WorldWideScience

Sample records for activation loop phosphorylation

  1. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    OpenAIRE

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr9...

  2. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    Science.gov (United States)

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  3. Serine phosphorylation of NPM-ALK, which is dependent on the auto-activation of the kinase activation loop, contributes to its oncogenic potential.

    Science.gov (United States)

    Wang, Peng; Wu, Fang; Zhang, Jingdong; McMullen, Todd; Young, Leah C; Ingham, Robert J; Li, Liang; Lai, Raymond

    2011-02-01

    It is well established that the tumorigenic potential of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, is dependent on its tyrosine phosphorylation. Using tandem affinity purification-mass spectrometry, we found evidence of phosphorylation of three serine residues of NPM-ALK (Serine¹³⁵, Serine¹⁶⁴ and Serine⁴⁹⁷) ectopically expressed in GP293 cells. Using a specific anti-phosphoserine antibody and immunoprecipitation, we confirmed the presence of serine phosphorylation of NPM-ALK in all three NPM-ALK-expressing cell lines examined. Similar to the tyrosine phosphorylation, phosphorylation of these serine residues was dependent on the activation status of the kinase activation loop of ALK. All of these three serine residues are biologically important as mutation of any one of these residues resulted in a significant reduction in the tumorigenicity of NPM-ALK (assessed by cell viability and clonogenic assay), which correlated with a substantial reduction in the phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinase and signal transducer and activator of transcription 6. Serine phosphorylation of NPM-ALK appears to be regulated by multiple serine kinases since it was markedly reduced by pharmacologic inhibitors for glycogen synthase kinase-3, casein kinase I or mitogen-activated protein kinases. In summary, our study is the first to identify serine phosphorylation of NPM-ALK and to provide evidence that it enhances the tumorigenic potential of this oncogenic protein.

  4. An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes

    DEFF Research Database (Denmark)

    Papaleo, Elena; Ranzani, Valeria; Tripodi, Farida;

    2011-01-01

    elements in one of the larger families of E2 enzymes: an acidic insertion in β4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 µs molecular......E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous...... mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature...

  5. C-terminal Src kinase-mediated EPIYA phosphorylation of Pragmin creates a feed-forward C-terminal Src kinase activation loop that promotes cell motility.

    Science.gov (United States)

    Senda, Yoshie; Murata-Kamiya, Naoko; Hatakeyama, Masanori

    2016-07-01

    Pragmin is one of the few mammalian proteins containing the Glu-Pro-Ile-Tyr-Ala (EPIYA) tyrosine-phosphorylation motif that was originally discovered in the Helicobacter pylori CagA oncoprotein. Following delivery into gastric epithelial cells by type IV secretion and subsequent tyrosine phosphorylation at the EPIYA motifs, CagA serves as an oncogenic scaffold/adaptor that promiscuously interacts with SH2 domain-containing mammalian proteins such as the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) and the C-terminal Src kinase (Csk), a negative regulator of Src family kinases. Like CagA, Pragmin also forms a physical complex with Csk. In the present study, we found that Pragmin directly binds to Csk by the tyrosine-phosphorylated EPIYA motif. The complex formation potentiates kinase activity of Csk, which in turn phosphorylates Pragmin on tyrosine-238 (Y238), Y343, and Y391. As Y391 of Pragmin comprises the EPIYA motif, Pragmin-Csk interaction creates a feed-forward regulatory loop of Csk activation. Together with the finding that Pragmin and Csk are colocalized to focal adhesions, these observations indicate that the Pragmin-Csk interaction, triggered by Pragmin EPIYA phosphorylation, robustly stimulates the kinase activity of Csk at focal adhesions, which direct cell-matrix adhesion that regulates cell morphology and cell motility. As a consequence, expression of Pragmin and/or Csk in epithelial cells induces an elongated cell shape with elevated cell scattering in a manner that is mutually dependent on Pragmin and Csk. Deregulation of the Pragmin-Csk axis may therefore induce aberrant cell migration that contributes to tumor invasion and metastasis. PMID:27116701

  6. Histone 3 s10 phosphorylation: "caught in the R loop!".

    Science.gov (United States)

    Skourti-Stathaki, Konstantina; Proudfoot, Nicholas J

    2013-11-21

    In this issue of Molecular Cell, Castellano-Pozo et al. (2013) describe a connection between R loop structures and histone 3 S10 phosphorylation (H3S10P), a mark of chromatin compaction. Their results constitute a significant advance in our understanding of the role of R loops in genomic instability.

  7. Ser⁄ Thr residues at α3⁄β5 loop of Gαs are important in morphine-induced adenylyl cyclase sensitization but not mitogen-activated protein kinase phosphorylation

    Science.gov (United States)

    Seyedabadi, Mohammad; Ostad, Seyed Nasser; Albert, Paul R.; Dehpour, Ahmad R.; Rahimian, Reza; Ghazi-Khansari, Mahmoud; Ghahremani, Mohammad H.

    2015-01-01

    The signaling switch of β2-adrenergic and μ1-opioid receptors from stimulatory G-protein (Gαs) to inhibitory G-protein (Gαi) (and vice versa) influences adenylyl cyclase (AC) and extracellular-regulated kinase (ERK)1 ⁄ 2 activation. Post-translational modifications, including dephosphorylation of Gαs, enhance opioid receptor coupling to Gαs. In the present study, we substituted the Ser ⁄ Thr residues of Gαs at the α3 ⁄ β5 and α4 ⁄ β6 loops aiming to study the role of Gαs lacking Ser ⁄ Thr phosphorylation with respect to AC sensitization and mitogen-activated protein kinase activation. Isoproterenol increased the cAMP concentration (EC50 = 22.8 ± 3.4 μM) in Gαs-transfected S49 cyc– cells but not in nontransfected cells. However, there was no significant difference between the Gαs-wild-type (wt) and mutants. Morphine (10 μM) inhibited AC activity more efficiently in cyc– compared to Gαs-wt introduced cells (P < 0.05); however, we did not find a notable difference between Gαs-wt and mutants. Interestingly, Gαs-wt transfected cells showed more sensitization with respect to AC after chronic morphine compared to nontransfected cells (101 ± 12% versus 34 ± 6%; P < 0.001); μ1-opioid receptor interacted with Gαs, and both co-immunoprecipitated after chronic morphine exposure. Furthermore, mutation of T270A and S272A (P < 0.01), as well as T270A, S272A and S261A (P < 0.05), in α3 ⁄ β5, resulted in a higher level of AC supersensitization. ERK1⁄ 2 phosphorylation was rapidly induced by isoproterenol (by 9.5 ± 2.4-fold) and morphine (22 ± 2.2-fold) in Gαs-transfected cells; mutations of α3 ⁄ β5 and α4 ⁄ β6 did not affect the pattern or extent of mitogen-activated protein kinase activation. The findings of the present study show that Gαs interacts with the μ1-opioid receptor, and the Ser ⁄ Thr mutation to Ala at the α3 ⁄ β5 loop of Gαs enhances morphine-induced AC sensitization. In addition, Gαs was required for

  8. Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

    Science.gov (United States)

    Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A; Brunner, Michael R; Davidson, Iain F; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

    2016-05-10

    Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

  9. R loops are linked to histone H3 S10 phosphorylation and chromatin condensation.

    Science.gov (United States)

    Castellano-Pozo, Maikel; Santos-Pereira, José M; Rondón, Ana G; Barroso, Sonia; Andújar, Eloisa; Pérez-Alegre, Mónica; García-Muse, Tatiana; Aguilera, Andrés

    2013-11-21

    R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.

  10. Structural basis for Mep2 ammonium transceptor activation by phosphorylation.

    Science.gov (United States)

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C

    2016-04-18

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

  11. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    Science.gov (United States)

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-04-27

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  12. Phosphorylation regulates coilin activity and RNA association

    Directory of Open Access Journals (Sweden)

    Hanna J. Broome

    2013-02-01

    The Cajal body (CB is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation.

  13. An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

    Science.gov (United States)

    Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D'Amelio, Nicola; Gervasio, Francesco Luigi

    2016-04-01

    Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

  14. Bypassing AMPK Phosphorylation

    OpenAIRE

    Viollet, Benoit; Foretz, Marc; Schlattner, Uwe

    2014-01-01

    AMP-activated protein kinase (AMPK) functions as a signaling hub to balance energy supply with demand. Phosphorylation of activation loop Thr172 has been considered as an essential step in AMPK activation. In this issue of Chemistry & Biology, Scott and colleagues show that the small molecule direct AMPK activator, A-769662, bypasses this phosphorylation event, and acts synergistically with AMP on naive AMPK.

  15. A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

    DEFF Research Database (Denmark)

    Frödin, Morten; Antal, Torben L; Dümmler, Bettina A;

    2002-01-01

    The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific...... docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind...... the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism...

  16. Activating PER repressor through a DBT-directed phosphorylation switch.

    Directory of Open Access Journals (Sweden)

    Saul Kivimäe

    2008-07-01

    Full Text Available Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT, a Drosophila ortholog of human casein kinase I (CKIepsilon/delta. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER. DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The per(S mutation, which is associated with short-period (19-h circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function.

  17. Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened.

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    Yongbin Chen

    2011-06-01

    Full Text Available Hedgehog (Hh signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo, but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.

  18. Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth; Bourand, Alexa; Darbon, Emmanuelle; Joly, Nathalie; Briozzo, Pierre; Deutscher, Josef; (Pitt); (CNRS-CRMD)

    2009-09-11

    Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of

  19. Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange.

    Science.gov (United States)

    Oliver, Antony W; Paul, Angela; Boxall, Katherine J; Barrie, S Elaine; Aherne, G Wynne; Garrett, Michelle D; Mittnacht, Sibylle; Pearl, Laurence H

    2006-07-12

    The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine-glutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation.

  20. Mitotic chromosome compaction via active loop extrusion

    Science.gov (United States)

    Goloborodko, Anton; Imakaev, Maxim; Marko, John; Mirny, Leonid; MIT-Northwestern Team

    During cell division, two copies of each chromosome are segregated from each other and compacted more than hundred-fold into the canonical X-shaped structures. According to earlier microscopic observations and the recent Hi-C study, chromosomes are compacted into arrays of consecutive loops of ~100 kilobases. Mechanisms that lead to formation of such loop arrays are largely unknown. Here we propose that, during cell division, chromosomes can be compacted by enzymes that extrude loops on chromatin fibers. First, we use computer simulations and analytical modeling to show that a system of loop-extruding enzymes on a chromatin fiber self-organizes into an array of consecutive dynamic loops. Second, we model the process of loop extrusion in 3D and show that, coupled with the topo II strand-passing activity, it leads to robust compaction and segregation of sister chromatids. This mechanism of chromosomal condensation and segregation does not require additional proteins or specific DNA markup and is robust against variations in the number and properties of such loop extruding enzymes. Work at NU was supported by the NSF through Grants DMR-1206868 and MCB-1022117, and by the NIH through Grants GM105847 and CA193419. Work at MIT was supported by the NIH through Grants GM114190 R01HG003143.

