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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 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

  2. 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

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

    PubMed Central

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

    2016-01-01

    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

  4. 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…

  5. Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

    PubMed

    Lee, Su-Lin; Chou, Chih-Chien; Chuang, Hsiao-Ching; Hsu, En-Chi; Chiu, Po-Chen; Kulp, Samuel K; Byrd, John C; Chen, Ching-Shih

    2013-01-01

    Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA

  6. Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines

    PubMed Central

    Chuang, Hsiao-Ching; Hsu, En-Chi; Chiu, Po-Chen; Kulp, Samuel K.; Byrd, John C.; Chen, Ching-Shih

    2013-01-01

    Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA

  7. Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

    PubMed

    Lee, Su-Lin; Chou, Chih-Chien; Chuang, Hsiao-Ching; Hsu, En-Chi; Chiu, Po-Chen; Kulp, Samuel K; Byrd, John C; Chen, Ching-Shih

    2013-01-01

    Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA

  8. mTORC2-PKBα/Akt1 Serine 473 phosphorylation axis is essential for regulation of FOXP3 Stability by chemokine CCL3 in psoriasis.

    PubMed

    Chen, Ling; Wu, Jinjin; Pier, Eric; Zhao, Yun; Shen, Zhu

    2013-02-01

    The connection between infections and acute guttate psoriasis (AGP) outbreaks/chronic plaque psoriasis (CPP) exacerbation has been known for years. Impaired function of FOXP3+Tregs in psoriasis has been identified. However, the mechanisms behind these two observations have not been fully interpreted. In the present study, we provide evidence to support chemokine CCL3 as one of the vital links between infections and FOXP3 stability in the psoriatic microenvironment. We found that serum CCL3, strongly induced by microorganism infections including streptococcus, was closely correlated with FOXP3 levels in CD4+CD25+T cells of patients with psoriasis. CCL3 manipulated FOXP3 stability in a concentration-dependent bidirectional manner. High-concentration CCL3 decreased FOXP3 stability by promoting FOXP3's degradation through K48-linkage ubiquitination. This degradation was mainly dependent on upregulation of Serine 473 phosphorylation of the PKBα/Akt1 isoform, and almost independent of mTORC1 (mammalian target of rapamycin complex 1) activity. On the other hand, low-concentration CCL3 could enhance FOXP3 stability by the maintenance of the PKC pathway and the restriction of the PKB/Akt pathway. We further demonstrated that enhancing FOXP3 stability by low-concentration CCL3 attributed, at least partly, to the prevention of cytoplasmic Sin1, a vital component of mTORC2, nuclear translocation. Our results suggest vital roles for CCL3-mTORC2-isoform PKB/Akt1 S473 phosphorylation axis in FOXP3+Tregs and the development of psoriasis.

  9. 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

  10. 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

  11. 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

  12. 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…

  13. Stimulation of phosphorylation of ERK and CREB by phellopterin and auraptene isolated from Citrusjunos.

    PubMed

    Nakamura, Mitsuhiro; Suzuki, Tomoko; Takagi, Mai; Tamura, Hirotoshi; Masuda, Toshiya

    2014-10-01

    Bioactive compounds from citrus fruits contribute many benefits to human health. Extracellular signal-regulated kinase (ERK) signaling plays an important role in the regulation of multiple cellular processes. Activation of the ERK-cAMP response element binding protein (CREB) signaling is required for long- term memory formation. In this study, auraptene, phellopterin, thymol, coniferyl alcohol 9-methyl ether and methyl ferulate were isolated from Citrus junos. Among the five compounds isolated, auraptene and phellopterin increased the phosphorylation of ERK and CREB. This study provides, to our knowledge, the first evidence that phellopterin potently stimulates the phosphorylation of ERK and CREB. Phellopterin could be a novel neuroprotective agent. PMID:25522543

  14. Differences in hippocampal CREB phosphorylation in trace fear conditioning of two inbred mouse strains.

    PubMed

    Hwang, Yoo Kyeong; Song, Jae-Chun; Han, Seol-Heui; Cho, Jeiwon; Smith, Dani R; Gallagher, Michela; Han, Jung-Soo

    2010-07-23

    The effects of genetic background on fear trace conditioning were evaluated in relation to phosphorylated levels of cAMP response element-binding protein (CREB) in the hippocampus using two different inbred strains of mice, C57BL/6 and DBA/2. The male mice received a trace fear conditioning protocol and unpaired control groups were included to assess nonassociative effects on test performance. Both C57BL/6 and DBA/2 mice with paired training displayed higher freezing responses during testing than those with unpaired training, respectively. The C57BL/6 mice with paired training also displayed higher freezing responses to the tone-CS during testing than the DBA/2 mice with paired training. Because much evidence implicates the hippocampus as an important neural substrate for trace fear conditioning, the engagement of the hippocampus was examined after testing by measuring levels of CREB and phosphorylated CREB (pCREB). The results revealed that hippocampal CREB levels in both strains of mice were not significantly altered according to the type of training (unpaired vs. paired). However, the hippocampal pCREB levels were significantly higher in the paired training group than the unpaired control group in C57BL/6 mice, but not in DBA/2 mice. These findings indicate that hippocampal pCREB is closely tied to this form of associative conditioning only in C57BL/6 mice and that different neural substrates may support trace conditioning in C57BL/6 and DBA/2 strains. PMID:20501325

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

    PubMed Central

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

    2009-01-01

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

  16. 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.

  17. Basal variability in CREB phosphorylation predicts trait-like differences in amygdala-dependent memory.

    PubMed

    Cowansage, Kiriana K; Bush, David E A; Josselyn, Sheena A; Klann, Eric; Ledoux, Joseph E

    2013-10-01

    Much of what is known about the neurobiology of learning and memory comes from studies of the average behavior. In contrast, intersubject differences that emerge within groups are difficult to study systematically and are often excluded from scientific discussion. Nevertheless, population-wide variability is a virtually universal feature of both complex traits, such as intelligence, and hardwired responses, such as defensive behaviors. Here, we use outbred rats to investigate if cAMP response element-binding protein (CREB), a transcription factor that has long been known in experimental settings to be crucial for associative plasticity, participates in natural memory phenotypes. Using a combination of behavioral, biochemical, and viral techniques, we show that a subset of rats with trait-like deficits in aversive memory have basally reduced CREB activity in the lateral amygdala but can be induced to perform at average levels by directly or indirectly enhancing pretraining CREB phosphorylation. These data suggest that endogenous CREB activity in the amygdala may set a critical threshold for plasticity during memory formation. PMID:24062441

  18. Methoxychlor inhibits brain mitochondrial respiration and increases hydrogen peroxide production and CREB phosphorylation.

    PubMed

    Schuh, Rosemary A; Kristián, Tibor; Gupta, Rupesh K; Flaws, Jodi A; Fiskum, Gary

    2005-12-01

    The organochlorine insecticide methoxychlor (mxc) is an established reproductive toxicant that affects other systems including the central nervous system (CNS), possibly by mechanisms involving oxidative stress. This study tested the hypothesis that mxc inhibits brain mitochondrial respiration, resulting in increased production of reactive oxygen species (ROS). Oxygen electrode measurements of mitochondrial respiration and Amplex Red measurements of H(2)O(2) production were performed with rat brain mitochondria exposed in vitro to mxc (0-10 microg/ml) and with brain mitochondria from mice chronically exposed in vivo to mxc (0-64 mg/kg/day) for 20 days by intraperitoneal injection. In vitro mxc exposure inhibited ADP-dependent respiration (state 3) using both complex I- and II-supported substrates. Similarly, state 3 respiration was inhibited following in vivo mxc exposure using complex I substrates. H(2)O(2) production was stimulated after in vitro mxc treatment in the presence of complex I substrates, but not in mitochondria isolated from in vivo mxc-treated mice. Because previous studies demonstrated a relationship between oxidative stress and CREB phosphorylation, we also tested the hypothesis that mxc elevates phosphorylated CREB (pCREB) in mitochondria. Enzyme-linked immunosorbent assay (ELISA) measurements demonstrated that pCREB immunoreactivity was elevated by in vitro mxc exposure in the presence or absence of respiratory substrates, indicating that stimulation of H(2)O(2) production is not necessary for this effect. These multiple effects of mxc on mitochondria may play an important role in its toxicity, particularly in the CNS.

  19. Calcium release by ryanodine receptors mediates hydrogen peroxide-induced activation of ERK and CREB phosphorylation in N2a cells and hippocampal neurons.

    PubMed

    Kemmerling, Ulrike; Muñoz, Pablo; Müller, Marioly; Sánchez, Gina; Aylwin, María L; Klann, Eric; Carrasco, M Angélica; Hidalgo, Cecilia

    2007-05-01

    Hydrogen peroxide, which stimulates ERK phosphorylation and synaptic plasticity in hippocampal neurons, has also been shown to stimulate calcium release in muscle cells by promoting ryanodine receptor redox modification (S-glutathionylation). We report here that exposure of N2a cells or rat hippocampal neurons in culture to 200 microM H2O2 elicited calcium signals, increased ryanodine receptor S-glutathionylation, and enhanced both ERK and CREB phosphorylation. In mouse hippocampal slices, H2O2 (1 microM) also stimulated ERK and CREB phosphorylation. Preincubation with ryanodine (50 microM) largely prevented the effects of H2O2 on calcium signals and ERK/CREB phosphorylation. In N2a cells, the ERK kinase inhibitor U0126 suppressed ERK phosphorylation and abolished the stimulation of CREB phosphorylation produced by H2O2, suggesting that H2O2 enhanced CREB phosphorylation via ERK activation. In N2a cells in calcium-free media, 200 microM H2O2 stimulated ERK and CREB phosphorylation, while preincubation with thapsigargin prevented these enhancements. These combined results strongly suggest that H2O2 promotes ryanodine receptors redox modification; the resulting calcium release signals, by enhancing ERK activity, would increase CREB phosphorylation. We propose that ryanodine receptor stimulation by activity-generated redox species produces calcium release signals that may contribute significantly to hippocampal synaptic plasticity, including plasticity that requires long-lasting ERK-dependent CREB phosphorylation. PMID:17074386

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

    SciTech Connect

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

    2008-05-23

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

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

    PubMed Central

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

    1992-01-01

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

  2. 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...

  3. Calcium-sensing receptor-dependent activation of CREB phosphorylation in HEK293 cells and human parathyroid cells.

    PubMed

    Avlani, Vimesh A; Ma, Wenting; Mun, Hee-Chang; Leach, Katie; Delbridge, Leigh; Christopoulos, Arthur; Conigrave, Arthur D

    2013-05-15

    In addition to its acute effects on hormone secretion, epithelial transport, and shape change, the calcium-sensing receptor (CaSR) modulates the expression of genes that control cell survival, proliferation, and differentiation as well as the synthesis of peptide hormones and enzymes. In the present study, we investigated the impacts of a CaSR agonist and several CaSR modulators on phosphorylation of transcription factor CREB residue Ser(133) in CaSR-expressing HEK293 (HEK-CaSR) cells and human adenomatous parathyroid cells. Elevated Ca(2+)o concentration had no effect on CREB phosphorylation (p-CREB) in control HEK293 cells but stimulated p-CREB in both HEK-CaSR cells and human parathyroid cells. In addition, p-CREB was stimulated by the positive modulator cinacalcet and inhibited by the negative modulator NPS 2143 in both CaSR-expressing cell types. Two positive modulators that bind in the receptor's Venus Fly Trap domain, l-phenylalanine and S-methylglutathione, had no effect on p-CREB in HEK-CaSR cells, demonstrating the existence of pronounced signaling bias. Analysis of the signaling pathways using specific inhibitors demonstrated that phosphoinositide-specific phospholipase C and conventional protein kinase C isoforms make major contributions to Ca(2+)o-induced p-CREB in both cell-types, suggesting key roles for Gq/11. In addition, in parathyroid cells but not HEK-CaSR cells, activation of p-CREB was dependent on Gi/o, demonstrating the existence of cell type-specific signaling.

  4. TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules

    PubMed Central

    Jevtov, Irena; Zacharogianni, Margarita; van Oorschot, Marinke M.; van Zadelhoff, Guus; Aguilera-Gomez, Angelica; Vuillez, Igor; Braakman, Ineke; Hafen, Ernst; Stocker, Hugo; Rabouille, Catherine

    2015-01-01

    ABSTRACT The kinase TOR is found in two complexes, TORC1, which is involved in growth control, and TORC2, whose roles are less well defined. Here, we asked whether TORC2 has a role in sustaining cellular stress. We show that TORC2 inhibition in Drosophila melanogaster leads to a reduced tolerance to heat stress, whereas sensitivity to other stresses is not affected. Accordingly, we show that upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for maintaining the level of its known target, Akt1 (also known as PKB). We show that the phosphorylation of the stress-activated protein kinases is not modulated by TORC2 nor is the heat-induced upregulation of heat-shock proteins. Instead, we show, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears to be less efficient in the absence of TORC2 function. We propose that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules. PMID:26054799

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

    PubMed Central

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

    2000-01-01

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

  6. Diminished Phosphorylation of CREB Is a Key Event in the Dysregulation of Gluconeogenesis and Glycogenolysis in PCB126 Hepatotoxicity.

    PubMed

    Gadupudi, Gopi S; Klingelhutz, Aloysius J; Robertson, Larry W

    2016-09-19

    The dioxin-like PCB126 elicits toxicity in various target organs. In rat liver, an alteration in the transcript levels of several genes involved in glucose and fatty acid metabolism provides insights into the origin of its hepatotoxicity. To explore the mechanisms, male Sprague-Dawley rats, fed an AIN-93G diet, were injected with PCB126 (1 or 5 μmol/kg) or corn oil and euthanized after 2 weeks. PCB126 significantly decreased serum glucose levels and the transcript levels of genes of many gluconeogenic and glycogenolytic enzymes under the transcriptional control of a nuclear transcription factor, cAMP response element-binding protein (CREB). As a novel finding, we show that PCB126 significantly decreases CREB phosphorylation, which is important for regulating both gluconeogenesis and fatty acid oxidation in the liver and explains CREB's integrative effects on both carbohydrate and lipid metabolism in PCB126 toxicity. PMID:27509375

  7. 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

  8. 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

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

    PubMed

    Morris, Ken A; Gold, Paul E

    2013-02-01

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

  10. 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

  11. 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.

  12. 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

  13. Environmental Enrichment Alters Nicotine-Mediated Locomotor Sensitization and Phosphorylation of DARPP-32 and CREB in Rat Prefrontal Cortex

    PubMed Central

    Gomez, Adrian M.; Midde, Narasimha M.; Mactutus, Charles F.; Booze, Rosemarie M.; Zhu, Jun

    2012-01-01

    Exposure within an environmental enrichment paradigm results in neurobiological adaptations and decreases the baseline of locomotor activity. The current study determined activation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32) and CREB (cAMP response element binding protein), and locomotor activity in rats raised in enriched (EC), impoverished (IC), and standard (SC) conditions following repeated administration of nicotine or saline. In the saline-control group, the basal phosphorylation state of DARPP-32 at Threonine-34 site (pDARPP-32 Thr34) in the prefrontal cortex (PFC) was lower in EC compared to IC and SC rats, which was positively correlated with their respective baseline activities. While nicotine (0.35 mg/kg, freebase) produced locomotor sensitization across all housing conditions when the nicotine-mediated locomotor activity was expressed as a percent change from their respective saline control, EC rats displayed greater sensitization to nicotine than IC and SC rats. Consistent with the behavioral findings, repeated nicotine injection increased pDARPP-32 Thr34 in PFC of EC and IC rats and in nucleus accumbens of EC rats; however, the magnitude of change from saline control in nicotine-induced enhancement of pDARPP-32 Thr34 in PFC was strikingly increased in EC rats relative to IC rats. Moreover, EC rats had lower basal phosphorylation levels of CREB at serine 133 in PFC and nucleus accumbens compared to IC and SC rats, whereas the nicotine-induced increase in phosphorylated CREB-Ser133 was more pronounced in PFC of EC rats relative to IC and SC rats. Collectively, these findings suggest innovative insights into advancing our understanding of the molecular mechanisms of enrichment-induced changes in the motivational effects of nicotine, and aiding in the identification of new therapeutic strategies for tobacco smokers. PMID:22952905

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-04-01

    Cadmium is a widely used heavy metal in industry and affects the male reproductive system of animals, including humans, as a result of occupational and environmental exposures. However, the molecular mechanism underlying its effect on steroidogenesis in gonads remains unclear. In this study, we demonstrated that exposure of K28 mouse testicular Leydig tumor cells to cadmium led to a significant increase in the mRNA level, promoter activity and protein level of the steroidogenic acute regulatory protein (StAR), an essential factor for steroid biosynthesis. It has been well documented that StAR gene transcription is regulated by multiple transcription factors, including cAMP-responsive element binding protein (CREB) family members and SF-1. Cadmium treatment caused an increase in CREB phosphorylation but did not alter the CREB protein level in the nucleus. EMSA studies revealed that cadmium-induced phosphorylated CREB formed specific complexes with the proximal region of the StAR gene promoter. Furthermore, co-transfection with a CREB expression plasmid significantly increased cadmium-induced StAR promoter activity. However, the nuclear level and the affinity of SF-1 protein for the StAR proximal promoter were dramatically decreased upon exposure to cadmium. Taken together, these results suggest that cadmium up-regulates StAR gene expression through phosphorylated CREB rather than through SF-1 in mouse testicular Leydig cells. PMID:25786521

  16. 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

  17. Effects of neonatal corticosterone and environmental enrichment on retinal ERK1/2 and CREB phosphorylation in adult mice.

    PubMed

    Matteucci, Andrea; Ceci, Chiara; Mallozzi, Cinzia; Macrì, Simone; Malchiodi-Albedi, Fiorella; Laviola, Giovanni

    2014-11-01

    Exposure to Stimulating Environments (SE) during development may improve neuroplasticity in central nervous system, protect against neurotoxic damage, and promote neuronal recovery in adult life. While biochemical mechanisms of SE-promoted neuronal plasticity are well known in the brain, much less is known on the signaling cascade governing plasticity and neuroprotection in the retina. In order to investigate if in the retina signaling molecules involved in neuronal plasticity are affected by SE, neonatal CD-1 mice were exposed to moderate corticosterone levels (NC), supplemented through maternal milk during the first postnatal week, or to environmental enrichment (EE) conditions (physical and social stimuli) from early adolescence. Our results showed that both NC and EE increased the phosphorylation level of Extracellularly Regulated Kinase 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) in the adult retinal tissue. Furthermore, we observed that activated ERK1/2 was restricted to Müller cells, while pCREB was mostly present in the nuclei of retinal neurons. Neither NC, nor EE modified the expression of GFAP, a marker of Müller cells activation. In conclusion our results indicate that both NC and EE activate ERK1/2 and CREB in the retina and provide a biochemical background for the neuroprotective activity exerted by SE against retinal damage. Furthermore, they support the role of Müller glia as a key cell determinant of retinal neuroplasticity.

  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. PI3K/Akt promotes feedforward mTORC2 activation through IKKα.

    PubMed

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

    2016-04-19

    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

  1. Distinct Changes in CREB Phosphorylation in Frontal Cortex and Striatum During Contingent and Non-Contingent Performance of a Visual Attention Task

    PubMed Central

    Pozzi, Laura; Sacchetti, Giuseppina; Agnoli, Laura; Mainolfi, Pierangela; Invernizzi, Roberto W.; Carli, Mirjana

    2011-01-01

    The cyclic-adenosine monophosphate response element-binding protein (CREB) family of transcription factors has been implicated in numerous forms of behavioral plasticity. We investigated CREB phosphorylation along some nodes of corticostriatal circuitry such as frontal cortex (FC) and dorsal (caudate–putamen, CPu) and ventral (nucleus accumbens, NAC) striatum in response to the contingent or non-contingent performance of the five-choice serial reaction time task (5-CSRTT) used to assess visuospatial attention. Three experimental manipulations were used; an attentional performance group (contingent, “master”), a group trained previously on the task but for whom the instrumental contingency coupling responding with stimulus detection and reward was abolished (non-contingent, “yoked”) and a control group matched for food deprivation and exposure to the test apparatus (untrained). Rats trained on the 5-CSRTT (both master and yoked) had higher levels of CREB protein in the FC, CPu, and NAC compared to untrained controls. Despite the divergent behavior of “master” and “yoked” rats CREB activity in the FC was not substantially different. In rats performing the 5-CSRTT (“master”), CREB activity was completely abolished in the CPu whereas in the NAC it remained unchanged. In contrast, CREB phosphorylation in CPu and NAC increased only when the contingency changed from goal-dependent to goal-independent reinforcement (“yoked”). The present results indicate that up-regulation of CREB protein expression across cortical and striatal regions possibly reflects the extensive instrumental learning and performance whereas increased CREB activity in striatal regions may signal the unexpected change in the relationship between instrumental action and reinforcement. PMID:22016726

  2. 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.

  3. 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

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

    PubMed Central

    Cho, Woo-Hyun; Han, Jung-Soo

    2016-01-01

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

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

    PubMed Central

    Cho, Woo-Hyun; Han, Jung-Soo

    2016-01-01

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

  6. GPR40 receptor activation leads to CREB phosphorylation and improves cognitive performance in an Alzheimer's disease mouse model.

    PubMed

    Khan, Muhammad Zahid; Zhuang, Xuxu; He, Ling

    2016-05-01

    Alzheimer's disease (AD) is a very complex neurodegenerative disorder as neuronal loss is a prominent and initial feature of AD. This loss correlates with cognitive deficits more closely than amyloid load. GPR40 receptor belongs to the class of G-protein coupled receptors, is expressed in wide parts of the brain including the hippocampus which is involved in spatial learning and memory. Till now, there are few studies investigating the functional role of GPR40 in brain. In this study, we evaluated the functional role of GPR40 receptor in the A-beta AD mice model. Administration of Aβ1-42 (410pmol) intracerebroventricularly (i.c.v.) once at the beginning of experiment significantly impaired cognitive performance (in step-through passive test), the ability of spatial learning and memory in (Morris water maze test), working memory, attention, anxiety in (Novel object recognition test), and spatial working and reference-memory in (Hole board discrimination test) compared with the control group. The results revealed that GPR40 receptor treatment groups significantly ameliorated model mice cognitive performance. All GPR40 receptor agonist GW9508, treatment groups enhanced the learning and memory ability in Step-through passive test, Morris water maze test, Hole board discrimination test, Novel object recognition test. Furthermore, we have observed that activation of GPR40 receptor provoked the phosphorylation of the cAMP response element binding protein (CREB) and significant increase in neurotropic factors including brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrohin-4 (NT-4) in mouse hippocampal neurons and contribute to neurogenesis. These results suggest that GPR40 is a suitable therapeutic candidate for neurogenesis and neuroprotection in the treatment and prevention of AD.

  7. 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

  8. 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.

  9. Rictor/mTORC2 facilitates central regulation of energy and glucose homeostasis

    PubMed Central

    Kocalis, Heidi E.; Hagan, Scott L.; George, Leena; Turney, Maxine K.; Siuta, Michael A.; Laryea, Gloria N.; Morris, Lindsey C.; Muglia, Louis J.; Printz, Richard L.; Stanwood, Gregg D.; Niswender, Kevin D.

    2014-01-01

    Insulin signaling in the central nervous system (CNS) regulates energy balance and peripheral glucose homeostasis. Rictor is a key regulatory/structural subunit of the mTORC2 complex and is required for hydrophobic motif site phosphorylation of Akt at serine 473. To examine the contribution of neuronal Rictor/mTORC2 signaling to CNS regulation of energy and glucose homeostasis, we utilized Cre-LoxP technology to generate mice lacking Rictor in all neurons, or in either POMC or AgRP expressing neurons. Rictor deletion in all neurons led to increased fat mass and adiposity, glucose intolerance and behavioral leptin resistance. Disrupting Rictor in POMC neurons also caused obesity and hyperphagia, fasting hyperglycemia and pronounced glucose intolerance. AgRP neuron specific deletion did not impact energy balance but led to mild glucose intolerance. Collectively, we show that Rictor/mTORC2 signaling, especially in POMC-expressing neurons, is important for central regulation of energy and glucose homeostasis. PMID:24944899

  10. Apolipoprotein E4 impairs in vivo hippocampal long-term synaptic plasticity by reducing the phosphorylation of CaMKIIα and CREB.

    PubMed

    Qiao, Feng; Gao, Xiu-Ping; Yuan, Li; Cai, Hong-Yan; Qi, Jin-Shun

    2014-01-01

    Inheritance of the apolipoprotein E genotype ε4 (APOE4) is a powerful risk factor for most cases of late-onset Alzheimer's disease (AD). However, the effects of ApoE4 on the long-term synaptic plasticity and its underlying mechanism have not clearly investigated. In the present study, we examined the effects of ApoE4 on the hippocampal late-phase long-term potentiation (L-LTP) and investigated its probable molecular mechanisms by using in vivo field potential recording, immunohistochemistry, and western blotting. The results showed that: (1) intra-hippocampal injection of 0.2 μg ApoE4, but not ApoE2, before high frequency stimulations (HFSs) attenuated the induction of hippocampal L-LTP in the CA1 region, while injection of the same concentration of ApoE4 after HFSs, even at a higher concentration (2 μg), did not affect the long term synaptic plasticity; (2) ApoE4 injection did not affect the paired pulse facilitation in the hippocampal CA1 region; (3) ApoE4 injection before, not after, HFSs significantly decreased the levels of phosphorylated Ca2+/calmodulin-dependent protein kinase IIα (p-CaMKIIα) and phosphorylated cAMP response element-binding protein (p-CREB) in the hippocampus. These results demonstrated for the first time that ApoE4 could impair hippocampal L-LTP by reducing p-CaMKIIα and p-CREB, suggesting that the ApoE4-induced suppression of hippocampal long-term synaptic plasticity may contribute to the cognitive impairments in genetic AD; and both CaMKIIα and CREB are important intracellular targets of the neurotoxic ApoE4.

  11. Regulation of Endothelial Cell Proliferation and Vascular Assembly through Distinct mTORC2 Signaling Pathways

    PubMed Central

    Wang, Shan; Amato, Katherine R.; Song, Wenqiang; Youngblood, Victoria; Lee, Keunwook; Boothby, Mark; Brantley-Sieders, Dana M.

    2015-01-01

    Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a diverse array of cellular processes, including cell growth, survival, metabolism, and cytoskeleton dynamics. mTOR functions in two distinct complexes, mTORC1 and mTORC2, whose activities and substrate specificities are regulated by complex specific cofactors, including Raptor and Rictor, respectively. Little is known regarding the relative contribution of mTORC1 versus mTORC2 in vascular endothelial cells. Using mouse models of Raptor or Rictor gene targeting, we discovered that Rictor ablation inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation and assembly in vitro and angiogenesis in vivo, whereas the loss of Raptor had only a modest effect on endothelial cells (ECs). Mechanistically, the loss of Rictor reduced the phosphorylation of AKT, protein kinase Cα (PKCα), and NDRG1 without affecting the mTORC1 pathway. In contrast, the loss of Raptor increased the phosphorylation of AKT despite inhibiting the phosphorylation of S6K1, a direct target of mTORC1. Reconstitution of Rictor-null cells with myristoylated AKT (Myr-AKT) rescued vascular assembly in Rictor-deficient endothelial cells, whereas PKCα rescued proliferation defects. Furthermore, tumor neovascularization in vivo was significantly decreased upon EC-specific Rictor deletion in mice. These data indicate that mTORC2 is a critical signaling node required for VEGF-mediated angiogenesis through the regulation of AKT and PKCα in vascular endothelial cells. PMID:25582201

  12. Selective ATP-competitive inhibitors of TOR suppress rapamycin-insensitive function of TORC2 in Saccharomyces cerevisiae.

    PubMed

    Liu, Qingsong; Ren, Tao; Fresques, Tara; Oppliger, Wolfgang; Niles, Brad J; Hur, Wooyoung; Sabatini, David M; Hall, Michael N; Powers, Ted; Gray, Nathanael S

    2012-06-15

    The target of rapamycin (TOR) is a critical regulator of growth, survival, and energy metabolism. The allosteric TORC1 inhibitor rapamycin has been used extensively to elucidate the TOR related signal pathway but is limited by its inability to inhibit TORC2. We used an unbiased cell proliferation assay of a kinase inhibitor library to discover QL-IX-55 as a potent inhibitor of S. cerevisiae growth. The functional target of QL-IX-55 is the ATP-binding site of TOR2 as evidenced by the discovery of resistant alleles of TOR2 through rational design and unbiased selection strategies. QL-IX-55 is capable of potently inhibiting both TOR complex 1 and 2 (TORC1 and TORC2) as demonstrated by biochemical IP kinase assays (IC(50) <50 nM) and cellular assays for inhibition of substrate YPK1 phosphorylation. In contrast to rapamycin, QL-IX-55 is capable of inhibiting TORC2-dependent transcription, which suggests that this compound will be a powerful probe to dissect the Tor2/TORC2-related signaling pathway in yeast.

  13. 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

  14. Broad-spectrum efficacy across cognitive domains by alpha7 nicotinic acetylcholine receptor agonism correlates with activation of ERK1/2 and CREB phosphorylation pathways.

    PubMed

    Bitner, Robert S; Bunnelle, William H; Anderson, David J; Briggs, Clark A; Buccafusco, Jerry; Curzon, Peter; Decker, Michael W; Frost, Jennifer M; Gronlien, Jens Halvard; Gubbins, Earl; Li, Jinhe; Malysz, John; Markosyan, Stella; Marsh, Kennan; Meyer, Michael D; Nikkel, Arthur L; Radek, Richard J; Robb, Holly M; Timmermann, Daniel; Sullivan, James P; Gopalakrishnan, Murali

    2007-09-26

    The alpha7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel alpha7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (Ki = 10.8 nM) and human (Ki = 16.7 nM) alpha7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the alpha7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that alpha7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of alpha7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked alpha7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that alpha7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia. PMID:17898229

  15. Broad-spectrum efficacy across cognitive domains by alpha7 nicotinic acetylcholine receptor agonism correlates with activation of ERK1/2 and CREB phosphorylation pathways.

    PubMed

    Bitner, Robert S; Bunnelle, William H; Anderson, David J; Briggs, Clark A; Buccafusco, Jerry; Curzon, Peter; Decker, Michael W; Frost, Jennifer M; Gronlien, Jens Halvard; Gubbins, Earl; Li, Jinhe; Malysz, John; Markosyan, Stella; Marsh, Kennan; Meyer, Michael D; Nikkel, Arthur L; Radek, Richard J; Robb, Holly M; Timmermann, Daniel; Sullivan, James P; Gopalakrishnan, Murali

    2007-09-26

    The alpha7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel alpha7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (Ki = 10.8 nM) and human (Ki = 16.7 nM) alpha7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the alpha7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that alpha7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of alpha7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked alpha7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that alpha7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia.

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

    PubMed

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

    2015-06-01

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

  17. 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

  18. 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

  19. mTORC2 is the hydrophobic motif kinase for SGK1.

    PubMed

    Yan, Lijun; Mieulet, Virginie; Lamb, Richard F

    2008-12-15

    The activation of the AGC (protein kinase A/protein kinase G/protein kinase C)-family kinase SGK1 (serum- and glucocorticoid-induced kinase 1) by insulin via PI3K (phosphoinositide 3-kinase) signalling has been appreciated for almost 10 years. PDK1 (phosphoinositide-dependent protein kinase 1), a kinase that phosphorylates the SGK1 catalytic domain at Thr(256), is known to play a critical role in SGK1 activation. However, the identity of the protein kinase(s) responsible for phosphorylation of Ser(422), a site outside the catalytic domain (the so-called hydrophobic motif, or HM) that promotes activation of the kinase by PDK1, was unclear. In work reported in this issue of the Biochemical Journal, García-Martínez and Alessi have revealed the identity of a 'PDK2' kinase that catalyses Ser(422) phosphorylation as mTORC2 (mammalian target of rapamycin complex 2), a multiprotein kinase that phosphorylates a similar site in PKB (protein kinase B). PMID:19025518

  20. Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

    PubMed Central

    Yasuda, Kunihiko; Takahashi, Mayumi; Mori, Nozomu

    2015-01-01

    NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCαS657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCαS657 and FoxO1. PMID:26600389

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

    PubMed Central

    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. DOI: http://dx.doi.org/10.7554/eLife.09336.001 PMID:26274562

  2. Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: prodigiosin vs. obatoclax.

