phosphorylation cycles are ubiquitous in cell regulation systems and are studied at multiple levels of complexity, from molecules to organisms, with the ultimate goal of establishing predictive understanding of the effects of genetic and pharmacological perturbations of protein phosphorylation in vivo. Achieving this goal is essentially impossible without mathematical models, which provide a systematic framework for exploring dynamic interactions of multiple network components. Most of the models studied to date do not discriminate between the distinct partially phosphorylated forms and focus on two limiting reaction regimes, distributive and processive, which differ in the number of enzyme-substrate binding events needed for complete phosphorylation or dephosphorylation. Here we use a minimal model of extracellular signal-related kinase regulation to explore the dynamics of a reaction network that includes all essential phosphorylation forms and arbitrary levels of reaction processivity. In addition to bistability, which has been studied extensively in distributive mechanisms, this network can generate periodic oscillations. Both bistability and oscillations can be realized at high levels of reaction processivity. Our work provides a general framework for systematic analysis of dynamics in multisite phosphorylation systems. PMID:27226482

  4. Compartment-Specific Phosphorylation of Squid Neurofilaments.

    PubMed

    Grant, Philip; Pant, Harish C

    2016-01-01

    Studies of the giant axon and synapse of third-order neurons in the squid stellate ganglion have provided a vast literature on neuronal physiology and axon transport. Large neuronal size also lends itself to comparative biochemical studies of cell body versus axon. These have focused on the regulation of synthesis, assembly, posttranslational modification and function of neuronal cytoskeletal proteins (microtubules (MTs) and neurofilaments (NFs)), the predominant proteins in axoplasm. These contribute to axonal organization, stability, transport, and impulse transmission responsible for rapid contractions of mantle muscles underlying jet propulsion. Studies of vertebrate NFs have established an extensive literature on NF structure, organization, and function; studies of squid NFs, however, have made it possible to compare compartment-specific regulation of NF synthesis, assembly, and function in soma versus axoplasm. Since NFs contain over 100 eligible sites for phosphorylation by protein kinases, the compartment-specific patterns of phosphorylation have been a primary focus of biochemical studies. We have learned that NF phosphorylation is tightly compartmentalized; extensive phosphorylation occurs only in the axonal compartment in squid and in vertebrate neurons. This extensive phosphorylation plays a key role in organizing NFs, in association with microtubules (MTs), into a stable, dynamic functional lattice that supports axon growth, diameter, impulse transmission, and synaptic activity. To understand how cytoskeletal phosphorylation is topographically regulated, the kinases and phosphatases, bound to NFs isolated from cell bodies and axoplasm, have also been studied. PMID:26795486

  5. Phosphorylation meets nuclear import: a review

    PubMed Central

    2010-01-01

    Phosphorylation is the most common and pleiotropic modification in biology, which plays a vital role in regulating and finely tuning a multitude of biological pathways. Transport across the nuclear envelope is also an essential cellular function and is intimately linked to many degeneration processes that lead to disease. It is therefore not surprising that phosphorylation of cargos trafficking between the cytoplasm and nucleus is emerging as an important step to regulate nuclear availability, which directly affects gene expression, cell growth and proliferation. However, the literature on phosphorylation of nucleocytoplasmic trafficking cargos is often confusing. Phosphorylation, and its mirror process dephosphorylation, has been shown to have opposite and often contradictory effects on the ability of cargos to be transported across the nuclear envelope. Without a clear connection between attachment of a phosphate moiety and biological response, it is difficult to fully understand and predict how phosphorylation regulates nucleocytoplasmic trafficking. In this review, we will recapitulate clue findings in the field and provide some general rules on how reversible phosphorylation can affect the nuclear-cytoplasmic localization of substrates. This is only now beginning to emerge as a key regulatory step in biology. PMID:21182795

  6. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    SciTech Connect

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.