  1. Molecular Profiling of Activated Neurons by Phosphorylated Ribosome Capture

    OpenAIRE

    Knight, Zachary A.; Tan, Keith; Birsoy, Kivanc; Schmidt, Sarah; Garrison, Jennifer L.; Wysocki, Robert W.; Emiliano, Ana; Ekstrand, Mats I.; Friedman, Jeffrey M.

    2012-01-01

    The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, there...

  2. Hierarchical curiosity loops and active sensing.

    Science.gov (United States)

    Gordon, Goren; Ahissar, Ehud

    2012-08-01

    A curious agent acts so as to optimize its learning about itself and its environment, without external supervision. We present a model of hierarchical curiosity loops for such an autonomous active learning agent, whereby each loop selects the optimal action that maximizes the agent's learning of sensory-motor correlations. The model is based on rewarding the learner's prediction errors in an actor-critic reinforcement learning (RL) paradigm. Hierarchy is achieved by utilizing previously learned motor-sensory mapping, which enables the learning of other mappings, thus increasing the extent and diversity of knowledge and skills. We demonstrate the relevance of this architecture to active sensing using the well-studied vibrissae (whiskers) system, where rodents acquire sensory information by virtue of repeated whisker movements. We show that hierarchical curiosity loops starting from optimally learning the internal models of whisker motion and then extending to object localization result in free-air whisking and object palpation, respectively. PMID:22386787

  3. Phosphorylation of Ubc9 by Cdk1 enhances SUMOylation activity.

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    Yee-Fun Su

    Full Text Available Increasing evidence has pointed to an important role of SUMOylation in cell cycle regulation, especially for M phase. In the current studies, we have obtained evidence through in vitro studies that the master M phase regulator CDK1/cyclin B kinase phosphorylates the SUMOylation machinery component Ubc9, leading to its enhanced SUMOylation activity. First, we show that CDK1/cyclin B, but not many other cell cycle kinases such as CDK2/cyclin E, ERK1, ERK2, PKA and JNK2/SAPK1, specifically enhances SUMOylation activity. Second, CDK1/cyclin B phosphorylates the SUMOylation machinery component Ubc9, but not SAE1/SAE2 or SUMO1. Third, CDK1/cyclin B-phosphorylated Ubc9 exhibits increased SUMOylation activity and elevated accumulation of the Ubc9-SUMO1 thioester conjugate. Fourth, CDK1/cyclin B enhances SUMOylation activity through phosphorylation of Ubc9 at serine 71. These studies demonstrate for the first time that the cell cycle-specific kinase CDK1/cyclin B phosphorylates a SUMOylation machinery component to increase its overall SUMOylation activity, suggesting that SUMOylation is part of the cell cycle program orchestrated by CDK1 through Ubc9.

  4. Raf activation is regulated by tyrosine 510 phosphorylation in Drosophila.

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    Fan Xia

    2008-05-01

    Full Text Available The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf, which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved.

  5. Activation of purified calcium channels by stoichiometric protein phosphorylation

    International Nuclear Information System (INIS)

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45Ca2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45Ca2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd2+, Ni2+, and Mg2+. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels

  6. A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filaments

    OpenAIRE

    Brito, Reicy; Alamo, Lorenzo; Lundberg, Ulf; Guerrero, José R.; Pinto, Antonio; Sulbarán, Guidenn; Gawinowicz, Mary Ann; Craig, Roger; Padrón, Raúl

    2011-01-01

    Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high resolution structure is known. In the relaxed state, tarantula RLCs are ~50% non- and 50% mono-phosphorylated, while on activation mono-phosphorylation increases and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphor...

  7. Molecular profiling of activated neurons by phosphorylated ribosome capture.

    Science.gov (United States)

    Knight, Zachary A; Tan, Keith; Birsoy, Kivanc; Schmidt, Sarah; Garrison, Jennifer L; Wysocki, Robert W; Emiliano, Ana; Ekstrand, Mats I; Friedman, Jeffrey M

    2012-11-21

    The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue. PMID:23178128

  8. 90-kDa ribosomal S6 kinase is phosphorylated and activated by 3-phosphoinositide-dependent protein kinase-1

    DEFF Research Database (Denmark)

    Jensen, Claus Antonio Juel; Buch, M B; Krag, T O;

    1999-01-01

    90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of th...... of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.......90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation...... of the linker region at Ser(369), catalyzed by extracellular signal-regulated kinase (ERK), and at Ser(386), catalyzed by the C-terminal kinase, after its activation by ERK. In addition, the N-terminal kinase must be phosphorylated at Ser(227) in the activation loop by an as yet unidentified kinase. Here, we...

  9. Time-resolved in silico modeling of fine-tuned cAMP signaling in platelets: feedback loops, titrated phosphorylations and pharmacological modulation

    Directory of Open Access Journals (Sweden)

    Dandekar Thomas

    2011-10-01

    . Downstream cyclic nucleotide signaling events target different phosphorylation sites for cAMP- and cGMP-dependent protein kinases (PKA, PKG in the vasodilator-stimulated phosphoprotein (VASP. VASP phosphorylation as well as cAMP levels resulting from different drug strengths and combined stimulants were quantitatively modeled. These predictions were again experimentally validated. High sensitivity of the signaling pathway at low concentrations is involved in a fine-tuned balance as well as stable activation of this inhibitory cyclic nucleotide pathway. Conclusions On the basis of experimental data, literature mining and database screening we established a dynamic in silico model of cyclic nucleotide signaling and probed its signaling sensitivity. Thoroughly validated, it successfully predicts drug combination effects on platelet function, including synergism, antagonism and regulatory loops.

  10. Cooling Active Region Loops Observed With SXT and TRACE

    CERN Document Server

    Winebarger, A R; Winebarger, Amy R.; Warren, Harry P.

    2005-01-01

    An Impulsive Heating Multiple Strand (IHMS) Model is able to reproduce the observational characteristics of EUV (~ 1 MK) active region loops. This model implies that some of the loops must reach temperatures where X-ray filters are sensitive (> 2.5 MK) before they cool to EUV temperatures. Hence, some bright EUV loops must be preceded by bright X-ray loops. Previous analysis of X-ray and EUV active region observations, however, have concluded that EUV loops are not preceded by X-ray loops. In this paper, we examine two active regions observed in both X-ray and EUV filters and analyze the evolution of five loops over several hours. These loops first appear bright in the X-ray images and later appear bright in the EUV images. The delay between the appearance of the loops in the X-ray and EUV filters is as little as 1 hour and as much as 3 hours. All five loops appear as single ``monolithic'' structures in the X-ray images, but are resolved into many smaller structures in the (higher resolution) EUV images. The ...

  11. The structure of a dual-specificity tyrosine phosphorylation-regulated kinase 1A-PKC412 complex reveals disulfide-bridge formation with the anomalous catalytic loop HRD(HCD) cysteine.

    Science.gov (United States)

    Alexeeva, Marina; Åberg, Espen; Engh, Richard A; Rothweiler, Ulli

    2015-05-01

    Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase associated with neuronal development and brain physiology. The DYRK kinases are very unusual with respect to the sequence of the catalytic loop, in which the otherwise highly conserved arginine of the HRD motif is replaced by a cysteine. This replacement, along with the proximity of a potential disulfide-bridge partner from the activation segment, implies a potential for redox control of DYRK family activities. Here, the crystal structure of DYRK1A bound to PKC412 is reported, showing the formation of the disulfide bridge and associated conformational changes of the activation loop. The DYRK kinases represent emerging drug targets for several neurological diseases as well as cancer. The observation of distinct activation states may impact strategies for drug targeting. In addition, the characterization of PKC412 binding offers new insights for DYRK inhibitor discovery. PMID:25945585

  12. Effect of phosphorylation on antioxidant activities of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide.

    Science.gov (United States)

    Song, Yi; Ni, Yuanying; Hu, Xiaosong; Li, Quanhong

    2015-11-01

    Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system. PMID:26231331

  13. Cell cycle regulation by feed-forward loops coupling transcription and phosphorylation

    DEFF Research Database (Denmark)

    Csikász-Nagy, Attila; Kapuy, Orsolya; Tóth, Attila;

    2009-01-01

    ) from Cdk1. By mathematical modelling, we show that such FFLs can activate EPs at different phases of the cell cycle depending of the effective signs (+ or -) of the regulatory steps of the FFL. We provide several case studies of EPs that are controlled by FFLs exactly as our models predict. The signal......-transduction properties of FFLs allow one (or a few) Cdk signal(s) to drive a host of cell cycle responses in correct temporal sequence.......The eukaryotic cell cycle requires precise temporal coordination of the activities of hundreds of 'executor' proteins (EPs) involved in cell growth and division. Cyclin-dependent protein kinases (Cdks) play central roles in regulating the production, activation, inactivation and destruction...