    PubMed

    Espona-Fiedler, M; Soto-Cerrato, V; Hosseini, A; Lizcano, J M; Guallar, V; Quesada, R; Gao, T; Pérez-Tomás, R

    2012-02-15

    The PI3K/AKT/mTOR signaling pathway regulates cell proliferation, survival and angiogenesis. The mammalian target of rapamycin (mTOR) is a protein kinase ubiquitously expressed within cells that regulates cell growth and survival by integrating nutrient and hormonal signals. mTOR exists in two complexes, mTORC1 and mTORC2. Hyperactivation of the mTOR protein has been linked to development of cancer, raising mTOR as an attractive target for cancer therapy. Prodigiosin (PG) and obatoclax (OBX), two members of the prodiginines family, are small molecules with anticancer properties which are currently under clinical trials. In the present paper, we demonstrate that mTOR is a molecular target of both prodiginines in melanoma, a highly drug-resistant cancer model. The inhibition of mTORC1 and mTORC2 complexes by PG or OBX resulted in a loss of AKT phosphorylation at S473, preventing its full activation, with no significant effect on T308. The strongest activity inhibition (89%) was induced by PG on mTORC2. Binding assays using Surface Plasmon Resonance (SPR) provide kinetic and affinity data of the interaction of these small molecules with mTOR. In addition, in silico modeling produced a detailed atomic description of the binding modes. These results provide new data to understand the mechanism of action of these molecules, and provide new structural data that will allow the development of more specific mTOR inhibitors for cancer treatment.

  3. Rictor/mTORC2 signaling mediates TGFβ1-induced fibroblast activation and kidney fibrosis.

    PubMed

    Li, Jianzhong; Ren, Jiafa; Liu, Xin; Jiang, Lei; He, Weichun; Yuan, Weiping; Yang, Junwei; Dai, Chunsun

    2015-09-01

    The mammalian target of rapamycin (mTOR) was recently identified in two structurally distinct multiprotein complexes: mTORC1 and mTORC2. Previously, we found that Rictor/mTORC2 protects against cisplatin-induced acute kidney injury, but the role and mechanisms for Rictor/mTORC2 in TGFβ1-induced fibroblast activation and kidney fibrosis remains unknown. To study this, we initially treated NRK-49F cells with TGFβ1 and found that TGFβ1 could activate Rictor/mTORC2 signaling in cultured cells. Blocking Rictor/mTORC2 signaling with Rictor or Akt1 small interfering RNAs markedly inhibited TGFβ1-induced fibronection and α-smooth muscle actin expression. Ensuing western blotting or immunostaining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureteral obstruction. Next, a mouse model with fibroblast-specific deletion of Rictor was generated. These knockout mice were normal at birth and had no obvious kidney dysfunction or kidney morphological abnormality within 2 months of birth. Compared with control littermates, the kidneys of Rictor knockout mice developed less interstitial extracellular matrix deposition and inflammatory cell infiltration at 1 or 2 weeks after ureteral obstruction. Thus our study suggests that Rictor/mTORC2 signaling activation mediates TGFβ1-induced fibroblast activation and contributes to the development of kidney fibrosis. This may provide a therapeutic target for chronic kidney diseases.

  4. Rictor/mTORC2 signaling mediates TGFβ1-induced fibroblast activation and kidney fibrosis

    PubMed Central

    Li, Jianzhong; Ren, Jiafa; Liu, Xin; Jiang, Lei; He, Weichun; Yuan, Weiping; Yang, Junwei; Dai, Chunsun

    2015-01-01

    The mammalian target of rapamycin (mTOR) was recently identified in two structurally distinct multiprotein complexes: mTORC1 and mTORC2. Previously, we found that Rictor/mTORC2 protects against cisplatin-induced acute kidney injury, but the role and mechanisms for Rictor/mTORC2 in TGFβ1-induced fibroblast activation and kidney fibrosis remains unknown. To study this, we initially treated NRK-49F cells with TGFβ1 and found that TGFβ1 could activate Rictor/mTORC2 signaling in cultured cells. Blocking Rictor/mTORC2 signaling with Rictor or Akt1 small interfering RNAs markedly inhibited TGFβ1-induced fibronection and α-smooth muscle actin expression. Ensuing western blotting or immunostaining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureteral obstruction. Next, a mouse model with fibroblast-specific deletion of Rictor was generated. These knockout mice were normal at birth and had no obvious kidney dysfunction or kidney morphological abnormality within 2 months of birth. Compared with control littermates, the kidneys of Rictor knockout mice developed less interstitial extracellular matrix deposition and inflammatory cell infiltration at 1 or 2 weeks after ureteral obstruction. Thus our study suggests that Rictor/mTORC2 signaling activation mediates TGFβ1-induced fibroblast activation and contributes to the development of kidney fibrosis. This may provide a therapeutic target for chronic kidney diseases. PMID:25970154

  5. mTORC2 Signaling Regulates Nox4-Induced Podocyte Depletion in Diabetes

    PubMed Central

    Eid, Stéphanie; Boutary, Suzan; Braych, Kawthar; Sabra, Ramzi; Massaad, Charbel; Hamdy, Ahmed; Rashid, Awad; Moodad, Sarah; Block, Karen; Gorin, Yves; Abboud, Hanna E.

    2016-01-01

    Abstract Aim: Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. We explored the hypothesis that mammalian target of rapamycin complex 2 (mTORC2) mediates podocyte injury in diabetes. Results: High glucose (HG)-induced podocyte injury reflected by alterations in the slit diaphragm protein podocin and podocyte depletion/apoptosis. This was paralleled by activation of the Rictor/mTORC2/Akt pathway. HG also increased the levels of Nox4 and NADPH oxidase activity. Inhibition of mTORC2 using small interfering RNA (siRNA)-targeting Rictor in vitro decreased HG-induced Nox1 and Nox4, NADPH oxidase activity, restored podocin levels, and reduced podocyte depletion/apoptosis. Inhibition of mTORC2 had no effect on mammalian target of rapamycin complex 1 (mTORC1) activation, described by our group to be increased in diabetes, suggesting that the mTORC2 activation by HG could mediate podocyte injury independently of mTORC1. In isolated glomeruli of OVE26 mice, there was a similar activation of the Rictor/mTORC2/Akt signaling pathway with increase in Nox4 and NADPH oxidase activity. Inhibition of mTORC2 using antisense oligonucleotides targeting Rictor restored podocin levels, reduced podocyte depletion/apoptosis, and attenuated glomerular injury and albuminuria. Innovation: Our data provide evidence for a novel function of mTORC2 in NADPH oxidase-derived reactive oxygen species generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. Conclusion: mTORC2 and/or NADPH oxidase inhibition may represent a therapeutic modality for diabetic kidney disease. Antioxid. Redox Signal. 25, 703–719. PMID:27393154

  6. In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.

    PubMed

    Bulley, Simon J; Droubi, Alaa; Clarke, Jonathan H; Anderson, Karen E; Stephens, Len R; Hawkins, Phillip T; Irvine, Robin F

    2016-09-20

    Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP3, its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal. PMID:27601656

  7. 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

  8. A dual mTORC1 and mTORC2 inhibitor shows antitumor activity in esophageal squamous cell carcinoma cells and sensitizes them to cisplatin.

    PubMed

    Huang, Yu; Xi, Qingsong; Chen, Yu; Wang, Jing; Peng, Ping; Xia, Shu; Yu, Shiying

    2013-10-01

    The mammalian target of rapamycin (mTOR) signaling pathway is critical for the growth and proliferation of various malignant tumors, including esophageal squamous cell carcinoma (ESCC). Therefore, targeting of mTOR protein is a promising strategy for therapy in this disease. In the present study, we examined the antitumor effects of a specific mTOR kinase inhibitor, PP242, which blocks both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) pathways, in two ESCC cell lines: Eca-109 and TE-1. We showed that PP242, but not rapamycin, attenuated the activities of both mTORC1 and mTORC2 signaling in ESCC. PP242 inhibited 4E-binding protein-1 phosphorylation and abrogated mTORC1-dependent PI3K/Akt feedback activation. Significantly, PP242 effectively suppressed ESCC cell proliferation, induced apoptosis, and arrested the cell cycle. Furthermore, PP242 promoted cisplatin-induced apoptosis and enhanced the antitumor efficacy of cisplatin in ESCC cells, which was likely to be associated with inhibition of Akt activity. Our results show that simultaneous targeting of both mTORC1 and mTORC2 pathways leads to effective antitumor actions in ESCC, and strongly suggest that dual mTORC1/2 inhibitors should be developed as potential agents for the treatment of ESCC.

  9. mTORC2 controls actin polymerization required for consolidation of long-term memory.

    PubMed

    Huang, Wei; Zhu, Ping Jun; Zhang, Shixing; Zhou, Hongyi; Stoica, Loredana; Galiano, Mauricio; Krnjević, Krešimir; Roman, Gregg; Costa-Mattioli, Mauro

    2013-04-01

    A major goal of biomedical research is the identification of molecular and cellular mechanisms that underlie memory storage. Here we report a previously unknown signaling pathway that is necessary for the conversion from short- to long-term memory. The mammalian target of rapamycin (mTOR) complex 2 (mTORC2), which contains the regulatory protein Rictor (rapamycin-insensitive companion of mTOR), was discovered only recently and little is known about its function. We found that conditional deletion of Rictor in the postnatal murine forebrain greatly reduced mTORC2 activity and selectively impaired both long-term memory (LTM) and the late phase of hippocampal long-term potentiation (L-LTP). We also found a comparable impairment of LTM in dTORC2-deficient flies, highlighting the evolutionary conservation of this pathway. Actin polymerization was reduced in the hippocampus of mTORC2-deficient mice and its restoration rescued both L-LTP and LTM. Moreover, a compound that promoted mTORC2 activity converted early LTP into late LTP and enhanced LTM. Thus, mTORC2 could be a therapeutic target for the treatment of cognitive dysfunction.

  10. 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

  11. Sox4 cooperates with CREB in myeloid transformation

    PubMed Central

    Sandoval, Salemiz; Kraus, Christina; Cho, Er-Chieh; Cho, Michelle; Bies, Juraj; Manara, Elena; Accordi, Benedetta; Landaw, Elliot M.; Wolff, Linda; Pigazzi, Martina

    2012-01-01

    The cAMP response element-binding protein (CREB) is a nuclear transcription factor that is critical for normal and neoplastic hematopoiesis. Previous studies have demonstrated that CREB is a proto-oncogene whose overexpression promotes cellular proliferation in hematopoietic cells. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukemia. In this study, we used retroviral insertional mutagenesis to identify genes that accelerate leukemia in CREB transgenic mice. Our mutagenesis screen identified several integration sites, including oncogenes Gfi1, Myb, and Ras. The Sox4 transcription factor was identified by our screen as a gene that cooperates with CREB in myeloid leukemogenesis. We show that the transduction of CREB transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells in vitro. Furthermore, leukemic blasts from the majority of acute myeloid leukemia patients have higher CREB, phosphorylated CREB, and Sox 4 protein expression. Sox4 transduction of mouse bone marrow cells results in increased expression of CREB target genes. We also demonstrate that CREB is a direct target of Sox4 by chromatin immunoprecipitation assays. These results indicate that Sox4 and CREB cooperate and contribute to increased proliferation of hematopoietic progenitor cells. PMID:22627767

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    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

  14. Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

    PubMed Central

    2014-01-01

    Introduction The Runt-related transcription factor Runx2 is critical for skeletal development but is also aberrantly expressed in breast cancers, and promotes cell growth and invasion. A de-regulated serine/threonine kinase Akt signaling pathway is implicated in mammary carcinogenesis and cell survival; however, the mechanisms underlying Runx2 role in survival of invasive breast cancer cells are still unclear. Methods The phenotypic analysis of Runx2 function in cell survival was performed by gene silencing and flow cytometric analysis in highly invasive MDA-MB-231 and SUM-159-PT mammary epithelial cell lines. The expression analysis of Runx2 and pAkt (serine 473) proteins in metastatic breast cancer specimens was performed by immunohistochemistry. The mRNA and protein levels of kinases and phosphatases functional in Akt signaling were determined by real-time PCR and Western blotting, while DNA-protein interaction was studied by chromatin immunoprecipitation assays. Results The high Runx2 levels in invasive mammary epithelial cell lines promoted cell survival in Akt phosphorylation (pAkt-serine 473) dependent manner. The analysis of kinases and phosphatases associated with pAkt regulation revealed that Runx2 promotes pAkt levels via mammalian target of rapamycin complex-2 (mTORC2). The recruitment of Runx2 on mTOR promoter coupled with Runx2-dependent expression of mTORC2 component Rictor defined Runx2 function in pAkt-mediated survival of invasive breast cancer cells. Conclusions Our results identified a novel mechanism of Runx2 regulatory crosstalk in Akt signaling that could have important consequences in targeting invasive breast cancer-associated cell survival. PMID:24479521

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

    PubMed Central

    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

    2016-01-01

    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. PMID:26026051

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

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

    2007-04-20

    Adenosine A(2A) 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 D(2) dopamine (DA) receptors. Behavioral and biochemical studies indicate an antagonistic relationship between A(2A) and D(2) receptors. Previous studies have demonstrated that food-restricted (FR) rats display behavioral and striatal cellular hypersensitivity to D(1) and D(2) 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 A(2A) 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 A(2A) agonist, and suggest the involvement of an upregulated A(2A) receptor-linked signaling pathway in NAc. Medications targeting the A(2A) receptor may have utility in the treatment of maladaptive behaviors associated with FR

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

    PubMed

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

    2014-07-16

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

  2. Potent dual inhibitors of TORC1 and TORC2 complexes (KU-0063794 and KU-0068650) demonstrate in vitro and ex vivo anti-keloid scar activity.

    PubMed

    Syed, Farhatullah; Sanganee, Hitesh J; Singh, Subir; Bahl, Ashwani; Bayat, Ardeshir

    2013-05-01

    Mammalian target of rapamycin (mTOR) is essential in controlling several cellular functions. This pathway is dysregulated in keloid disease (KD). KD is a common fibroproliferative dermal lesion with an ill-defined treatment strategy. KD demonstrates excessive matrix deposition, angiogenesis, and inflammatory cell infiltration. In KD, both total and phosphorylated forms of mTOR and p70(S6K)(Thr421/Ser424) are upregulated. Therefore, the aim of this study was to investigate adenosine triphosphate-competitive inhibitors of mTOR kinase previously unreported in keloid and their comparative efficacy with Rapamycin. Here, we present two mTOR kinase inhibitors, KU-0063794 and KU-0068650, that target both mTORC1 and mTORC2 signaling. Treatment with either KU-0063794 or KU-0068650 resulted in complete suppression of Akt, mTORC1, and mTORC2, and inhibition of keloid cell spreading, proliferation, migration, and invasive properties at a very low concentration (2.5 μmol  l(-1)). Both KU-0063794 and KU-0068650 significantly (P<0.05) inhibited cell cycle regulation and HIF1-α expression compared with that achieved with Rapamycin alone. In addition, both compounds induced shrinkage and growth arrest in KD, associated with the inhibition of angiogenesis, induction of apoptosis, and reduction in keloid phenotype-associated markers. In contrast, Rapamycin induced minimal antitumor activity. In conclusion, potent dual mTORC1 and mTORC2 inhibitors display therapeutic potential for the treatment of KD.

  3. 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

  4. mTORC2 regulates mechanically induced cytoskeletal reorganization and lineage selection in marrow derived mesenchymal stem cells

    PubMed Central

    Sen, Buer; Xie, Zhihui; Case, Natasha; Thompson, William R.; Uzer, Gunes; Styner, Maya; Rubin, Janet

    2013-01-01

    The cell cytoskeleton interprets and responds to physical cues from the microenvironment. Applying mechanical force to mesenchymal stem cells induces formation of a stiffer cytoskeleton, which biases against adipogenic differentiation and toward osteoblastogenesis. mTORC2, the mTOR complex defined by its binding partner rictor, is implicated in resting cytoskeletal architecture and is activated by mechanical force. We asked if mTORC2 played a role in mechanical adaptation of the cytoskeleton. We found that during bi-axial strain induced cytoskeletal restructuring, mTORC2 and Akt co-localize with newly assembled focal adhesions (FA). Disrupting the function of mTORC2, or that of its downstream substrate Akt, prevented mechanically-induced F-actin stress fiber development. mTORC2 becomes associated with vinculin during strain, and knock-down of vinculin prevents mTORC2 activation. In contrast, mTORC2 is not recruited to the FA complex during its activation by insulin, nor does insulin alter cytoskeletal structure. Further, when rictor was knocked down, the ability of MSC to enter the osteoblastic lineage was reduced, and when cultured in adipogenic medium, rictor-deficient MSC showed accelerated adipogenesis. This indicated that cytoskeletal remodeling promotes osteogenesis over adipogenesis. In sum, our data show that mTORC2 is involved in stem cell responses to biophysical stimuli, regulating both signaling and cytoskeletal reorganization. As such, mechanical activation of mTORC2 signaling participates in mesenchymal stem cell lineage selection, preventing adipogenesis by preserving β-catenin and stimulating osteogenesis by generating a stiffer cytoskeleton. PMID:23821483

  5. mTORC1 and mTORC2 selectively regulate CD8+ T cell differentiation

    PubMed Central

    Pollizzi, Kristen N.; Patel, Chirag H.; Sun, Im-Hong; Oh, Min-Hee; Waickman, Adam T.; Wen, Jiayu; Delgoffe, Greg M.; Powell, Jonathan D.

    2015-01-01

    Activation of mTOR-dependent pathways regulates the specification and differentiation of CD4+ T effector cell subsets. Herein, we show that mTOR complex 1 (mTORC1) and mTORC2 have distinct roles in the generation of CD8+ T cell effector and memory populations. Evaluation of mice with a T cell–specific deletion of the gene encoding the negative regulator of mTORC1, tuberous sclerosis complex 2 (TSC2), resulted in the generation of highly glycolytic and potent effector CD8+ T cells; however, due to constitutive mTORC1 activation, these cells retained a terminally differentiated effector phenotype and were incapable of transitioning into a memory state. In contrast, CD8+ T cells deficient in mTORC1 activity due to loss of RAS homolog enriched in brain (RHEB) failed to differentiate into effector cells but retained memory characteristics, such as surface marker expression, a lower metabolic rate, and increased longevity. However, these RHEB-deficient memory-like T cells failed to generate recall responses as the result of metabolic defects. While mTORC1 influenced CD8+ T cell effector responses, mTORC2 activity regulated CD8+ T cell memory. mTORC2 inhibition resulted in metabolic reprogramming, which enhanced the generation of CD8+ memory cells. Overall, these results define specific roles for mTORC1 and mTORC2 that link metabolism and CD8+ T cell effector and memory generation and suggest that these functions have the potential to be targeted for enhancing vaccine efficacy and antitumor immunity. PMID:25893604

  6. 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

  7. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals.

    PubMed

    Altarejos, Judith Y; Montminy, Marc

    2011-03-01

    The cyclic AMP-responsive element-binding protein (CREB) is phosphorylated in response to a wide variety of signals, yet target gene transcription is only increased in a subset of cases. Recent studies indicate that CREB functions in concert with a family of latent cytoplasmic co-activators called cAMP-regulated transcriptional co-activators (CRTCs), which are activated through dephosphorylation. A dual requirement for CREB phosphorylation and CRTC dephosphorylation is likely to explain how these activator-co-activator cognates discriminate between different stimuli. Following their activation, CREB and CRTCs mediate the effects of fasting and feeding signals on the expression of metabolic programmes in insulin-sensitive tissues.

  8. Prenatal auditory stimulation alters the levels of CREB mRNA, p-CREB and BDNF expression in chick hippocampus.

    PubMed

    Chaudhury, Sraboni; Wadhwa, Shashi

    2009-10-01

    Prenatal auditory stimulation influences the development of the chick auditory pathway and the hippocampus showing an increase in various morphological parameters as well as expression of calcium-binding proteins. Calcium regulates the activity of cyclic adenosine monophosphate-response element binding (CREB) protein. CREB is known to play a role in development, undergo phosphorylation with neural activity as well as regulate transcription of BDNF. BDNF is important for the survival of neurons and regulates synaptic strength. Hence in the present study, we have evaluated the levels of CREB mRNA and protein along with p-CREB protein as well as BDNF mRNA and protein levels in the chick hippocampus at embryonic days (E) 12, E16, E20 and post-hatch day (PH) 1 following activation by prenatal auditory stimulation. Fertilized eggs were exposed to species-specific sound or sitar music (frequency range: 100-6300Hz) at 65dB levels for 15min/h over 24h from E10 till hatching. The control chick hippocampus showed higher CREB mRNA and p-CREB protein in the early embryonic stages, which later decline whereas BDNF mRNA and BDNF protein levels increase until PH1. The CREB mRNA and p-CREB protein were significantly increased at E12, E16 and PH1 in the auditory stimulated groups as compared to control group. A significant increase in the level of BDNF mRNA was observed from E12 and the protein expression from E16 onwards in both auditory stimulated groups. Therefore, enhanced phosphorylation of CREB during development following prenatal sound stimulation may be responsible for cell survival. Increased levels of p-CREB again at PH1 may trigger synthesis of proteins necessary for synaptic plasticity. Further, the increased levels of BDNF may also help in regulating synaptic plasticity. PMID:19559781

  9. 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

  10. 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

  11. 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

  12. 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

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

    PubMed

    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

  14. 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…

  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. Rapamycin prevents cadmium-induced neuronal cell death via targeting both mTORC1 and mTORC2 pathways.

    PubMed

    Xu, Chong; Liu, Chunxiao; Liu, Lei; Zhang, Ruijie; Zhang, Hai; Chen, Sujuan; Luo, Yan; Chen, Long; Huang, Shile

    2015-10-01

    Cadmium (Cd), a toxic environmental contaminant, contributes to neurodegeneration. Rapamycin, a macrocyclic lactone, has shown preventive effect on Cd-induced neuronal cell death. However, the underlying mechanism is not fully understood. Here, we show that rapamycin prevented Cd-induced apoptotic cell death in neuronal cells. Coincidently, rapamycin markedly blocked Cd-induced phosphorylation of Akt, S6K1 and 4E-BP1 in the cells. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE), conferred resistance to rapamycin inhibition of Cd-induced cell death, implying that the preventive effect of rapamycin on Cd-induced neurotoxicity is mTOR kinase activity-dependent. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor, rictor or raptor/rictor enhanced rapamycin's blockage of Cd-induced cell death. Furthermore, downregulation of S6K1, ectopic expression of constitutively hypophosphorylated 4E-BP1 or dominant negative Akt, or co-treatment with Akt inhibitor also potentiated the rapamycin's inhibitory effect. The findings indicate that rapamycin prevents Cd-induced neuronal cell death via suppressing both mTORC1 and mTORC2 pathways. Our results highlight that rapamycin may be exploited for the prevention of Cd-induced neurodegenerative disorders.

  17. 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.

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

    PubMed Central

    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

  19. 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

  20. Ablation of the mTORC2 component rictor in brain or Purkinje cells affects size and neuron morphology.

    PubMed

    Thomanetz, Venus; Angliker, Nico; Cloëtta, Dimitri; Lustenberger, Regula M; Schweighauser, Manuel; Oliveri, Filippo; Suzuki, Noboru; Rüegg, Markus A

    2013-04-15

    The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. Whereas mTORC1 is known to regulate cell and organismal growth, the role of mTORC2 is less understood. We describe two mouse lines that are devoid of the mTORC2 component rictor in the entire central nervous system or in Purkinje cells. In both lines neurons were smaller and their morphology and function were strongly affected. The phenotypes were accompanied by loss of activation of Akt, PKC, and SGK1 without effects on mTORC1 activity. The striking decrease in the activation and expression of several PKC isoforms, the subsequent loss of activation of GAP-43 and MARCKS, and the established role of PKCs in spinocerebellar ataxia and in shaping the actin cytoskeleton strongly suggest that the morphological deficits observed in rictor-deficient neurons are mediated by PKCs. Together our experiments show that mTORC2 has a particularly important role in the brain and that it affects size, morphology, and function of neurons.

  1. 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

  2. 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

  3. Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity

    PubMed Central

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

    2015-01-01

    Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been demonstrated to lower blood glucose, blood lipid, and body weight in patients with type 2 diabetes mellitus. The anti-obesity effect of berberine has been attributed to its anti-adipogenic activity. However, the underlying molecular mechanism remains largely unknown. In the present study, we found that berberine significantly suppressed the expressions of CCAAT/enhancer-binding protein (C/EBP)α, peroxisome proliferators-activated receptor γ2 (PPARγ2), and other adipogenic genes in the process of adipogenesis. Berberine decreased cAMP-response element-binding protein (CREB) phosphorylation and C/EBPβ expression at the early stage of 3T3-L1 preadipocyte differentiation. In addition, CREB phosphorylation and C/EBPβ expression induced by 3-isobutyl-1-methylxanthine (IBMX) and forskolin were also attenuated by berberine. The binding activities of cAMP responsive element (CRE) stimulated by IBMX and forskolin were inhibited by berberine. The binding of phosphorylated CREB to the promoter of C/EBPβ was abrogated by berberine after the induction of preadipocyte differentiation. These results suggest that berberine blocks adipogenesis mainly via suppressing CREB activity, which leads to a decrease in C/EBPβ-triggered transcriptional cascades. PMID:25928058

  4. Dynamic O-GlcNAc modification regulates CREB-mediated gene expression and memory formation.

    PubMed

    Rexach, Jessica E; Clark, Peter M; Mason, Daniel E; Neve, Rachael L; Peters, Eric C; Hsieh-Wilson, Linda C

    2012-01-22

    The transcription factor cyclic AMP-response element binding protein (CREB) is a key regulator of many neuronal processes, including brain development, circadian rhythm and long-term memory. Studies of CREB have focused on its phosphorylation, although the diversity of CREB functions in the brain suggests additional forms of regulation. Here we expand on a chemoenzymatic strategy for quantifying glycosylation stoichiometries to characterize the functional roles of CREB glycosylation in neurons. We show that CREB is dynamically modified with an O-linked β-N-acetyl-D-glucosamine sugar in response to neuronal activity and that glycosylation represses CREB-dependent transcription by impairing its association with CREB-regulated transcription coactivator (CRTC; also known as transducer of regulated CREB activity). Blocking glycosylation of CREB alters cellular function and behavioral plasticity, enhancing both axonal and dendritic growth and long-term memory consolidation. Our findings demonstrate a new role for O-glycosylation in memory formation and provide a mechanistic understanding of how glycosylation contributes to critical neuronal functions. Moreover, we identify a previously unknown mechanism for the regulation of activity-dependent gene expression, neural development and memory.

  5. Morphoproteomics identifies constitutive activation of the mTORC2/Akt and NF-κB pathways and expressions of IGF-1R, Sirt1, COX-2, and FASN in peripheral T-cell lymphomas: pathogenetic implications and therapeutic options

    PubMed Central

    Quesada, Andrés E; Nguyen, Nghia D; Rios, Adan; Brown, Robert E

    2014-01-01

    Background: Gaining a better understanding of the molecular circuitries and pathways implicated in the malignant growth and biological behavior of T cell lymphomas may identify potential cellular targets with clinical therapeutic potential. The immunohistochemical characterization of key cellular proteins participating in these pathways can provide surrogate markers of biological activity. The mammalian target of rapamycin complex (mTORC) signaling pathway has been implicated in T-cell lymphopoiesis. The mTORC2 pathway involves downstream activation of nuclear factor (NF)-κB and p-Akt (Ser 473). Fatty acid synthase (FASN) and insulin-like growth factor-1 receptor (IGF-1R) are expressed upstream of the mTORC and NF-κB signaling pathways. Cyclooxygenase (COX)-2 products influence these pathways. Our goal was to use morphoproteomics to characterize the expression patterns of the proteins in various peripheral T-cell lymphomas. Design: Ten cases of peripheral T-cell lymphoma (PTCL) were examined for expression of proteins along the mTORC, Akt and NF-κB pathways and affiliated tumorigenic molecules. These included two angioimmunoblastic PTCL, one natural killer/PTCL, one anaplastic large PTCL, and six PTCL not otherwise specified. Immunostaining for phosphorylated (p) mTOR (Ser 2448), p-Akt (Ser 473), p-NF-κBp65 (Ser 536), IGF-1R (Tyr1165/1166), silent mating type information regulation 2 homolog 1 (Sirt1), COX-2 and FASN was performed on paraffin-embedded tissue for each case. Percent expression was scored using bright-field microscopy with high expression designated as more than 50% of the cells with positive stain in the appropriate subcellular compartment. Results: All ten cases demonstrated nuclear staining for p-mTOR (Ser 2448) corresponding to mTORC 2, and all cases showed strong, diffuse nuclear staining for p-NF-κBp65 (Ser 536). All ten also showed nuclear and cytoplasmic staining for p-Akt (Ser 473) and cytoplasmic staining for IGF-1R. High expressions

  6. NMDA receptor-dependent CREB activation in survival of cerebellar granule cells during in vivo and in vitro development.

    PubMed

    Monti, Barbara; Marri, Lucia; Contestabile, Antonio

    2002-10-01

    During both in vivo and in vitro development, cerebellar granule cells depend on the activity of the NMDA glutamate receptor subtype for survival and full differentiation. With the present results, we demonstrate that CREB activation, downstream of the NMDA receptor, is a necessary step to ensure survival of these neurons. The levels of CREB expression and activity increase progressively during the second week of postnatal cerebellar development and the phosphorylated form of CREB is localized selectively to cerebellar granule cells during the critical developmental stages examined. Chronically blocking the NMDA receptor through systemic administration of the competitive antagonist, CGP 39551, during the in vivo critical developmental period, between 7-11 postnatal days, results in increased apoptotic elimination of differentiating granule neurons in the cerebellum [Monti & Contestabile, Eur. J. Neurosci., 12, 3117-3123 (2000)]. We report here that this event is accompanied by a significant decrease of CREB phosphorylation in the cerebellum of treated rat pups. When cerebellar granule neurons are explanted and maintained in dissociated cultures, the levels of CREB phosphorylation increase with differentiation, similar to that which happens during in vivo development. When granule cells are kept in non-trophic conditions, their viability is affected and both CREB phosphorylation and transcriptional activity are decreased significantly. The neuronal viability and the deficiency of CREB activity, are both rescued by the pharmacological activation of the NMDA receptor. These results provide good circumstantial evidence for a functional link between the NMDA receptor and CREB activity in promoting neuronal survival during development.

  7. [CREB activation is a key player for ischemic tolerance in the brain].

    PubMed

    Kitagawa, Kazuo; Sasaki, Tsutomu; Terasaki, Yasukazu; Yagita, Yoshiki; Mochizuki, Hideki

    2012-01-01

    Ischemic tolerance is as powerful and reproducible for neuro-protection as hypothermia. Several pathways could be involved in acquisition of ischemic tolerance. CREB is an abundant transcription factor in the brain and plays critical role on synaptic plasticity and neuronal survival. CREB activation has been also shown to be involved in ischemic tolerance. Ischemia or oxygen-glucose deprivation leads to release of glutamate, which binds to synaptic NMDA receptor. Then, influx of calcium ions into intracellular space activates calcium-calmodulin dependent protein kinase (CaMK). CaMK I/IV phosphorylates Ser 133 of CREB, and Thr 484 of salt-inducible kinase (SIK). Phosphorylation of SIK2 at Thr 484 triggers degradation of SIK2 through ubiquitin proteasome system. SIK2 maintains the phosphorylation level of CREB-regulated transcriptional co-activator (CRTC). Degradation of SIK2 induces dephosphorylation of CRTC1, and moves CRTC1 from cytoplasm into nucleus. Thus CRTC1 binds to basic ZIP domain of CREB. Both Ser133 phosphorylation and CRTC1 bound to the basic ZIP domain of CREB enhances CRE-mediated transcription, induces gene expression of survival factors, and renders the neurons resistant to subsequent severe ischemia.

  8. Discovery of AZD3147: a potent, selective dual inhibitor of mTORC1 and mTORC2.

    PubMed

    Pike, Kurt G; Morris, Jeff; Ruston, Linette; Pass, Sarah L; Greenwood, Ryan; Williams, Emma J; Demeritt, Julie; Culshaw, Janet D; Gill, Kristy; Pass, Martin; Finlay, M Raymond V; Good, Catherine J; Roberts, Craig A; Currie, Gordon S; Blades, Kevin; Eden, Jonathan M; Pearson, Stuart E

    2015-03-12

    High throughput screening followed by a lead generation campaign uncovered a novel series of urea containing morpholinopyrimidine compounds which act as potent and selective dual inhibitors of mTORC1 and mTORC2. We describe the continued compound optimization campaign for this series, in particular focused on identifying compounds with improved cellular potency, improved aqueous solubility, and good stability in human hepatocyte incubations. Knowledge from empirical SAR investigations was combined with an understanding of the molecular interactions in the crystal lattice to improve both cellular potency and solubility, and the composite parameters of LLE and pIC50-pSolubility were used to assess compound quality and progress. Predictive models were employed to efficiently mine the attractive chemical space identified resulting in the discovery of 42 (AZD3147), an extremely potent and selective dual inhibitor of mTORC1 and mTORC2 with physicochemical and pharmacokinetic properties suitable for development as a potential clinical candidate.

  9. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.

    PubMed

    Jacinto, Estela; Facchinetti, Valeria; Liu, Dou; Soto, Nelyn; Wei, Shiniu; Jung, Sung Yun; Huang, Qiaojia; Qin, Jun; Su, Bing

    2006-10-01

    Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Recent biochemical studies suggested that TORC2 is the elusive PDK2 for Akt/PKB Ser473 phosphorylation in the hydrophobic motif. Phosphorylation at Ser473, along with Thr308 of its activation loop, is deemed necessary for Akt function, although the regulatory mechanisms and physiological importance of each phosphorylation site remain to be fully understood. Here, we report that SIN1/MIP1 is an essential TORC2/PDK2 subunit. Genetic ablation of sin1 abolished Akt-Ser473 phosphorylation and disrupted rictor-mTOR interaction but maintained Thr308 phosphorylation. Surprisingly, defective Ser473 phosphorylation affected only a subset of Akt targets in vivo, including FoxO1/3a, while other Akt targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, were unaffected. Our findings reveal that the SIN1-rictor-mTOR function in Akt-Ser473 phosphorylation is required for TORC2 function in cell survival but is dispensable for TORC1 function. PMID:16962653

  10. The Rac1 inhibitor NSC23766 suppresses CREB signaling by targeting NMDA receptor function.

    PubMed

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

    2014-10-15

    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.

  11. 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

  12. ATP and noradrenaline activate CREB in astrocytes via noncanonical Ca(2+) and cyclic AMP independent pathways.

    PubMed

    Carriba, Paulina; Pardo, Luis; Parra-Damas, Arnaldo; Lichtenstein, Mathieu P; Saura, Carlos A; Pujol, Aurora; Masgrau, Roser; Galea, Elena

    2012-09-01

    In neurons, it is well established that CREB contributes to learning and memory by orchestrating the translation of experience into the activity-dependent (i.e., driven by neurotransmitters) transcription of plasticity-related genes. The activity-dependent CREB-triggered transcription requires the concerted action of cyclic AMP/protein kinase A and Ca(2+) /calcineurin via the CREB-regulated transcription co-activator (CRTC). It is not known, however, whether a comparable molecular sequence occurs in astrocytes, despite the unquestionable contribution of these cells to brain plasticity. Here we sought to determine whether and how ATP and noradrenaline cause CREB-dependent transcription in rat cortical astrocyte cultures. Both transmitters induced CREB phosphorylation (Western Blots), CREB-dependent transcription (CRE-luciferase reporter assays), and the transcription of Bdnf, a canonical regulator of synaptic plasticity (quantitative RT-PCR). We indentified a Ca(2+) and diacylglycerol-independent protein kinase C at the uppermost position of the cascade leading to CREB-dependent transcription. Notably, CREB-dependent transcription was partially dependent on ERK1/2 and CRTC, but independent of cyclic AMP/protein kinase A or Ca(2+) /calcineurin. We conclude that ATP and noradrenaline activate CREB-dependent transcription in cortical astrocytes via an atypical protein kinase C. It is of relevance that the signaling involved be starkly different to the one described in neurons since there is no convergence of Ca(2+) and cyclic AMP-dependent pathways on CRTC, which, moreover, exerts a modulatory rather than a central role. Our data thus point to the existence of an alternative, non-neuronal, glia-based role of CREB in plasticity.

  13. 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

  14. 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

  15. Identification, Synthesis and Evaluation of Substituted Benzofurazans as Inhibitors of CREB-mediated Gene Transcription

    PubMed Central

    Xie, Fuchun; Li, Bingbing X.; Broussard, Candice; Xiao, Xiangshu

    2013-01-01

    Cyclic-AMP response-element binding protein (CREB) is a stimulus-activated transcription factor. Its transcription activity requires its binding with CREB-binding protein (CBP) after CREB is phosphorylated at Ser133. The domains involved for CREB-CBP interaction are kinase-inducible domain (KID) from CREB and KID-interacting domain (KIX) from CBP. Recent studies suggest that CREB is an attractive target for novel cancer therapeutics. To identify novel chemotypes as inhibitors of KIX-KID interaction, we screened the NCI-diversity set of compounds using a split renilla luciferase assay and identified 2-[(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio]pyridine 1-oxide (compound 1) was identified as a potent inhibitor of KIX-KID interaction. However, compound 1 was not particularly selective against CREB-mediated gene transcription in living HEK 293T cells. Further structure-activity relationship studies identified 4-aniline substituted nitrobenzofurazans with improved selectivity. PMID:23953193

  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.

  18. Clock and light regulation of the CREB coactivator CRTC1 in the suprachiasmatic circadian clock.

    PubMed

    Sakamoto, Kensuke; Norona, Frances E; Alzate-Correa, Diego; Scarberry, Daniel; Hoyt, Kari R; Obrietan, Karl

    2013-05-22

    The CREB/CRE transcriptional pathway has been implicated in circadian clock timing and light-evoked clock resetting. To date, much of the work on CREB in circadian physiology has focused on how changes in the phosphorylation state of CREB regulate the timing processes. However, beyond changes in phosphorylation, CREB-dependent transcription can also be regulated by the CREB coactivator CRTC (CREB-regulated transcription coactivator), also known as TORC (transducer of regulated CREB). Here we profiled both the rhythmic and light-evoked regulation of CRTC1 and CRTC2 in the murine suprachiasmatic nucleus (SCN), the locus of the master mammalian clock. Immunohistochemical analysis revealed rhythmic expression of CRTC1 in the SCN. CRTC1 expression was detected throughout the dorsoventral extent of the SCN in the middle of the subjective day, with limited expression during early night, and late night expression levels intermediate between mid-day and early night levels. In contrast to CRTC1, robust expression of CRTC2 was detected during both the subjective day and night. During early and late subjective night, a brief light pulse induced strong nuclear accumulation of CRTC1 in the SCN. In contrast with CRTC1, photic stimulation did not affect the subcellular localization of CRTC2 in the SCN. Additionally, reporter gene profiling and chromatin immunoprecipitation analysis indicated that CRTC1 was associated with CREB in the 5' regulatory region of the period1 gene, and that overexpression of CRTC1 leads to a marked upregulation in period1 transcription. Together, these data raise the prospect that CRTC1 plays a role in fundamental aspects of SCN clock timing and entrainment.

  19. 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

  20. 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

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

    PubMed

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

    1999-05-15

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

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

    PubMed

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

    1999-05-15

    We have investigated the participation of cAMP response element-binding protein (CREB) in the response of the songbird brain to a natural auditory stimulus, a conspecific song. The cells in the two song control nuclei, the higher vocal center (HVC) and area X of zebra finches (Taeniopygia guttata), were intensely stained with an anti-CREB monoclonal antibody. Double-labeling studies showed that CREB immunoreactivity was detected only in area X-projecting neurons in the HVC. The cloned CREB cDNA from zebra finches (zCREB) is highly homologous to mammalian delta CREB. Phosphorylation of zCREB at Ser119 in area X-projecting HVC neurons was induced by hearing tape-recorded conspecific songs of zebra finches, but not by birdsongs of another species or white noise. These results raise the possibility that zCREB plays a crucial role in the sensory process of song learning. PMID:10234027

  3. GRM7 Regulates Embryonic Neurogenesis via CREB and YAP

    PubMed Central

    Xia, Wenlong; Liu, YanLi; Jiao, Jianwei

    2015-01-01

    Summary 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. Brain expression of pCREB in rats exposed to consummatory successive negative contrast.

    PubMed

    Glueck, Amanda C; Dennis, Torry S; Perrotti, Linda I; Torres, Carmen; Papini, Mauricio R

    2015-02-01

    A 32-to-4% sucrose devaluation leads to suppression of consummatory behavior relative to unshifted 4% sucrose controls. This is accompanied by an emotional response inducing memory consolidation. Expression levels of phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB, a marker of synaptic plasticity) were higher after the first devaluation session than after the second in prelimbic cortex, anterior cingulate cortex, and dorso-medial striatum. The central nucleus of the amygdala showed a tendency to differential pCREB expression. This evidence contributes to identifying the brain circuit for one form of traumatic memory involving reward loss. PMID:25529194

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

    PubMed

    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

  6. 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

  7. Effects of carvedilol treatment on cardiac cAMP response element binding protein expression and phosphorylation in acute coxsackievirus B3-induced myocarditis

    PubMed Central

    2013-01-01

    Background The role of β-adrenergic stimulation on viral myocarditis has been investigated in animal models of viral myocarditis. Excess stimulation of β-adrenergic receptors by catecholamines causes phosphorylation/activation of cAMP response element binding protein (CREB) by the cAMP signaling pathway. CREB as an important regulator of gene expression mediates the cardiovascular remodeling process and promotes anti-inflammatory immune responses. However, the CREB expression and phosphorylation have not been studied, and the effects of carvedilol (a nonselective β-adrenoceptor antagonist) on the CREB has not been investigated in the setting of acute viral myocarditis. Methods This study was therefore designed to examine the effects of carvedilol on the transcriptional factor CREB in a murine model of acute viral myocarditis. In a coxsackievirus B3 murine myocarditis model (Balb/c), effects of carvedilol on plasma noradrenaline, heart rate and blood pressure, myocardial histopathological changes and fibrosis, cardiomyocyte apoptosis, cardiac CREB and phosphorylated CREB, cytokine levels, and viral RNA were studied. Results The expression and phosphorylation of CREB were decreased with concomitant increase of IL-6 and TNF-α in murine coxsackievirus-induced acute viral myocarditis. The levels of IL-6 and TNF-α were correlated with the expression of CREB or phosphorylated CREB. Carvedilol increased the cardiac CREB expression and phosphorylation and decreased the plasma catecholamine levels and the production of IL-6 and TNF-α with amelioration of acute viral myocarditis. Conclusion These results show that CREB may be involved in the pathophysiology of viral myocarditis and carvedilol exerts some of its beneficial effects by increasing the CREB expression and phosphorylation. PMID:24225056

  8. 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.

  9. 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

  10. 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.

  11. Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2.

    PubMed

    Hogan, Meghan F; Ravnskjaer, Kim; Matsumura, Shigenobu; Huising, Mark O; Hull, Rebecca L; Kahn, Steven E; Montminy, Marc

    2015-10-23

    Under fasting conditions, increases in circulating concentrations of glucagon maintain glucose homeostasis via the induction of hepatic gluconeogenesis. Triggering of the cAMP pathway in hepatocytes stimulates the gluconeogenic program via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP-regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where it contributes to the attendant hyperglycemia. Whether selective activation of the hepatic CREB/CRTC pathway is sufficient to trigger metabolic changes in other tissues is unclear, however. Modest hepatic expression of a phosphorylation-defective and therefore constitutively active CRTC2S171,275A protein increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A-expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice, demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis.

  12. Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2*

    PubMed Central

    Hogan, Meghan F.; Ravnskjaer, Kim; Matsumura, Shigenobu; Huising, Mark O.; Hull, Rebecca L.; Kahn, Steven E.; Montminy, Marc

    2015-01-01

    Under fasting conditions, increases in circulating concentrations of glucagon maintain glucose homeostasis via the induction of hepatic gluconeogenesis. Triggering of the cAMP pathway in hepatocytes stimulates the gluconeogenic program via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP-regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where it contributes to the attendant hyperglycemia. Whether selective activation of the hepatic CREB/CRTC pathway is sufficient to trigger metabolic changes in other tissues is unclear, however. Modest hepatic expression of a phosphorylation-defective and therefore constitutively active CRTC2S171,275A protein increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A-expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice, demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis. PMID:26342077

  13. The Phosphorylation Status of a Cyclic AMP-Responsive Activator Is Modulated via a Chromatin-Dependent Mechanism

    PubMed Central

    Michael, Laura F.; Asahara, Hiroshi; Shulman, Andrew I.; Kraus, W. Lee; Montminy, Marc

    2000-01-01

    Cyclic AMP (cAMP) stimulates the expression of numerous genes via the protein kinase A (PKA)-mediated phosphorylation of CREB at Ser133. Ser133 phosphorylation, in turn, promotes recruitment of the coactivator CREB binding protein and its paralog p300, histone acetyltransferases (HATs) that have been proposed to mediate target gene activation, in part, by destabilizing promoter bound nucleosomes and thereby allowing assembly of the transcriptional apparatus. Here we show that although histone deacetylase (HDAC) inhibitors potentiate target gene activation via cAMP, they do not stimulate transcription over the early burst phase, during which CREB phosphorylation and CBP/p300 recruitment are maximal. Rather, HDAC inhibitors augment CREB activity during the late attenuation phase by prolonging CREB phosphorylation on chromosomal but, remarkably, not on extrachromosomal templates. In reconstitution studies, assembly of periodic nucleosomal arrays on a cAMP-responsive promoter template potently inhibited CREB phosphorylation by PKA, and acetylation of these template-bound nucleosomes by p300 partially rescued CREB phosphorylation by PKA. Our results suggest a novel regulatory mechanism by which cellular HATs and HDACs modulate the phosphorylation status of nuclear activators in response to cellular signals. PMID:10669737

  14. A neurotoxic glycerophosphocholine impacts PtdIns-4, 5-bisphosphate and TORC2 signaling by altering ceramide biosynthesis in yeast.

    PubMed

    Kennedy, Michael A; Gable, Kenneth; Niewola-Staszkowska, Karolina; Abreu, Susana; Johnston, Anne; Harris, Linda J; Reggiori, Fulvio; Loewith, Robbie; Dunn, Teresa; Bennett, Steffany A L; Baetz, Kristin

    2014-01-01

    Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-β42 signals the intraneuronal accumulation of PC(O-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(O-16:0/2:0) is also toxic to Saccharomyces cerevisiae, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding protein, was relocalized in response to PC(O-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(O-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P2 leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(O-16:0/2:0) on the distribution of PM PtdIns(4,5)P2 pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(O-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.

  15. The cooperation of CREB and NFAT is required for PTHrP-induced RANKL expression in mouse osteoblastic cells.

    PubMed

    Park, Hyun-Jung; Baek, Kyunghwa; Baek, Jeong-Hwa; Kim, Hyung-Ryong

    2015-03-01

    Parathyroid hormone-related protein (PTHrP) is known to induce the expression of receptor activator of NF-κB ligand (RANKL) in stromal cells/osteoblasts. However, the signaling pathways involved remain controversial. In the present study, we investigated the role of cAMP/protein kinase A (PKA) and calcineurin/NFAT pathways in PTHrP-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. PTHrP-mediated induction of RANKL expression was significantly inhibited by H89 and FK506, an inhibitor of PKA and calcineurin, respectively. PTHrP upregulated CREB phosphorylation and the transcriptional activity of NFAT. Knockdown of CREB or NFATc1 blocked PTHrP-induced RANKL expression. PTHrP increased the activity of the RANKL promoter reporter that contains approximately 2 kb mouse RANKL promoter DNA sequences. Insertions of mutations in CRE-like element or in NFAT-binding element abrogated PTHrP-induced RANKL promoter activity. Chromatin immunoprecipitation assays showed that PTHrP increased the binding of CREB and NFATc1/NFATc3 to their cognate binding elements in the RANKL promoter. Inhibition of cAMP/PKA and its downstream ERK activity suppressed PTHrP-induced expression and transcriptional activity of NFATc1. CREB knockdown prevented PTHrP induction of NFATc1 expression. Furthermore, NFATc1 and CREB were co-immunoprecipitated. Mutations in CRE-like element completely blocked NFATc1-induced transactivation of the RANKL promoter reporter; however, mutations in NFAT-binding element partially suppressed CREB-induced RANKL promoter activity. Overexpression of CREB increased NFATc1 binding to the RANKL promoter and vice versa. These results suggest that PTHrP-induced RANKL expression depends on the activation of both cAMP/PKA and calcineurin/NFAT pathways, and subsequently, CREB and NFAT cooperate to transactivate the mouse RANKL gene.

  16. 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

  17. 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

  18. Enhancement of Behavioral Sensitization, Anxiety-Like Behavior, and Hippocampal and Frontal Cortical CREB Levels Following Cocaine Abstinence in Mice Exposed to Cocaine during Adolescence

    PubMed Central

    Valzachi, Maria Cristina; Teodorov, Elizabeth; Marcourakis, Tania; Bailey, Alexis; Camarini, Rosana

    2013-01-01

    Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p.) for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC) and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous system. PMID:24205196

  19. Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2.

    PubMed

    Yu, Ker; Shi, Celine; Toral-Barza, Lourdes; Lucas, Judy; Shor, Boris; Kim, Jae Eun; Zhang, Wei-Guo; Mahoney, Robert; Gaydos, Christine; Tardio, Luanna; Kim, Sung Kyoo; Conant, Roger; Curran, Kevin; Kaplan, Joshua; Verheijen, Jeroen; Ayral-Kaloustian, Semiramis; Mansour, Tarek S; Abraham, Robert T; Zask, Arie; Gibbons, James J

    2010-01-15

    The mammalian target of rapamycin (mTOR) is a major component of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway that is dysregulated in 50% of all human malignancies. Rapamycin and its analogues (rapalogs) partially inhibit mTOR through allosteric binding to mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report WYE-125132 (WYE-132), a highly potent, ATP-competitive, and specific mTOR kinase inhibitor (IC(50): 0.19 +/- 0.07 nmol/L; >5,000-fold selective versus PI3Ks). WYE-132 inhibited mTORC1 and mTORC2 in diverse cancer models in vitro and in vivo. Importantly, consistent with genetic ablation of mTORC2, WYE-132 targeted P-AKT(S473) and AKT function without significantly reducing the steady-state level of the PI3K/PDK1 activity biomarker P-AKT(T308), highlighting a prominent and direct regulation of AKT by mTORC2 in cancer cells. Compared with the rapalog temsirolimus/CCI-779, WYE-132 elicited a substantially stronger inhibition of cancer cell growth and survival, protein synthesis, cell size, bioenergetic metabolism, and adaptation to hypoxia. Oral administration of WYE-132 to tumor-bearing mice showed potent single-agent antitumor activity against MDA361 breast, U87MG glioma, A549 and H1975 lung, as well as A498 and 786-O renal tumors. An optimal dose of WYE-132 achieved a substantial regression of MDA361 and A549 large tumors and caused complete regression of A498 large tumors when coadministered with bevacizumab. Our results further validate mTOR as a critical driver for tumor growth, establish WYE-132 as a potent and profound anticancer agent, and provide a strong rationale for clinical development of specific mTOR kinase inhibitors as new cancer therapy.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. Mechanism of CREB recognition and coactivation by the CREB-regulated transcriptional coactivator CRTC2.

    PubMed

    Luo, Qianyi; Viste, Kristin; Urday-Zaa, Janny Concha; Senthil Kumar, Ganesan; Tsai, Wen-Wei; Talai, Afsaneh; Mayo, Kelly E; Montminy, Marc; Radhakrishnan, Ishwar

    2012-12-18

    Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.

  7. cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.

    PubMed

    Ahn, J I; Yoo, J-Y; Kim, T H; Kim, Y I; Ferguson, S D; Fazleabas, A T; Young, S L; Lessey, B A; Ahn, J Y; Lim, J M; Jeong, J-W

    2016-01-01

    Endometriosis is a major cause of infertility and pelvic pain, affecting more than 10% of reproductive-aged women. Progesterone resistance has been observed in the endometrium of women with this disease, as evidenced by alterations in progesterone-responsive gene and protein expression. cAMPResponse Element-Binding 3-like protein 1 (Creb3l1) has previously been identified as a progesterone receptor (PR) target gene in mouse uterus via high density DNA microarray analysis. However, CREB3L1 function has not been studied in the context of endometriosis and uterine biology. In this study, we validated progesterone (P4) regulation of Creb3l1 in the uteri of wild-type and progesterone receptor knockout (PRKO) mice. Furthermore, we observed that CREB3L1 expression was significantly higher in secretory phase human endometrium compared to proliferative phase and that CREB3L1 expression was significantly decreased in the endometrium of women with endometriosis. Lastly, by transfecting CREB3L1 siRNA into cultured human endometrial stromal cells (hESCs) prior to hormonal induction of in vitro decidualization, we showed that CREB3L1 is required for the decidualization process. Interestingly, phosphorylation of ERK1/2, critical factor for decidualization, was also significantly reduced in CREB3L1-silenced hESCs. It is known that hESCs from patients with endometriosis show impaired decidualization and that dysregulation of the P4-PR signaling axis is linked to a variety of endometrial diseases including infertility and endometriosis. Therefore, these results suggest that CREB3L1 is required for decidualization in mice and humans and may be linked to the pathogenesis of endometriosis in a P4-dependent manner. PMID:26917262

  8. Co-administration of the mTORC1/TORC2 inhibitor INK128 and the Bcl-2/Bcl-xL antagonist ABT-737 kills human myeloid leukemia cells through Mcl-1 down-regulation and AKT inactivation

    PubMed Central

    Rahmani, Mohamed; Aust, Mandy Mayo; Hawkins, Elisa; Parker, Rebecca E.; Ross, Masey; Kmieciak, Maciej; Reshko, Leonid Borisovich; Rizzo, Kathryn A.; Dumur, Catherine I.; Ferreira-Gonzalez, Andrea; Grant, Steven

    2015-01-01

    Effects of concurrent inhibition of mTORC1/2 and Bcl-2/Bcl-xL in human acute myeloid leukemia cells were examined. Tetracycline-inducible Bcl-2/Bcl-xL dual knockdown markedly sensitized acute myeloid leukemia cells to the dual TORC1/2 inhibitor INK128 in vitro as well as in vivo. Moreover, INK128 co-administered with the Bcl-2/xL antagonist ABT-737 sharply induced cell death in multiple acute myeloid leukemia cell lines, including TKI-resistant FLT3-ITD mutants and primary acute myeloid leukemia blasts carrying various genetic aberrations e.g., FLT3, IDH2, NPM1, and Kras, while exerting minimal toxicity toward normal hematopoietic CD34+ cells. Combined treatment was particularly active against CD34+/CD38−/CD123+ primitive leukemic progenitor cells. The INK128/ABT-737 regimen was also effective in the presence of a protective stromal microenvironment. Notably, INK128 was more potent than the TORC1 inhibitor rapamycin in down-regulating Mcl-1, diminishing AKT and 4EBP1 phosphorylation, and potentiating ABT-737 activity. Mcl-1 ectopic expression dramatically attenuated INK128/ABT-737 lethality, indicating an important functional role for Mcl-1 down-regulation in INK128/ABT-737 actions. Immunoprecipitation analysis revealed that combined treatment markedly diminished Bax, Bak, and Bim binding to all major anti-apoptotic Bcl-2 members (Bcl-2/Bcl-xL/Mcl-1), while Bax/Bak knockdown reduced cell death. Finally, INK128/ABT-737 co-administration sharply attenuated leukemia growth and significantly prolonged survival in a systemic acute myeloid leukemia xenograft model. Analysis of subcutaneous acute myeloid leukemia-derived tumors revealed significant decrease in 4EBP1 phosphorylation and Mcl-1 protein level, consistent with results obtained in vitro. These findings demonstrate that co-administration of dual mTORC1/mTORC2 inhibitors and BH3-mimetics exhibits potent anti-leukemic activity in vitro and in vivo, arguing that this strategy warrants attention in acute myeloid

  9. Involuntary, Forced and Voluntary Exercises Equally Attenuate Neurocognitive Deficits in Vascular Dementia by the BDNF-pCREB Mediated Pathway.

    PubMed

    Lin, Yangyang; Lu, Xiao; Dong, Juntao; He, Xiaokuo; Yan, Tiebin; Liang, Huiying; Sui, Minghong; Zheng, Xiuyuan; Liu, Huihua; Zhao, Jingpu; Lu, Xinxin

    2015-09-01

    A rat model of vascular dementia was used to compare the effects of involuntary exercise induced by functional electrical stimulation (FES), forced exercise and voluntary exercise on the recovery of cognitive function recovery and its underlying mechanisms. In an involuntary exercise (I-EX) group, FES was used to induce involuntary gait-like running on ladder at 12 m/min. A forced exercise group (F-EX) and a voluntary exercise group (V-EX) exercised by wheel running. The Barnes maze was used for behavioral assessment. Brain-derived neurotrophic factor (BDNF), phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP response element binding protein (CREB) positive cells in hippocampal CA1, CA2/3 and dentate gyrus (DG) regions were evaluated using immunohistochemical methods. Western blotting was used to assess the levels of BDNF, phosphorylated protein kinase B (Akt), tropomyosin receptor kinase B (TrkB), mitogen-activated protein kinase 1 and 2 (MEK1/2), ERK1/2 and CREB in BDNF-pCREB signaling in the hippocampus and prefrontal cortex. Involuntary, forced and voluntary exercises were all found to reverse the cognitive deficits of vascular dementia with about equal effectiveness. The number of BDNF, pCREB and pERK1/2 immunopositive cells was significantly increased in the hippocampal CA1, CA2/3 and DG regions in all three exercise groups. In addition, involuntary exercise activated BDNF and the phosphorylation of Akt, TrkB, MEK1/2, ERK1/2 and CREB in the hippocampus and prefrontal cortex equally as well as voluntary or forced exercise. These results suggest that involuntary exercise induced by FES may be as beneficial for alleviating cognitive deficits after cerebral ischemia. PMID:26240057

  10. 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

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

    PubMed

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

    2015-01-01

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

  12. Reactive oxygen species decrease cAMP response element binding protein expression in cardiomyocytes via a protein kinase D1-dependent mechanism that does not require Ser133 phosphorylation.

    PubMed

    Ozgen, Nazira; Guo, Jianfen; Gertsberg, Zoya; Danilo, Peter; Rosen, Michael R; Steinberg, Susan F

    2009-10-01

    Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser(133) in cardiomyocytes. Although CREB-Ser(133) phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser(133) phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser(133) phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

  13. 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

  14. 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

  15. 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.

  16. 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…

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

    PubMed

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

    2013-04-01

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

  18. 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

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

    PubMed

    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.

  20. Transcriptional control of the inflammatory response: a role for the CREB-binding protein (CBP).

    PubMed

    Matt, Theresia

    2002-01-01

    The cellular pathophysiology of septic shock is characterized by the activation of genes in response to exposure of cells to bacterial lipopolysaccharide. Tumour necrosis factor-alpha (TNF-alpha) or endotoxin induce the activation of two major transcription factors, NF-kappa B (nuclear factor-kappaB) and AP-1 (activating protein-1), which in turn induce genes involved in chronic and acute inflammatory responses. The activity of both of them is regulated by phosphorylation and subsequent interaction with the coactivator protein CBP (CREB-binding protein). Thus, the limiting CBP may play an important role in the development of critical illness.

  1. 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.

  2. 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.

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

    PubMed

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

    2015-01-01

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

  5. 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

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

    PubMed Central

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

    2016-01-01

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

  7. Activation of TORC1 transcriptional coactivator through MEKK1-induced phosphorylation.

    PubMed

    Siu, Yeung-Tung; Ching, Yick-Pang; Jin, Dong-Yan

    2008-11-01

    CREB is a prototypic bZIP transcription factor and a master regulator of glucose metabolism, synaptic plasticity, cell growth, apoptosis, and tumorigenesis. Transducers of regulated CREB activity (TORCs) are essential transcriptional coactivators of CREB and an important point of regulation on which various signals converge. In this study, we report on the activation of TORC1 through MEKK1-mediated phosphorylation. MEKK1 potently activated TORC1, and this activation was independent of downstream effectors MEK1/MEK2, ERK2, JNK, p38, protein kinase A, and calcineurin. MEKK1 induced phosphorylation of TORC1 both in vivo and in vitro. Expression of the catalytic domain of MEKK1 alone in cultured mammalian cells sufficiently caused phosphorylation and subsequent activation of TORC1. MEKK1 physically interacted with TORC1 and stimulated its nuclear translocation. An activation domain responsive to MEKK1 stimulation was mapped to amino acids 431-650 of TORC1. As a physiological activator of CREB, interleukin 1alpha triggered MEKK1-dependent phosphorylation of TORC1 and its consequent recruitment to the cAMP response elements in the interleukin 8 promoter. Taken together, our findings suggest a new mechanism for regulated activation of TORC1 transcriptional coactivator and CREB signaling.

  8. 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.

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

    PubMed Central

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

    2015-01-01

    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

  10. 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

  11. Screening of the Human Kinome Identifies MSK1/2-CREB1 as an Essential Pathway Mediating Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication during Primary Infection

    PubMed Central

    Cheng, Fan; Sawant, Tanvee Vinod; Lan, Ke; Lu, Chun; Jung, Jae U.

    2015-01-01

    ABSTRACT Viruses often hijack cellular pathways to facilitate infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus etiologically associated with Kaposi's sarcoma, a vascular tumor of endothelial cells. Despite intensive studies, cellular pathways mediating KSHV infection and replication are still not well defined. Using an antibody array approach, we examined cellular proteins phosphorylated during primary KSHV infection of primary human umbilical vein endothelial cells. Enrichment analysis identified integrin/mitogen-activated protein kinase (integrin/MAPK), insulin/epidermal growth factor receptor (insulin/EGFR), and JAK/STAT as the activated networks during primary KSHV infection. The transcriptional factor CREB1 (cyclic AMP [cAMP]-responsive element-binding protein 1) had the strongest increase in phosphorylation. While knockdown of CREB1 had no effect on KSHV entry and trafficking, it drastically reduced the expression of lytic transcripts and proteins and the production of infectious virions. Chemical activation of CREB1 significantly enhanced viral lytic replication. In contrast, CREB1 neither influenced the expression of the latent gene LANA nor affected KSHV infectivity. Mechanistically, CREB1 was not activated through the classic cAMP/protein kinase A (cAMP/PKA) pathway or via the AKT, MK2, and RSK pathways. Rather, CREB1 was activated by the mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Consequently, chemical inhibition or knockdown of MSKs significantly inhibited the KSHV lytic replication program; however, it had a minimal effect on LANA expression and KSHV infectivity. Together, these results identify the MSK1/2-CREB1 proteins as novel essential effectors of KSHV lytic replication during primary infection. The differential effect of the MSK1/2-CREB1 pathway on the expression of viral latent and lytic genes might control the robustness of viral lytic replication, and therefore the

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. Unrestrained mammalian target of rapamycin complexes 1 and 2 increase expression of phosphatase and tensin homolog deleted on chromosome 10 to regulate phosphorylation of Akt kinase.

    PubMed

    Das, Falguni; Ghosh-Choudhury, Nandini; Dey, Nirmalya; Mandal, Chandi Charan; Mahimainathan, Lenin; Kasinath, Balakuntalam S; Abboud, Hanna E; Choudhury, Goutam Ghosh

    2012-02-01

    Tuberous sclerosis complex 2 (TSC2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) function to block growth factor-induced mammalian target of rapamycin (mTOR) signaling and are mutated in autosomal dominant hamartoma syndromes. mTOR binds to a spectrum of common and different proteins to form TOR complex 1 (TORC1) and TORC2, which regulate cell growth, division, and metabolism. TSC2 deficiency induces constitutive activation of mTOR, leading to a state of insulin resistance due to a negative feedback regulation, resulting in reduced Akt phosphorylation. We have recently described an alternative mechanism showing that in TSC2 deficiency, enhanced PTEN expression contributes to reduced Akt phosphorylation. To explore the mechanism of PTEN regulation, we used rapamycin and constitutively active mTOR to show that TORC1 increases the expression of PTEN mRNA and protein. We found that in TSC2(-/-) mouse embryonic fibroblasts expression of a kinase-dead mutant of mTOR, which inhibits both TORC1 and TORC2, decreases the expression of PTEN via transcriptional mechanism. Furthermore, kinase-dead mTOR increased and decreased phosphorylation of Akt at catalytic loop site Thr-308 and hydrophobic motif site Ser-473, respectively. Moreover, inhibition of deregulated TORC1 in TSC2-null mouse embryonic fibroblasts or in 293 cells by down-regulation of raptor decreased the levels of the transcription factor Hif1α and blocked PTEN expression, resulting in enhanced phosphorylation of Akt at Thr-308 and Ser-473. Finally, knockdown of rictor or mSin1 attenuated the expression of Hif1α, which decreased transcription of PTEN. These results unravel a previously unrecognized cell-autonomous function of TORC1 and TORC2 in the up-regulation of PTEN, which prevents phosphorylation of Akt and may shield against the development of malignancy in TSC patients. PMID:22184110

  19. The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

    PubMed

    Ma, Xiuying; Wu, Deqing; Zhou, Shu; Wan, Feng; Liu, Hua; Xu, Xiaorong; Xu, Xuanfu; Zhao, Yan; Tang, Maochun

    2016-01-01

    In the periphery of pancreatic ductal adenocarcinoma (PDAC), high accumulation of tumor-associated macrophages (TAMs), which exhibit M2 phenotype, has been shown to be correlated with extra-pancreatic invasion, lymph vessel invasion, lymph node involvement and shortened survival time. However, mechanisms by which tumor cells educate and reprogram TAMs remain largely unclear. The phenotype of TAMs in PDAC tissues was confirmed by immunofluoresence and confocal microscopy. Human CD14+ monocytes were incubated with recombinant human REG4 (rREG4) before being stimulated with LPS and IL-10 and IL-6 were measured with ELISA. A panel of M1 and M2 genes were measured by quantitative real-time PCR. Panc1, AsPC1 and BxPC3 cells were cultured in the conditioned medium (CM) and treated with REG4. The macrophages were infected with CREB shRNA or cultured by the CM of Panc1 cells infected with REG4 shRNA. The expression of CD163, CD206 and REG4 and the phosphorylation levels of epidermal growth factor receptor (EGFR), AKT and cAMP response element-binding protein (CREB) in cells were assessed with western blotting. Cell proliferation and invasiveness were also assessed. The rREG4 or the conditioned medium of Panc1 cells which secreted REG4 induced the polarization macrophages to M2 phenotype. Treatment of human macrophages with REG4 resulted in phosphorylation of EGFR, AKT and CREB. The latter was responsible for REG4-mediated macrophage polarization to M2. The conditioned medium of macrophages treated with rREG4 promoted the proliferation and invasion of pancreatic cancer cell lines. REG4, overexpressed in PDAC and secreted by cancer cells, promoted macrophage polarization to M2, through at least in part, activation of ERK1/2 and CREB and changed the microenvironment to facilitate cancer growth and metastasis.