  14. Altered myoelectric activity in the experimental blind loop syndrome.

    Science.gov (United States)

    Justus, P G; Fernandez, A; Martin, J L; King, C E; Toskes, P P; Mathias, J R

    1983-09-01

    Nutrient malabsorption and diarrhea are characteristic of the blind loop syndrome. Alterations in motility have been implicated as a cause of bacterial overgrowth, but the possibility that altered motility may result from alterations in the flora has not been explored. The purpose of this study was to characterize the myoelectric activity of the small intestine in the blind loop rat model. Eight groups of rats were studied: rats with self-filling blind loops, which develop bacterial overgrowth; rats with self-emptying blind loops, which are surgical controls that do not develop overgrowth; unoperated litter mates; rats with self-filling blind loops and unoperated controls treated with chloramphenicol, 200 mg/d i.p.; rats with surgically removed self-filling blind loops; operated control rats; and gnotobiotic rats with self-filling blind loops. In the untreated rats with self-filling blind loops, there was altered myoelectric activity characterized by an increased percentage of slow waves occupied by action potentials and by organized activity similar to the migrating action potential complex. Migrating action potential complex activity and percentage of slow waves occupied by action potentials were significantly decreased with chloramphenicol therapy; that decrease correlated with a decrease in aerobes and anaerobes. Migrating action potential complex activity was abolished in rats with surgically removed self-filling blind loops; they also showed a significant decrease in percentage of slow waves occupied by action potentials. Gnotobiotic rats with self-filling blind loops showed no alteration in myoelectric activity. These data indicate: (a) bacterial overgrowth is associated with a significant increase in percentage of slow waves occupied by action potentials and migrating action potential complex activity; (b) chloramphenicol significantly reduced both percentage of slow waves occupied by action potentials and migrating action potential complex activity; and (c

  15. CXCL3 contributes to CD133+ CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

    Science.gov (United States)

    Zhang, Lin; Zhang, Lixing; Li, Hong; Ge, Chao; Zhao, Fangyu; Tian, Hua; Chen, Taoyang; Jiang, Guoping; Xie, Haiyang; Cui, Ying; Yao, Ming; Li, Jinjun

    2016-01-01

    Although the chemotactic cytokine CXCL3 is thought to play an important role in tumor initiation and invasion, little is known about its function in hepatocellular carcinoma (HCC). In our previous study, we found that Ikaros inhibited CD133 expression via the MAPK pathway in HCC. Here, we showed that Ikaros may indirectly down-regulate CXCL3 expression in HCC cells, which leads to better outcomes in patients with CD133+ cancer stem cell (CSC) populations. CD133 overexpression induced CXCL3 expression, and silencing of CD133 down-regulated CXCL3 in HCC cells. Knockdown of CXCL3 inhibited CD133+ HCC CSCs’ self-renewal and tumorigenesis. The serum CXCL3 level was higher in HCC patients’ samples than that in healthy individual. HCC patients with higher CXCL3 expression displayed a poor prognosis, and a high level of CXCL3 was significantly associated with vascular invasion and tumor capsule formation. Exogenous CXCL3 induced Erk1/2 and ETS1 phosphorylation and promoted CD133 expression, indicating a positive feedback loop between CXCL3 and CD133 gene expression in HCC cells via Erk1/2 activation. Together, our findings indicated that CXCL3 might be a potent therapeutic target for HCC. PMID:27255419

  16. The double-loop feedback for active learning with understanding

    DEFF Research Database (Denmark)

    Christensen, Hans Peter

    2004-01-01

    Learning is an active process, and in engineering education authentic projects is often used to activate the students and promote learning. However, it is not all activity that leads to deep learning; and in a rapid changing society deep understanding is necessary for life-long learning. Empirical...... findings at DTU question the direct link between high activity and a deep approach to learning. Active learning is important to obtain engineering competencies, but active learning requires more than activity. Feedback and reflection is crucial to the learning process, since new knowledge is built...... on the student’s existing understanding. A model for an active learning process with a double-loop feedback is suggested - the first loop gives the student experience through experimentation, the second conceptual understanding through reflection. Students often miss the second loop, so it is important...

  17. Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity.

    Science.gov (United States)

    Huguenin-Dezot, Nicolas; De Cesare, Virginia; Peltier, Julien; Knebel, Axel; Kristaryianto, Yosua Adi; Rogerson, Daniel T; Kulathu, Yogesh; Trost, Matthias; Chin, Jason W

    2016-07-26

    Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages. PMID:27425610

  18. Sequential Myosin Phosphorylation Activates Tarantula Thick Filament via a Disorder-Order Transition

    OpenAIRE

    Espinoza-Fonseca, L Michel; Alamo, Lorenzo; Pinto, Antonio; Thomas, David D.; Padrón, Raúl

    2015-01-01

    Phosphorylation of myosin regulatory light chain (RLC) N-terminal extension (NTE) activates myosin in thick filaments. RLC phosphorylation plays a primary regulatory role in smooth muscle and a secondary (modulatory) role in striated muscle, which is regulated by Ca2+ via TnC/TM on the thin filament. Tarantula striated muscle exhibits both regulatory systems: one switches on/off contraction through thin filament regulation, and another through PKC constitutively Ser35 phosphorylated swaying f...

  19. Two putative protein kinase CK2 phosphorylation sites are important for Myf-5 activity

    DEFF Research Database (Denmark)

    Winter, B; Kautzner, I; Issinger, O G;

    1997-01-01

    Myf-5, a member of a family of muscle-specific transcription factors, is important for myogenic cell determination and differentiation. Here, we report that Myf-5 protein constitutes a substrate for phosphorylation in vitro by protein kinase CK2. We identified two potential phosphorylation sites...... localization and/or protein stability. Our data suggest that CK2-mediated phosphorylation of Myf-5 is required for Myf-5 activity....

  20. Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5.

    Science.gov (United States)

    Matusiak, Magdalena; Van Opdenbosch, Nina; Vande Walle, Lieselotte; Sirard, Jean-Claude; Kanneganti, Thirumala-Devi; Lamkanfi, Mohamed

    2015-02-01

    The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1β secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1β secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5. PMID:25605939

  1. Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

    DEFF Research Database (Denmark)

    Bañuelos, Sonia; Omaetxebarria, Miren J; Ramos, Isbaal;

    2007-01-01

    Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified...

  2. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    Science.gov (United States)

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  3. Phosphorylation of Serine422 increases the stability and transactivation activities of human Osterix

    DEFF Research Database (Denmark)

    Xu, Yuexin; Yao, Bing; Shi, Kaikai;

    2015-01-01

    Osterix (Osx) is an essential regulator for osteoblast differentiation and bone formation. Although phosphorylation has been reported to be involved in the regulation of Osx activity, the precise underlying mechanisms remain to be elucidated. Here we identified S422 as a novel phosphorylation sit...

  4. AMP-activated protein kinase phosphorylation in brain is dependent on method of sacrifice and tissue preparation

    OpenAIRE

    Scharf, Matthew T.; Mackiewicz, Miroslaw; Naidoo, Nirinjini; O'Callaghan, James P.; Pack, Allan I.

    2007-01-01

    AMP-activated protein kinase is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α-AMP-activated protein kinase phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMP-activated protein kinase phosphorylation in the mouse brain, we compared different methods of sacrifice and tissue preparation. We found that fre...

  5. IN VITRO ANALYSIS OF τ PHOSPHORYLATION SITES AND ITS BIOLOGICAL ACTIVITY

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective.To explore the association between the abnormal phosphorylation sites found in Alzheimer disease (AD) τ and the inhibition of its biological activity. Methods.Ultracentrifugation,chromatography,manual Edman degradation and autosequence techniques were used to prepare and phosphorylate human recombinant τ ,isolate and purify 32P τ peptides and determine phosphorylation sites. Results.Phosphorylation of τ by casein kinase 1 (CK 1),cyclic AMP dependent protein kinase (PKA) and glycogen synthetase kinase 3 (GSK 3) separately inhibited its biological activity and the inhibition of this activity by GSK 3 was significantly increased if τ was prephosphorylated by CK 1 or PKA.The most potent inhibition was seen by a combined phosphorylation of τ with PKA and GSK 3.The treatment of τ by PKA and GSK 3 combination induced phosphorylation of τ at Ser 195,Ser 198,Ser 199,Ser 202,Thr 205,Thr 231,Ser 235,Ser 262,Ser 356,Ser 404,whereas Thr 181,Ser 184,Ser 262,Ser 356 and Ser 400 were phosphorylated by GSK 3 alone under the same condition. Conclusion.Phosphorylation of τ by PKA plus GSK 3 at Thr 205 might play a key role in τ pathology in AD.

  6. Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity.

    Science.gov (United States)

    Kathum, Omer Abid; Schräder, Tobias; Anhlan, Darisuren; Nordhoff, Carolin; Liedmann, Swantje; Pande, Amit; Mellmann, Alexander; Ehrhardt, Christina; Wixler, Viktor; Ludwig, Stephan

    2016-06-01

    Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed. PMID:26687707

  7. Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation.

    Science.gov (United States)

    Chen, Shaoyong; Gulla, Sarah; Cai, Changmeng; Balk, Steven P

    2012-03-01

    Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

  8. Allosteric activation mechanism of the cys-loop receptors

    Institute of Scientific and Technical Information of China (English)

    Yong-chang CHANG; Wen WU; Jian-liang ZHANG; Yao HUANG

    2009-01-01

    Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of amino-terminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

  9. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    Science.gov (United States)

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators. PMID:27226582

  10. NOMAGE4 activities 2011, Part II, Supercritical water loop

    DEFF Research Database (Denmark)

    Vierstraete, Pierre; Van Nieuwenhove, Rudi; Lauritzen, Bent

    The supercritical water reactor (SCWR) is one of the six different reactor technologies selected for research and development under the Generation IV program. Several countries have shown interest to this concept but up to now, there exist no in-pile facilities to perform the required material...... and fuel tests. Working on this direction, the Halden Reactor Project has started an activity in collaboration with Risoe-DTU (with Mr. Rudi Van Nieuwenhove as the project leader) to study the feasibility of a SCW loop in the Halden Reactor, which is a Heavy Boiling Water Reactor (HBWR). The ultimate goal...... of the project is to design a loop allowing material and fuel test studies at significant mass flow with in-core instrumentation and chemistry control possibilities. The present report focusses on the main heat exchanger required for such a loop in the Halden Reactor. The goal of this heat exchanger is to assure...

  11. Sequential myosin phosphorylation activates tarantula thick filament via a disorder-order transition.

    Science.gov (United States)

    Espinoza-Fonseca, L Michel; Alamo, Lorenzo; Pinto, Antonio; Thomas, David D; Padrón, Raúl

    2015-08-01

    Phosphorylation of myosin regulatory light chain (RLC) N-terminal extension (NTE) activates myosin in thick filaments. RLC phosphorylation plays a primary regulatory role in smooth muscles and a secondary (modulatory) role in striated muscles, which is regulated by Ca(2+)via TnC/TM on the thin filament. Tarantula striated muscle exhibits both regulatory systems: one switches on/off contraction through thin filament regulation, and another through PKC constitutively Ser35 phosphorylated swaying free heads in the thick filaments that produces quick force on twitches regulated from 0 to 50% and modulation is accomplished recruiting additional force-potentiating free and blocked heads via Ca(2+)4-CaM-MLCK Ser45 phosphorylation. We have used microsecond molecular dynamics (MD) simulations of tarantula RLC NTE to understand the structural basis for phosphorylation-based regulation in tarantula thick filament activation. Trajectory analysis revealed that an inter-domain salt bridge network (R39/E58,E61) facilitates the formation of a stable helix-coil-helix (HCH) motif formed by helices P and A in the unphosphorylated NTE of both myosin heads. Phosphorylation of the blocked head on Ser45 does not induce any substantial structural changes. However, phosphorylation of the free head on Ser35 disrupts this salt bridge network and induces a partial extension of helix P along RLC helix A. While not directly participating in the HCH folding, phosphorylation of Ser35 unlocks a compact structure and allows the NTE to spontaneously undergo coil-helix transitions. The modest structural change induced by the subsequent Ser45 diphosphorylation monophosphorylated Ser35 free head facilitates full helix P extension into a single structurally stable α-helix through a network of intra-domain salt bridges (pS35/R38,R39,R42). We conclude that tarantula thick filament activation is controlled by sequential Ser35-Ser45 phosphorylation via a conserved disorder-to-order transition. PMID

  12. Phosphorylation by PKA potentiates retinoic acid receptor α activity by means of increasing interaction with and phosphorylation by cyclin H/cdk7

    OpenAIRE

    Gaillard, Emilie; Bruck, Nathalie; Brelivet, Yann; Bour, Gaétan; Lalevée, Sébastien; Bauer, Annie; Poch, Olivier; Moras, Dino; Rochette-Egly, Cécile

    2006-01-01

    Nuclear retinoic acid receptors (RARs) work as ligand-dependent heterodimeric RAR/retinoid X receptor transcription activators, which are targets for phosphorylations. The N-terminal activation function (AF)-1 domain of RARα is phosphorylated by the cyclin-dependent kinase (cdk) 7/cyclin H complex of the general transcription factor TFIIH and the C-terminal AF-2 domain by the cAMP-dependent protein kinase A (PKA). Here, we report the identification of a molecular pathway by which phosphorylat...