  20. Spatial learning associated with stimulus response in goldfish Carassius auratus: relationship to activation of CREB signalling.

    PubMed

    Rajan, Koilmani Emmanuvel; Thangaleela, Subramanian; Balasundaram, Chellam

    2015-06-01

    Earlier, we reported spatial learning ability in goldfish (Carassius auratus) by using spatial paradigm with food reward. Therefore, we hypothesized that goldfish may use associated cue to integrate "where" and "what" for spatial memory. To test our hypothesis, we first trained goldfish to learn to cross the gate1, which is associated with spatial task. Subsequently, they were trained to learn to enter the task chamber and to identify the food reward chamber associated with visual cue (red/green light). Red and green lights were positioned randomly for each trial but always the food reward was kept in green chamber. In addition, to elucidate the role of the signalling cascade in spatial memory associated with visual cue, nicotinamide (NAM, 1000 mg/kg, i.p), a NAD(+) precursor, was used to inhibit the Sirtuin 1 (SIRT1) cyclic AMP response element binding protein (CREB) pathway. Fishes were trained for 5 days in a maze after treating with either vehicle (VEH, DD H2O) or NAM, and then, they were individually tested for memory. We found that VEH-treated fish learned and recalled the task successfully by showing less latency and making more correct choices than NAM-treated group. Subsequent analysis showed that NAM treatment significantly down-regulated the phosphorylation of extracellular signal-regulated kinase (ERK1/2), CREB, expression of SirT1 and brain-derived neurotrophic factor (Bdnf) in telencephalon. Taken together, our results provide behavioural evidence of spatial memory associated with visual cue in C. auratus, which could be regulated by ERK1/2-CREB-SirT1-Bdnf pathway.

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

    PubMed

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

    2016-03-01

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

  2. IL-10 Production in Macrophages Is Regulated by a TLR-Driven CREB-Mediated Mechanism That Is Linked to Genes Involved in Cell Metabolism

    PubMed Central

    Sanin, David E.; Prendergast, Catriona T.

    2015-01-01

    IL-10 is produced by macrophages in diverse immune settings and is critical in limiting immune-mediated pathology. In helminth infections, macrophages are an important source of IL-10; however, the molecular mechanism underpinning production of IL-10 by these cells is poorly characterized. In this study, bone marrow–derived macrophages exposed to excretory/secretory products released by Schistosoma mansoni cercariae rapidly produce IL-10 as a result of MyD88-mediated activation of MEK/ERK/RSK and p38. The phosphorylation of these kinases was triggered by TLR2 and TLR4 and converged on activation of the transcription factor CREB. Following phosphorylation, CREB is recruited to a novel regulatory element in the Il10 promoter and is also responsible for regulating a network of genes involved in metabolic processes, such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Moreover, skin-resident tissue macrophages, which encounter S. mansoni excretory/secretory products during infection, are the first monocytes to produce IL-10 in vivo early postinfection with S. mansoni cercariae. The early and rapid release of IL-10 by these cells has the potential to condition the dermal microenvironment encountered by immune cells recruited to this infection site, and we propose a mechanism by which CREB regulates the production of IL-10 by macrophages in the skin, but also has a major effect on their metabolic state. PMID:26116503

  3. GPR26-deficient mice display increased anxiety- and depression-like behaviors accompanied by reduced phosphorylated cyclic AMP responsive element-binding protein level in central amygdala.

    PubMed

    Zhang, L-L; Wang, J-J; Liu, Y; Lu, X-B; Kuang, Y; Wan, Y-H; Chen, Y; Yan, H-M; Fei, J; Wang, Z-G

    2011-11-24

    Anxiety disorders are among the most common and well studied psychiatric disorders in humans. A number of animal models have been established to study the mechanisms of anxiety and to test putative anxiolytic drugs. Gpr26 belongs to the G-protein-coupled receptor family and is exclusively expressed in brain tissue. To investigate the biological function of Gpr26 in vivo, we have generated Gpr26 knockout mice. The mutant mice grew and developed normally but displayed increased levels of anxiety-like behaviors in the open field and elevated plus maze tests, as well as a higher level of depression-like behaviors in the forced-swim and tail-suspension tests. Meanwhile, no significant alteration in spatial learning and memory abilities were found for Gpr26-deficient mice in the Morris water maze test. Previous studies demonstrated that lower protein kinase A (PKA)-cAMP responsive element-binding protein (CREB)-neuropeptide Y (NPY) signaling in the amygdala is linked to higher anxiety and excessive alcohol-drinking behaviors in rats. Therefore, we further examined the phosphorylated CREB (pCREB) and CREB levels in the brains of Gpr26-deficient mice. Reduced pCREB levels were observed in the central amygdala but not in the other regions, while total CREB levels remained comparable between wild-type and mutant mice. Combined, our data indicate that Gpr26 is important for emotion regulation in mice, a function probably mediated by the phosphorylation of CREB in the central amygdala.

  4. The Transcription Factor CREB Enhances Interleukin-17A Production and Inflammation in a Mouse Model of Atherosclerosis

    PubMed Central

    Kotla, Sivareddy; Singh, Nikhlesh K.; Heckle, Mark R.; Tigyi, Gabor J.; Rao, Gadiparthi N.

    2014-01-01

    It has been demonstrated that 15-lipoxygenase (15-LO) plays a role in atherogenesis, but the underlying mechanisms were unclear. Therefore, the purpose of the present study is to explore the mechanisms of 15-LO role in atherogenesis. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO-dependent metabolite of arachidonic acid (AA), stimulated the production of reactive oxygen species (ROS) by monocytes through the xanthine oxidase–mediated activation of NADPH oxidase, which led to the Syk-, Pyk2-, mitogen-activated protein kinase (MAPK)–, and cyclic adenosine monophosphate response element–binding protein (CREB)–dependent production of the pro-inflammatory cytokine interleukin-17A (IL-17A). In addition, this pathway was required for the 15(S)-HETE–dependent migration and adhesion of monocytes to endothelial cells. Consistent with these observations, we found that peritoneal macrophages from ApoE−/− mice fed a high-fat diet (a mouse model of atherosclerosis) exhibited increased xanthine oxidase and NADPH oxidase activities, ROS production, phosphorylation of Syk, Pyk2, MAPK, and CREB, and enhanced IL-17A production compared to those from ApoE−/−:12/15-LO−/− mice. These events correlated with increased lipid deposits and numbers of monocytes and macrophages in the aortic arches of these mice, which resulted in atherosclerotic plaque formation. Together, these observations suggest that 15(S)-HETE exacerbates atherogenesis by enhancing CREB-dependent IL-17A production. PMID:24045154

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

    PubMed

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

    2005-01-01

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

  6. Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 beta gene.

    PubMed Central

    Tsukada, J; Saito, K; Waterman, W R; Webb, A C; Auron, P E

    1994-01-01

    A site located between -2782 and -2729 of the human prointerleukin-1 beta (IL1B) gene functions as a strong lipopolysaccharide (LPS)-responsive enhancer independent of the previously identified enhancer located between -2896 and -2846 (F. Shirakawa, K. Saito, C.A. Bonagura, D.L. Galson, M. J. Fenton, A. C. Webb, and P. E. Auron, Mol. Cell. Biol. 13:1332-1344, 1993). Although these two enhancers appear to function cooperatively in the native sequence context, they function independently as LPS-responsive elements upon removal of an interposed silencer sequence. The new enhancer is not induced by dibutyryl cyclic AMP (dbcAMP) alone but is superinduced by costimulation with LPS-dbcAMP. This pattern of induction depends upon the nature of the sequence, a composite NF-IL6-cAMP response element (CRE) binding site. This pseudosymmetrical sequence is shown to contrast with a classical symmetric CRE which responds to dbcAMP but not LPS. DNA binding studies using in vivo nuclear extract, recombinant proteins, and specific antibodies show that LPS induces the formation of two different complexes at the enhancer: (i) an NF-IL6-CREB heterodimer and (ii) a heterodimer consisting of NF-IL6 and a non-CREB, CRE-binding protein. Cotransfection studies using NF-IL6 and CREB expression vectors show that NF-IL6 transactivates the enhancer in the presence of LPS, whereas CREB acts either positively or negatively, depending upon its cAMP-regulated phosphorylation state. Our data demonstrate that the newly identified enhancer is a specialized LPS-responsive sequence which can be modulated by cAMP as a result of the involvement of NF-IL6-CRE-binding protein heterodimers. Images PMID:7935442

  7. Coupling gene expression to cAMP signalling: role of CREB and CREM.

    PubMed

    Sassone-Corsi, P

    1998-01-01

    Several endocrine and neuronal functions are governed by the cAMP-dependent pathway. Transcriptional regulation upon stimulation of this pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members, which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). CRE-binding protein (CREBs) function is modulated by phosphorylation by several kinases. Direct activation of gene expression by CREB requires phosphorylation by the cAMP-dependent PKA to serine 133. Among the repressors, ICER (Inducible cAMP Early Repressor) deserves special mention. ICER is generated from an alternative CREM promoter and is the only inducible CRE-binding protein. ICER negatively autoregulates the alternative promoter, generating a feedback loop. ICER expression is tissue specific and developmentally regulated. The kinetics of ICER expression are characteristic of an early response gene. CREM plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. The transcriptional activator CREM is highly expressed in postmeiotic cells. The role of CREM in spermiogenesis was addressed using CREM knock-out mice. Spermatogenesis stops at the first step of spermiogenesis in the mutants and there is a significant increase in apoptotic germ cells. This phenotype is reminiscent of cases of human infertility. ICER is regulated in a circadian manner in the pineal gland, the site of the hormone melatonin production. This night-day oscillation is driven by the endogenous clock (located in the suprachiasmatic nucleus). The synthesis of melatonin is regulated by a rate-limiting enzyme, serotonin N-acetyltransferase (NAT). Analysis of the CREM-null mice and of the promoter of the NAT gene revealed that ICER controls the amplitude and rhythmicity of NAT, and thus the oscillation in the hormonal synthesis of melatonin.

  8. Phosphorylation of chemoattractant receptors regulates chemotaxis, actin reorganization and signal relay.

    PubMed

    Brzostowski, Joseph A; Sawai, Satoshi; Rozov, Orr; Liao, Xin-Hua; Imoto, Daisuke; Parent, Carole A; Kimmel, Alan R

    2013-10-15

    Migratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.

  9. 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

  10. 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

  11. 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

  12. Lithium enhances CRTC oligomer formation and the interaction between the CREB coactivators CRTC and CBP--implications for CREB-dependent gene transcription.

    PubMed

    Heinrich, Annette; von der Heyde, Anne Sophie; Böer, Ulrike; Phu, Do Thanh; Tzvetkov, Mladen; Oetjen, Elke

    2013-01-01

    Lithium salts are important drugs to treat bipolar disorder. Previous work showed that lithium by enforcing the interaction between the transcription factor CREB and its coactivator CRTC1 enhanced cAMP-stimulated CREB-dependent gene transcription. Both CREB and CRTC have been implicated in neuronal adaptation, which might underlie lithium's therapeutic action. In the present study the mechanisms of lithium action on cAMP-induced CREB-dependent gene transcription were further elucidated. Transient transfection assays revealed that all three CRTC isoforms conferred lithium responsiveness to CREB whereas their intrinsic transcriptional activities remained unchanged by lithium, suggesting a conformational change of CREB or CRTC by lithium. In in vitro protein-protein interaction assays lithium enhanced the interaction between CREB and both coactivators CRTC and CBP. Furthermore, lithium enforced the oligomerization of CRTC, a prerequisite for CREB interaction. For further evaluation it was investigated whether lithium competes with magnesium, which coordinates the conformation of the CREB basic region leucine zipper (bZip). Mutational analysis of the magnesium coordinating lysine-290 within the bZip, in vitro and intracellular interaction assays and luciferase reporter-gene assays revealed that the effect of lithium on the CREB-CRTC interaction or on the transcriptional activity, respectively, was not affected by the mutation, thus excluding a magnesium-lithium competition. However, the CREB-CRTC interaction was strongly increased in lysine-290-mutants thereby extending the CRTC-CREB interaction domain. Taken together the results exclude a competition between lithium and magnesium at the bZip, but suggest that lithium by enforcing the CRTC-oligomer formation and the interaction of CREB-CBP-CRTC enhances cAMP-induced CREB-dependent gene transcription.

  13. 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

  14. 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

  15. CREB binding protein (CBP) activation is required for luteinizing hormone beta expression and normal fertility in mice.

    PubMed

    Miller, Ryan S; Wolfe, Andrew; He, Ling; Radovick, Sally; Wondisford, Fredric E

    2012-07-01

    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.

  16. 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…

  17. Acid activation of Trpv1 leads to an up-regulation of calcitonin gene-related peptide expression in dorsal root ganglion neurons via the CaMK-CREB cascade: a potential mechanism of inflammatory pain.

    PubMed

    Nakanishi, Masako; Hata, Kenji; Nagayama, Tomotaka; Sakurai, Teruhisa; Nishisho, Toshihiko; Wakabayashi, Hiroki; Hiraga, Toru; Ebisu, Shigeyuki; Yoneda, Toshiyuki

    2010-08-01

    Increased production of calcitonin gene-related peptide (CGRP) in sensory neurons is implicated in inflammatory pain. The inflammatory site is acidic due to proton release from infiltrating inflammatory cells. Acid activation of peripheral nociceptors relays pain signals to the CNS. Here, we examined whether acid activated the transient receptor potential vanilloid subtype 1 (Trpv1), a widely recognized acid-sensing nociceptor and subsequently increased CGRP expression. Chemically induced inflammation was associated with thermal hyperalgesia and increased CGRP expression in dorsal root ganglion (DRG) in rats. In organ cultures of DRG, acid (pH 5.5) elevated CGRP expression and the selective Trpv1 antagonist 5'-Iodoresiniferatoxin decreased it. Trpv1-deficient DRG showed reduced CGRP increase by acid. Of note, many of CGRP/Trpv1-positive DRG neurons exhibited the phosphorylation of cAMP response element-binding protein (CREB), a nociceptive transcription factor. Knockdown of CREB by small interfering RNA or a dominant-negative form of CREB diminished acid-elevated CGRP expression. Acid elevated the transcriptional activity of CREB, which in turn stimulated CGRP gene promoter activity. These effects were inhibited by a Ca(2+)/calmodulin-dependent protein kinase (CaMK) inhibitor KN-93. In conclusion, our results suggest that inflammatory acidic environments activate Trpv1, leading to an up-regulation of CGRP expression via CaMK-CREB cascade, a series of events that may be associated with inflammatory pain.

  18. 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

  19. 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

  20. 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

  1. 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

  2. The VGF-derived peptide TLQP62 produces antidepressant-like effects in mice via the BDNF/TrkB/CREB signaling pathway.

    PubMed

    Lin, Peipei; Wang, Chuang; Xu, Bing; Gao, Siyun; Guo, Jiejie; Zhao, Xin; Huang, Huihui; Zhang, Junfang; Chen, Xiaowei; Wang, Qinwen; Zhou, Wenhua

    2014-05-01

    Recent studies demonstrate that the neuropeptide VGF (nonacronymic)-derived peptide is regulated in the hippocampus by antidepressant therapies. Brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB), cAMP response element-binding protein (CREB) signaling, and monoamine transmitter pathways mediate the behavioral effects of antidepressants, but it is not known if these pathways also contribute to the antidepressant-like effects of VGF-derived peptide TLQP62. Here the antidepressant-like effects of TLQP62 were evaluated by measuring immobility time in the forced swimming and tail suspension tests (FST and TST) following acute microinjection of the TLQP62 (0.25, 0.5 and 1 nmol/side) into the hippocampal CA1 regions. This treatment dose-dependently reduced immobility in the FST and TST compared to phosphate-buffered saline (PBS) infusion without affecting locomotor activity in the open field test (OFT). In addition, daily intrahippocampal microinfusion of TLQP62 (1 nmol/side/day; 21 days) also upregulated the expression of BDNF and the phosphorylation of CREB (pCREB) and TrkB (pTrkB) without altering CREB or TrkB. Blocking tissue plasminogen activator (tPA) by microinfusion of tPASTOP or TrkB activation by microinfusion of K252a 60 min prior to TLQP62 infusion almost completely abolished TLQP62-induced antidepressant-like effects, BDNF upregulation, and CREB/TrkB phosphorylation. In contrast, none of these effects were diminished by pretreatment with the non-specific 5-HT receptor antagonist metergoline, the selective 5-HT1A receptor antagonist NAN-190, the 5-HT synthase inhibitor parachlorophenylalanine, the selective α1-adrenoceptor antagonist prazosin, the β receptor antagonist propranolol, or the D2 receptor antagonist raclopride. Moreover, our study was also to investigate the antidepressant-like effects of TLQP62 (50, 250 and 500 nmol/kg; i.p.) on depression-related behaviors in comparison with fluoxetine (10mg/kg; i.p.). While TLQP62

  3. Nobiletin protects against cerebral ischemia via activating the p-Akt, p-CREB, BDNF and Bcl-2 pathway and ameliorating BBB permeability in rat.

    PubMed

    Zhang, Lan; Zhao, Huiying; Zhang, Xiangjian; Chen, Linyu; Zhao, Xumeng; Bai, Xue; Zhang, Jian

    2013-07-01

    There is cumulative evidence that the serine-threonine kinase Akt and its downstream nuclear transcription factor CREB are involved in neuronal survival and protection. The Akt activates and phosphorylates CREB at Ser133, resulting in the up-regulation of pro-survival CREB target genes such as BDNF and Bcl-2. Thus, Akt/CREB signaling pathway may be one propitious target for treatment of ischemic cerebral injury. Nobiletin (NOB) exhibits a wide spectrum of beneficial biological properties including anti-inflammatory, antioxidant, anti-carcinogenic actions and contributes to reverse learning impairment in Alzheimer's disease rat. However, little is currently known regarding the exact role of NOB in ischemic stroke. Here, we designed to evaluate its possible therapeutic effect on cerebral ischemia. Adult male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (pMCAO) and randomly divided into five groups: Sham (sham-operated+0.05% Tween-80), MCAO (pMCAO+0.9% saline), Vehicle group (pMCAO+0.05% Tween-80), NOB-L (pMCAO+NOB 10 mg/kg) and NOB-H (pMCAO+NOB 25 mg/kg) groups. Rats were pre-administered intraperitoneally once daily for 3 days before surgery and then received once again immediately after surgery. Neurological deficit scores, brain water content and infarct volume were evaluated at 24 h after stroke. Additionally, the activities of Akt, CREB, BDNF, Bcl-2 and claudin-5 in ischemic brain cortex were analyzed by the methods of immunohistochemistry, western blot and RT-qPCR. Compared with Vehicle group, neurological deficits and brain edema were relieved in NOB-H group (P<0.05), infarct volume was lessened in both NOB-L and NOB-H groups (P<0.05) at 24 h after stroke. Immunohistochemistry, western blot and RT-qPCR analysis indicated that NOB dramatically promoted the activities of Akt, CREB, BDNF and Bcl-2 (P<0.05). Meanwhile, claudin-5 expression was also enhanced. On the basis of these findings, we concluded that NOB protected the

  4. 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

  5. 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…

  6. CREB pathway links PGE2 signaling with macrophage polarization.

    PubMed

    Luan, Bing; Yoon, Young-Sil; Le Lay, John; Kaestner, Klaus H; Hedrick, Susan; Montminy, Marc

    2015-12-22

    Obesity is thought to promote insulin resistance in part via activation of the innate immune system. Increases in proinflammatory cytokine production by M1 macrophages inhibit insulin signaling in white adipose tissue. In contrast, M2 macrophages have been found to enhance insulin sensitivity in part by reducing adipose tissue inflammation. The paracrine hormone prostaglandin E2 (PGE2) enhances M2 polarization in part through activation of the cAMP pathway, although the underlying mechanism is unclear. Here we show that PGE2 stimulates M2 polarization via the cyclic AMP-responsive element binding (CREB)-mediated induction of Krupple-like factor 4 (KLF4). Targeted disruption of CREB or the cAMP-regulated transcriptional coactivators 2 and 3 (CRTC2/3) in macrophages down-regulated M2 marker gene expression and promoted insulin resistance in the context of high-fat diet feeding. As re-expression of KLF4 rescued M2 marker gene expression in CREB-depleted cells, our results demonstrate the importance of the CREB/CRTC pathway in maintaining insulin sensitivity in white adipose tissue via its effects on the innate immune system.

  7. CREB pathway links PGE2 signaling with macrophage polarization

    PubMed Central

    Luan, Bing; Yoon, Young-Sil; Le Lay, John; Kaestner, Klaus H.; Hedrick, Susan; Montminy, Marc

    2015-01-01

    Obesity is thought to promote insulin resistance in part via activation of the innate immune system. Increases in proinflammatory cytokine production by M1 macrophages inhibit insulin signaling in white adipose tissue. In contrast, M2 macrophages have been found to enhance insulin sensitivity in part by reducing adipose tissue inflammation. The paracrine hormone prostaglandin E2 (PGE2) enhances M2 polarization in part through activation of the cAMP pathway, although the underlying mechanism is unclear. Here we show that PGE2 stimulates M2 polarization via the cyclic AMP-responsive element binding (CREB)-mediated induction of Krupple-like factor 4 (KLF4). Targeted disruption of CREB or the cAMP-regulated transcriptional coactivators 2 and 3 (CRTC2/3) in macrophages down-regulated M2 marker gene expression and promoted insulin resistance in the context of high-fat diet feeding. As re-expression of KLF4 rescued M2 marker gene expression in CREB-depleted cells, our results demonstrate the importance of the CREB/CRTC pathway in maintaining insulin sensitivity in white adipose tissue via its effects on the innate immune system. PMID:26644581

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

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

    PubMed

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

    2013-09-10

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

  19. 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

  20. 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

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

    PubMed

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

    2016-05-13

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

  2. 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.

  3. Striatal microRNA controls cocaine intake through CREB signalling.

    PubMed

    Hollander, Jonathan A; Im, Heh-In; Amelio, Antonio L; Kocerha, Jannet; Bali, Purva; Lu, Qun; Willoughby, David; Wahlestedt, Claes; Conkright, Michael D; Kenny, Paul J

    2010-07-01

    Cocaine addiction is characterized by a gradual loss of control over drug use, but the molecular mechanisms regulating vulnerability to this process remain unclear. Here we report that microRNA-212 (miR-212) is upregulated in the dorsal striatum of rats with a history of extended access to cocaine. Striatal miR-212 decreases responsiveness to the motivational properties of cocaine by markedly amplifying the stimulatory effects of the drug on cAMP response element binding protein (CREB) signalling. This action occurs through miR-212-enhanced Raf1 activity, resulting in adenylyl cyclase sensitization and increased expression of the essential CREB co-activator TORC (transducer of regulated CREB; also known as CRTC). Our findings indicate that striatal miR-212 signalling has a key role in determining vulnerability to cocaine addiction, reveal new molecular regulators that control the complex actions of cocaine in brain reward circuitries and provide an entirely new direction for the development of anti-addiction therapeutics based on the modulation of noncoding RNAs.

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

    PubMed

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

    2011-11-01

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

  5. Downregulation of CREB Promotes Cell Proliferation by Mediating G1/S Phase Transition in Hodgkin Lymphoma.

    PubMed

    Lu, Fangjin; Zheng, Ying; Donkor, Paul Owusu; Zou, Peng; Mu, Ping

    2016-01-01

    The cyclic-AMP response element-binding protein (CREB), a well-known nuclear transcription factor, has been shown to play an essential role in many cellular processes, including differentiation, cell survival, and cell proliferation, by regulating the expression of downstream genes. Recently, increased expression of CREB was frequently found in various tumors, indicating that CREB is implicated in the process of tumorigenesis. However, the effects of CREB on Hodgkin lymphoma (HL) remain unknown. To clarify the role of CREB in HL, we performed knockdown experiments in HL. We found that downregulation of CREB by short hairpin RNA (shRNA) resulted in enhancement of cell proliferation and promotion of G1/S phase transition, and these effects can be rescued by expression of shRNA-resistant CREB. Meanwhile, the expression level of cell cycle-related proteins, such as cyclin D1, cyclin E1, cyclin-dependent kinase 2 (CDK2), and CDK4, was elevated in response to depletion of CREB. Furthermore, we performed chromatin immunoprecipitation (ChIP) assay and confirmed that CREB directly bound to the promoter regions of these genes, which consequently contributed to the regulation of cell cycle. Consistent with our results, a clinical database showed that high expression of CREB correlates with favorable prognosis in B-cell lymphoma patients, which is totally different from the function of CREB in other cancers such as colorectal cancer, acute myeloid leukemia, and some endocrine cancers. Taken together, all of these features of CREB in HL strongly support its role as a tumor suppressor gene that can decelerate cell proliferation by inhibiting the expression of several cell cycle-related genes. Our results provide new evidence for prognosis prediction of HL and a promising therapeutic strategy for HL patients. PMID:27458098

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

    PubMed

    Akhmedov, Dmitry; Rajendran, Kavitha; Mendoza-Rodriguez, Maria G; Berdeaux, Rebecca

    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

  7. 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

  8. Role of cAMP-responsive element-binding protein (CREB)-regulated transcription coactivator 3 (CRTC3) in the initiation of mitochondrial biogenesis and stress response in liver cells.

    PubMed

    Than, Tin Aung; Lou, Huan; Ji, Cheng; Win, Sanda; Kaplowitz, Neil

    2011-06-24

    Peroxisome proliferator-activated receptor α, coactivator 1α (PGC-1α) is the master regulator of mitochondrial biogenesis. PGC-1α expression is under the control of the transcription factor, cAMP-responsive element-binding protein (CREB). In searching for candidate transcription factors that mediate mitochondrial stress-initiated mitochondria-to-nucleus signaling in the regulation of mitochondrial biogenesis, we assessed the effect of silencing CREB-regulated transcription co-activators (CRTC). CRTC isoforms are co-activators of CREB-regulated transcription by a CREB phosphorylation-independent pathway. Using cultured HepG2 cells and primary mouse hepatocytes, we determined that mitochondrial stress imposed by the complex I inhibitor rotenone elicited mitochondrial biogenesis, which was dependent on an induction of PGC-1α, which was inhibited by silencing PGC-1α. PGC-1α induction in response to rotenone was inhibited by silencing the expression of CRTC3, which blocked downstream mitochondria biogenesis. In contrast, silencing CRTC2 did not affect the induction of this pathway in response to rotenone. Thus, CRTC3 plays a selective role in mitochondrial biogenesis in response to rotenone.

  9. 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

  10. 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

  11. 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

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

    PubMed

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

    2016-01-01

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

  13. The activation of melanogenesis by p-CREB and MITF signaling with extremely low-frequency electromagnetic fields on B16F10 melanoma.

    PubMed

    Kim, Yu-Mi; Cho, Sang-Eun; Seo, Young-Kwon

    2016-10-01

    Melanin in the skin determines the skin color, and decreased melanin causes many hypopigmentation disorders and increased damage to skin by ultraviolet B (UVB) light irradiation. Here, we stimulate melanogenesis in B16F10 melanoma cells by using specific frequencies of ELF-EMFs. In this study, we focus on the melanogenesis of EMF-ELFs and find that 60-75Hz ELF-EMFs upregulate melanin synthesis by stimulated expression of tyrosinase and TRP-1 through inhibition of phosphorylation ERK, activation of CREB, and MITF up-regulation in B16F10 melanoma cells. The results show that 60-75Hz ELF-EMFs significantly increase secreted melanin, cellular melanin content, and tyrosinase activity, and the cell mitochondria activity, cell viability, and cell membrane condition are unchanged. Furthermore, the protein expression level of MITF and p-CREB signaling pathway are significantly increased. Moreover, 60Hz ELF-EMFs reduce the phosphorylate of ERK in B16F10 melanoma cells. These findings indicate that stimulation of melanogenesis by using ELF-EMFs has therapeutic potential for treating hypopigmentation disorders such as vitiligo. PMID:27543340

  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. Identification of Creb3l4 as an essential negative regulator of adipogenesis

    PubMed Central

    Kim, T-H; Jo, S-H; Choi, H; Park, J-M; Kim, M-Y; Nojima, H; Kim, J-W; Ahn, Y-H

    2014-01-01

    Understanding the molecular networks that regulate adipogenesis is crucial for combating obesity. However, the identity and molecular actions of negative regulators that regulate the early development of adipocytes remain poorly understood. In this study, we investigated the role of CREB3L4, a member of the CREB3-like family, in the regulation of adiposity. Constitutive overexpression of CREB3L4 resulted in the inhibition of adipocyte differentiation, whereas knockdown of Creb3l4 expression caused differentiation of preadipocytes into mature adipocytes, bypassing the mitotic clonal expansion step. In 3T3-L1 preadipocytes, Creb3l4 knockdown resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ2) and CCAAT/enhancer binding protein (C/EBPα), either by increasing the protein stability of C/EBPβ or by decreasing the expression of GATA3, a negative regulator of PPARγ2 expression. Consequently, increased PPARγ2 and C/EBPα levels induced adipocyte differentiation, even in the presence of minimal hormonal inducer. Thus, it can be speculated that CREB3L4 has a role as gatekeeper, inhibiting adipogenesis in 3T3-L1 preadipocytes. Moreover, adipocytes of Creb3l4-knockout mice showed hyperplasia caused by increased adipogenesis, and exhibited improved glucose tolerance and insulin sensitivity, as compared with littermate wild-type mice. These results raise the possibility that Creb3l4 could be a useful therapeutic target in the fight against obesity and metabolic syndrome. PMID:25412305

  17. Control of CREB expression in tumors: from molecular mechanisms and signal transduction pathways to therapeutic target

    PubMed Central

    Steven, André; Seliger, Barbara

    2016-01-01

    The cyclic AMP response element binding (CREB) protein has pleiotropic activities in physiologic processes. Due to its central position downstream of many growth signaling pathways CREB has the ability to influence cell survival, growth and differentiation of normal, but also of tumor cells suggesting an oncogenic potential of CREB. Indeed, increased CREB expression and activation is associated with tumor progression, chemotherapy resistance and reduced patients' survival. We summarize here the different cellular functions of CREB in tumors of distinct histology as well as its use as potential prognostic marker. In addition, the underlying molecular mechanisms to achieve constitutive activation of CREB including structural alterations, such as gene amplification and chromosomal translocation, and deregulation, which could occur at the transcriptional, post-transcriptional and post-translational level, will be described. Since downregulation of CREB by different strategies resulted in inhibition of cell proliferation, invasion and induction of apoptosis, the role of CREB as a promising target for cancer therapy will be also discussed. PMID:26934558

  18. A CREB-Sirt1-Hes1 Circuitry Mediates Neural Stem Cell Response to Glucose Availability.

    PubMed

    Fusco, Salvatore; Leone, Lucia; Barbati, Saviana Antonella; Samengo, Daniela; Piacentini, Roberto; Maulucci, Giuseppe; Toietta, Gabriele; Spinelli, Matteo; McBurney, Michael; Pani, Giovambattista; Grassi, Claudio

    2016-02-01

    Adult neurogenesis plays increasingly recognized roles in brain homeostasis and repair and is profoundly affected by energy balance and nutrients. We found that the expression of Hes-1 (hairy and enhancer of split 1) is modulated in neural stem and progenitor cells (NSCs) by extracellular glucose through the coordinated action of CREB (cyclic AMP responsive element binding protein) and Sirt-1 (Sirtuin 1), two cellular nutrient sensors. Excess glucose reduced CREB-activated Hes-1 expression and results in impaired cell proliferation. CREB-deficient NSCs expanded poorly in vitro and did not respond to glucose availability. Elevated glucose also promoted Sirt-1-dependent repression of the Hes-1 promoter. Conversely, in low glucose, CREB replaced Sirt-1 on the chromatin associated with the Hes-1 promoter enhancing Hes-1 expression and cell proliferation. Thus, the glucose-regulated antagonism between CREB and Sirt-1 for Hes-1 transcription participates in the metabolic regulation of neurogenesis. PMID:26804914

  19. Targeted activation of CREB in reactive astrocytes is neuroprotective in focal acute cortical injury.

    PubMed

    Pardo, Luis; Schlüter, Agatha; Valor, Luis M; Barco, Angel; Giralt, Mercedes; Golbano, Arantxa; Hidalgo, Juan; Jia, Peilin; Zhao, Zhongming; Jové, Mariona; Portero-Otin, Manuel; Ruiz, Montserrat; Giménez-Llort, Lydia; Masgrau, Roser; Pujol, Aurora; Galea, Elena

    2016-05-01

    The clinical challenge in acute injury as in traumatic brain injury (TBI) is to halt the delayed neuronal loss that occurs hours and days after the insult. Here we report that the activation of CREB-dependent transcription in reactive astrocytes prevents secondary injury in cerebral cortex after experimental TBI. The study was performed in a novel bitransgenic mouse in which a constitutively active CREB, VP16-CREB, was targeted to astrocytes with the Tet-Off system. Using histochemistry, qPCR, and gene profiling we found less neuronal death and damage, reduced macrophage infiltration, preserved mitochondria, and rescued expression of genes related to mitochondrial metabolism in bitransgenic mice as compared to wild type littermates. Finally, with meta-analyses using publicly available databases we identified a core set of VP16-CREB candidate target genes that may account for the neuroprotective effect. Enhancing CREB activity in astrocytes thus emerges as a novel avenue in acute brain post-injury therapeutics.

  20. 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

  1. 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

  2. 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

  3. Molecular mechanisms of environmental enrichment: impairments in Akt/GSK3β, neurotrophin-3 and CREB signaling.

    PubMed

    Hu, Yuan-Shih; Long, Nancy; Pigino, Gustavo; Brady, Scott T; Lazarov, Orly

    2013-01-01

    Experience of mice in a complex environment enhances neurogenesis and synaptic plasticity in the hippocampus of wild type and transgenic mice harboring familial Alzheimer's disease (FAD)-linked APPswe/PS1ΔE9. In FAD mice, this experience also reduces levels of tau hyperphosphorylation and oligomeric β-amyloid. Although environmental enrichment has significant effects on brain plasticity and neuropathology, the molecular mechanisms underlying these effects are unknown. Here we show that environmental enrichment upregulates the Akt pathway, leading to the downregulation of glycogen synthase kinase 3β (GSK3β), in wild type but not FAD mice. Several neurotrophic signaling pathways are activated in the hippocampus of both wild type and FAD mice, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and this increase is accompanied by the upregulation of the BDNF receptor, tyrosine kinase B (TrkB). Interestingly, neurotrophin-3 (NT-3) is upregulated in the brains of wild type mice but not FAD mice, while insulin growth factor-1 (IGF-1) is upregulated exclusively in the brains of FAD mice. Upregulation of neurotrophins is accompanied by the increase of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus following environmental enrichment. Most importantly, we observed a significant increase in levels of cAMP response element- binding (CREB) transcripts in the hippocampus of wild type and FAD mice following environmental enrichment. However, CREB phosphorylation, a critical step for the initiation of learning and memory-required gene transcription, takes place in the hippocampus of wild type but not of FAD mice. These results suggest that experience of wild type mice in a complex environmental upregulates critical signaling that play a major role in learning and memory in the hippocampus. However, in FAD mice, some of these pathways are impaired and cannot be rescued by environmental enrichment.

  4. Upregulation of adenylate cyclase 3 (ADCY3) increases the tumorigenic potential of cells by activating the CREB pathway

    PubMed Central

    Lee, Sang Jin; Hwang, Jung-Ah; Lee, Jieun; Choi, Il-Ju; Seo, Hyehyun; Park, Jong-Hoon; Suzuki, Hiromu; Yamamoto, Eiichiro; Kim, In-Hoo; Jeong, Jin Sook; Ju, Mi Ha; Lee, Dong-Hee; Lee, Yeon-Su

    2013-01-01

    Adenylate cyclase 3 (ADCY3) is a widely expressed membrane-associated protein in human tissues, which catalyzes the formation of cyclic adenosine-3′,5′-monophosphate (cAMP). However, our transcriptome analysis of gastric cancer tissue samples (NCBI GEO GSE30727) revealed that ADCY3 expression was specifically altered in cancer samples. Here we investigated the tumor-promoting effects of ADCY3 overexpression and confirmed a significant correlation between the upregulation of ADCY3 and Lauren's intestinal-type gastric cancers. ADCY3 overexpression increased cell migration, invasion, proliferation, and clonogenicity in HEK293 cells; conversely, silencing ADCY3 expression in SNU-216 cells reduced these phenotypes. Interestingly, ADCY3 overexpression increased both the mRNA level and activity of matrix metalloproteinase 2 (MMP2) and MMP9 by increasing the levels of cAMP and phosphorylated cAMP-responsive element-binding protein (CREB). Consistent with these findings, treatment with a protein kinase A (PKA) inhibitor decreased MMP2 and MMP9 expression levels in ADCY3-overexpressing cells. Knockdown of ADCY3 expression by stable shRNA in human gastric cancer cells suppressed tumor growth in a tumor xenograft model. Thus, ADCY3 overexpression may exert its tumor-promoting effects via the cAMP/PKA/CREB pathway. Additionally, bisulfite sequencing of the ADCY3 promoter region revealed that gene expression was reduced by hypermethylation of CpG sites, and increased by 5-Aza-2′-deoxycytidine (5-Aza-dC)-induced demethylation. Our study is the first to report an association of ADCY3 with gastric cancer as well as its tumorigenic potentials. In addition, we demonstrate that the expression of ADCY3 is regulated through an epigenetic mechanism. Further study on the mechanism of ADCY3 in tumorigenesis will provide the basis as a new molecular target of gastric cancer. PMID:24113161

  5. CREB-regulated transcription co-activator family stimulates promoter II-driven aromatase expression in preadipocytes.

    PubMed

    Samarajeewa, Nirukshi U; Docanto, Maria M; Simpson, Evan R; Brown, Kristy A

    2013-08-01

    The dramatically increased prevalence of breast cancer after menopause is of great concern and is correlated with elevated local levels of estrogens. This is mainly due to an increase in aromatase expression driven by its proximal promoter II (PII). We have previously demonstrated that the CREB co-activator CRTC2 binds directly to PII and stimulates its activity via mechanisms involving LKB1-AMPK in response to prostaglandin E(2) (PGE(2)). There are three members of the CRTC family (CRTC1-3) and this study aimed to characterize the role of other CRTCs in the activation of aromatase PII. The expression and subcellular localization of CRTCs were examined in preadipocytes using qPCR and immunofluorescence. Under basal conditions, CRTC1 expression was the lowest, whereas CRTC3 transcripts were present at higher levels. Basally, CRTC2 and CRTC3 were mainly cytoplasmic and PGE(2) caused their nuclear translocation. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of CRTCs on PII activity and binding. Basal PII activity was significantly increased with all CRTCs. Forskolin (FSK)/phorbol 12-myristate 13-acetate (PMA), to mimic PGE(2), resulted in a further significant increase in PII activity with all CRTCs, with CRTC2 and CRTC3 having greater effects. This was consistent with ChIP data showing an increased binding of CRTCs to PII with FSK/PMA. Moreover, gene silencing of CRTC2 and CRTC3 significantly reduced the FSK/PMA-mediated stimulation of aromatase activity. Interestingly, CRTCs acted cooperatively with CREB1 to increase PII activity, and both CREs were found to be essential for the maximal induction of PII activity by CRTCs. Phosphorylation of CRTC2 at its AMPK target site, Ser 171, dictated its subcellular localization, and the activation of aromatase PII in preadipocytes. In conclusion, this study demonstrates that aromatase regulation in primary human breast preadipocytes involves more than one CRTC.

  6. Molecular Mechanisms of Environmental Enrichment: Impairments in Akt/GSK3β, Neurotrophin-3 and CREB Signaling

    PubMed Central

    Hu, Yuan-Shih; Long, Nancy; Pigino, Gustavo; Brady, Scott T.; Lazarov, Orly

    2013-01-01

    Experience of mice in a complex environment enhances neurogenesis and synaptic plasticity in the hippocampus of wild type and transgenic mice harboring familial Alzheimer's disease (FAD)-linked APPswe/PS1ΔE9. In FAD mice, this experience also reduces levels of tau hyperphosphorylation and oligomeric β-amyloid. Although environmental enrichment has significant effects on brain plasticity and neuropathology, the molecular mechanisms underlying these effects are unknown. Here we show that environmental enrichment upregulates the Akt pathway, leading to the downregulation of glycogen synthase kinase 3β (GSK3β), in wild type but not FAD mice. Several neurotrophic signaling pathways are activated in the hippocampus of both wild type and FAD mice, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and this increase is accompanied by the upregulation of the BDNF receptor, tyrosine kinase B (TrkB). Interestingly, neurotrophin-3 (NT-3) is upregulated in the brains of wild type mice but not FAD mice, while insulin growth factor-1 (IGF-1) is upregulated exclusively in the brains of FAD mice. Upregulation of neurotrophins is accompanied by the increase of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus following environmental enrichment. Most importantly, we observed a significant increase in levels of cAMP response element- binding (CREB) transcripts in the hippocampus of wild type and FAD mice following environmental enrichment. However, CREB phosphorylation, a critical step for the initiation of learning and memory-required gene transcription, takes place in the hippocampus of wild type but not of FAD mice. These results suggest that experience of wild type mice in a complex environmental upregulates critical signaling that play a major role in learning and memory in the hippocampus. However, in FAD mice, some of these pathways are impaired and cannot be rescued by environmental enrichment. PMID:23700479

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

    PubMed

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

    2014-10-01

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

  8. Intra-amygdala injections of CREB antisense impair inhibitory avoidance memory: Role of norepinephrine and acetylcholine

    PubMed Central

    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 brain sites, through mechanisms that include the release of norepinephrine and acetylcholine within the amygdala. Thus, CREB antisense injections may affect memory by interfering with mechanisms of modulation, rather than storage, of memory. In the present experiment, rats received bilateral intra-amygdala infusions of CREB antisense (2 nmol/1 μL) 6 h prior to inhibitory avoidance training. In vivo microdialysis samples were collected from the right amygdala before, during, and following training. CREB antisense produced amnesia tested at 48 h after training. In addition, CREB antisense infusions dampened the training-related release of norepinephrine, and to a lesser extent of acetylcholine, in the amygdala. Furthermore, intra-amygdala infusions of the β-adrenergic receptor agonist clenbuterol administered immediately after training attenuated memory impairments induced by intra-amygdala injections of CREB antisense. These findings suggest that intra-amygdala treatment with CREB antisense may affect processes involved in modulation of memory in part through interference with norepinephrine and acetylcholine neurotransmission in the amygdala. PMID:18772255

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

    PubMed Central

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

    2014-01-01

    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response. PMID:25154887

  10. 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

  11. 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.

  12. 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

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

    PubMed Central

    Abe, Kentaro; Matsui, Sumiko; Watanabe, Dai

    2015-01-01

    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

  14. Silymarin suppresses the PGE2 -induced cell migration through inhibition of EP2 activation; G protein-dependent PKA-CREB and G protein-independent Src-STAT3 signal pathways.

    PubMed

    Woo, Seon Min; Min, Kyoung-Jin; Chae, In Gyeong; Chun, Kyung-Soo; Kwon, Taeg Kyu

    2015-03-01

    Silymarin has been known as a chemopreventive agent, and possesses multiple anti-cancer activities including induction of apoptosis, inhibition of proliferation and growth, and blockade of migration and invasion. However, whether silymarin could inhibit prostaglandin (PG) E2 -induced renal cell carcinoma (RCC) migration and what are the underlying mechanisms are not well elucidated. Here, we found that silymarin markedly inhibited PGE2 -stimulated migration. PGE2 induced G protein-dependent CREB phosphorylation via protein kinase A (PKA) signaling, and PKA inhibitor (H89) inhibited PGE2 -mediated migration. Silymarin reduced PGE2 -induced CREB phosphorylation and CRE-promoter activity. PGE2 also activated G protien-independent signaling pathways (Src and STAT3) and silymarin reduced PGE2 -induced phosphorylation of Src and STAT3. Inhibitor of Src (Saracatinib) markedly reduced PGE2 -mediated migration. We found that EP2, a PGE2 receptor, is involved in PGE2 -mediated cell migration. Down regulation of EP2 by EP2 siRNA and EP2 antagonist (AH6809) reduced PGE2 -inudced migration. In contrast, EP2 agonist (Butaprost) increased cell migration and silymarin effectively reduced butaprost-mediated cell migration. Moreover, PGE2 increased EP2 expression through activation of positive feedback mechanism, and PGE2 -induced EP2 expression, as well as basal EP2 levels, were reduced in silymarin-treated cells. Taken together, our study demonstrates that silymarin inhibited PGE2 -induced cell migration through inhibition of EP2 signaling pathways (G protein dependent PKA-CREB and G protein-independent Src-STAT3).

  15. Identification of cholinergic and non-cholinergic neurons in the pons expressing phosphorylated cyclic adenosine monophosphate response element-binding protein as a function of rapid eye movement sleep.

    PubMed

    Datta, S; Siwek, D F; Stack, E C

    2009-09-29

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

  16. Effects of A-CREB, a dominant negative inhibitor of CREB, on the expression of c-fos and other immediate early genes in the rat SON during hyperosmotic stimulation in vivo

    PubMed Central

    Lubelski, Daniel; Ponzio, Todd A.; Gainer, Harold

    2016-01-01

    Intraperitoneal administration of hypertonic saline to the rat supraoptic nucleus (SON) increases the expression of several immediate early genes (IEG) and the vasopressin gene. These increases have usually been attributed to action of the cyclic-AMP Response Element Binding Protein (CREB). In this paper, we study the role of CREB in these events in vivo by delivering a potent dominant-negative form of CREB, known as A-CREB, to the rat SON through the use of an adeno-associated viral (AAV) vector. Preliminary experiments on HEK 293 cells in vitro showed that the A-CREB vector that we used completely eliminated CREB-induced c-fos expression. We stereotaxically injected this AAV-A-CREB into one SON and a control AAV into the contralateral SON of the same rat. Two weeks following these injections we injected hypertonic saline intraperitoneally into the rat. Using this paradigm, we could measure the relative effects of inhibiting CREB on the induced expression of c-fos, ngfi-a, ngfi-b, and vasopressin genes in the A-CREB AAV injected SON versus the control AAV injected SON in the same rat. We found only a small (20%) decrease of c-fos expression and a 30% decrease of ngfi-b expression in the presence of the A-CREB. There were no significant changes in expression found in the other IEGs nor in vasopressin that were produced by the A-CREB. This suggests that CREB may play only a minor role in the expression of IEGs and vasopressin in the osmotically activated SON in vivo. PMID:22079318

  17. 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.

  18. Phosphodiesterase type IV inhibition prevents sequestration of CREB binding protein, protects striatal parvalbumin interneurons and rescues motor deficits in the R6/2 mouse model of Huntington's disease.

    PubMed

    Giampà, Carmela; Middei, Silvia; Patassini, Stefano; Borreca, Antonella; Marullo, Fabrizia; Laurenti, Daunia; Bernardi, Giorgio; Ammassari-Teule, Martine; Fusco, Francesca R

    2009-03-01

    The phosphodiesterase type IV inhibitor rolipram increases cAMP response element-binding protein (CREB) phosphorylation and exerts neuroprotective effects in both the quinolinic acid rat model of Huntington's disease (DeMarch et al., 2007) and the R6/2 mouse including sparing of striatal neurons, prevention of neuronal intranuclear inclusion formation and attenuation of microglial reaction (DeMarch et al., 2008). In this study, we sought to determine if rolipram has a beneficial role in the altered distribution of CREB binding protein in striatal spiny neurons and in the motor impairments shown by R6/2 mutants. Moreover, we investigated whether rolipram treatment altered the degeneration of parvalbuminergic interneurons typical of Huntington's disease (Fusco et al., 1999). Transgenic mice and their wild-type controls from a stable colony maintained in our laboratory were treated with rolipram (1.5 mg/kg) or saline daily starting from 4 weeks of age. The cellular distribution of CREB binding protein in striatal spiny neurons was assessed by immunofluorescence, whereas parvalbuminergic neuron degeneration was evaluated by cell counts of immunohistochemically labeled tissue. Motor coordination and motor activity were also examined. We found that rolipram was effective in preventing CREB binding protein sequestration into striatal neuronal intranuclear inclusions, sparing parvalbuminergic interneurons of R6/2 mice, and rescuing their motor coordination and motor activity deficits. Our findings demonstrate the possibility of reversing pharmacologically the behavioral and neuropathological abnormalities of symptomatic R6/2 mice and underline the potential therapeutic value of phosphodiesterase type IV inhibitors in Huntington's disease.

  19. 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

  20. NF-κB Inhibition Resolves Cognitive Deficits in Experimental Type 2 Diabetes Mellitus through CREB and Glutamate/GABA Neurotransmitters Pathway.

    PubMed

    Datusalia, Ashok Kumar; Sharma, Shyam Sunder

    2016-01-01

    Diabetes is associated with deficits in memory and cognitive functions and sustained inflammation. Recently, involvement of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) has been postulated in many cognitive functions, immune system and inflammation. Despite of role of NF-κB in inflammation, a large gap remains in understanding of the mechanisms and consequences of NF-κB activation in the central nervous system.In this study, we have evaluated the effects of NF-κB activation inhibitor on memory function, neurotransmitter levels changes and brain inflammatory cytokines in type-2 diabetic rats. BAY 11-7082 (BAY) was used as a pharmacological inhibitor of IκBα (inhibitor of kappa B alpha) phosphorylation to block NF-κB activation. Type-2 diabetic rats showed significant memory impairment at 15(th) week. Three weeks BAY treatment produced significant increase in Morris water maze test learning and memory performance. Diabetic animals also showed improved performance in passive avoidance and Y-maze test paradigm following treatment with NF-κB inhibitor BAY. BAY treatment did not show any significant effect on blood glucose and insulin levels. NF-κB inhibition significantly reduced neuroinflammation as evidenced by decrease in IL-6 and TNF-α levels. BAY treatment in diabetic rats also increased the phosphorylation of CREB which indicates that the NF-κB activation inhibitor engage a CREB regulated mechanism in-vivo. Moreover, BAY also reversed the alterations in brain glutamate and GABA levels in diabetic rats. These findings corroborate that NF-κB inhibition may be an effective treatment strategy in diabetes associated cognitive deficits. PMID:26517200

  1. NF-κB Inhibition Resolves Cognitive Deficits in Experimental Type 2 Diabetes Mellitus through CREB and Glutamate/GABA Neurotransmitters Pathway.

    PubMed

    Datusalia, Ashok Kumar; Sharma, Shyam Sunder

    2016-01-01

    Diabetes is associated with deficits in memory and cognitive functions and sustained inflammation. Recently, involvement of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) has been postulated in many cognitive functions, immune system and inflammation. Despite of role of NF-κB in inflammation, a large gap remains in understanding of the mechanisms and consequences of NF-κB activation in the central nervous system.In this study, we have evaluated the effects of NF-κB activation inhibitor on memory function, neurotransmitter levels changes and brain inflammatory cytokines in type-2 diabetic rats. BAY 11-7082 (BAY) was used as a pharmacological inhibitor of IκBα (inhibitor of kappa B alpha) phosphorylation to block NF-κB activation. Type-2 diabetic rats showed significant memory impairment at 15(th) week. Three weeks BAY treatment produced significant increase in Morris water maze test learning and memory performance. Diabetic animals also showed improved performance in passive avoidance and Y-maze test paradigm following treatment with NF-κB inhibitor BAY. BAY treatment did not show any significant effect on blood glucose and insulin levels. NF-κB inhibition significantly reduced neuroinflammation as evidenced by decrease in IL-6 and TNF-α levels. BAY treatment in diabetic rats also increased the phosphorylation of CREB which indicates that the NF-κB activation inhibitor engage a CREB regulated mechanism in-vivo. Moreover, BAY also reversed the alterations in brain glutamate and GABA levels in diabetic rats. These findings corroborate that NF-κB inhibition may be an effective treatment strategy in diabetes associated cognitive deficits.

  2. 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

  3. Neuronal activity controls Bdnf expression via Polycomb de-repression and CREB/CBP/JMJD3 activation in mature neurons.

    PubMed

    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

  4. Altered LKB1/CREB-regulated transcription co-activator (CRTC) signaling axis promotes esophageal cancer cell migration and invasion.

    PubMed

    Gu, Y; Lin, S; Li, J-L; Nakagawa, H; Chen, Z; Jin, B; Tian, L; Ucar, D A; Shen, H; Lu, J; Hochwald, S N; Kaye, F J; Wu, L

    2012-01-26

    LKB1 is a tumor susceptibility gene for the Peutz-Jeghers cancer syndrome and is a target for mutational inactivation in sporadic human malignancies. LKB1 encodes a serine/threonine kinase that has critical roles in cell growth, polarity and metabolism. A novel and important function of LKB1 is its ability to regulate the phosphorylation of CREB-regulated transcription co-activators (CRTCs) whose aberrant activation is linked with oncogenic activities. However, the roles and mechanisms of LKB1 and CRTC in the pathogenesis of esophageal cancer have not been previously investigated. In this study, we observed altered LKB1-CRTC signaling in a subset of human esophageal cancer cell lines and patient samples. LKB1 negatively regulates esophageal cancer cell migration and invasion in vitro. Mechanistically, we determined that CRTC signaling becomes activated because of LKB1 loss, which results in the transcriptional activation of specific downstream targets including LYPD3, a critical mediator for LKB1 loss-of-function. Our data indicate that de-regulated LKB1-CRTC signaling might represent a crucial mechanism for esophageal cancer progression.

  5. 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

  6. Involvement of the CREB5 regulatory network in colorectal cancer metastasis.

    PubMed

    Qi, Lu; Ding, Yanqing

    2014-07-01

    The signal regulatory network involved in colorectal cancer metastasis is complicated and thus the search for key control steps in the network is of great significance for unraveling colorectal cancer metastasis mechanism and finding drug-target site. Previous studies suggested that CREB5 (cAMP responsive element binding protein 5) might play key role in the metastatic signal network of colorectal cancer. Through colorectal cancer expression profile and enriching analysis of the effect of CREB5 gene expression levels on colorectal cancer molecular events, we found that these molecular events are correlated with tumor metastasis. Based on the feature that CREB5 could combine with c-Jun to form heterodimer, together with enriched binding sites for transcription factor AP-1, we identified 16 genes which were up-regulated in the CREB5 high-expression group, contained AP-1 binding sites, and participated in cancer pathway. The molecular network involving these 16 genes, in particular, CSF1R, MMP9, PDGFRB, FIGF and IL6, regulates cell migration. Therefore, CREB5 might accelerate the metastasis of colorectal cancer by regulating these five key genes.

  7. Limonin, a Component of Dictamni Radicis Cortex, Inhibits Eugenol-Induced Calcium and cAMP Levels and PKA/CREB Signaling Pathway in Non-Neuronal 3T3-L1 Cells.

    PubMed

    Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho

    2015-12-10

    Limonin, one of the major components in dictamni radicis cortex (DRC), has been shown to play various biological roles in cancer, inflammation, and obesity in many different cell types and tissues. Recently, the odorant-induced signal transduction pathway (OST) has gained attention not only because of its function in the perception of smell but also because of its numerous physiological functions in non-neuronal cells. However, little is known about the effects of limonin and DRC on the OST pathway in non-neuronal cells. We investigated odorant-stimulated increases in Ca(2+) and cAMP, major second messengers in the OST pathway, in non-neuronal 3T3-L1 cells pretreated with limonin and ethanol extracts of DRC. Limonin and the extracts significantly decreased eugenol-induced Ca(2+) and cAMP levels and upregulated phosphorylation of CREB and PKA. Our results demonstrated that limonin and DRC extract inhibit the OST pathway in non-neuronal cells by modulating Ca(2+) and cAMP levels and phosphorylation of CREB.

  8. Involvement of AMPA receptor phosphorylation in antidepressant actions with special reference to tianeptine.

    PubMed

    Svenningsson, Per; Bateup, Helen; Qi, Hongshi; Takamiya, Kogo; Huganir, Richard L; Spedding, Michael; Roth, Bryan L; McEwen, Bruce S; Greengard, Paul

    2007-12-01

    Depression is associated with abnormal neuronal plasticity. AMPA receptors mediate transmission and plasticity at excitatory synapses in a manner which is positively regulated by phosphorylation at Ser831-GluR1, a CaMKII/PKC site, and Ser845-GluR1, a PKA site. Treatment with the selective serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitor fluoxetine increases P-Ser845-GluR1 but not P-Ser831-GluR1. Here, it was found that treatment with another antidepressant, tianeptine, increased P-Ser831-GluR1 in the frontal cortex and the CA3 region of hippocampus and P-Ser845-GluR1 in the CA3 region of hippocampus. A receptorome profile detected no affinity for tianeptine at any monaminergic receptors or transporters, confirming an atypical profile for this compound. Behavioural analyses showed that mice bearing point mutations at both Ser831- and Ser845-GluR1, treated with saline, exhibited increased latency to enter the centre of an open field and increased immobility in the tail-suspension test compared to their wild-type counterparts. Chronic tianeptine treatment increased open-field locomotion and reduced immobility in wild-type mice but not in phosphomutant GluR1 mice. P-Ser133-CREB was reduced in the CA3 region of hippocampus in phosphomutant mice, and tianeptine decreased P-Ser133-CREB in this region in wild-type, but not in phosphomutant, mice. Tianeptine increased P-Ser133-CREB in the CA1 region in wild-type mice but not in phosphomutant GluR1 mice. There were higher basal P-Ser133-CREB and c-fos levels in frontal and cingulate cortex in phosphomutant GluR1 mice; these changes in level were counteracted by tianeptine in a GluR1-independent manner. Using phosphorylation assays and phosphomutant GluR1 mice, this study provides evidence that AMPA receptor phosphorylation mediates certain explorative and antidepressant-like actions under basal conditions and following tianeptine treatment.

  9. A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction

    PubMed Central

    Fusco, Salvatore; Ripoli, Cristian; Podda, Maria Vittoria; Ranieri, Sofia Chiatamone; Leone, Lucia; Toietta, Gabriele; McBurney, Michael W.; Schütz, Günther; Riccio, Antonella; Grassi, Claudio; Galeotti, Tommaso; Pani, Giovambattista

    2012-01-01

    Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD+-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes relevant to neuronal metabolism and survival in the cortex and hippocampus of dietary-restricted animals. Biochemical studies reveal a complex interplay between CREB and Sirt-1: CREB directly regulates the transcription of the sirtuin in neuronal cells by binding to Sirt-1 chromatin; Sirt-1, in turn, is recruited by CREB to DNA and promotes CREB-dependent expression of target gene peroxisome proliferator-activated receptor-γ coactivator-1α and neuronal NO Synthase. Accordingly, expression of these CREB targets is markedly reduced in the brain of Sirt KO mice that are, like CREB-deficient mice, poorly responsive to calorie restriction. Thus, the above circuitry, modulated by nutrient availability, links energy metabolism with neurotrophin signaling, participates in brain adaptation to nutrient restriction, and is potentially relevant to accelerated brain aging by overnutrition and diabetes. PMID:22190495

  10. A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction.

    PubMed

    Fusco, Salvatore; Ripoli, Cristian; Podda, Maria Vittoria; Ranieri, Sofia Chiatamone; Leone, Lucia; Toietta, Gabriele; McBurney, Michael W; Schütz, Günther; Riccio, Antonella; Grassi, Claudio; Galeotti, Tommaso; Pani, Giovambattista

    2012-01-10

    Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD(+)-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes relevant to neuronal metabolism and survival in the cortex and hippocampus of dietary-restricted animals. Biochemical studies reveal a complex interplay between CREB and Sirt-1: CREB directly regulates the transcription of the sirtuin in neuronal cells by binding to Sirt-1 chromatin; Sirt-1, in turn, is recruited by CREB to DNA and promotes CREB-dependent expression of target gene peroxisome proliferator-activated receptor-γ coactivator-1α and neuronal NO Synthase. Accordingly, expression of these CREB targets is markedly reduced in the brain of Sirt KO mice that are, like CREB-deficient mice, poorly responsive to calorie restriction. Thus, the above circuitry, modulated by nutrient availability, links energy metabolism with neurotrophin signaling, participates in brain adaptation to nutrient restriction, and is potentially relevant to accelerated brain aging by overnutrition and diabetes. PMID:22190495

  11. Spatio-Temporal in vivo Recording of dCREB2 Dynamics in Drosophila Long-Term Memory Processing

    PubMed Central

    Zhang, Jiabin; Tanenhaus, Anne K.; Davis, John C.; Hanlon, Bret M.; Yin, Jerry C. P.

    2014-01-01

    CREB (cAMP response element-binding protein) is an evolutionarily conserved transcription factor, playing key roles in synaptic plasticity, intrinsic excitability and long-term memory (LTM) formation. The Drosophila homologue of mammalian CREB, dCREB2, is also important for LTM. However, the spatio-temporal nature of dCREB2 activity during memory consolidation is poorly understood. Using an in vivo reporter system, we examined dCREB2 activity continuously in specific brain regions during LTM processing. Two brain regions that have been shown to be important for Drosophila LTM are the ellipsoid body (EB) and the mushroom body (MB). We found that dCREB2 reporter activity is persistently elevated in EB R2/R4m neurons, but not neighboring R3/R4d neurons, following LTM-inducing training. In multiple subsets of MB neurons, dCREB2 reporter activity is suppressed immediately following LTM-specific training, and elevated during late windows. In addition, we observed heterogeneous responses across different subsets of neurons in MB αβ lobe during LTM processing. All of these changes suggest that dCREB2 functions in both the EB and MB for LTM formation, and that this activity contributes to the process of systems consolidation. PMID:25460038

  12. 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,…

  13. 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.

  14. 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

  15. Expression of Iron Regulatory Protein 1 Is Regulated not only by HIF-1 but also pCREB under Hypoxia

    PubMed Central

    Luo, Qian-Qian; Qian, Zhong-Ming; Zhou, Yu-Fu; Zhang, Meng-Wan; Wang, Dang; Zhu, Li; Ke, Ya

    2016-01-01

    The inconsistent of responses of IRP1 and HIF-1 alpha to hypoxia and the similar tendencies in the changes of IRP1 and pCREB contents led us to hypothesize that pCREB might be involved in the regulation of IRP1 under hypoxia. Here, we investigated the role of pCREB in IRP1 expression in HepG2 cells under hypoxia using quantitative PCR, western blot, immunofluorescence, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). We demonstrated that 1) Hypoxia increased pCREB levels inside of the nucleus; 2) Putative CREs were found in the IRP1 gene; 3) Nuclear extracts of HepG2 cells treated with hypoxia could bind to CRE1 and CRE3, and 100-fold competitor of putative CREs could abolish the binding activity to varying degrees; 4) pCREB was found in the CRE1 and CRE3 DNA-protein complexes of EMSA; 5) CRE1 and CRE3 binding activity of IRP1 depended on CREB activation but not on HIF-1; 6) Increased IRP1 expression under hypoxia could be prevented by LY294002; 7) ChIP assays demonstrated that pCREB binds to IRP1 promoter; and 8) HIF-1 and/or HIF-2 siRNA had no effect on the expression of pCREB and IRP1 proteins in cells treated with hypoxia for 8 hours. Our findings evidenced for the involvement of pCREB in IRP1 expression and revealed a dominant role of PI3K/Akt pathway in CREB activation under hypoxia and also suggested that dual-regulation of IRP1 expression by HIF-1 and pCERB or other transcription factor(s) under hypoxia might be a common mechanism in most if not all of hypoxia-inducible genes. PMID:27766034

  16. Oxidative phosphorylation revisited.

    PubMed

    Nath, Sunil; Villadsen, John

    2015-03-01

    The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic explanation for the energy coupling and ATP synthesis carried out in mitochondria and chloroplast thylakoids. The mechanism does not suffer from the flaws in Mitchell's chemiosmotic theory that have been pointed out in many studies since its first appearance 50 years ago, when it was hailed as a ground-breaking mechanistic explanation of what is perhaps the most important process in cellular energetics. The new findings fit very well with the predictions of Nath's torsional mechanism of energy transduction and ATP synthesis. It is argued that this mechanism, based on at least 15 years of experimental and theoretical work by Sunil Nath, constitutes a fundamentally different theory of the energy conversion process that eliminates all the inconsistencies in Mitchell's chemiosmotic theory pointed out by other authors. It is concluded that the energy-transducing complexes in oxidative phosphorylation and photosynthesis are proton-dicarboxylic acid anion cotransporters and not simply electrogenic proton translocators. These results necessitate revision of previous theories of biological energy transduction, coupling, and ATP synthesis. The novel molecular mechanism is extended to cover ATP synthesis in prokaryotes, in particular to alkaliphilic and haloalkaliphilic bacteria, essentially making it a complete theory addressing mechanistic, kinetic, and thermodynamic details. Finally, based on the new interpretation of oxidative phosphorylation, quantitative values for the P/O ratio, the amount of ATP generated per redox package of the reduced substrates, are calculated and compared with experimental values for fermentation on different substrates. It is our hope that the presentation of

  17. Renal proximal tubule Na,K-ATPase is controlled by CREB-regulated transcriptional coactivators as well as salt-inducible kinase 1.