  13. Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Amaya Blanco-Rivero

    Full Text Available BACKGROUND: Cah3 is the only carbonic anhydrase (CA isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO(2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO(2 conditions. RESULTS/CONCLUSIONS: In the present work we demonstrate that after transfer to low CO(2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO(2 conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO(2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO(2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO(2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. SIGNIFICANCE: This is the first report of a CA being post

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

    Science.gov (United States)

    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.

  15. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    Science.gov (United States)

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  16. NOMAGE4 activities 2011. Part II, Supercritical water loop

    Energy Technology Data Exchange (ETDEWEB)

    Vierstraete, P. (Ecole Nationale Superieure des mines, Paris (France)); Van Nieuwenhove, R. (Institutt for Energiteknikk, OECD Halden Reactor Project (HRP), Kjeller (Norway)); Lauritzen, B. (Technical Univ. of Denmark, Risoe National Lab. for Sustainable Energy, Roskilde (Denmark))

    2012-01-15

    The supercritical water reactor (SCWR) is one of the six different reactor technologies selected for research and development under the Generation IV program. Several countries have shown interest to this concept but up to now, there exist no in-pile facilities to perform the required material and fuel tests. Working on this direction, the Halden Reactor Project has started an activity in collaboration with Risoe-DTU (with Mr. Rudi Van Nieuwenhove as the project leader) to study the feasibility of a SCW loop in the Halden Reactor, which is a Heavy Boiling Water Reactor (HBWR). The ultimate goal of the project is to design a loop allowing material and fuel test studies at significant mass flow with in-core instrumentation and chemistry control possibilities. The present report focusses on the main heat exchanger required for such a loop in the Halden Reactor. The goal of this heat exchanger is to assure a supercritical flow state inside the test section (the core side) and a subcritical flow state inside the pump section. The objective is to design the heat exchanger in order to optimize the efficiency of the heat transfer and to respect several requirements as the room available inside the reactor hall, the maximal total pressure drop allowed and so on. (Author)

  17. Kin28 Is Found within TFIIH and a Kin28-Ccl1-Tfb3 Trimer Complex with Differential Sensitivities to T-Loop Phosphorylation

    OpenAIRE

    Keogh, Michael-Christopher; Cho, Eun-Jung; Podolny, Vladimir; Buratowski, Stephen

    2002-01-01

    Basal transcription factor TFIIH phosphorylates the RNA polymerase II (RNApII) carboxy-terminal domain (CTD) within the transcription initiation complex. The catalytic kinase subunit of TFIIH is a member of the cyclin-dependent kinase (Cdk) family, designated Kin28 in Saccharomyces cerevisiae and Cdk7 in higher eukaryotes. Together with TFIIH subunits cyclin H and Mat1, Cdk7 kinase is also found in a trimer complex known as Cdk activating kinase (CAK). A yeast trimer complex has not previousl...

  18. Structural basis of how stress-induced MDMX phosphorylation activates p53.

    Science.gov (United States)

    Chen, X; Gohain, N; Zhan, C; Lu, W-Y; Pazgier, M; Lu, W

    2016-04-14

    The tumor-suppressor protein p53 is tightly controlled in normal cells by its two negative regulators--the E3 ubiquitin ligase MDM2 and its homolog MDMX. Under stressed conditions such as DNA damage, p53 escapes MDM2- and MDMX-mediated functional inhibition and degradation, acting to prevent damaged cells from proliferating through induction of cell cycle arrest, DNA repair, senescence or apoptosis. Ample evidence suggests that stress signals induce phosphorylation of MDM2 and MDMX, leading to p53 activation. However, the structural basis of stress-induced p53 activation remains poorly understood because of the paucity of technical means to produce site-specifically phosphorylated MDM2 and MDMX proteins for biochemical and biophysical studies. Herein, we report total chemical synthesis, via native chemical ligation, and functional characterization of (24-108)MDMX and its Tyr99-phosphorylated analog with respect to their ability to interact with a panel of p53-derived peptide ligands and PMI, a p53-mimicking but more potent peptide antagonist of MDMX, using FP and surface plasmon resonance techniques. Phosphorylation of MDMX at Tyr99 weakens peptide binding by approximately two orders of magnitude. Comparative X-ray crystallographic analyses of MDMX and of pTyr99 MDMX in complex with PMI as well as modeling studies reveal that the phosphate group of pTyr99 imposes extensive steric clashes with the C-terminus of PMI or p53 peptide and induces a significant lateral shift of the peptide ligand, contributing to the dramatic decrease in the binding affinity of MDMX for p53. Because DNA damage activates c-Abl tyrosine kinase that phosphorylates MDMX at Tyr99, our findings afford a rare glimpse at the structural level of how stress-induced MDMX phosphorylation dislodges p53 from the inhibitory complex and activates it in response to DNA damage. PMID:26148237

  19. Carboxyl terminal of rhodopsin kinase is required for the phosphorylation of photo—activated rhodopsin

    Institute of Scientific and Technical Information of China (English)

    YUQINGMING; LANMA; 等

    1998-01-01

    Human rhodopsin kinase (RK) and a carboxyl terminus-truncated mutant RK lacking the last 59 amino acids (RKC) were expressed in human embryonic kidney 293 cells to investigate the role of the carboxyl terminus of RK in recognition and phosphorylation of rhodopsin.RKC,like the wild-type RK,was detected in both plasma membranes and cytosolic fractions.The Cterminal truncated rhodopsin kinase was unable to phosphorylate photo-activated rhodopsin,but possesses kinase activity similar to the wild-type RK in phosphorylation of small peptide substrate.It suggests that the truncation did not disturb the gross structures of RK catalytic domain.Our results also show that RKC failed to translocate to photo-activated rod out segments.Taken together,our study demonstrate the carboxyl terminus of RK is required for phosphorylation of photo-activated rhodopsin and strongly indicate that carboxyl-terminus of RK may be involved in interaction with photo-activated rhodopsin.

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

    Science.gov (United States)

    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

  1. Studies on Synthesis and Antitumor Activity of Phosphorylated Achyranthes bidentata Polysaccharide (P-AbPS)

    Institute of Scientific and Technical Information of China (English)

    CHEN,Xiao-Ming(陈晓明); ZHANG,Jian(张健); TIAN,Geng-Yuan(田庚元)

    2002-01-01

    The synthesis of phosphorylated Achyranthes bidentata polysaccharide (P-AbPS) was reported based on different strategies.The P-AbPS with high degree of substitution (D.S. >0.5) was obtained when phosphorus oxychloride (POCl3) was used as a phosphorylating agent and trimethyl phosphate-pyridine or dimethyl formamide was used as solvent. The influences of different solvents and reaction conditins were discussed. Thepharmacology assay shows that P-AbPS possesses antitumor activity against sarcoma 180 and Lewis lung carner in mice.

  2. Dehydration stress activates Arabidopsis MPK6 to signal DCP1 phosphorylation

    OpenAIRE

    Xu, Jun; Chua, Nam-Hai

    2012-01-01

    The pathways regulating mRNA stability in response to environment stress in plants are not well understood. The stress responsive MAPK, MPK6, phosphorylates the mRNA decapping enzyme DCP1 to enhance its activity and regulate mRNA stability.

  3. AGC kinases regulate phosphorylation and activation of eukaryotic translation initiation factor 4B

    NARCIS (Netherlands)

    van Gorp, A. G. M.; van der Vos, K. E.; Brenkman, A. B.; Bremer, A.; van den Broek, N.; Zwartkruis, F.; Hershey, J. W.; Burgering, B. M. T.; Calkhoven, C. F.; Coffer, P. J.

    2009-01-01

    Eukaryotic translation initiation factor 4B (eIF4B) plays a critical role during the initiation of protein synthesis and its activity can be regulated by multiple phosphorylation events. In a search for novel protein kinase B (PKB/c-akt) substrates, we identified eIF4B as a potential target. Using a

  4. Talin1 phosphorylation activates β1 integrins: a novel mechanism to promote prostate cancer bone metastasis.

    Science.gov (United States)

    Jin, J-K; Tien, P-C; Cheng, C-J; Song, J H; Huang, C; Lin, S-H; Gallick, G E

    2015-04-01

    Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that β1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how β1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of β1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in β1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of β1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1(S425D) led to increased β1 integrin activation and generated biologic effects opposite to talin1(S425A) expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1(S425D) restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent β1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading

  5. Protein kinase C {alpha} activity is important for contraction-induced FXYD1 phosphorylation in skeletal muscle

    DEFF Research Database (Denmark)

    Thomassen, Martin; Rose, Adam John; Jensen, Thomas Elbenhardt;

    2011-01-01

    Exercise induced phosphorylation of FXYD1 is a potential important regulator of Na(+), K(+) pump activity. It was investigated if skeletal muscle contractions induce phosphorylation of FXYD1 and if Protein Kinase C a (PKCa) activity is a prerequisite for this possible mechanism. In part 1, human...

  6. THE CHARACTERIZATION OF MYOSIN LIGHT CHAIN PHOSPHORYLATION BY THE CONSTITUTIVELY ACTIVE FRAGMENT OF MLCK

    Institute of Scientific and Technical Information of China (English)

    杨静娴; 王晓明; 唐泽耀; 陈华; 戴淑芳; 林原

    2003-01-01

    Objective. To study the exact effects and characteristics of a constitutively active myosin light chain kinase fragment (MLCKF) on Ca2+-CaM dependent phosphorylation of myosin light chains (CDPM) of smooth muscle. Methods. Proteolysis of myosin light chain kinase (MLCK) by trypsin was used to obtain the constitutively active fragment of MLCK; Western blot was applied to demonstrate the homogeneity of typsin-digested MLCK fragments and intact MLCK. The phosphorylation of myosin light chains was detected using SDS-PAGE and Scoin Image Software, and myosin Mg2+-ATPase activities were measured using spectrophotometry to observe the characteristics of MLCKF in phosphorylating myosin light chains and stimulating myosin Mg2+-ATPase activities compared with intact MLCK. Results. Our assay demonstrated that MLCKF had a specific activity on CDPM. We also found that the extent of CDPM by MLCKF was greater than that by intact MLCK at different concentrations, different incubation time and different incubation temperatures. CDPM by MLCKF was also less influenced by the change of ionic strength of KC1 than CDPM by intact MLCK. Mg2+--ATPase activities of phosphorylat- ed myosin light chains by MLCKF were higher than those by MLCK at different concentrations and different reaction time. These differences were statistically significant (**P<0.01, *P<0.05). Conclusion. MLCKF not only possessed a specific activity on CDPM but also was more efficient than MLCK in phosphorylating myosin light chains and stimulating myosin Mg2*-ATPase activities. Our results suggested that MLCKF possibly plays a certain role in smooth muscle contraction-relaxation cycle.

  7. The temperature structure and pressure balance of magnetic loops in active regions. [in solar atmosphere

    Science.gov (United States)

    Foukal, P.

    1975-01-01

    EUV observations show many active region loops in lines formed at temperatures between 10,000 and 2,000,000 K. The brightest loops are associated with flux tubes leading to the umbrae of sunspots. It is shown that the high visibility of certain loops in transition region lines is due principally to a sharp radial decrease of temperature to chromospheric values toward the loop axis. The plasma density of these cool loops is not significantly greater than in the hot gas immediately surrounding it. Consequently, the internal gas pressure of the cool material is clearly lower. The hot material immediately surrounding the cool loops is generally denser than the external corona by a factor 3-4. When the active region is examined in coronal lines, this hot high pressure plasma shows up as loops that are generally parallel to the cool loops but significantly displaced laterally.

  8. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    Science.gov (United States)

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

  9. Determination of the starch-phosphorylating enzyme activity in plant extracts

    DEFF Research Database (Denmark)

    Ritte, G.; Steup, M.; Kossmann, J.;

    2003-01-01

    For quantification of alpha-glucan, water dikinase(GWD) activity in crude extracts of plant tissues a radio-labeling assay was established that uses soluble starch and P-33-labeled ATP as phosphate acceptor and donor, respectively. A constant rate of starch labeling was observed only if the ATP...... applied was labeled at the beta position. In wild-type extracts from leaves of Arabidopsis thaliana (L.) Heynh. the maximum rate of starch phosphorylation was approximately 27 pmol min(-1) (mg protein)(-1). Leaf extracts from the GWD-deficient sex1 mutants of Arabidopsis showed no significant...... incorporation of phosphate whereas extracts from potato (Solanum tuberosum L.) tuber expressing a GWD antisense construct exhibited less activity than the wild-type control. To our knowledge this is the first time that a quantification of the starch-phosphorylating activity has been achieved in plant crude...

  10. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis

    Science.gov (United States)

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKYY115E phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  11. Functional characterization of the kinase activation loop in nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) using tandem affinity purification and liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of >or=1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.

  12. PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2.

    Science.gov (United States)

    Hanafusa, Hiroshi; Kedashiro, Shin; Tezuka, Motohiro; Funatsu, Motoki; Usami, Satoshi; Toyoshima, Fumiko; Matsumoto, Kunihiro

    2015-08-01

    Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation. PMID:26192437

  13. Tau phosphorylation at Alzheimer's disease-related Ser356 contributes to tau stabilization when PAR-1/MARK activity is elevated.

    Science.gov (United States)

    Ando, Kanae; Oka, Mikiko; Ohtake, Yosuke; Hayashishita, Motoki; Shimizu, Sawako; Hisanaga, Shin-Ichi; Iijima, Koichi M

    2016-09-16

    Abnormal phosphorylation of the microtubule-associated protein tau is observed in many neurodegenerative diseases, including Alzheimer's disease (AD). AD-related phosphorylation of two tau residues, Ser262 and Ser356, by PAR-1/MARK stabilizes tau in the initial phase of mismetabolism, leading to subsequent phosphorylation events, accumulation, and toxicity. However, the relative contribution of phosphorylation at each of these sites to tau stabilization has not yet been elucidated. In a Drosophila model of human tau toxicity, we found that tau was phosphorylated at Ser262, but not at Ser356, and that blocking Ser262 phosphorylation decreased total tau levels. By contrast, when PAR-1 was co-overexpressed with tau, tau was hyperphosphorylated at both Ser262 and Ser356. Under these conditions, the protein levels of tau were significantly elevated, and prevention of tau phosphorylation at both residues was necessary to completely suppress this elevation. These results suggest that tau phosphorylation at Ser262 plays the predominant role in tau stabilization when PAR-1/MARK activity is normal, whereas Ser356 phosphorylation begins to contribute to this process when PAR-1/MARK activity is abnormally elevated, as in diseased brains. PMID:27520376

  14. Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

    Science.gov (United States)

    Zeymer, Cathleen; Werbeck, Nicolas D; Zimmermann, Sabine; Reinstein, Jochen; Hansen, D Flemming

    2016-09-12

    States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions. PMID:27534930

  15. MHC class I signaling in T cells leads to tyrosine kinase activity and PLC-gamma 1 phosphorylation

    DEFF Research Database (Denmark)

    Skov, S; Odum, Niels; Claesson, M H

    1995-01-01

    phosphorylation and the subsequent calcium response. The early tyrosine kinase activity was found to be dependent on expression of the TCR/CD3 complex and the CD45 molecule on the surface of the T cells. Furthermore, MHC-I cross-linking was shown to tyrosine phosphorylate PLC-gamma 1 (phospholipase C-gamma 1...

  16. Polycomb Group Protein Displacement and Gene Activation through MSK-Dependent H3K27me3S28 Phosphorylation

    DEFF Research Database (Denmark)

    Gehani, Simmi Suman; Agrawal-Singh, Shuchi; Dietrich, Nikolaj;

    2010-01-01

    phosphorylation, and displacement of PcG proteins, lead to gene activation. We present evidence that the H3K27me3S28 phosphorylation is functioning in response to stress signaling, mitogenic signaling, and retinoic acid (RA)-induced neuronal differentiation. We propose that MSK-mediated H3K27me3S28...

  17. Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

    Science.gov (United States)

    Callender, Tracy L; Laureau, Raphaelle; Wan, Lihong; Chen, Xiangyu; Sandhu, Rima; Laljee, Saif; Zhou, Sai; Suhandynata, Ray T; Prugar, Evelyn; Gaines, William A; Kwon, YoungHo; Börner, G Valentin; Nicolas, Alain; Neiman, Aaron M; Hollingsworth, Nancy M

    2016-08-01

    During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1. PMID:27483004

  18. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase

    Directory of Open Access Journals (Sweden)

    Ly-Thuy-Tram Le

    2013-02-01

    Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

  19. Protein kinase D regulates RhoA activity via rhotekin phosphorylation.

    Science.gov (United States)

    Pusapati, Ganesh V; Eiseler, Tim; Rykx, An; Vandoninck, Sandy; Derua, Rita; Waelkens, Etienne; Van Lint, Johan; von Wichert, Götz; Seufferlein, Thomas

    2012-03-16

    The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin. PMID:22228765

  20. EMT phenotype is induced by increased Src kinase activity via Src-mediated caspase-8 phosphorylation.

    Science.gov (United States)

    Zhao, Yang; Li, XiaoJun; Sun, XiangFei; Zhang, YunFeng; Ren, Hong

    2012-01-01

    Caspase-8 governs multiple cell responses to the microenvironmental cues. However, its integration of "death-life" signalings remains elusive. In our study, the role of caspase-8-Src is well-established as a promoter for migration or metastasis in Casp8(+)Src(+) A549/H226 cells in vivo and in vitro. In particular for nude mice models, mice implanted with Casp8(+)Src(+) A459/H226 cells remarkably increased spontaneous tumor metastatic burden with a significant survival disadvantage. Additionally, we detect that Src-mediated caspase-8 phosphorylation stimulates Src phosphorylation at Tyr-416 via the linkage of Src SH2 domain with phosph-Tyr-380 site of caspase-8. In turn, activated Src can efficiently induce epithelial-mesenchymal transition (EMT) phenotypic features to promote tumor cells metastasis. Surprisingly, RXDLL motif deletion in the DEDa of caspase-8 attenuates tumor cell migration or metastasis via impairing the recruitment of caspase-8 into the cellular periphery where activated Src is subject to caspase-8 phosphorylation. Together, a simple model is that the peripherization of caspase-8 is well-poised to facilitate Src-mediated caspase-8 phosphrylation at Tyr-380, then binding of phospho-Tyr380 of caspase-8 to Src SH2 domain may maintain Src in an active conformation to induce EMT phenotype, a key step toward cancer metastasis. PMID:22508042

  1. Masoprocol decreases rat lipolytic activity by decreasing the phosphorylation of HSL.

    Science.gov (United States)

    Gowri, M S; Azhar, R K; Kraemer, F B; Reaven, G M; Azhar, S

    2000-09-01

    Masoprocol (nordihydroguaiaretic acid), a lipoxygenase inhibitor isolated from the creosote bush, has been shown to decrease adipose tissue lipolytic activity both in vivo and in vitro. The present study was initiated to test the hypothesis that the decrease in lipolytic activity by masoprocol resulted from modulation of adipose tissue hormone-sensitive lipase (HSL) activity. The results indicate that oral administration of masoprocol to rats with fructose-induced hypertriglyceridemia significantly decreased their serum free fatty acid (FFA; P HSL activity were significantly lower (P HSL protein. Incubation of masoprocol with adipocytes from chow-fed rats significantly inhibited isoproterenol-induced lipolytic activity and HSL activity, associated with a decrease in the ability of isoproterenol to phosphorylate HSL. Masoprocol had no apparent effect on adipose tissue phosphatidylinositol 3-kinase activity, but okadaic acid, a serine/threonine phosphatase inhibitor, blocked the antilipolytic effect of masoprocol. The results of these in vitro and in vivo experiments suggest that the antilipolytic activity of masoprocol is secondary to its ability to inhibit HSL phosphorylation, possibly by increasing phosphatase activity. As a consequence, masoprocol administration results in lower serum FFA and TG concentrations in hypertriglyceridemic rodents.