    PubMed

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

    2015-12-01

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

  18. 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

  19. 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

  20. 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 Central

    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

  1. LIMK1 Regulates Long-Term Memory and Synaptic Plasticity via the Transcriptional Factor CREB

    PubMed Central

    Todorovski, Zarko; Asrar, Suhail; Liu, Jackie; Saw, Ner Mu Nar; Joshi, Krutika; Cortez, Miguel A.; Snead, O. Carter; Xie, Wei

    2015-01-01

    Deletion of the LIMK1 gene is associated with Williams syndrome, a unique neurodevelopmental disorder characterized by severe defects in visuospatial cognition and long-term memory (LTM). However, whether LIMK1 contributes to these deficits remains elusive. Here, we show that LIMK1-knockout (LIMK1−/−) mice are drastically impaired in LTM but not short-term memory (STM). In addition, LIMK1−/− mice are selectively defective in late-phase long-term potentiation (L-LTP), a form of long-lasting synaptic plasticity specifically required for the formation of LTM. Furthermore, we show that LIMK1 interacts and regulates the activity of cyclic AMP response element-binding protein (CREB), an extensively studied transcriptional factor critical for LTM. Importantly, both L-LTP and LTM deficits in LIMK1−/− mice are rescued by increasing the activity of CREB. These results provide strong evidence that LIMK1 deletion is sufficient to lead to an LTM deficit and that this deficit is attributable to CREB hypofunction. Our study has identified a direct gene-phenotype link in mice and provides a potential strategy to restore LTM in patients with Williams syndrome through the enhancement of CREB activity in the adult brain. PMID:25645926

  2. 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…

  3. LIMK1 regulates long-term memory and synaptic plasticity via the transcriptional factor CREB.

    PubMed

    Todorovski, Zarko; Asrar, Suhail; Liu, Jackie; Saw, Ner Mu Nar; Joshi, Krutika; Cortez, Miguel A; Snead, O Carter; Xie, Wei; Jia, Zhengping

    2015-04-01

    Deletion of the LIMK1 gene is associated with Williams syndrome, a unique neurodevelopmental disorder characterized by severe defects in visuospatial cognition and long-term memory (LTM). However, whether LIMK1 contributes to these deficits remains elusive. Here, we show that LIMK1-knockout (LIMK1(-/-)) mice are drastically impaired in LTM but not short-term memory (STM). In addition, LIMK1(-/-) mice are selectively defective in late-phase long-term potentiation (L-LTP), a form of long-lasting synaptic plasticity specifically required for the formation of LTM. Furthermore, we show that LIMK1 interacts and regulates the activity of cyclic AMP response element-binding protein (CREB), an extensively studied transcriptional factor critical for LTM. Importantly, both L-LTP and LTM deficits in LIMK1(-/-) mice are rescued by increasing the activity of CREB. These results provide strong evidence that LIMK1 deletion is sufficient to lead to an LTM deficit and that this deficit is attributable to CREB hypofunction. Our study has identified a direct gene-phenotype link in mice and provides a potential strategy to restore LTM in patients with Williams syndrome through the enhancement of CREB activity in the adult brain.

  4. 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…

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

    PubMed

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

    2011-02-17

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

  6. 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

  7. 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

  8. 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

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

    PubMed Central

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

    2014-01-01

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

  10. 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

  11. Elevation of BDNF exon I-specific transcripts in the frontal cortex and midbrain of rat during spontaneous morphine withdrawal is accompanied by enhanced pCreb1 occupancy at the corresponding promoter.

    PubMed

    Peregud, Danil I; Panchenko, Leonid F; Gulyaeva, Natalia V

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) is believed to play a crucial role in the mechanisms underlying opiate dependence; however, little is known about specific features and mechanisms regulating its expression in the brain under these conditions. The aim of this study was to investigate the effects of acute morphine intoxication and withdrawal from chronic intoxication on expression of BDNF exon I-, II-, IV-, VI- and IX-containing transcripts in the rat frontal cortex and midbrain. We also have studied whether alterations of BDNF exon-specific transcripts are accompanied by changes in association of well-known transcriptional regulators of BDNF gene-phosphorylated (active form) cAMP response element binding protein (pCreb1) and methyl-CpG binding protein 2 (MeCP2) with corresponding regulatory regions of the BDNF gene. Acute morphine intoxication did not affect levels of BDNF exons in brain regions, while spontaneous morphine withdrawal in dependent rats was accompanied by an elevation of the BDNF exon I-containing mRNAs both in the frontal cortex and midbrain. During spontaneous morphine withdrawal, increased associations of pCreb1 were found with promoter of exon I in the frontal cortex and promoters of exon I, IV and VI in the midbrain. The association of MeCP2 with BDNF promoters during spontaneous morphine withdrawal did not change. Thus, BDNF exon-specific transcripts are differentially expressed in brain regions during spontaneous morphine withdrawal in dependent rats and pCreb1 may be at least partially responsible for these alterations.

  12. Focused microwave irradiation of the brain preserves in vivo protein phosphorylation: comparison with other methods of sacrifice and analysis of multiple phosphoproteins.

    PubMed

    O'Callaghan, James P; Sriram, Krishnan

    2004-05-30

    At any point in time, net protein phosphorylation represents the contribution of protein kinase and protein phosphatase activities affecting a specific site on a given substrate. Preservation of phosphorylated proteins in neural tissues has traditionally included flash-freezing or fresh tissue processing following tissue isolation. Rapid heat inactivation of protein kinases and phosphatases by focused microwave irradiation sacrifice represents another method to preserve, in vivo, brain protein phosphorylation state. In this study, we compared preservation of the phosphorylation state of a variety of phosphoproteins in the brain following sacrifice of mice by decapitation, decapitation into liquid nitrogen and focused microwave irradiation. We found that microwave irradiation generally provided the highest and most consistent levels of protein phosphorylation, regardless of the substrates examined in striatum and hippocampus. In general, flash-freezing resulted in the least preservation of phospho-state with ERK1/2 and CREB showing almost complete dephosphorylation. When regions of freshly decapitated brains were homogenized and incubated on ice for 30 min, ERK1/2 phosphorylation was completely lost, whereas it was well preserved in microwaved samples left at room temperature for 2 h. Loss of ERK1/2 phosphorylation in the fresh samples could not be attributed to substrate proteolysis. Our results indicate that focused microwave irradiation sacrifice may be required to achieve biologically relevant data for the in vivo protein phosphorylation state of many phosphoproteins.

  13. Differential contribution of CBP:CREB binding to corticotropin-releasing hormone expression in the infant and adult hypothalamus

    PubMed Central

    Korosi, Aniko; Rice, Courtney J.; Ji, Sung; Rogge, George A.; Wood, Marcelo A.; Baram, Tallie Z.

    2013-01-01

    Corticotropin-releasing hormone (CRH) contributes crucially to the regulation of central and peripheral responses to stress. Because of the importance of a finely tuned stress system, CRH expression is tightly regulated in an organ- and brain region-specific manner. Thus, in the hypothalamus, CRH is constitutively expressed and this expression is further enhanced by stress; however, the underlying regulatory mechanisms are not fully understood. The regulatory region of the crh gene contains several elements, including the cyclic-AMP response element (CRE), and the role of the CRE interaction with the cyclic-AMP response element binding protein (CREB) in CRH expression has been a focus of intensive research. Notably, whereas thousands of genes contain a CRE, the functional regulation of gene expression by the CRE:CREB system is limited to ~100 genes, and likely requires additional proteins. Here, we investigated the role of a member of the CREB complex, CREB binding protein (CBP), in basal and stress-induced CRH expression during development and in the adult. Using mice with a deficient CREB-binding site on CBP, we found that CBP:CREB interaction is necessary for normal basal CRH expression at the mRNA and protein level in the nine-day-old mouse, prior to onset of functional regulation of hypothalamic CRH expression by glucocorticoids. This interaction, which functions directly on crh or indirectly via regulation of other genes, was no longer required for maintenance of basal CRH expression levels in the adult. However, CBP:CREB binding contributed to stress-induced CRH expression in the adult, enabling rapid CRH synthesis in hypothalamus. CBP:CREB binding deficiency did not disrupt basal corticosterone plasma levels or acute stress-evoked corticosterone release. Because dysregulation of CRH expression occurs in stress-related disorders including depression, a full understanding of the complex regulation of this gene is important in both health and disease. PMID

  14. Effects of curcumin (Curcuma longa) on learning and spatial memory as well as cell proliferation and neuroblast differentiation in adult and aged mice by upregulating brain-derived neurotrophic factor and CREB signaling.

    PubMed

    Nam, Sung Min; Choi, Jung Hoon; Yoo, Dae Young; Kim, Woosuk; Jung, Hyo Young; Kim, Jong Whi; Yoo, Miyoung; Lee, Sanghee; Kim, Chul Jung; Yoon, Yeo Sung; Hwang, In Koo

    2014-06-01

    Aging is a progressive process, and it may lead to the initiation of neurological diseases. In this study, we investigated the effects of wild Indian Curcuma longa using a Morris water maze paradigm on learning and spatial memory in adult and D-galactose-induced aged mice. In addition, the effects on cell proliferation and neuroblast differentiation were assessed by immunohistochemistry for Ki67 and doublecortin (DCX) respectively. The aging model in mice was induced through the subcutaneous administration of D-galactose (100 mg/kg) for 10 weeks. C. longa (300 mg/kg) or its vehicle (physiological saline) was administered orally to adult and D-galactose-treated mice for the last three weeks before sacrifice. The administration of C. longa significantly shortened the escape latency in both adult and D-galactose-induced aged mice and significantly ameliorated D-galactose-induced reduction of cell proliferation and neuroblast differentiation in the subgranular zone of hippocampal dentate gyrus. In addition, the administration of C. longa significantly increased the levels of phosphorylated CREB and brain-derived neurotrophic factor in the subgranular zone of dentate gyrus. These results indicate that C. longa mitigates D-galactose-induced cognitive impairment, associated with decreased cell proliferation and neuroblast differentiation, by activating CREB signaling in the hippocampal dentate gyrus.

  15. Effects of Curcumin (Curcuma longa) on Learning and Spatial Memory as Well as Cell Proliferation and Neuroblast Differentiation in Adult and Aged Mice by Upregulating Brain-Derived Neurotrophic Factor and CREB Signaling

    PubMed Central

    Nam, Sung Min; Choi, Jung Hoon; Yoo, Dae Young; Kim, Woosuk; Jung, Hyo Young; Kim, Jong Whi; Yoo, Miyoung; Lee, Sanghee; Kim, Chul Jung; Yoon, Yeo Sung

    2014-01-01

    Abstract Aging is a progressive process, and it may lead to the initiation of neurological diseases. In this study, we investigated the effects of wild Indian Curcuma longa using a Morris water maze paradigm on learning and spatial memory in adult and D-galactose-induced aged mice. In addition, the effects on cell proliferation and neuroblast differentiation were assessed by immunohistochemistry for Ki67 and doublecortin (DCX) respectively. The aging model in mice was induced through the subcutaneous administration of D-galactose (100 mg/kg) for 10 weeks. C. longa (300 mg/kg) or its vehicle (physiological saline) was administered orally to adult and D-galactose-treated mice for the last three weeks before sacrifice. The administration of C. longa significantly shortened the escape latency in both adult and D-galactose-induced aged mice and significantly ameliorated D-galactose-induced reduction of cell proliferation and neuroblast differentiation in the subgranular zone of hippocampal dentate gyrus. In addition, the administration of C. longa significantly increased the levels of phosphorylated CREB and brain-derived neurotrophic factor in the subgranular zone of dentate gyrus. These results indicate that C. longa mitigates D-galactose-induced cognitive impairment, associated with decreased cell proliferation and neuroblast differentiation, by activating CREB signaling in the hippocampal dentate gyrus. PMID:24712702

  16. 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

  17. 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

  18. 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

  19. 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

  20. CREB-regulated transcription coactivator 1: important roles in neurodegenerative disorders.

    PubMed

    Xue, Zhan-Cheng; Wang, Chuang; Wang, Qin-Wen; Zhang, Jun-Fang

    2015-04-25

    The cAMP-responsive element binding protein (CREB)-regulated transcription coactivator, CRTC (also known as transducer of regulated CREB, TORC), is identified as a potent modulator of cAMP response element (CRE)-driven gene transcription. The CRTC family consists of three members (CRTC1-3), among which the CRTC1 shows the highest expression in the brain. Several studies have demonstrated that the CRTC1 plays critical roles in neuronal dendritic growth, long-term synaptic plasticity, memory consolidation and reconsolidation etc., whereas dysfunction of CRTC1 is mainly involved in neurodegenerative disorders. In light of these findings, we aim to review recent research reports that indicate the CRTC1 dysfunction and its underlying mechanisms in the neurodegenerative disorders.

  1. Phosphorylation of yeast hexokinases.

    PubMed

    Vojtek, A B; Fraenkel, D G

    1990-06-20

    We show by the use of 32P-labeling in vivo that hexokinase 2 and hexokinase 1 in Saccharomyces cerevisiae are phosphoproteins. The highest labeling was after incubation in medium with a low concentration of glucose, when labeling appears to be predominant even without use of immunoprecipitation. The nature of the modification is not known, but it has properties consistent with a phosphomonoester of serine or threonine. The cAMP-dependent protein kinase plays a negative role in hexokinase phosphorylation, in that there was reduced labeling in strains (bcy1) lacking a regulatory subunit, and increased labeling during growth with high concentrations of glucose in a strain attenuated in the catalytic subunit (tpk1w1). The function of the modification is not known, but there was a correlation between the extent of labeling and the expression of kinase-dependent high-affinity glucose uptake.

  2. CREB-dependent gene regulation by prion protein: impact on MMP-9 and beta-dystroglycan.

    PubMed

    Pradines, Elodie; Loubet, Damien; Schneider, Benoît; Launay, Jean-Marie; Kellermann, Odile; Mouillet-Richard, Sophie

    2008-11-01

    Corruption of the normal function of the cellular prion protein (PrP(C)) by the scrapie isoform (PrP(Sc)) emerges as a critical causal event in Transmissible Spongiform Encaphalopathies (TSE) pathogenesis. However, PrP(C) physiological role remains unclear. By exploiting the properties of the 1C11 neuroectodermal cell line, able to convert into 1C11(5-HT) serotonergic or 1C11(NE) noradrenergic neuronal cells, we assigned a signaling function to PrP(C). Here, we establish that antibody-mediated PrP(C) ligation promotes the recruitment of the cAMP responsive element binding protein (CREB) transcription factor downstream from the MAPK ERK1/2, in 1C11 precursor cells and their 1C11(5-HT) and 1C11(NE) neuronal progenies. Whatever the differentiation state of 1C11 cells, the PrP(C)-dependent CREB activation triggers Egr-1 and c-fos transcription, two immediate early genes that relay CREB's role in cell survival and proliferation as well as in neuronal plasticity. Furthermore, in 1C11-derived neuronal cells, we draw a link between the PrP(C)-CREB coupling and a transcriptional regulation of the metalloproteinase MMP-9 and its inhibitor TIMP-1, which play pivotal roles in neuronal pathophysiology. Finally, the PrP(C)-dependent control on MMP-9 impacts on the processing of the transmembrane protein, beta-dystroglycan. Taken together, our data define molecular mechanisms that likely mirror PrP(C) ubiquitous contribution to cytoprotection and its involvement in neuronal plasticity.

  3. 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

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

    PubMed Central

    Hansen, Rolf T.; Zhang, Han-Ting

    2013-01-01

    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

  5. Antidepressant Effect of Crocus sativus Aqueous Extract and its Effect on CREB, BDNF, and VGF Transcript and Protein Levels in Rat Hippocampus.

    PubMed

    Ghasemi, T; Abnous, K; Vahdati, F; Mehri, S; Razavi, B M; Hosseinzadeh, H

    2015-07-01

    Crocus sativus L., commonly known as saffron, is a perennial stemless herb in Iridaceae family. It has been used in traditional medicine as well as in modern pharmacological studies for variety of conditions including depression. Recent studies have suggested brain-derived neurotrophic factor (BDNF), VGF Neuropeptide, Cyclic-AMP Response Element Binding Protein (CREB) and phospho-CREB (p-CREB) may play roles in depression. In this research the molecular mechanism of antidepressant effect of aqueous extract of saffron and its effect on the levels of BDNF, VGF, CREB and p-CREB in rat hippocampus, were investigated. The aqueous extract of saffron (40, 80 and 160 mg/kg/day) and imipramine 10 mg/kg/day were injected intraperitoneally (i.p.) for 21 days to rats. The FST (forced swimming test) was performed on the days 1(st) and 21(st). The protein expression and transcript levels of BDNF, VGF CREB and phospho-CREB in rat hippocampus, were evaluated using western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results of FST showed that saffron reduced the immobility time. The protein levels of BDNF, CREB and p-CREB were significantly increased in saffron treated rats. VGF protein expression was also increased, but not significantly. The transcript levels of BDNF significantly increased. No significant changes in CREB and VGF transcript levels were observed. It was concluded that aqueous extract of saffron has antidepressant effects and the mechanism of its antidepressant effect may be due to increasing the levels of BDNF, VGF, CREB and P-CREB in rat hippocampus.

  6. 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

  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. Regulation of the NRSF/REST gene by methylation and CREB affects the cellular phenotype of small-cell lung cancer.

    PubMed

    Kreisler, A; Strissel, P L; Strick, R; Neumann, S B; Schumacher, U; Becker, C-M

    2010-10-28

    The neuron-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) is a negative regulator of gene expression restricting the expression of neuronal genes to the nervous system. NRSF/REST is highly expressed in non-neuronal tissues like the lung. In previous work, we identified small-cell lung cancer (SCLC) cell lines with no detectable NRSF/REST expression that, as a consequence, expressed neuronal markers like L1-cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (NCAM). The loss of NRSF/REST expression was linked to malignant progression; however, its mechanistic role remained elusive. Here, we show that NRSF/REST itself, rather than one of its regulated genes, acts like a classic tumour suppressor, being in part regulated by methylation. In SCLCs, NRSF/REST is positively regulated by CREB, with an NRSF/REST promoter fragment showing cell type specificity. Downstream, NRSF/REST directly regulates AKT2, in which NRSF/REST loss leads to an epidermal growth factor-mediated de-regulation of AKT-Serine473 phosphorylation, important for cellular proliferation and survival. Assaying anchorage-independent growth, we observed that with reduced NRSF/REST expression, proliferation was significantly enhanced, whereas NRSF/REST rescue decreased the potential of cells to grow anchorage independently. Our observations support the fact that NRSF/REST may act as an important modulator of malignant progression in SCLC. PMID:20697351

  9. High mobility group Box-1 inhibits cancer cell motility and metastasis by suppressing activation of transcription factor CREB and nWASP expression.

    PubMed

    Zuo, Zhenghong; Che, Xun; Wang, Yulei; Li, Bowen; Li, Jingxia; Dai, Wei; Lin, Charles P; Huang, Chuanshu

    2014-09-15

    The ability to metastasize is a hallmark of malignant tumors, and metastasis is the principal cause of death of cancer patients. The High Mobility Group Box-1 (HMGB1) is a multifunction protein that serves as both a chromatin protein and an extracellular signaling molecule. Our current study demonstrated a novel mechanism of HMGB1 in the regulation of cancer cell actin polymerization, cell skeleton formation, cancer cell motility and metastasis. We found that knockdown of HMGB1 in human lung cancer A549 cells significantly increased cell β-actin polymerization, cell skeleton formation, cancer cell migration and invasion in vitro, as well as metastasis in vivo. And this increase could be inhibited by treatment of HMGB1 knockdown cells with recombinant human HMGB1. Further studies discovered that HMGB1 suppressed phosphorylation, nuclear translocation, and activation of CREB, by inhibiting nuclear translocation of PKA catalytic subunit. This reduces nWASP mRNA transcription and expression, further impairing cancer cell motility. Our findings on the novel mechanism underlying the HMGB1 anti-metastatic effect on cancer provides significant insight into the understanding of the nature of HMGB1 in cancer invasion and metastasis, further serving as key information for utilization of HMGB1 and its regulated downstream components as new targets for cancer therapy. PMID:25277185

  10. Participation of microRNA 124-CREB pathway: a parallel memory enhancing mechanism of standardised extract of Bacopa monniera (BESEB CDRI-08).

    PubMed

    Preethi, Jayakumar; Singh, Hemant K; Charles, Prisila Dulcy; Rajan, Koilmani Emmanuvel

    2012-10-01

    Bacosides, the effective component of standardised leaf extract of Bacopa monniera (BESEB CDRI-08) has been reported to have memory enhancing effect. Our previous reports suggested that BESEB CDRI-08 (BME) improves memory in postnatal rats by enhancing serotonin [5-hydroxytryptamine (5-HT)] metabolism, its transportation and subsequently activates 5-HT(3A) receptor during hippocampus-dependent learning. In this study, we examine whether the up-regulated 5-HT(3A) receptor activity by BME modulate microRNA 124-CREB pathway to enhance synaptic plasticity. Wistar rat pups received single dose of vehicle solution (0.5 % gum acacia + 0.9 % saline)/BME (80 mg/kg)/mCPBG (10 mg/kg)/BME + mCPBG during the postnatal days (PND) 15-29. On PND 30, individuals were trained at brightness discrimination task and 24 h later, they were tested on the task. The BME treated group exhibited significantly lower percentage of errors during retention than acquisition. In addition, pre-miR-124 expression in hippocampus was significantly down-regulated in the BME and mCPBG + BME treated groups combined with a significant increase in the plasticity related genes, cAMP response element-binding protein, its phosphorylation and postsynaptic density protein 95. Our results suggest that this may be one of the mechanisms of bacosides present in BME for the memory enhancement.

  11. Participation of microRNA 124-CREB pathway: a parallel memory enhancing mechanism of standardised extract of Bacopa monniera (BESEB CDRI-08).

    PubMed

    Preethi, Jayakumar; Singh, Hemant K; Charles, Prisila Dulcy; Rajan, Koilmani Emmanuvel

    2012-10-01

    Bacosides, the effective component of standardised leaf extract of Bacopa monniera (BESEB CDRI-08) has been reported to have memory enhancing effect. Our previous reports suggested that BESEB CDRI-08 (BME) improves memory in postnatal rats by enhancing serotonin [5-hydroxytryptamine (5-HT)] metabolism, its transportation and subsequently activates 5-HT(3A) receptor during hippocampus-dependent learning. In this study, we examine whether the up-regulated 5-HT(3A) receptor activity by BME modulate microRNA 124-CREB pathway to enhance synaptic plasticity. Wistar rat pups received single dose of vehicle solution (0.5 % gum acacia + 0.9 % saline)/BME (80 mg/kg)/mCPBG (10 mg/kg)/BME + mCPBG during the postnatal days (PND) 15-29. On PND 30, individuals were trained at brightness discrimination task and 24 h later, they were tested on the task. The BME treated group exhibited significantly lower percentage of errors during retention than acquisition. In addition, pre-miR-124 expression in hippocampus was significantly down-regulated in the BME and mCPBG + BME treated groups combined with a significant increase in the plasticity related genes, cAMP response element-binding protein, its phosphorylation and postsynaptic density protein 95. Our results suggest that this may be one of the mechanisms of bacosides present in BME for the memory enhancement. PMID:22837048

  12. Drosophila salt-inducible kinase (SIK) regulates starvation resistance through cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC).

    PubMed

    Choi, Sekyu; Kim, Wonho; Chung, Jongkyeong

    2011-01-28

    Salt-inducible kinase (SIK), one of the AMP-activated kinase (AMPK)-related kinases, has been suggested to play important functions in glucose homeostasis by inhibiting the cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC). To examine the role of SIK in vivo, we generated Drosophila SIK mutant and found that the mutant flies have higher amounts of lipid and glycogen stores and are resistant to starvation. Interestingly, SIK transcripts are highly enriched in the brain, and we found that neuron-specific expression of exogenous SIK fully rescued lipid and glycogen storage phenotypes as well as starvation resistance of the mutant. Using genetic and biochemical analyses, we demonstrated that CRTC Ser-157 phosphorylation by SIK is critical for inhibiting CRTC activity in vivo. Furthermore, double mutants of SIK and CRTC became sensitive to starvation, and the Ser-157 phosphomimetic mutation of CRTC reduced lipid and glycogen levels in the SIK mutant, suggesting that CRTC mediates the effects of SIK signaling. Collectively, our results strongly support the importance of the SIK-CRTC signaling axis that functions in the brain to maintain energy homeostasis in Drosophila.

  13. 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

  14. Saponarin activates AMPK in a calcium-dependent manner and suppresses gluconeogenesis and increases glucose uptake via phosphorylation of CRTC2 and HDAC5.

    PubMed

    Seo, Woo-Duck; Lee, Ji Hae; Jia, Yaoyao; Wu, Chunyan; Lee, Sung-Joon

    2015-11-15

    This study investigated the molecular mechanism of saponarin, a flavone glucoside, in the regulation of insulin sensitivity. Saponarin suppressed the rate of gluconeogenesis and increased cellular glucose uptake in HepG2 and TE671 cells by regulating AMPK. Using an in vitro kinase assay, we showed that saponarin did not directly interact with the AMPK protein. Instead, saponarin increased intracellular calcium levels and induced AMPK phosphorylation, which was diminished by co-stimulation with STO-609, an inhibitor of CAMKKβ. Transcription of hepatic gluconeogenesis genes was upregulated by nuclear translocation of CRTC2 and HDAC5, coactivators of CREB and FoxO1 transcription factors, respectively. This nuclear translocation was inhibited by increased phosphorylation of CRTC2 and HDAC5 by saponarin-induced AMPK in HepG2 cells and suppression of CREB and FoxO1 transactivation activities in cells stimulated by saponarin. The results from a chromatin immunoprecipitation assay confirmed the reduced binding of CRTC2 on the PEPCK and G6Pase promoters. In TE671 cells, AMPK phosphorylated HDAC5, which suppressed nuclear penetration and upregulated GLUT4 transcription, leading to enhanced glucose uptake. Collectively, these results suggest that saponarin activates AMPK in a calcium-dependent manner, thus regulating gluconeogenesis and glucose uptake.

  15. Tau accumulation induces synaptic impairment and memory deficit by calcineurin-mediated inactivation of nuclear CaMKIV/CREB signaling.

    PubMed

    Yin, Yaling; Gao, Di; Wang, Yali; Wang, Zhi-Hao; Wang, Xin; Ye, Jinwang; Wu, Dongqin; Fang, Lin; Pi, Guilin; Yang, Ying; Wang, Xiao-Chuan; Lu, Chengbiao; Ye, Keqiang; Wang, Jian-Zhi

    2016-06-28

    Intracellular accumulation of wild-type tau is a hallmark of sporadic Alzheimer's disease (AD), but the molecular mechanisms underlying tau-induced synapse impairment and memory deficit are poorly understood. Here we found that overexpression of human wild-type full-length tau (termed hTau) induced memory deficits with impairments of synaptic plasticity. Both in vivo and in vitro data demonstrated that hTau accumulation caused remarkable dephosphorylation of cAMP response element binding protein (CREB) in the nuclear fraction. Simultaneously, the calcium-dependent protein phosphatase calcineurin (CaN) was up-regulated, whereas the calcium/calmodulin-dependent protein kinase IV (CaMKIV) was suppressed. Further studies revealed that CaN activation could dephosphorylate CREB and CaMKIV, and the effect of CaN on CREB dephosphorylation was independent of CaMKIV inhibition. Finally, inhibition of CaN attenuated the hTau-induced CREB dephosphorylation with improved synapse and memory functions. Together, these data indicate that the hTau accumulation impairs synapse and memory by CaN-mediated suppression of nuclear CaMKIV/CREB signaling. Our findings not only reveal new mechanisms underlying the hTau-induced synaptic toxicity, but also provide potential targets for rescuing tauopathies. PMID:27298345

  16. Reciprocal Regulation of Reactive Oxygen Species and Phospho-CREB Regulates Voltage Gated Calcium Channel Expression during Mycobacterium tuberculosis Infection

    PubMed Central

    Selvakumar, Arti; Antony, Cecil; Singhal, Jhalak; Tiwari, Brijendra K.; Singh, Yogendra; Natarajan, Krishnamurthy

    2014-01-01

    Our previous work has demonstrated the roles played by L-type Voltage Gated Calcium Channels (VGCC) in regulating Mycobacterium tuberculosis (M. tb) survival and pathogenesis. Here we decipher mechanisms and pathways engaged by the pathogen to regulate VGCC expression in macrophages. We show that M. tb and its antigen Rv3416 use phospho-CREB (pCREB), Reactive Oxygen Species (ROS), Protein Kinase C (PKC) and Mitogen Activated Protein Kinase (MAPK) to modulate VGCC expression in macrophages. siRNA mediated knockdown of MyD88, IRAK1, IRAK2 or TRAF6 significantly inhibited antigen mediated VGCC expression. Inhibiting Protein Kinase C (PKC) or MEK-ERK1/2 further increased VGCC expression. Interestingly, inhibiting intracellular calcium release upregulated antigen mediated VGCC expression, while inhibiting extracellular calcium influx had no significant effect. siRNA mediated knockdown of transcription factors c-Jun, SOX5 and CREB significantly inhibited Rv3416 mediated VGCC expression. A dynamic reciprocal cross-regulation between ROS and pCREB was observed that in turn governed VGCC expression with ROS playing a limiting role in the process. Further dissection of the mechanisms such as the interplay between ROS and pCREB would improve our understanding of the regulation of VGCC expression during M. tb infection. PMID:24797940

  17. 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)

  18. 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.

  19. 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

  20. Desipramine improves depression-like behavior and working memory by up-regulating p-CREB in Alzheimer's disease associated mice.

    PubMed

    Wang, Dan-Dan; Li, Jia; Yu, Li-Peng; Wu, Mei-Na; Sun, Li-Na; Qi, Jin-Shun

    2016-06-01

    Aggregation of amyloid [Formula: see text] protein (A[Formula: see text] and progressive loss of memory are the main characteristics of Alzheimer's disease (AD). It is noteworthy that approximately 40% of AD patients have depressive symptom. The close correlation between cognitive deficits and mental depression suggests a possibility that antidepression treatment might be beneficial to cognitive improvement in AD. The present study, by using tail-suspension test (TST), forced swimming, alternative electro-stimulus Y maze test and immunohistochemistry, examined the neuroprotective effects of desipramine, a newer generation tricyclic antidepressants (TCA), and investigated its possible molecular mechanism. The results showed that: (1) intra-hippocampal injection of A[Formula: see text] induced depression-like behavior and associative learning deficits in mice, with an increased mean immobility time in tail-suspension and forced swimming test and an increased mean error times in Y maze test; (2) after treatment with desipramine (10[Formula: see text]mg/kg, i.p.), the average immobility time significantly decreased, from [Formula: see text][Formula: see text]s in A[Formula: see text] group to [Formula: see text][Formula: see text]s in A[Formula: see text] plus desipramine group ([Formula: see text]) in TST and from [Formula: see text][Formula: see text]s to [Formula: see text][Formula: see text]s ([Formula: see text] or 9, [Formula: see text]) in forced swimming test, respectively;the mean error times of mice in Y maze test also significantly decreased, from [Formula: see text] in A[Formula: see text] group to [Formula: see text] in A[Formula: see text] plus desipramine group ([Formula: see text], [Formula: see text]); (3) desipramine administration significantly prevented against A[Formula: see text]-induced down-regulation of phosphorylated cAMP response element binding protein (p-CREB) in the hippocampus. These results indicate that A[Formula: see text] could

  1. 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

  2. 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.

  3. 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

  4. Phosphorylation mechanisms in chemical evolution

    NASA Astrophysics Data System (ADS)

    Schoffstall, Allen M.; Laing, Euton M.

    1985-06-01

    An objective of this work is to elucidate the mechanism of phosphorylation of nucleosides in amide solvents and in urea. A second objective is to assess the importance of phosphorylation and dephosphorylation of nucleotide derivatives in amide environments. Although the most complex amide studied here was N-methylacetamide, inferences are made on the importance of dephosphorylation for nucleotides in oligopeptide environments. Phosphorylations in amide solvents and in urea are suggested to proceed through monomeric metaphosphate, which was first postulated as a reaction intermediate thirty years ago (Butcher and Westheimer, 1955). Phosphorylation of nucleosides and nucleotides and dephosphorylation of nucleotide derivatives have been studied in formamide, N-methylformamide, urea and N-methylacetamide. Hydrated forms of 5'-ADP and 5'ATP are unstable in hot amide solvents and in urea. They decompose to a mixture of adenosine and its phosphorylated derivatives. The rate of decomposition is much slower in N-methylacetamide than in formamide or urea. Experiments designed to prepare oligonucleotides in the presence of oligopeptides have been reported (White, 1983). According to the present study, it is not unreasonable to expect that nucleotide derivatives can be condensed with nucleosides to form oligonucleotides in a peptide environment. However, nucleotide monomers such as 5'-ATP, 5'-ADP or 5'AMP will suffer isomerization or decomposition during condensation use of activated phosphate derivatives is preferable. Monomeric metaphosphate has not been isolated or characterized in amide solvents. It is proposed here as a reaction intermediate, probably in a complexed form with the amide.

  5. Glycogen phosphorylation and Lafora disease.

    PubMed

    Roach, Peter J

    2015-12-01

    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease.