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

    Science.gov (United States)

    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. Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish triggering from activation of human neutrophils

    International Nuclear Information System (INIS)

    Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O2-) which are potent mediators of inflammation. Various agents elicit different responses. In contrast, phorbol myristate acetate (PMA, 1.6 μM) acting directly via protein kinase C is a potent stimulus for O2-. The authors compared the kinetics of appearance of various second messengers with the capacity of these ligands to elicit O2- generation. Kinetic analysis showed a two-phase response to membrane ligands; both an early (≥ 15 s) and a late (>15 s) increase in [3H]- and [14C]diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB4 elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Moreover, comparison of increases in [3H]DG versus those of [14C]DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 M/sub r/ protein was phosphorylated with kinetics consistent with the production of O2- and DG in response to fMLP and PMA. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only triggering (the rapid generation of a signal) but also activation (the maintenance of a signal) to sustain responses

  4. Identification of a protein kinase activity that phosphorylates connexin43 in a pH-dependent manner

    Directory of Open Access Journals (Sweden)

    P. Yahuaca

    2000-04-01

    Full Text Available The carboxyl-terminal (CT domain of connexin43 (Cx43 has been implicated in both hormonal and pH-dependent gating of the gap junction channel. An in vitro assay was utilized to determine whether the acidification of cell extracts results in the activation of a protein kinase that can phosphorylate the CT domain. A glutathione S-transferase (GST-fusion protein was bound to Sephadex beads and used as a target for protein kinase phosphorylation. A protein extract produced from sheep heart was allowed to bind to the fusion protein-coated beads. The bound proteins were washed and then incubated with 32P-ATP. Phosphorylation was assessed after the proteins were resolved by SDS-PAGE. Incubation at pH 7.5 resulted in a minimal amount of phosphorylation while incubation at pH 6.5 resulted in significant phosphorylation reaction. Maximal activity was achieved when both the binding and kinase reactions were performed at pH 6.5. The protein kinase activity was stronger when the incubations were performed with manganese rather than magnesium. Mutants of Cx43 which lack the serines between amino acids 364-374 could not be phosphorylated in the in vitro kinase reaction, indicating that this is a likely target of this reaction. These results indicate that there is a protein kinase activity in cells that becomes more active at lower pH and can phosphorylate Cx43.

  5. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M;

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated......Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show...... on histidine residues, however, only the B isoform appeared to be serine phosphorylated....

  6. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

    Science.gov (United States)

    Chang, Audrey N; Mahajan, Pravin; Knapp, Stefan; Barton, Hannah; Sweeney, H Lee; Kamm, Kristine E; Stull, James T

    2016-07-01

    The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties. PMID:27325775

  7. KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting Aurora B kinase activity.

    Science.gov (United States)

    Caldas, Gina V; DeLuca, Keith F; DeLuca, Jennifer G

    2013-12-23

    Aurora B kinase phosphorylates kinetochore proteins during early mitosis, increasing kinetochore–microtubule (MT) turnover and preventing premature stabilization of kinetochore–MT attachments. Phosphorylation of kinetochore proteins during late mitosis is low, promoting attachment stabilization, which is required for anaphase onset. The kinetochore protein KNL1 recruits Aurora B–counteracting phosphatases and the Aurora B–targeting factor Bub1, yet the consequences of KNL1 depletion on Aurora B phospho-regulation remain unknown. Here, we demonstrate that the KNL1 N terminus is essential for Aurora B activity at kinetochores. This region of KNL1 is also required for Bub1 kinase activity at kinetochores, suggesting that KNL1 promotes Aurora B activity through Bub1-mediated Aurora B targeting. However, ectopic targeting of Aurora B to kinetochores does not fully rescue Aurora B activity in KNL1-depleted cells, suggesting KNL1 influences Aurora B activity through an additional pathway. Our findings establish KNL1 as a requirement for Aurora B activity at kinetochores and for wild-type kinetochore–MT attachment dynamics.

  8. Cdk1 Phosphorylates SPAT-1/Bora to Promote Plk1 Activation in C. elegans and Human Cells

    Directory of Open Access Journals (Sweden)

    Yann Thomas

    2016-04-01

    Full Text Available The conserved Bora protein is a Plk1 activator, essential for checkpoint recovery after DNA damage in human cells. Here, we show that Bora interacts with Cyclin B and is phosphorylated by Cyclin B/Cdk1 at several sites. The first 225 amino acids of Bora, which contain two Cyclin binding sites and three conserved phosphorylated residues, are sufficient to promote Plk1 phosphorylation by Aurora A in vitro. Mutating the Cyclin binding sites or the three conserved phosphorylation sites abrogates the ability of the N terminus of Bora to promote Plk1 activation. In human cells, Bora-carrying mutations of the three conserved phosphorylation sites cannot sustain mitotic entry after DNA damage. In C. elegans embryos, mutation of the three conserved phosphorylation sites in SPAT-1, the Bora ortholog, results in a severe mitotic entry delay. Our results reveal a crucial and conserved role of phosphorylation of the N terminus of Bora for Plk1 activation and mitotic entry.

  9. Glycolysis and oxidative phosphorylation in neurons and astrocytes during network activity in hippocampal slices.

    Science.gov (United States)

    Ivanov, Anton I; Malkov, Anton E; Waseem, Tatsiana; Mukhtarov, Marat; Buldakova, Svetlana; Gubkina, Olena; Zilberter, Misha; Zilberter, Yuri

    2014-03-01

    Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-triggered energy metabolism. These effects are not induced by changes in ATP content, pH(i), [Ca(2+)](i) or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices--instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes.

  10. PKC-dependent Phosphorylation of the H1 Histamine Receptor Modulates TRPC6 Activity.

    Science.gov (United States)

    Chen, Xingjuan; Egly, Christian; Riley, Ashley M; Li, Wennan; Tewson, Paul; Hughes, Thomas E; Quinn, Anne Marie; Obukhov, Alexander G

    2014-01-01

    Transient receptor potential canonical 6 (TRPC6) is a cation selective, DAG-regulated, Ca2+-permeable channel activated by the agonists of Gq-protein-coupled heptahelical receptors. Dysfunctions of TRPC6 are implicated in the pathogenesis of various cardiovascular and kidney conditions such as vasospasm and glomerulosclerosis. When stimulated by agonists of the histamine H1 receptor (H1R), TRPC6 activity decays to the baseline despite the continuous presence of the agonist. In this study, we examined whether H1R desensitization contributes to regulating the decay rate of TRPC6 activity upon receptor stimulation. We employed the HEK expression system and a biosensor allowing us to simultaneously detect the changes in intracellular diacylglycerol (DAG) and Ca2+ concentrations. We found that the histamine-induced DAG response was biphasic, in which a transient peak was followed by maintained elevated plateau, suggesting that desensitization of H1R takes place in the presence of histamine. The application of PKC inhibitor Gö6983 slowed the decay rate of intracellular DAG concentration. Activation of the mouse H1R mutant lacking a putative PKC phosphorylation site, Ser399, responsible for the receptor desensitization, resulted in a prolonged intracellular DAG increase and greater Mn2+ influx through the TRPC6 channel. Thus, our data support the hypothesis that PKC-dependent H1R phosphorylation leads to a reduced production of intracellular DAG that contributes to TRPC6 activity regulation.

  11. PKC-dependent Phosphorylation of the H1 Histamine Receptor Modulates TRPC6 Activity

    Directory of Open Access Journals (Sweden)

    Xingjuan Chen

    2014-04-01

    Full Text Available Transient receptor potential canonical 6 (TRPC6 is a cation selective, DAG-regulated, Ca2+-permeable channel activated by the agonists of Gq-protein-coupled heptahelical receptors. Dysfunctions of TRPC6 are implicated in the pathogenesis of various cardiovascular and kidney conditions such as vasospasm and glomerulosclerosis. When stimulated by agonists of the histamine H1 receptor (H1R, TRPC6 activity decays to the baseline despite the continuous presence of the agonist. In this study, we examined whether H1R desensitization contributes to regulating the decay rate of TRPC6 activity upon receptor stimulation. We employed the HEK expression system and a biosensor allowing us to simultaneously detect the changes in intracellular diacylglycerol (DAG and Ca2+ concentrations. We found that the histamine-induced DAG response was biphasic, in which a transient peak was followed by maintained elevated plateau, suggesting that desensitization of H1R takes place in the presence of histamine. The application of PKC inhibitor Gö6983 slowed the decay rate of intracellular DAG concentration. Activation of the mouse H1R mutant lacking a putative PKC phosphorylation site, Ser399, responsible for the receptor desensitization, resulted in a prolonged intracellular DAG increase and greater Mn2+ influx through the TRPC6 channel. Thus, our data support the hypothesis that PKC-dependent H1R phosphorylation leads to a reduced production of intracellular DAG that contributes to TRPC6 activity regulation.

  12. Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase, at Ser-446.

    Science.gov (United States)

    Masuda, Kouhei; Shima, Hiroshi; Katagiri, Chiaki; Kikuchi, Kunimi

    2003-08-22

    We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK > p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK. PMID:12794087

  13. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    Science.gov (United States)

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  14. SAD-B Phosphorylation of CAST Controls Active Zone Vesicle Recycling for Synaptic Depression.

    Science.gov (United States)

    Mochida, Sumiko; Hida, Yamato; Tanifuji, Shota; Hagiwara, Akari; Hamada, Shun; Abe, Manabu; Ma, Huan; Yasumura, Misato; Kitajima, Isao; Sakimura, Kenji; Ohtsuka, Toshihisa

    2016-09-13

    Short-term synaptic depression (STD) is a common form of activity-dependent plasticity observed widely in the nervous system. Few molecular pathways that control STD have been described, but the active zone (AZ) release apparatus provides a possible link between neuronal activity and plasticity. Here, we show that an AZ cytomatrix protein CAST and an AZ-associated protein kinase SAD-B coordinately regulate STD by controlling reloading of the AZ with release-ready synaptic vesicles. SAD-B phosphorylates the N-terminal serine (S45) of CAST, and S45 phosphorylation increases with higher firing rate. A phosphomimetic CAST (S45D) mimics CAST deletion, which enhances STD by delaying reloading of the readily releasable pool (RRP), resulting in a pool size decrease. A phosphonegative CAST (S45A) inhibits STD and accelerates RRP reloading. Our results suggest that the CAST/SAD-B reaction serves as a brake on synaptic transmission by temporal calibration of activity and synaptic depression via RRP size regulation. PMID:27626661

  15. The mitogen-activated protein (MAP) kinase ERK induces tRNA synthesis by phosphorylating TFIIIB

    OpenAIRE

    Felton-Edkins, Zoe A.; Fairley, Jennifer A.; Graham, Emma L.; Johnston, Imogen M.; White, Robert J.; Scott, Pamela H.

    2003-01-01

    RNA polymerase (pol) III transcription increases within minutes of serum addition to growth-arrested fibroblasts. We show that ERK mitogen-activated protein kinases regulate pol III output by directly binding and phosphorylating the BRF1 subunit of transcription factor TFIIIB. Blocking the ERK signalling cascade inhibits TFIIIB binding to pol III and to transcription factor TFIIIC2. Chromatin immunoprecipitation shows that the association of BRF1 and pol III with tRNALeu genes in cells decrea...

  16. Phosphorylation and activation of the plasma membrane Na+/H+ exchanger (NHE1 during osmotic cell shrinkage.

    Directory of Open Access Journals (Sweden)

    Robert R Rigor

    Full Text Available The Na(+/H(+Exchanger isoform 1 (NHE1 is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na(+/H(+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS. We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC. Na(+/H(+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1 that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA increase Na(+ transport capacity without affecting transport affinity (K(m=44 mM in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na(+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.

  17. Type III effector activation via nucleotide binding, phosphorylation, and host target interaction.

    Directory of Open Access Journals (Sweden)

    Darrell Desveaux

    2007-03-01

    Full Text Available The Pseudomonas syringae type III effector protein avirulence protein B (AvrB is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]-interacting protein. RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 A resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s. We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function.

  18. Phosphorylated heat shock protein 27 promotes lipid clearance in hepatic cells through interacting with STAT3 and activating autophagy.

    Science.gov (United States)

    Shen, Lei; Qi, Zhilin; Zhu, Yanyan; Song, Xiaomeng; Xuan, Chunxia; Ben, Peiling; Lan, Lei; Luo, Lan; Yin, Zhimin

    2016-08-01

    Nonalcoholic fatty liver disease (NAFLD) has become the major liver disease worldwide. Recently, several studies have identified that the activation of autophagy attenuates hepatic steatosis. Heat shock protein 27 (Hsp27) is involved in autophagy in response to various stimuli. In this study, we demonstrate that phosphorylated Hsp27 stimulates autophagy and lipid droplet clearance and interacts with STAT3. In vivo study showed that high fat diet (HFD) feeding increased Hsp25 (mouse orthology of Hsp27) phosphorylation and autophagy in mouse livers. Inhibition of Hsp25 phosphorylation exacerbated HFD-induced hepatic steatosis in mice. In vitro study showed that palmitate-induced lipid overload in hepatic cells was enhanced by Hsp27 knockdown, KRIBB3 treatment and Hsp27-3A (non-phosphorylatable) overexpression but was prevented by Hsp27-WT (wild type) and Hsp27-3D (phosphomimetic) overexpression. Mechanism analysis demonstrated that palmitate could induce Hsp27 phosphorylation which promoted palmitate-induced autophagy. Phosphorylated Hsp27 interacted with STAT3 in response to palmitate treatment, and disrupted the STAT3/PKR complexes, facilitated PKR-dependent eIF2α phosphorylation, and thus stimulated autophagy. To conclude, our study provides a novel mechanism by which the phosphorylated Hsp27 promotes hepatic lipid clearance and suggests a new insight for therapy of steatotic diseases such as nonalcoholic fatty liver disease (NAFLD). PMID:27185187

  19. Preparation, characterization and antibacterial activity of chitosan and phosphorylated chitosan from cuttlebone of Sepia kobiensis (Hoyle, 1885)

    OpenAIRE

    Annaian Shanmugam; Kandasamy Kathiresan; Lakshman Nayak

    2016-01-01

    Chitosan is a commercially available derivative of chitin that has been extensively studied for its antimicrobial properties. In order to improve the water solubility and its biological activity, the chemical modification or derivatisation is attempted. In the present investigation, the chitosan prepared from the cuttlebone of Sepia kobiensis was being chemically modified by reacting it with orthophosphoric acid so as to obtain phosphorylated chitosan. Then the chitosan and phosphorylated chi...

  20. Cell proliferation and migration are modulated by Cdk-1-phosphorylated endothelial-monocyte activating polypeptide II.

    Directory of Open Access Journals (Sweden)

    Margaret A Schwarz

    Full Text Available BACKGROUND: Endothelial-Monocyte Activating Polypeptide (EMAP II is a secreted protein with well-established anti-angiogenic activities. Intracellular EMAP II expression is increased during fetal development at epithelial/mesenchymal boundaries and in pathophysiologic fibroproliferative cells of bronchopulmonary dysplasia, emphysema, and scar fibroblast tissue following myocardial ischemia. Precise function and regulation of intracellular EMAP II, however, has not been explored to date. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that high intracellular EMAP II suppresses cellular proliferation by slowing progression through the G2M cell cycle transition in epithelium and fibroblast. Furthermore, EMAP II binds to and is phosphorylated by Cdk1, and exhibits nuclear/cytoplasmic partitioning, with only nuclear EMAP II being phosphorylated. We observed that extracellular secreted EMAP II induces endothelial cell apoptosis, where as excess intracellular EMAP II facilitates epithelial and fibroblast cells migration. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that EMAP II has specific intracellular effects, and that this intracellular function appears to antagonize its extracellular anti-angiogenic effects during fetal development and pulmonary disease progression.

  1. Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling.

    Science.gov (United States)

    Yabu, T; Shiba, H; Shibasaki, Y; Nakanishi, T; Imamura, S; Touhata, K; Yamashita, M

    2015-02-01

    Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.

  2. Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning.

    Science.gov (United States)

    Crow, T; Xue-Bian, J J; Siddiqi, V; Kang, Y; Neary, J T

    1998-05-01

    The pathway supporting the conditioned stimulus (CS) is one site of plasticity that has been studied extensively in conditioned Hermissenda. Several signal transduction pathways have been implicated in classical conditioning of this preparation, although the major emphasis has been on protein kinase C. Here we provide evidence for the activation and phosphorylation of a mitogen-activated protein kinase (MAPK) pathway by one-trial and multi-trial conditioning. A one-trial in vitro conditioning procedure consisting of light (CS) paired with the application of 5-HT results in the increased incorporation of 32PO4 into proteins detected with two-dimensional gel electrophoresis. Two of the phosphoproteins have molecular weights of 44 and 42 kDa, consistent with extracellular signal-regulated protein kinases (ERK1 and ERK2). Phosphorylation of the 44 and 42 kDa proteins by one-trial conditioning was inhibited by pretreatment with PD098059, A MEK1 (ERK-Activating kinase) inhibitor. Assays of ERK activity with brain myelin basic protein as a substrate revealed greater ERK activity for the group that received one-trial conditioning compared with an unpaired control group. Western blot analysis of phosphorylated ERK using antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2 showed an increase in phosphorylation after one-trial conditioning compared with unpaired controls. The increased phosphorylation of ERK after one-trial conditioning was blocked by pretreatment with PD098059. Hermissenda that received 10 or 15 conditioning trials showed significant behavioral suppression compared with pseudo-random controls. After conditioning and behavioral testing, the conditioned animals showed significantly greater phosphorylation of ERK compared with the pseudo-random controls. These results show that the ERK-MAPK signaling pathway is activated in Pavlovian conditioning of Hermissenda.

  3. CD133 promotes gallbladder carcinoma cell migration through activating Akt phosphorylation

    Science.gov (United States)

    Zhen, Jiaojiao; Ai, Zhilong

    2016-01-01

    Gallbladder carcinoma (GBC) is the fifth most common malignancy of gastrointestinal tract. The prognosis of gallbladder carcinoma is extremely terrible partially due to metastasis. However, the mechanisms underlying gallbladder carcinoma metastasis remain largely unknown. CD133 is a widely used cancer stem cell marker including in gallbladder carcinoma. Here, we found that CD133 was highly expressed in gallbladder carcinoma as compared to normal tissues. CD133 was located in the invasive areas in gallbladder carcinoma. Down-regulation expression of CD133 inhibited migration and invasion of gallbladder carcinoma cell without obviously reducing cell proliferation. Mechanism analysis revealed that down-regulation expression of CD133 inhibited Akt phosphorylation and increased PTEN protein level. The inhibitory effect of CD133 down-regulation on gallbladder carcinoma cell migration could be rescued by Akt activation. Consistent with this, addition of Akt inhibitor Wortmannin markedly inhibited the migration ability of CD133-overexpressing cells. Thus, down-regulation of CD133 inhibits migration of gallbladder carcinoma cells through reducing Akt phosphorylation. These findings explore the fundamental biological aspect of CD133 in gallbladder carcinoma progression, providing insights into gallbladder carcinoma cell migration. PMID:26910892

  4. Hematopoietic Stem Cell Activity Is Regulated by Pten Phosphorylation Through a Niche-Dependent Mechanism.

    Science.gov (United States)

    Li, Jing; Zhang, Jun; Tang, Minghui; Xin, Junping; Xu, Yan; Volk, Andrew; Hao, Caiqin; Hu, Chenglong; Sun, Jiewen; Wei, Wei; Cao, Quichan; Breslin, Peter; Zhang, Jiwang

    2016-08-01

    The phosphorylated form of Pten (p-Pten) is highly expressed in >70% of acute myeloid leukemia samples. However, the role of p-Pten in normal and abnormal hematopoiesis has not been studied. We found that Pten protein levels are comparable among long-term (LT) hematopoietic stem cells (HSCs), short-term (ST) HSCs, and multipotent progenitors (MPPs); however, the levels of p-Pten are elevated during the HSC-to-MPP transition. To study whether p-Pten is involved in regulating self-renewal and differentiation in HSCs, we compared the effects of overexpression of p-Pten and nonphosphorylated Pten (non-p-Pten) on the hematopoietic reconstitutive capacity (HRC) of HSCs. We found that overexpression of non-p-Pten enhances the LT-HRC of HSCs, whereas overexpression of p-Pten promotes myeloid differentiation and compromises the LT-HRC of HSCs. Such phosphorylation-regulated Pten functioning is mediated by repressing the cell:cell contact-induced activation of Fak/p38 signaling independent of Pten's lipid phosphatase activity because both p-Pten and non-p-Pten have comparable activity in repressing PI3K/Akt signaling. Our studies suggest that, in addition to repressing PI3K/Akt/mTor signaling, non-p-Pten maintains HSCs in bone marrow niches via a cell-contact inhibitory mechanism by inhibiting Fak/p38 signaling-mediated proliferation and differentiation. In contrast, p-Pten promotes the proliferation and differentiation of HSCs by enhancing the cell contact-dependent activation of Src/Fak/p38 signaling. Stem Cells 2016;34:2130-2144. PMID:27096933

  5. Protein kinase A increases type-2 inositol 1,4,5-trisphosphate receptor activity by phosphorylation of serine 937.

    Science.gov (United States)

    Betzenhauser, Matthew J; Fike, Jenna L; Wagner, Larry E; Yule, David I

    2009-09-11

    Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) represents a mechanism for shaping intracellular Ca(2+) signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca(2+) release in cells that express predominantly InsP(3)R2. PKA is known to phosphorylate InsP(3)R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP(3)R2 in DT40-3KO cells that are devoid of endogenous InsP(3)R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca(2+) signals and augmented the single channel open probability of InsP(3)R2. A PKA phosphorylation site unique to the InsP(3)R2 was identified at Ser(937). The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser(937), since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca(2+) signaling following PKA activation in cells that express predominantly InsP(3)R2.