  6. 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

  7. A novel epigenetic CREB-miR-373 axis mediates ZIP4-induced pancreatic cancer growth.

    PubMed

    Zhang, Yuqing; Yang, Jingxuan; Cui, Xiaobo; Chen, Yong; Zhu, Vivian F; Hagan, John P; Wang, Huamin; Yu, Xianjun; Hodges, Sally E; Fang, Jing; Chiao, Paul J; Logsdon, Craig D; Fisher, William E; Brunicardi, F Charles; Chen, Changyi; Yao, Qizhi; Fernandez-Zapico, Martin E; Li, Min

    2013-09-01

    Changes in the intracellular levels of the essential micronutrient zinc have been implicated in multiple diseases including pancreatic cancer; however, the molecular mechanism is poorly understood. Here, we report a novel mechanism where increased zinc mediated by the zinc importer ZIP4 transcriptionally induces miR-373 in pancreatic cancer to promote tumour growth. Reporter, expression and chromatin immunoprecipitation assays demonstrate that ZIP4 activates the zinc-dependent transcription factor CREB and requires this transcription factor to increase miR-373 expression through the regulation of its promoter. miR-373 induction is necessary for efficient ZIP4-dependent enhancement of cell proliferation, invasion, and tumour growth. Further analysis of miR-373 in vivo oncogenic function reveals that it is mediated through its negative regulation of TP53INP1, LATS2 and CD44. These results define a novel ZIP4-CREB-miR-373 signalling axis promoting pancreatic cancer growth, providing mechanistic insights explaining in part how a zinc transporter functions in cancer cells and may have broader implications as inappropriate regulation of intracellular zinc levels plays an important role in many other diseases.

  8. The CREB/CRTC2 Pathway Modulates Autoimmune Disease by Promoting Th17 Differentiation

    PubMed Central

    Hernandez, Jeniffer B.; Chang, Christina; LeBlanc, Mathias; Grimm, David; Le Lay, John; Kaestner, Klaus H.; Zheng, Ye; Montminy, Marc

    2015-01-01

    Following their activation in response to inflammatory signals, innate immune cells secrete T cell polarizing cytokines that promote the differentiation of naïve CD4 T cells into T helper (Th) cell subsets. Amongst these, Th17 cells play a prominent role in the development of a number of autoimmune diseases. Although regarded primarily as an immunosuppressant signal, cAMP has been found to mediate pro-inflammatory effects of macrophage-derived prostaglandin E2 (PGE2) on Th17 cells. Here we show that PGE2 enhances Th17 cell differentiation via the activation of the CREB co-activator CRTC2. Following its dephosphorylation, CRTC2 stimulates the expression of the cytokines IL-17A and IL-17F by binding to CREB over both promoters. CRTC2 mutant mice have decreased Th17 cell numbers, and they are protected from experimental autoimmune encephalitis, a model for multiple sclerosis. Our results suggest that small molecule inhibitors of CRTC2 may provide therapeutic benefit to individuals with autoimmune disease. PMID:26031354

  9. Creb coactivators direct anabolic responses and enhance performance of skeletal muscle.

    PubMed

    Bruno, Nelson E; Kelly, Kimberly A; Hawkins, Richard; Bramah-Lawani, Mariam; Amelio, Antonio L; Nwachukwu, Jerome C; Nettles, Kendall W; Conkright, Michael D

    2014-05-01

    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.

  10. Nucleoside phosphorylation by phosphate minerals.

    PubMed

    Costanzo, Giovanna; Saladino, Raffaele; Crestini, Claudia; Ciciriello, Fabiana; Di Mauro, Ernesto

    2007-06-01

    In the presence of formamide, crystal phosphate minerals may act as phosphate donors to nucleosides, yielding both 5'- and, to a lesser extent, 3'-phosphorylated forms. With the mineral Libethenite the formation of 5'-AMP can be as high as 6% of the adenosine input and last for at least 10(3) h. At high concentrations, soluble non-mineral phosphate donors (KH(2)PO(4) or 5'-CMP) afford 2'- and 2':3'-cyclic AMP in addition to 5'-and 3'-AMP. The phosphate minerals analyzed were Herderite Ca[BePO(4)F], Hureaulite Mn(2+)(5)(PO(3)(OH)(2)(PO(4))(2)(H(2)O)(4), Libethenite Cu(2+)(2)(PO(4))(OH), Pyromorphite Pb(5)(PO(4))(3)Cl, Turquoise Cu(2+)Al(6)(PO(4))(4)(OH)(8)(H(2)O)(4), Fluorapatite Ca(5)(PO(4))(3)F, Hydroxylapatite Ca(5)(PO(4))(3)OH, Vivianite Fe(2+)(3)(PO(4))(2)(H(2)O)(8), Cornetite Cu(2+)(3)(PO(4))(OH)(3), Pseudomalachite Cu(2+)(5)(PO(4))(2)(OH)(4), Reichenbachite Cu(2+)(5)(PO(4))(2)(OH)(4), and Ludjibaite Cu(2+)(5)(PO(4))(2)(OH)(4)). Based on their behavior in the formamide-driven nucleoside phosphorylation reaction, these minerals can be characterized as: 1) inactive, 2) low level phosphorylating agents, or 3) active phosphorylating agents. Instances were detected (Libethenite and Hydroxylapatite) in which phosphorylation occurs on the mineral surface, followed by release of the phosphorylated compounds. Libethenite and Cornetite markedly protect the beta-glycosidic bond. Thus, activated nucleic monomers can form in a liquid non-aqueous environment in conditions compatible with the thermodynamics of polymerization, providing a solution to the standard-state Gibbs free energy change (DeltaG degrees ') problem, the major obstacle for polymerizations in the liquid phase in plausible prebiotic scenarios.

  11. 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

  12. BzpF is a CREB-like transcription factor that regulates spore maturation and stability in Dictyostelium

    PubMed Central

    Huang, Eryong; Talukder, Shaheynoor; Hughes, Timothy R.; Curk, Tomaz; Zupan, Blaz; Shaulsky, Gad; Katoh-Kurasawa, Mariko

    2011-01-01

    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

  13. GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

    PubMed

    He, Ximiao; Syed, Khund Sayeed; Tillo, Desiree; Mann, Ishminder; Weirauch, Matthew T; Vinson, Charles

    2015-07-16

    To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABPα and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS ((C)/GCGGAA GT: ) and CRE ( GT: GACGTCAC) motifs overlap precisely, producing the ETS↔CRE motif ((C)/GCGGAA GT: GACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABPα and CREB1 upon binding the ETS↔CRE motif. The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes. CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding. We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding.

  14. Not all stress is equal: CREB is not necessary for restraint stress reinstatement of cocaine-conditioned reward

    PubMed Central

    Briand, Lisa A.; Blendy, Julie A.

    2013-01-01

    Stress elicits relapse to cocaine seeking in humans and in animal models. Cyclic AMP response element binding protein (CREB) is required for swim stress-induced reinstatement of cocaine conditioned place preference. However, the role of CREB in other stress-induced reinstatement models has not been examined. To determine whether CREB is required across different stressors we examined the ability of restraint to elicit reinstatement of cocaine-conditioned place preference in wild type and CREBαΔ mutant mice. In contrast to previously published differences in swim stress-induced reinstatement, both wildtype and CREBαΔ mutant mice demonstrated restraint stress elicited reinstatement of cocaine-conditioned reward. While CREB is necessary for swim stress-elicited zif268 expression within the nucleus accubmens (NAc) shell & prelimbic cortex (PrL), restraint-stress-elicited comparable increases in zif268 expression within these regions in both wildtype and CREBαΔ mutant mice. Our findings suggest that not all stressors engage the same circuits or molecular mechanisms to elicit reinstatement behavior. PMID:23458740

  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. 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

  17. 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.

  18. Phosphorylation stoichiometry determination in plant photosynthetic membranes.

    PubMed

    Ingelsson, Björn; Fristedt, Rikard; Turkina, Maria V

    2015-01-01

    This chapter describes different strategies for the study of phosphorylation dynamics and stoichiometry in photosynthetic membranes. Detailed procedures for the detection, large-scale identification, and quantification of phosphorylated proteins optimized for plant thylakoid proteins are given. PMID:25930698

  19. CREB-BDNF pathway influences alcohol cue-elicited activation in drinkers.

    PubMed

    Chen, Jiayu; Hutchison, Kent E; Calhoun, Vince D; Claus, Eric D; Turner, Jessica A; Sui, Jing; Liu, Jingyu

    2015-08-01

    Alcohol use disorder (AUD) 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 with alcohol cue-elicited brain activations in 315 heavy drinkers, where pICA-MR assesses multiple reference genes for their architecture and functional influences on neurobiological conditions. The genetic component derived from the cAMP-response element-binding protein and -brain derived neurotrophic factor (CREB-BDNF) pathway reference was significantly associated (r = -0.38, P = 3.98 × 10(-12) ) with an imaging component reflecting hyperactivation in precuneus, superior parietal lobule, and posterior cingulate for drinkers with more severe alcohol dependence symptoms. 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. Collectively, our findings suggest that genetic variations in various neural plasticity and signaling pathways partially explain the variance of precuneus reactivity to alcohol cues which appears to be associated with AUD severity. PMID:25939814

  20. 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

  1. 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.

  2. Tyrosine phosphorylation of WW proteins

    PubMed Central

    Reuven, Nina; Shanzer, Matan

    2015-01-01

    A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

  3. Phosphorylation of STAT3 mediates the induction of cyclooxygenase-2 by cortisol in the human amnion at parturition.

    PubMed

    Wang, Wangsheng; Guo, Chunming; Zhu, Ping; Lu, Jiangwen; Li, Wenjiao; Liu, Chao; Xie, Huiliang; Myatt, Leslie; Chen, Zi-Jiang; Sun, Kang

    2015-10-27

    The induction of cyclooxygenase-2 (COX-2) and subsequent production of prostaglandin E2 (PGE2) by cortisol in the amnion contrast with the effect of cortisol on most other tissues, but this proinflammatory effect of cortisol may be a key event in human parturition (labor). We evaluated the underlying mechanism activated by cortisol in primary human amnion fibroblasts. Exposure of the amnion fibroblasts to cortisol led to the activation of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, which induced the phosphorylation of the kinase SRC and STAT3 (signal transducer and activator of transcription 3). STAT3 interacted with the glucocorticoid receptor (GR) and the transcription factor CREB-1 (cAMP response element-binding protein 1) at the promoter of the gene encoding COX-2, which promoted the production of the secreted prostaglandin PGE2. PGE2 activates the prostaglandin receptors EP2 and EP4, which stimulate cAMP-PKA signaling. Thus, cortisol reinforced the activation of cAMP-PKA signaling through an SRC-STAT3-COX-2-PGE2-mediated feedback loop. Inhibiting STAT3, SRC, or the cAMP-PKA pathway attenuated the cortisol-stimulated induction of COX-2 and PGE2 production in amnion fibroblasts. In human amnion tissue, the amount of phosphorylated STAT3 correlated positively with that of cortisol, COX-2, and PGE2, and all were more abundant in tissue obtained after active labor than in tissue obtained from cesarean surgeries in the absence of labor. These results indicated that the coordinated recruitment of STAT3, CREB-1, and GR to the promoter of the gene encoding COX-2 contributes to the feed-forward induction of COX-2 activity and prostaglandin synthesis in the amnion during parturition.

  4. Tyrosine phosphorylation and bacterial virulence

    PubMed Central

    Whitmore, Sarah E; Lamont, Richard J

    2012-01-01

    Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria. In this article, we review the structure and function of bacterial tyrosine kinases and phosphatases. Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes (bacterial tyrosine (BY) kinases) that are characterized by the presence of Walker motifs. The reverse reaction is catalyzed by three classes of enzymes: the eukaryotic-like phosphatases (PTPs) and dual-specific phosphatases; the low molecular weight protein-tyrosine phosphatases (LMW-PTPs); and the polymerase–histidinol phosphatases (PHP). Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates, thereby contributing to bacterial pathogenicity. Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development. The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii. Ltp1 is upregulated by contact with S. gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2. PMID:22388693

  5. Gene Network Inference and Biochemical Assessment Delineates GPCR Pathways and CREB Targets in Small Intestinal Neuroendocrine Neoplasia

    PubMed Central

    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 D2 and Serotonin [5-HT2] 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. CREB Negatively Regulates IGF2R Gene Expression and Downstream Pathways to Inhibit Hypoxia-Induced H9c2 Cardiomyoblast Cell Death.

    PubMed

    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

  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. 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…

  13. 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

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

    PubMed

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

    2009-10-01

    cAMP can stimulate the transcription of many activity-dependent genes via activation of the transcription factor, cAMP response element-binding protein (CREB). However, in mouse cortical neuron cultures, prior to synaptogenesis, neither cAMP nor dopamine, which acts via cAMP, stimulated CREB-dependent gene transcription when NR2B-containing NMDA receptors (NMDARs) were blocked. Stimulation of transcription by cAMP was potentiated by inhibitors of excitatory amino acid uptake, suggesting a role for extracellular glutamate or aspartate in cAMP-induced transcription. Aspartate was identified as the extracellular messenger: enzymatic scavenging of l-aspartate, but not glutamate, blocked stimulation of CREB-dependent gene transcription by cAMP; moreover, cAMP induced aspartate but not glutamate release. Together, these results suggest that cAMP acts via an autocrine or paracrine pathway to release aspartate, which activates NR2B-containing NMDARs, leading to Ca(2+) entry and activation of transcription. This cAMP/aspartate/NMDAR signaling pathway may mediate the effects of transmitters such as dopamine on axon growth and synaptogenesis in developing neurons or on synaptic plasticity in mature neural networks.

  15. 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

  16. 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.

  17. 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.

  18. Salt stress-induced protein phosphorylation

    SciTech Connect

    Godoy, J.A.; Torres-Schumann, S.; Llobell, A.; Pintor-Toro, J.A.

    1989-04-01

    Protein phosphorylation induced by salt stress in tomato germinating seeds were investigated by two-dimensional polyacrilamide gel electrophoresis of proteins labeled in vivo with ({sup 32}P)-Phosphate. NaCl induced the phosphorylation of a 14 Kd polypeptide. Pulse-chase experiments revealed that the phosphorylated molecules of this polypeptide are only stable while the stress is present. Phosphorylated 14 Kd polypeptides could be detected in radicles of salt-shocked seedlings after 6 hours stress period. 14 Kd polypeptide phosphorylation was also observed in seeds germinating in the presence of abscisic acid (ABA). The amount of phosphorylated 14 Kd polypeptide was significantly increased in seeds treated simultaneously with NaCl and ABA.

  19. CREB Binding Protein Functions During Successive Stages of Eye Development in Drosophila

    PubMed Central

    Kumar, Justin P.; Jamal, Tazeen; Doetsch, Alex; Turner, F. Rudolf; Duffy, Joseph B.

    2004-01-01

    During the development of the compound eye of Drosophila several signaling pathways exert both positive and inhibitory influences upon an array of nuclear transcription factors to produce a near-perfect lattice of unit eyes or ommatidia. Individual cells within the eye are exposed to many extracellular signals, express multiple surface receptors, and make use of a large complement of cell-subtype-specific DNA-binding transcription factors. Despite this enormous complexity, each cell will make the correct developmental choice and adopt the appropriate cell fate. How this process is managed remains a poorly understood paradigm. Members of the CREB binding protein (CBP)/p300 family have been shown to influence development by (1) acting as bridging molecules between the basal transcriptional machinery and specific DNA-binding transcription factors, (2) physically interacting with terminal members of signaling cascades, (3) acting as transcriptional coactivators of downstream target genes, and (4) playing a key role in chromatin remodeling. In a screen for new genes involved in eye development we have identified the Drosophila homolog of CBP as a key player in both eye specification and cell fate determination. We have used a variety of approaches to define the role of CBP in eye development on a cell-by-cell basis. PMID:15514061

  20. 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

  1. 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

  2. Histone Acetylation and CREB Binding Protein Are Required for Neuronal Resistance against Ischemic Injury

    PubMed Central

    Yildirim, Ferah; Ji, Shengbo; Kronenberg, Golo; Barco, Angel; Olivares, Roman; Benito, Eva; Dirnagl, Ulrich; Gertz, Karen; Endres, Matthias

    2014-01-01

    Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT) and deacetylase activities (HDAC). Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB)–binding protein (CBP) as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD) in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min) subthreshold occlusion of the middle cerebral artery (MCA), followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons. PMID:24748101

  3. Enhanced activity of the CREB co-activator Crtc1 in LKB1 null lung cancer.

    PubMed

    Komiya, T; Coxon, A; Park, Y; Chen, W-D; Zajac-Kaye, M; Meltzer, P; Karpova, T; Kaye, F J

    2010-03-18

    Activation of Crtc1 (also known as Mect1/Torc1) by a t(11;19) chromosomal rearrangement underlies the etiology of malignant salivary gland tumors. As LKB1 is a target for mutational inactivation in lung cancer and was recently shown to regulate hepatic Crtc2/CREB transcriptional activity in mice, we now present evidence suggesting disruption of an LKB1/Crtc pathway in cancer. Although Crtc1 is preferentially expressed in adult brain tissues, we observed elevated levels of steady-state Crtc1 in thoracic tumors. In addition, we show that somatic loss of LKB1 is associated with underphosphorylation of endogenous Crtc1, enhanced Crtc1 nuclear localization and enhanced expression of the Crtc prototypic target gene, NR4A2/Nurr1. Inhibition of NR4A2 was associated with growth suppression of LKB1 null tumors, but showed little effect on LKB1-wildtype cells. These data strengthen the role of dysregulated Crtc as a bona fide cancer gene, present a new element to the complex LKB1 tumorigenic axis, and suggest that Crtc genes may be aberrantly activated in a wider range of common adult malignancies.

  4. Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation.

    PubMed

    Amelio, Antonio L; Caputi, Massimo; Conkright, Michael D

    2009-09-16

    The CREB regulated transcription co-activators (CRTCs) regulate many biological processes by integrating and converting environmental inputs into transcriptional responses. Although the mechanisms by which CRTCs sense cellular signals are characterized, little is known regarding how CRTCs contribute to the regulation of cAMP inducible genes. Here we show that these dynamic regulators, unlike other co-activators, independently direct either pre-mRNA splice-site selection or transcriptional activation depending on the cell type or promoter context. Moreover, in other scenarios, the CRTC co-activators coordinately regulate transcription and splicing. Mutational analyses showed that CRTCs possess distinct functional domains responsible for regulating either pre-mRNA splicing or transcriptional activation. Interestingly, the CRTC1-MAML2 oncoprotein lacks the splicing domain and is incapable of altering splice-site selection despite robustly activating transcription. The differential usage of these distinct domains allows CRTCs to selectively mediate multiple facets of gene regulation, indicating that co-activators are not solely restricted to coordinating alternative splicing with increase in transcriptional activity.

  5. Proline-Directed Androgen Receptor Phosphorylation

    PubMed Central

    Gao, Yanfei; Chen, Shaoyong

    2015-01-01

    The androgen receptor (AR) has been identified for decades and mediates essential steroid functions. Like most of biological molecules, AR functional activities are modulated by post-translational modifications. This review is focused on the reported activities and significance of AR phosphorylation, with particular emphasis on proline-directed serine/threonine phosphorylation that occurs predominantly on the receptor. The marked enrichment of AR phosphorylation in the most diverse N-terminal domain suggests that targeting AR phosphorylation can be synergistic to antagonizing the C-terminal domain by clinical antiandrogens. PMID:25866551

  6. FT-IR analysis of phosphorylated protein

    NASA Astrophysics Data System (ADS)

    Ishii, Katsunori; Yoshihashi, Sachiko S.; Chihara, Kunihiro; Awazu, Kunio

    2004-09-01

    Phosphorylation and dephosphorylation, which are the most remarkable posttranslational modifications, are considered to be important chemical reactions that control the activation of proteins. We examine the phosphorylation analysis method by measuring the infrared absorption peak of phosphate group that observed at about 1070cm-1 (9.4μm) with Fourier Transform Infrared Spectrometer (FT-IR). This study indicates that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm-1 with FT-IR method. As long as target peptides have the same amino acid sequence, it is possible to identify the phosphorylated sites (threonine, serine and tyrosine).

  7. 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.

  8. Charge environments around phosphorylation sites in proteins

    PubMed Central

    Kitchen, James; Saunders, Rebecca E; Warwicker, Jim

    2008-01-01

    Background Phosphorylation is a central feature in many biological processes. Structural analyses have identified the importance of charge-charge interactions, for example mediating phosphorylation-driven allosteric change and protein binding to phosphopeptides. Here, we examine computationally the prevalence of charge stabilisation around phosphorylated sites in the structural database, through comparison with locations that are not phosphorylated in the same structures. Results A significant fraction of phosphorylated sites appear to be electrostatically stabilised, largely through interaction with sidechains. Some examples of stabilisation across a subunit interface are evident from calculations with biological units. When considering the immediately surrounding environment, in many cases favourable interactions are only apparent after conformational change that accompanies phosphorylation. A simple calculation of potential interactions at longer-range, applied to non-phosphorylated structures, recovers the separation exhibited by phosphorylated structures. In a study of sites in the Phospho.ELM dataset, for which structural annotation is provided by non-phosphorylated proteins, there is little separation of the known phospho-acceptor sites relative to background, even using the wider interaction radius. However, there are differences in the distributions of patch polarity for acceptor and background sites in the Phospho.ELM dataset. Conclusion In this study, an easy to implement procedure is developed that could contribute to the identification of phospho-acceptor sites associated with charge-charge interactions and conformational change. Since the method gives information about potential anchoring interactions subsequent to phosphorylation, it could be combined with simulations that probe conformational change. Our analysis of the Phospho.ELM dataset also shows evidence for mediation of phosphorylation effects through (i) conformational change associated with

  9. Relationships between histone phosphorylation and cell proliferation

    SciTech Connect

    Gurley, L.R.; D'Anna, J.A.; Halleck, M.S.; Barham, S.S.; Walters, R.A.; Jett, J.H.; Tobey, R.A.

    1980-01-01

    From studies with various Peromyscus cell lines, correlations were made which led to the proposal that H2A phosphorylation is most active in constitutive heterochromatin. Recent studies on the two H2A variants found in these cells have revealed that the high level of H2A phosphorylation associated with heterochromatin is not the result of an increase in H2A phosphorylation rate or an increase in the number of phosphorylation sites, but rather, is due to an increase in the proportion of one of the H2A variants which is more highly phosphorylated than the other. If H2A phosphorylation is necessary for the constitutive heterochromatin state, it is reasonable that the cell would accomplish the generation of this structure by permanently installing a more highly phosphorylated H2A in the heterochromatin nucleosome rather than by trying to modulate the phosphorylation rate in such a condensed structure. The proposal that histone phosphorylation is involved with the condensed structures of chromatin is based primarily on correlations between histone phosphorylation measurements and cellular phenomena. One proof that this concept is correct ultimately rests in the ability to demonstrate these correlations in isolated chromosomes and chromatin fractions. This demonstration is presently limited by the excessive dephosphorylation of histones which occurs during the isolation of chromosomes and chromatin fractions. Thus, the demonstration of an effective inhibitor of histone dephosphorylation which is compatible with the isolation of nuclear structures and chromatin fractions having native morphologies is essential for future studies on the biological function of histone phosphorylation. (ERB)

  10. Distinct roles of methamphetamine in modulating spatial memory consolidation, retrieval, reconsolidation and the accompanying changes of ERK and CREB activation in hippocampus and prefrontal cortex.

    PubMed

    Cao, Guofen; Zhu, Jie; Zhong, Qing; Shi, Chaofeng; Dang, Yonghui; Han, Wei; Liu, Xinshe; Xu, Ming; Chen, Teng

    2013-04-01

    Drugs of abuse modulated learning and memory in humans yet the underlying mechanism remained unclear. The extracellular signal-regulated kinase (ERK) and the transcription factor cAMP response element-binding protein (CREB) were involved in neuroplastic changes associated with learning and memory. In the current study, we used a Morris water maze to examine the effect of methamphetamine (METH) on different processes of spatial memory in mice. We then investigated the status of ERK and CREB in the hippocampus and prefrontal cortex (PFC). We found that 1.0 mg/kg dose of METH facilitated spatial memory consolidation when it was injected immediately after the last learning trial. In contrast, the same dose of METH had no effect on spatial memory retrieval when it was injected 30 min before the test. Furthermore, 1.0 mg/kg dose of METH injected immediately after retrieval had no effect on spatial memory reconsolidation. Activation of both ERK and CREB in the hippocampus was found following memory consolidation but not after retrieval or reconsolidation in METH-treated mouse groups. In contrast, activation of both ERK and CREB in the PFC was found following memory retrieval but not other processes in METH-treated mouse groups. These results suggested that METH facilitated spatial memory consolidation but not retrieval or reconsolidation. Moreover, activation of the ERK and CREB signaling pathway in the hippocampus might be involved in METH-induced spatial memory changes.

  11. The CREB Coactivator CRTC2 is a Lymphoma Tumor Suppressor that Preserves Genome Integrity Through Transcription of DNA Mismatch Repair Genes

    PubMed Central

    Fang, Minggang; Pak, Magnolia L.; Chamberlain, Lynn; Xing, Wei; Yu, Hongbo; Green, Michael R.

    2015-01-01

    SUMMARY The CREB-regulated transcription coactivator CRTC2 stimulates CREB target gene expression and has a well-established role in modulating glucose and lipid metabolism. Here we find, unexpectedly, that loss of CRTC2, as well as CREB1 and its coactivator CREB-binding protein (CBP), results in a deficiency in DNA mismatch repair (MMR) and a resultant increased mutation frequency. We show that CRTC2, CREB1 and CBP are transcriptional activators of well-established MMR genes, including EXO1, MSH6, PMS1 and POLD2. Mining of expression profiling databases and analysis of patient samples reveal that CRTC2 and its target MMR genes are down-regulated in specific T-cell lymphoma subtypes, which are microsatellite unstable. The levels of acetylated histone H3 on the CRTC2 promoter are significantly reduced in lymphoma compared to normal tissue, explaining the decreased CRTC2 expression. Our results establish a role for CRTC2 as a lymphoma tumor suppressor gene that preserves genome integrity by stimulating transcription of MMR genes. PMID:26004186

  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. 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

  14. CREB-binding protein, p300, butyrate, and Wnt signaling in colorectal cancer.

    PubMed

    Bordonaro, Michael; Lazarova, Darina L

    2015-07-21

    This paper reviews the distinctive roles played by the transcriptional coactivators CREB-binding protein (CBP) and p300 in Wnt/β-catenin signaling and cell physiology in colorectal cancer (CRC). Specifically, we focus on the effects of CBP- and p300-mediated Wnt activity on (1) neoplastic progression; (2) the activities of butyrate, a breakdown product of dietary fiber, on cell signaling and colonic cell physiology; (3) the development of resistance to histone deacetylase inhibitors (HDACis), including butyrate and synthetic HDACis, in colonic cells; and (4) the physiology and number of cancer stem cells. Mutations of the Wnt/β-catenin signaling pathway initiate the majority of CRC cases, and we have shown that hyperactivation of this pathway by butyrate and other HDACis promotes CRC cell apoptosis. This activity by butyrate may in part explain the preventive action of fiber against CRC. However, individuals with a high-fiber diet may still develop neoplasia; therefore, resistance to the chemopreventive action of butyrate likely contributes to CRC. CBP or p300 may modify the ability of butyrate to influence colonic cell physiology since the two transcriptional coactivators affect Wnt signaling, and likely, its hyperactivation by butyrate. Also, CBP and p300 likely affect colonic tumorigenesis, as well as stem cell pluripotency. Improvement of CRC prevention and therapy requires a better understanding of the alterations in Wnt signaling and gene expression that underlie neoplastic progression, stem cell fate, and the development of resistance to butyrate and clinically relevant HDACis. Detailed knowledge of how CBP- and p300 modulate colonic cell physiology may lead to new approaches for anti-CRC prevention and therapeutics, particularly with respect to combinatorial therapy of CBP/p300 inhibitors with HDACis.

  15. The CREB binding protein inhibitor ICG-001 suppresses pancreatic cancer growth

    PubMed Central

    Arensman, Michael D.; Telesca, Donatello; Lay, Anna R.; Kershaw, Kathleen M.; Wu, Nanping; Donahue, Timothy R.; Dawson, David W.

    2014-01-01

    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 CREB-binding protein (CBP) to disrupt its interaction with β-catenin and inhibit CBP function as a co-activator 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, appeared 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

  16. Expression patterns of CREB binding protein (CREBBP) and its methylated species during zebrafish development.

    PubMed

    Batut, Julie; Duboé, Carine; Vandel, Laurence

    2015-01-01

    Proper embryonic development requires a fine-tuned control of gene expression, which is achieved in part through the activity of transcription coactivators or corepressors. The nuclear coactivator cAMP-response element-binding protein (CREB) binding protein (CREBBP or CBP) interacts with numerous transcription factors and thereby plays a key role in various signaling pathways. Interestingly, in cell-based studies CREBBP activity is modulated by post-translational modifications such as methylation on arginine residues which is catalyzed by coactivator-associated arginine methyltransferase 1 (CARM1). However, whether and where CREBBP, and in particular its methylated forms, are expressed during development in vertebrates has not been addressed so far. Here, we analyzed the expression of the two crebbp genes (crebbpa & crebbpb) during zebrafish development using both RT-qPCR and in situ hybridization. We found that while crebbpa expression is higher in posterior, caudal nascent somites during somitogenesis, crebbpb accumulates in anterior, rostral, and more mature somites. In addition, crebbpa mRNA is enriched in the central myotome at 24 hpf indicating that its expression is spatially and temporally controlled. We next characterized the expression of CREBBP protein from blastula to gastrula stages by immunohistochemistry. We found that while CREBBP is clearly cytoplasmic in the early blastula, it becomes both cytoplasmic and nuclear at 30% epiboly before turning mainly nuclear during gastrulation. Of interest, CREBBP methylated species appear to be mainly nuclear from 30% epiboly to 6-somite stage. This suggests that methylation may regulate CREBBP import to the nucleus during zebrafish development and could therefore participate in the control of early developmental processes.

  17. 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

  18. The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development.

    PubMed Central

    Goedert, M; Jakes, R; Crowther, R A; Six, J; Lübke, U; Vandermeeren, M; Cras, P; Trojanowski, J Q; Lee, V M

    1993-01-01

    Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8506352

  19. 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

  20. 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

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

    PubMed

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

    2014-12-08

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

  2. 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

  3. Oxidative phosphorylation in cancer cells.

    PubMed

    Solaini, Giancarlo; Sgarbi, Gianluca; Baracca, Alessandra

    2011-06-01

    Evidence suggests that mitochondrial metabolism may play a key role in controlling cancer cells life and proliferation. Recent evidence also indicates how the altered contribution of these organelles to metabolism and the resistance of cancer mitochondria against apoptosis-associated permeabilization are closely related. The hallmarks of cancer growth, increased glycolysis and lactate production in tumours, have raised attention due to recent observations suggesting a wide spectrum of oxidative phosphorylation deficit and decreased availability of ATP associated with malignancies and tumour cell expansion. More specifically, alteration in signal transduction pathways directly affects mitochondrial proteins playing critical roles in controlling the membrane potential as UCP2 and components of both MPTP and oxphos complexes, or in controlling cells life and death as the Bcl-2 proteins family. Moreover, since mitochondrial bioenergetics and dynamics, are also involved in processes of cells life and death, proper regulation of these mitochondrial functions is crucial for tumours to grow. Therefore a better understanding of the key pathophysiological differences between mitochondria in cancer cells and in their non-cancer surrounding tissue is crucial to the finding of tools interfering with these peculiar tumour mitochondrial functions and will disclose novel approaches for the prevention and treatment of malignant diseases. Here, we review the peculiarity of tumour mitochondrial bioenergetics and the mode it is linked to the cell metabolism, providing a short overview of the evidence accumulated so far, but highlighting the more recent advances.

  4. 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

  5. Fsp27/CIDEC is a CREB target gene induced during early fasting in liver and regulated by FA oxidation rate

    PubMed Central

    Vilà-Brau, Anna; De Sousa-Coelho, Ana Luísa; Gonçalves, Joana F.; Haro, Diego; Marrero, Pedro F.

    2013-01-01

    FSP27 [cell death-inducing DFFA-like effector c (CIDEC) in humans] is a protein associated with lipid droplets that downregulates the fatty acid oxidation (FAO) rate when it is overexpressed. However, little is known about its physiological role in liver. Here, we show that fasting regulates liver expression of Fsp27 in a time-dependent manner. Thus, during the initial stages of fasting, a maximal induction of 800-fold was achieved, whereas during the later phase of fasting, Fsp27 expression decreased. The early response to fasting can be explained by a canonical PKA-CREB-CRTC2 signaling pathway because: i) CIDEC expression was induced by forskolin, ii) Fsp27 promoter activity was increased by CREB, and iii) Fsp27 expression was upregulated in the liver of Sirt1 knockout animals. Interestingly, pharmacological (etomoxir) or genetic (Hmgcs2 interference) inhibition of the FAO rate increases the in vivo expression of Fsp27 during fasting. Similarly, CIDEC expression was upregulated in HepG2 cells by either etomoxir or HMGCS2 interference. Our data indicate that there is a kinetic mechanism of autoregulation between short- and long-term fasting, by which free FAs delivered to the liver during early fasting are accumulated/exported by FSP27/CIDEC, whereas over longer periods of fasting, they are degraded in the mitochondria through the carnitine palmitoyl transferase system. PMID:23220584

  6. 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

    2015-03-03

    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.

  7. Luman/CREB3 recruitment factor regulates glucocorticoid receptor activity and is essential for prolactin-mediated maternal instinct.

    PubMed

    Martyn, Amanda C; Choleris, Elena; Gillis, Daniel J; Armstrong, John N; Amor, Talya R; McCluggage, Adam R R; Turner, Patricia V; Liang, Genqing; Cai, Kimberly; Lu, Ray

    2012-12-01

    The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system in animal responses to stress. It is known that the HPA axis is attenuated at parturition to prevent detrimental effects of glucocorticoid secretion including inhibition of lactation and maternal responsiveness. Luman/CREB3 recruitment factor (LRF) was identified as a negative regulator of CREB3 which is involved in the endoplasmic reticulum stress response. Here, we report a LRF gene knockout mouse line that has a severe maternal behavioral defect. LRF(-/-) females lacked the instinct to tend pups; 80% of their litters died within 24 h, while most pups survived if cross-fostered. Prolactin levels were significantly repressed in lactating LRF(-/-) dams, with glucocorticoid receptor (GR) signaling markedly augmented. In cell culture, LRF repressed transcriptional activity of GR and promoted its protein degradation. LRF was found to colocalize with the known GR repressor, RIP140/NRIP1, which inhibits the activity by GR within specific nuclear punctates that are similar to LRF nuclear bodies. Furthermore, administration of prolactin or the GR antagonist RU486 restored maternal responses in mutant females. We thus postulate that LRF plays a critical role in the attenuation of the HPA axis through repression of glucocorticoid stress signaling during parturition and the postpartum period.

  8. Luman/CREB3 Recruitment Factor Regulates Glucocorticoid Receptor Activity and Is Essential for Prolactin-Mediated Maternal Instinct

    PubMed Central

    Martyn, Amanda C.; Choleris, Elena; Gillis, Daniel J.; Armstrong, John N.; Amor, Talya R.; McCluggage, Adam R. R.; Turner, Patricia V.; Liang, Genqing; Cai, Kimberly

    2012-01-01

    The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system in animal responses to stress. It is known that the HPA axis is attenuated at parturition to prevent detrimental effects of glucocorticoid secretion including inhibition of lactation and maternal responsiveness. Luman/CREB3 recruitment factor (LRF) was identified as a negative regulator of CREB3 which is involved in the endoplasmic reticulum stress response. Here, we report a LRF gene knockout mouse line that has a severe maternal behavioral defect. LRF−/− females lacked the instinct to tend pups; 80% of their litters died within 24 h, while most pups survived if cross-fostered. Prolactin levels were significantly repressed in lactating LRF−/− dams, with glucocorticoid receptor (GR) signaling markedly augmented. In cell culture, LRF repressed transcriptional activity of GR and promoted its protein degradation. LRF was found to colocalize with the known GR repressor, RIP140/NRIP1, which inhibits the activity by GR within specific nuclear punctates that are similar to LRF nuclear bodies. Furthermore, administration of prolactin or the GR antagonist RU486 restored maternal responses in mutant females. We thus postulate that LRF plays a critical role in the attenuation of the HPA axis through repression of glucocorticoid stress signaling during parturition and the postpartum period. PMID:23071095

  9. 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

  10. The chemical biology of protein phosphorylation.

    PubMed

    Tarrant, Mary Katherine; Cole, Philip A

    2009-01-01

    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.

  11. The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways.

    PubMed

    Fang, Z H; Visker, M H P W; Miranda, G; Delacroix-Buchet, A; Bovenhuis, H; Martin, P

    2016-10-01

    Casein (CN) phosphorylation is an important posttranslational modification and is one of the key factors responsible for constructing and stabilizing casein micelles. Variation in phosphorylation degree of αS-CN is of great interest because it is suggested to affect milk technological properties. This study aimed to investigate the variation in phosphorylation degree of αS-CN among milk of individual cows and to explore relationships among different phosphorylation isoforms of αS-CN. For this purpose, we analyzed morning milk samples from 529 French Montbéliarde cows using liquid chromatography coupled with electrospray ionization mass spectrometry. We detected 3 new phosphorylation isoforms: αS2-CN-9P, αS2-CN-14P, and αS2-CN-15P in bovine milk, in addition to the known isoforms αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, and αS2-CN-13P. The relative concentrations of each αS-CN phosphorylation isoform varied considerably among individual cows. Furthermore, the phenotypic correlations and hierarchical clustering suggest at least 2 regulatory systems for phosphorylation of αS-CN: one responsible for isoforms with lower levels of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P), and another responsible for isoforms with higher levels of phosphorylation (αS1-CN-9P, αS2-CN-12P, αS2-CN-13P, and αS2-CN-14P). Identifying all phosphorylation sites of αS2-CN and investigating the genetic background of different αS2-CN phosphorylation isoforms may provide further insight into the phosphorylation mechanism of caseins.

  12. The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways.

    PubMed

    Fang, Z H; Visker, M H P W; Miranda, G; Delacroix-Buchet, A; Bovenhuis, H; Martin, P

    2016-10-01

    Casein (CN) phosphorylation is an important posttranslational modification and is one of the key factors responsible for constructing and stabilizing casein micelles. Variation in phosphorylation degree of αS-CN is of great interest because it is suggested to affect milk technological properties. This study aimed to investigate the variation in phosphorylation degree of αS-CN among milk of individual cows and to explore relationships among different phosphorylation isoforms of αS-CN. For this purpose, we analyzed morning milk samples from 529 French Montbéliarde cows using liquid chromatography coupled with electrospray ionization mass spectrometry. We detected 3 new phosphorylation isoforms: αS2-CN-9P, αS2-CN-14P, and αS2-CN-15P in bovine milk, in addition to the known isoforms αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, and αS2-CN-13P. The relative concentrations of each αS-CN phosphorylation isoform varied considerably among individual cows. Furthermore, the phenotypic correlations and hierarchical clustering suggest at least 2 regulatory systems for phosphorylation of αS-CN: one responsible for isoforms with lower levels of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P), and another responsible for isoforms with higher levels of phosphorylation (αS1-CN-9P, αS2-CN-12P, αS2-CN-13P, and αS2-CN-14P). Identifying all phosphorylation sites of αS2-CN and investigating the genetic background of different αS2-CN phosphorylation isoforms may provide further insight into the phosphorylation mechanism of caseins. PMID:27522420

  13. 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.

  14. 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.

  15. Long-term dynamics of multisite phosphorylation

    PubMed Central

    Rubinstein, Boris Y.; Mattingly, Henry H.; Berezhkovskii, Alexander M.; Shvartsman, Stanislav Y.

    2016-01-01

    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

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose.

    PubMed

    Hatano, Tomoyuki; Morigasaki, Susumu; Tatebe, Hisashi; Ikeda, Kyoko; Shiozaki, Kazuhiro

    2015-01-01

    The Target Of Rapamycin (TOR) is an evolutionarily conserved protein kinase that forms 2 distinct protein complexes referred to as TOR complex 1 (TORC1) and 2 (TORC2). Recent extensive studies have demonstrated that TORC1 is under the control of the small GTPases Rheb and Rag that funnel multiple input signals including those derived from nutritional sources; however, information is scarce as to the regulation of TORC2. A previous study using the model system provided by the fission yeast Schizosaccharomyces pombe identified Ryh1, a Rab-family GTPase, as an activator of TORC2. Here, we show that the nucleotide-binding state of Ryh1 is regulated in response to glucose, mediating this major nutrient signal to TORC2. In glucose-rich growth media, the GTP-bound form of Ryh1 induces TORC2-dependent phosphorylation of Gad8, a downstream target of TORC2 in fission yeast. Upon glucose deprivation, Ryh1 becomes inactive, which turns off the TORC2-Gad8 pathway. During glucose starvation, however, Gad8 phosphorylation by TORC2 gradually recovers independently of Ryh1, implying an additional TORC2 activator that is regulated negatively by glucose. The paired positive and negative regulatory mechanisms may allow fine-tuning of the TORC2-Gad8 pathway, which is essential for growth under glucose-limited environment.

  1. 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

  2. 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…

  3. 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

  4. 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

  5. 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…

  6. Expression of PDZ-binding kinase (PBK) is regulated by cell cycle-specific transcription factors E2F and CREB/ATF.

    PubMed

    Nandi, Asit K; Rapoport, Aaron P

    2006-04-01

    Earlier we reported that a novel mitotic protein kinase, PDZ-binding kinase (PBK), is expressed in primary hematopoietic neoplasms. Recent reports have suggested a role for PBK in mitotic progression. In the present study, we demonstrate that PBK is down regulated during doxorubicin induced growth arrest of HL60 promyelocytic leukemia cells at least partly due to cell cycle-specific transcriptional regulation. Furthermore, we show that transcriptional control is mostly due to binding of transcription factors E2F and CREB/ATF to two distinct binding sites within the PBK promoter. This was demonstrated by: (i) electrophoretic mobility shift assays showing transcription factor binding within the PBK promoter at the putative E2F (-146bp) and CREB/ATF (-312bp) binding sites; (ii) Western immunoblot analysis of knockdown extracts from siRNA inhibition of transcription factor expression showing that PBK protein expression is dependent upon the presence of these transcription factors; (iii) codistribution of CREB factor and PBK in cell lines of disparate tissue origin; and (iv) luciferase reporter assays showing that PBK promoter activity is dependent on factor binding at intact E2F and CREB/ATF sites. These findings may provide insight into the mechanisms that upregulate PBK expression in proliferative hematologic malignancies and down regulate its expression following growth arrest of leukemic cells. PMID:16171862

  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. 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

  9. 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.

  10. Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

    PubMed

    Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

    2013-12-01

    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

  11. 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.

  12. 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.

  13. Phosphorylation Modulates Catalytic Activity of Mycobacterial Sirtuins

    PubMed Central

    Yadav, Ghanshyam S.; Ravala, Sandeep K.; Malhotra, Neha; Chakraborti, Pradip K.

    2016-01-01

    Sirtuins are NAD+-dependent deacetylases involved in the regulation of diverse cellular processes and are conserved throughout phylogeny. Here we report about in vitro transphosphorylation of the only NAD+-dependent deacetylase (mDAC) present in the genome of Mycobacterium tuberculosis by eukaryotic-type Ser/Thr kinases, particularly PknA. The phosphorylated mDAC displayed decreased deacetylase activity compared to its unphosphorylated counterpart. Mass-spectrometric study identified seven phosphosites in mDAC; however, mutational analysis highlighted major contribution of Thr-214 for phosphorylation of the protein. In concordance to this observation, variants of mDAC substituting Thr-214 with either Ala (phospho-ablated) or Glu (phosphomimic) exhibited significantly reduced deacetylase activity suggesting phosphorylation mediated control of enzymatic activity. To assess the role of phosphorylation towards functionality of mDAC, we opted for a sirtuin knock-out strain of Escherichia coli (Δdac), where interference of endogenous mycobacterial kinases could be excluded. The Δdac strain in nutrient deprived acetate medium exhibited compromised growth and complementation with mDAC reversed this phenotype. The phospho-ablated or phosphomimic variant, on the other hand, was unable to restore the functionality of mDAC indicating the role of phosphorylation per se in the process. We further over-expressed mDAC or mDAC-T214A as His-tagged protein in M. smegmatis, where endogenous eukaryotic-type Ser/Thr kinases are present. Anti-phosphothreonine antibody recognized both mDAC and mDAC-T214A proteins in western blotting. However, the extent of phosphorylation as adjudged by scanning the band intensity, was significantly low in the mutant protein (mDAC-T214A) compared to that of the wild-type (mDAC). Furthermore, expression of PknA in the mDAC complemented Δdac strain was able to phosphorylate M. tuberculosis sirtuin. The growth profile of this culture in acetate medium was

  14. Systematic Discovery of In Vivo Phosphorylation Networks

    PubMed Central

    Linding, Rune; Jensen, Lars Juhl; Ostheimer, Gerard J.; van Vugt, Marcel A.T.M.; Jørgensen, Claus; Miron, Ioana M.; Diella, Francesca; Colwill, Karen; Taylor, Lorne; Elder, Kelly; Metalnikov, Pavel; Nguyen, Vivian; Pasculescu, Adrian; Jin, Jing; Park, Jin Gyoon; Samson, Leona D.; Woodgett, James R.; Russell, Robert B.; Bork, Peer; Yaffe, Michael B.; Pawson, Tony

    2009-01-01

    Summary Protein kinases control cellular decision processes by phosphorylating specific substrates. Proteome-wide mapping has identified thousands of in vivo phosphorylation sites. However, systematically resolving which kinase targets each site is presently infeasible, due to the limited specificity of consensus motifs and the potential influence of contextual factors, such as protein scaffolds, localisation and expression, on cellular substrate specificity. We have therefore developed a computational method, NetworKIN, that augments motifs with context for kinases and phosphoproteins. This can pinpoint individual kinases responsible for specific in vivo phosphorylation events and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. We show that context provides 60–80% of the computational capability to assign in vivo substrate specificity. Applying this approach to a DNA damage signalling network, we extend its cell-cycle regulation by showing that 53BP1 is a CDK1 substrate, show that Rad50 is phosphorylated by ATM kinase under genotoxic stress, and suggest novel roles of ATM in apoptosis. Finally, we present a scalable strategy to validate our predictions and use it to support the prediction that BCLAF1 is a GSK3 substrate. PMID:17570479

  15. Motor Domain Phosphorylation Modulates Kinesin-1 Transport*

    PubMed Central

    DeBerg, Hannah A.; Blehm, Benjamin H.; Sheung, Janet; Thompson, Andrew R.; Bookwalter, Carol S.; Torabi, Seyed F.; Schroer, Trina A.; Berger, Christopher L.; Lu, Yi; Trybus, Kathleen M.; Selvin, Paul R.

    2013-01-01

    Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated. PMID:24072715

  16. Protein phosphorylation systems in postmortem human brain

    SciTech Connect

    Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P. )

    1989-01-01

    Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.

  17. TNFα and IFNγ Synergistically Enhance Transcriptional Activation of CXCL10 in Human Airway Smooth Muscle Cells via STAT-1, NF-κB, and the Transcriptional Coactivator CREB-binding Protein

    PubMed Central

    Clarke, Deborah L.; Clifford, Rachel L.; Jindarat, Sarawut; Proud, David; Pang, Linhua; Belvisi, Maria; Knox, Alan J.

    2010-01-01

    Asthmatic airway smooth muscle (ASM) expresses interferon-γ-inducible protein-10 (CXCL10), a chemokine known to mediate mast cell migration into ASM bundles that has been reported in the airways of asthmatic patients. CXCL10 is elevated in patients suffering from viral exacerbations of asthma and in patients with chronic obstructive pulmonary disease (COPD), diseases in which corticosteroids are largely ineffective. IFNγ and TNFα synergistically induce CXCL10 release from human ASM cells in a steroid-insensitive manner, via an as yet undefined mechanism. We report that TNFα activates the classical NF-κB (nuclear factor κB) pathway, whereas IFNγ activates JAK2/STAT-1α and that inhibition of the JAK/STAT pathway is more effective in abrogating CXCL10 release than the steroid fluticasone. The synergy observed with TNFα and IFNγ together, however, did not lie at the level of NF-κB activation, STAT-1α phosphorylation, or in vivo binding of these transcription factors to the CXCL10 promoter. Stimulation of human ASM cells with TNFα and IFNγ induced histone H4 but not histone H3 acetylation at the CXCL10 promoter, although no synergism was observed when both cytokines were combined. We show, however, that TNFα and IFNγ exert a synergistic effect on the recruitment of CREB-binding protein (CBP) to the CXCL10, which is accompanied by increased RNA polymerase II. Our results provide evidence that synergism between TNFα and IFNγ lies at the level of coactivator recruitment in human ASM and suggest that inhibition of JAK/STAT signaling may be of therapeutic benefit in steroid-resistant airway disease. PMID:20833730

  18. TNFα and IFNγ synergistically enhance transcriptional activation of CXCL10 in human airway smooth muscle cells via STAT-1, NF-κB, and the transcriptional coactivator CREB-binding protein.

    PubMed

    Clarke, Deborah L; Clifford, Rachel L; Jindarat, Sarawut; Proud, David; Pang, Linhua; Belvisi, Maria; Knox, Alan J

    2010-09-17

    Asthmatic airway smooth muscle (ASM) expresses interferon-γ-inducible protein-10 (CXCL10), a chemokine known to mediate mast cell migration into ASM bundles that has been reported in the airways of asthmatic patients. CXCL10 is elevated in patients suffering from viral exacerbations of asthma and in patients with chronic obstructive pulmonary disease (COPD), diseases in which corticosteroids are largely ineffective. IFNγ and TNFα synergistically induce CXCL10 release from human ASM cells in a steroid-insensitive manner, via an as yet undefined mechanism. We report that TNFα activates the classical NF-κB (nuclear factor κB) pathway, whereas IFNγ activates JAK2/STAT-1α and that inhibition of the JAK/STAT pathway is more effective in abrogating CXCL10 release than the steroid fluticasone. The synergy observed with TNFα and IFNγ together, however, did not lie at the level of NF-κB activation, STAT-1α phosphorylation, or in vivo binding of these transcription factors to the CXCL10 promoter. Stimulation of human ASM cells with TNFα and IFNγ induced histone H4 but not histone H3 acetylation at the CXCL10 promoter, although no synergism was observed when both cytokines were combined. We show, however, that TNFα and IFNγ exert a synergistic effect on the recruitment of CREB-binding protein (CBP) to the CXCL10, which is accompanied by increased RNA polymerase II. Our results provide evidence that synergism between TNFα and IFNγ lies at the level of coactivator recruitment in human ASM and suggest that inhibition of JAK/STAT signaling may be of therapeutic benefit in steroid-resistant airway disease.

  19. Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation

    PubMed Central

    Rigatti, Marc; Le, Andrew V.; Gerber, Claire; Moraru, Ion I.; Dodge-Kafka, Kimberly L.

    2016-01-01

    Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca2+ cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca2+ re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100–200nM) to regulate the phosphorylation of large quantities of PLB (50µM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100–200 fold lower concentrations. PMID:26027516

  20. Interactions between the Class II Transactivator and CREB Binding Protein Increase Transcription of Major Histocompatibility Complex Class II Genes

    PubMed Central

    Fontes, Joseph D.; Kanazawa, Satoshi; Jean, Dickson; Peterlin, B. Matija

    1999-01-01

    Class II major histocompatibility (class II) genes are regulated in a B-cell-specific and gamma interferon-inducible fashion. The master switch for the expression of these genes is the class II transactivator (CIITA). In this report, we demonstrate that one of the functions of CIITA is to recruit the CREB binding protein (CBP) to class II promoters. Not only functional but also specific binding interactions between CIITA and CBP were demonstrated. Moreover, a dominant negative form of CBP decreased the activity of class II promoters and levels of class II determinants on the surface of cells. Finally, the inhibition of class II gene expression by the glucocorticoid hormone could be attributed to the squelching of CBP by the glucocorticoid receptor. We conclude that CBP, a histone acetyltransferase, plays an important role in the transcription of class II genes. PMID:9858618

  1. Primary Renal Sclerosing Epithelioid Fibrosarcoma: Report of Two Cases with EWSR1-CREB3L1 Gene Fusion

    PubMed Central

    Argani, Pedram; Lewin, Jack R.; Edmonds, Pamela; Netto, George J.; Prieto-Granada, Carlos; Zhang, Lei; Jungbluth, Achim A.; Antonescu, Cristina R.

    2014-01-01

    We report the first two genetically confirmed cases of primary renal sclerosing epithelioid fibrosarcoma (SEF), occurring in a 17 year-old male and a 61 year-old female. In both cases, the tumors demonstrated the typical epithelioid clear cell morphology associated with extensive hyalinizing fibrosis, raising the differential diagnosis of solitary fibrous tumor, metanephric stromal tumor, and the sclerosing variant of clear cell sarcoma of the kidney. Both neoplasms demonstrated diffuse immunoreactivity for MUC4, a highly specific marker for SEF, and both demonstrated evidence of rearrangement of both the EWSR1 and CREB3L1 genes which have recently shown to be fused in this entity. Both neoplasms presented with metastatic disease. Primary renal SEF represents yet another translocation-associated sarcoma now shown to arise primarily in the kidney. PMID:25353281

  2. Phosphorylation of RACK1 in plants

    SciTech Connect

    Chen, Jay -Gui

    2015-08-31

    Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. In conclusion, these findings promote a new regulatory system in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.

  3. Phosphorylation of RACK1 in plants

    DOE PAGES

    Chen, Jay -Gui

    2015-08-31

    Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. In conclusion, these findings promote a new regulatory systemmore » in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.« less

  4. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . E-mail: biocrr@lsuhsc.edu

    2005-05-27

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.

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

    PubMed

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

    2014-09-01

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

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

    PubMed

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

    2015-12-01

    17β-Estradiol (E2)-dependent cell proliferation requires both estrogen receptor α (ERα)-based integrated control of gene transcription and kinase pathways activation. Such coordination of intracellular E2:ERα-dependent signaling mechanisms is finely tuned by receptor association with specific partner proteins. Recently, we identified the leucine (L) 429 and alanine (A) 430 within the ERα ligand binding domain as important residues for receptor non-covalent interaction to ubiquitinated species [i.e., ERα ubiquitin-binding surface (ERα UBS)] and for E2-induced ERα activation. To date, if these two ERα amino acids are involved in the control of E2-dependent pathways required for cell proliferation is unknown. Here, by using stably expressing ERα mutated in L429 and A430 (i.e., L429A,A430G-LAAG) cell lines, we show that L429 and A430 are critical for E2-induced cell proliferation, PI3K/AKT pathway activation, and ERα-mediated transcriptional changes. Moreover, we demonstrate that these two receptor structural determinants direct the E2-induced PI3K/AKT/CREB1 pathway activation and CREB1-mediated transcriptional activity that in turn control the hormone-induced cell proliferation. As a whole, our data demonstrate for the first time that the ERα UBS contributes to the modulation of E2-induced ERα-mediated cell proliferation and provide a novel connection between the receptor structure and the functional molecular mechanisms by which E2:ERα complex can regulate cell processes. PMID:26348925

  7. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation.

    PubMed

    Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang; Zhou, Zhen-Yu; Song, Cai-Juan

    2015-11-20

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer.

  8. Differences in long-term memory stability and AmCREB level between forward and backward conditioned honeybees (Apis mellifera)

    PubMed Central

    Felsenberg, Johannes; Dyck, Yan; Feige, Janina; Ludwig, Jenny; Plath, Jenny Aino; Froese, Anja; Karrenbrock, Melanie; Nölle, Anna; Heufelder, Karin; Eisenhardt, Dorothea

    2015-01-01

    In classical conditioning a predictive relationship between a neutral stimulus (conditioned stimulus; CS) and a meaningful stimulus (unconditioned stimulus; US) is learned when the CS precedes the US. In backward conditioning the sequence of the stimuli is reversed. In this situation animals might learn that the CS signals the end or the absence of the US. In honeybees 30 min and 24 h following backward conditioning a memory for the excitatory and inhibitory properties of the CS could be retrieved, but it remains unclear whether a late long-term memory is formed that can be retrieved 72 h following backward conditioning. Here we examine this question by studying late long-term memory formation in forward and backward conditioning of the proboscis extension response (PER). We report a difference in the stability of memory formed upon forward and backward conditioning with the same number of conditioning trials. We demonstrate a transcription-dependent memory 72 h after forward conditioning but do not observe a 72 h memory after backward conditioning. Moreover we find that protein degradation is differentially involved in memory formation following these two conditioning protocols. We report differences in the level of a transcription factor, the cAMP response element binding protein (CREB) known to induce transcription underlying long-term memory formation, following forward and backward conditioning. Our results suggest that these alterations in CREB levels might be regulated by the proteasome. We propose that the differences observed are due to the sequence of stimulus presentation between forward and backward conditioning and not to differences in the strength of the association of both stimuli. PMID:25964749

  9. Phosphorylation and actin activation of brain myosin.

    PubMed Central

    Barylko, B; Sobieszek, A

    1983-01-01

    A method is described for obtaining brain myosin that shows significant actin activation, after phosphorylation with chicken gizzard myosin light chain kinase. Myosin with this activity could be obtained only via the initial purification of brain actomyosin. The latter complex, isolated by a method similar to that used for smooth muscle, contained actin, myosin, tropomyosin of the non-muscle type and another actin-binding protein of approximately 100,000 daltons. From the presence of a specific myosin light chain kinase and phosphatase in brain tissue it is suggested that the regulation of actin-myosin interaction operates via phosphorylation and dephosphorylation of myosin. Images Fig. 1. Fig. 3. PMID:11894951

  10. ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis

    PubMed Central

    Parsa, Kishore V. L.; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K.; Misra, Parimal

    2013-01-01

    PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser298 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMTS298D) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMTS298D but not PIMTS298A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser298 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser298 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PMID:24358311

  11. ERK2-mediated phosphorylation of transcriptional coactivator binding protein PIMT/NCoA6IP at Ser298 augments hepatic gluconeogenesis.

    PubMed

    Kapadia, Bandish; Viswakarma, Navin; Parsa, Kishore V L; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K; Misra, Parimal

    2013-01-01

    PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser(298) and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMT(S298D)) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMT(S298D) but not PIMT(S298A) augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser(298) phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser(298) is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia.

  12. Rab-family GTPase regulates TOR complex 2 signaling in fission yeast

    PubMed Central

    Tatebe, Hisashi; Morigasaki, Susumu; Murayama, Shinichi; Zeng, Cui Tracy; Shiozaki, Kazuhiro

    2010-01-01

    Summary Background From yeast to human, TOR (Target Of Rapamycin) kinase plays pivotal roles in coupling extracellular stimuli to cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2), which phosphorylate and activate different AGC-family protein kinases. TORC1 is controlled by the small GTPase Rheb, but little is known about TORC2 regulators. Results We have identified the Ryh1 GTPase, a human Rab6 ortholog, as an activator of TORC2 signaling in the fission yeast Schizosaccharomyces pombe. Mutational inactivation of Ryh1 or its guanine nucleotide exchange factor compromises the TORC2-dependent phosphorylation of the AGC-family Gad8 kinase. In addition, the effector domain of Ryh1 is important for its physical interaction with TORC2 and for stimulation of TORC2 signaling. Thus, GTP-bound Ryh1 is likely to be the active form stimulatory to TORC2–Gad8 signaling. Consistently, expression of the GTP-locked mutant Ryh1 is sufficient to promote interaction between TORC2 and Gad8 and to induce Gad8 hyper-phosphorylation. The loss of functional Ryh1, TORC2 or Gad8 brings about similar vacuolar fragmentation and stress sensitivity, further corroborating their involvement in a common cellular process. Human Rab6 can substitute Ryh1 in S. pombe and therefore, Rab6 may be a potential activator of TORC2 in mammals. Conclusions In its GTP-bound form, Ryh1, an evolutionarily conserved Rab GTPase, activates TORC2 signaling to the AGC kinase Gad8. The Ryh1 GTPase and the TORC2–Gad8 pathway are required for vacuolar integrity and cellular stress resistance in S. pombe. PMID:21035342

  13. Nucleoside phosphorylation by the mineral schreibersite

    PubMed Central

    Gull, Maheen; Mojica, Mike A.; Fernández, Facundo M.; Gaul, David A.; Orlando, Thomas M.; Liotta, Charles L.; Pasek, Matthew A.

    2015-01-01

    Phosphorylation of the nucleosides adenosine and uridine by the simple mixing and mild heating of aqueous solutions of the organic compounds with synthetic analogs of the meteoritic mineral schreibersite, (Fe,Ni)3P under slightly basic conditions (pH ~9) is reported. These results suggest a potential role for meteoritic phosphorus in the origin and development of early life. PMID:26606901

  14. Ion channels, phosphorylation and mammalian sperm capacitation.

    PubMed

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto

    2011-05-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

  15. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling.

  16. Phosphoryl Transfer Reaction Snapshots in Crystals

    PubMed Central

    Gerlits, Oksana; Tian, Jianhui; Das, Amit; Langan, Paul; Heller, William T.; Kovalevsky, Andrey

    2015-01-01

    To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. The present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date. PMID:25925954

  17. Nucleoside phosphorylation by the mineral schreibersite.

    PubMed

    Gull, Maheen; Mojica, Mike A; Fernández, Facundo M; Gaul, David A; Orlando, Thomas M; Liotta, Charles L; Pasek, Matthew A

    2015-01-01

    Phosphorylation of the nucleosides adenosine and uridine by the simple mixing and mild heating of aqueous solutions of the organic compounds with synthetic analogs of the meteoritic mineral schreibersite, (Fe,Ni)3P under slightly basic conditions (pH ~9) is reported. These results suggest a potential role for meteoritic phosphorus in the origin and development of early life. PMID:26606901

  18. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana

    PubMed Central

    Lohscheider, Jens N.; Friso, Giulia; van Wijk, Klaas J.

    2016-01-01

    Plastoglobules (PGs) are plastid lipid–protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  19. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

    Pike, C.; Kopeck, K.; Sceppa, E. )

    1989-04-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with {sup 32}P-{gamma}-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of {sup 35}S-adenosine thiotriphosphate.

  20. Ion channels, phosphorylation and mammalian sperm capacitation

    PubMed Central

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto

    2011-01-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies. PMID:21540868

  1. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  2. Motexafin gadolinium modulates levels of phosphorylated Akt and synergizes with inhibitors of Akt phosphorylation.

    PubMed

    Ramos, Jason; Sirisawad, Mint; Miller, Richard; Naumovski, Louie

    2006-05-01

    Motexafin gadolinium (MGd, Xcytrin) is a tumor-selective expanded porphyrin that targets oxidative stress-related proteins. MGd treatment of the follicular lymphoma-derived cell line HF-1 resulted in growth suppression and apoptosis whereas MGd treatment of the Burkitt's lymphoma-derived cell line Ramos resulted in growth suppression but not apoptosis. Because phosphorylation status of Akt/protein kinase B is regulated by oxidative stress, we monitored total and phosphorylated Akt (pAkt) in MGd-treated HF-1 and Ramos cells. Levels of pAkt increased within 30 minutes after MGd treatment of HF-1 but after 4 hours began to show a progressive decline to below baseline levels before cells underwent apoptosis. In MGd-treated Ramos cells, pAkt increased approximately 2-fold within 4 hours and remained persistently elevated. Because pAkt activates survival pathways, we determined if MGd-induced cell death could be enhanced by inhibiting phosphorylation of Akt. The addition of specific inhibitors of Akt phosphorylation (Akt inhibitor 1 or SH-5) reduced pAkt levels in MGd-treated HF-1 and Ramos cells and synergistically enhanced MGd-induced cell death. MGd was also evaluated in combination with celecoxib, an inhibitor of Akt phosphorylation, or docetaxel, a microtubule inhibitor that can decrease Akt phosphorylation. The combination of MGd/celecoxib or MGd/docetaxel resulted in decreased Akt phosphorylation and in synergistic cytotoxicity compared with either agent alone. These data point to a potential protective role for pAkt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by combining it with agents that inhibit Akt phosphorylation.

  3. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    SciTech Connect

    Smiley, R.M. Columbia Univ College of Physicians and Surgeons, New York, NY ); Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J. )

    1990-01-01

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with {sup 32}PO{sub 4} in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the {beta}-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M{sub r} 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the {alpha}-2 agonist clonidine. Epinephrine, a combined {alpha} and {beta} agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the {alpha}-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

  4. Neurofilament Phosphorylation during Development and Disease: Which Came First, the Phosphorylation or the Accumulation?

    PubMed Central

    Dale, Jeffrey M.; Garcia, Michael L.

    2012-01-01

    Posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function. Some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments (NFs). NFs are type IV intermediate filaments (IFs) that can be composed of four subunits, neurofilament heavy (NF-H), neurofilament medium (NF-M), neurofilament light (NF-L), and α-internexin. Within wild type axons, NFs are responsible for mediating radial growth, a process that determines axonal diameter. NFs are phosphorylated on highly conserved lysine-serine-proline (KSP) repeats located along the C-termini of both NF-M and NF-H within myelinated axonal regions. Phosphorylation is thought to regulate aspects of NF transport and function. However, a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of NFs. The goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons. We review evidence that challenges the role of KSP phosphorylation as essential for radial growth and suggests an alternative role for NF phosphorylation in myelinated axons. Furthermore, we demonstrate that regulation of NF phosphorylation dynamics may be essential to avoiding NF accumulations. PMID:22570767

  5. Analysis of microRNA-203 function in CREB/MITF/RAB27a pathway: comparison between canine and human melanoma cells.

    PubMed

    Noguchi, S; Kumazaki, M; Mori, T; Baba, K; Ok