  6. Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

    Science.gov (United States)

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

    p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

  7. Novel STAT3 phosphorylation inhibitors exhibit potent growth suppressive activity in pancreatic and breast cancer cells

    Science.gov (United States)

    Lin, Li; Hutzen, Brian; Zuo, Mingxin; Ball, Sarah; Deangelis, Stephanie; Foust, Elizabeth; Pandit, Bulbul; Ihnat, Michael A.; Shenoy, Satyendra S.; Kulp, Samuel; Li, Pui-Kai; Li, Chenglong; Fuchs, James; Lin, Jiayuh

    2010-01-01

    The constitutive activation of Signal Transducer and Activator of Transcription 3 (STAT3) is frequently detected in most types of human cancer where it plays important roles in survival, drug-resistance, angiogenesis, and other functions. Targeting constitutive STAT3 signaling is thus an attractive therapeutic approach for these cancers. We have recently developed novel small molecule STAT3 inhibitors known as FLLL31 and FLLL32, which are derived from curcumin (the primary bioactive compound of turmeric). These compounds are designed to bind selectively to Janus Kinase 2 (JAK2) and the STAT3 SH2 domain, which serves crucial roles in STAT3 dimerization and signal transduction. Here we show that FLLL31 and FLLL32 are effective inhibitors of STAT3 phosphorylation, DNA binding activity, and transactivation in vitro, leading to the impediment of multiple oncogenic processes and the induction of apoptosis in pancreatic and breast cancer cell lines. FLLL31 and FLLL32 also inhibit colony formation in soft agar, cell invasion, and exhibit synergy with the anti-cancer drug doxorubicin against breast cancer cells. In addition, we show that FLLL32 can inhibit the induction of STAT3 phosphorylation by Interferon-α (IFNα) and Interleukin-6 (IL-6) in breast cancer cells. We also demonstrate that administration of FLLL32 can inhibit tumor growth and vascularity in chicken embryo xenografts as well as substantially reduce tumor volumes in mouse xenografts. Our findings highlight the potential of these new compounds and their efficacy in targeting pancreatic and breast cancers that exhibit constitutive STAT3 signaling. PMID:20215512

  8. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    Science.gov (United States)

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

  9. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    Full Text Available Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL from Geobacillus kaustophilus HTA426 (GkaP exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

  10. Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes.

    Science.gov (United States)

    Boulanger, Alice; Moon, Kyung; Decker, Kimberly B; Chen, Qing; Knipling, Leslie; Stibitz, Scott; Hinton, Deborah M

    2015-02-10

    Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed.

  11. Targeting notch pathway enhances rapamycin antitumor activity in pancreas cancers through PTEN phosphorylation

    Directory of Open Access Journals (Sweden)

    Vo Kevin

    2011-11-01

    Full Text Available Abstract Background Pancreas cancer is one of most aggressive human cancers with the survival rate for patients with metastatic pancreas cancer at 5-6 months. The poor survival demonstrates a clear need for better target identification, drug development and new therapeutic strategies. Recent discoveries have shown that the role for Notch pathway is important in both development and cancer. Its contribution to oncogenesis also involves crosstalks with other growth factor pathways, such as Akt and its modulator, PTEN. The mounting evidence supporting a role for Notch in cancer promotion and survival suggests that targeting this pathway alone or in combination with other therapeutics represents a promising therapeutic strategy. Results Using a pancreas cancer tissue microarray, we noted that Jagged1, Notch3 and Notch4 are overexpressed in pancreas tumors (26%, 84% and 31% respectively, whereas Notch1 is expressed in blood vessels. While there was no correlation between Notch receptor expression and survival, stage or tumor grade, Notch3 was associated with Jagged1 and EGFR expression, suggesting a unique relationship between Notch3 and Jagged1. Inhibition of the Notch pathway genetically and with gamma-secretase inhibitor (GSI resulted in tumor suppression and enhanced cell death. The observed anti-tumor activity appeared to be through Akt and modulation of PTEN phosphorylation. We discovered that transcriptional regulation of RhoA by Notch is important for PTEN phosphorylation. Finally, the mTOR inhibitor Rapamycin enhanced the effect of GSI on RhoA expression, resulting in down regulation of phospho-Akt and increased in vitro tumor cytotoxity. Conclusions Notch pathway plays an important role in maintaining pancreas tumor phenotype. Targeting this pathway represents a reasonable strategy for the treatment of pancreas cancers. Notch modulates the Akt pathway through regulation of PTEN phosphorylation, an observation that has not been made

  12. Potentiality of phosphorylation of BRCA1 at Ser 1524 to activate p21 in response to X-ray irradiation

    Institute of Scientific and Technical Information of China (English)

    LI Ning; ZHANG Hong; WANG Yanling; WANG Xiaohu; HAO Jifang; ZHAO Weiping

    2008-01-01

    The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. Many studies suggested that multiple functions of BRCA1 may contribute to its tumor suppressor activity, including roles in cell cycle checkpoints, apoptosis and transcription. It is postulated that phosphorylation of BRCA1 is an important means by which its cellular functions are regulated. In this study, we employed phospho-Ser-specific antibody recognizing Ser-1524 to study BRCA1 phosphorylation under conditions of DNA damage and the effects of phosphorylation on BRCA1 functions. The results showed that 10 Gy X-ray treatment significantly induced phosphorylation of Ser-1524 but not total BRCA1 protein levels. The expression both of p53 andp21 increased after irradiation, but ionizing radiation (IR)-induced activation of p21 was prior to that of p53. The percentages of G0/G1 phase remarkably increased after IR. In addition, no detectable levels of 89 kDa fragment of PARP, a marker of apoptotic cells, were observed. Data implied that IR-induced phosphorylation of BRCA1 at Ser-1524 might activate p21 protein, by which BRCA1 regulated cell cycle, but play no role in apoptosis.

  13. Primary WWOX phosphorylation and JNK activation during etoposide induces cytotoxicity in HEK293 cells

    Directory of Open Access Journals (Sweden)

    M Jamshidiha

    2010-06-01

    Full Text Available "n  "nBackground and the purpose of the study: Etoposide is an antineoplastic agent used in multiple cancers. It is known that etoposide induce cell death via interaction with topoisomerase II; however, the etopoisde cellular response is poorly understood. Upon etoposide induced DNA damage, many stress signaling pathways including JNK are activated. In response to DNA damage, it has been shown that WWOX, a recently introduced tumor suppressor, can be activated. In this study the activation of WWOX and JNK and their interaction following etoposide treatment were evaluated. "nMaterials and Methods:HEK293 cells treated with etoposide were lysed in a time course manner. The whole cell lysates were used to evaluate JNK and WWOX activation pattern using Phospho specific antibodies on western blots. The viability of cells treated with etoposide, JNK specific inhibitor and their combination was examined using MTT assay. "nResults:Findings of this study indicate that WWOX and JNK are activated in a simultaneous way in response to DNA damage. Moreover, JNK inhibition enhances etoposide induced cytotoxicity in HEK293. "nConclusion:Taken together, our results indicate that etoposide induces cytotoxicity and WWOX phosphorylation and the cytotoxicty is augmented by blocking JNK pathway.

  14. Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Svendsen, A.; Langberg, H.;

    1998-01-01

    reveal that the hinges of the active site lid are more flexible in the wild-type Hll than in S146A. In contrast, larger fluctuations are observed in the middle region of the active site loop in S 146A than in Hll. These findings reveal that the single mutation (S146A) of the active site serine leads......We have investigated the binding properties of and dynamics in Humicola lanuginosa lipase (HII) and the inactive mutant S146A (active Ser146 substituted with Ala) using fluorescence spectroscopy and molecular dynamics simulations, respectively. Hll and S146A show significantly different binding......, whereas only small changes are observed for I-Ill suggesting that the active site Lid in the latter opens more easily and hence more lipase molecules are bound to the liposomes. These observations are in agreement with molecular dynamics simulations and subsequent essential dynamics analyses. The results...

  15. Essential role of RelA Ser311 phosphorylation by ζPKC in NF-κB transcriptional activation

    OpenAIRE

    Duran, Angeles; Diaz-Meco, María T.; Moscat, Jorge

    2003-01-01

    The activation of the transcription factor NF-κB is central to the control of the cellular response triggered by many stimuli. Once released from the inhibitory molecule IκB, NF-κB is translocated to the nucleus, and it has to be phosphorylated to activate transcription. In ζ protein kinase C (PKC)-deficient cells, NF-κB is transcriptionally inactive and the phosphorylation of the RelA subunit in response to tumor necrosis factor (TNF-α) is severely impaired. In vitro assays showed that ζPKC ...

  16. Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

    Science.gov (United States)

    Xiao, Hongyan; Kovics, Richard; Jackson, Van; Remick, Daniel G

    2004-04-01

    Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation. PMID:15060414

  17. Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer's disease brain.

    Science.gov (United States)

    Kurbatskaya, Ksenia; Phillips, Emma C; Croft, Cara L; Dentoni, Giacomo; Hughes, Martina M; Wade, Matthew A; Al-Sarraj, Safa; Troakes, Claire; O'Neill, Michael J; Perez-Nievas, Beatriz G; Hanger, Diane P; Noble, Wendy

    2016-03-31

    Alterations in calcium homeostasis are widely reported to contribute to synaptic degeneration and neuronal loss in Alzheimer's disease. Elevated cytosolic calcium concentrations lead to activation of the calcium-sensitive cysteine protease, calpain, which has a number of substrates known to be abnormally regulated in disease. Analysis of human brain has shown that calpain activity is elevated in AD compared to controls, and that calpain-mediated proteolysis regulates the activity of important disease-associated proteins including the tau kinases cyclin-dependent kinase 5 and glycogen kinase synthase-3. Here, we sought to investigate the likely temporal association between these changes during the development of sporadic AD using Braak staged post-mortem brain. Quantification of protein amounts in these tissues showed increased activity of calpain-1 from Braak stage III onwards in comparison to controls, extending previous findings that calpain-1 is upregulated at end-stage disease, and suggesting that activation of calcium-sensitive signalling pathways are sustained from early stages of disease development. Increases in calpain-1 activity were associated with elevated activity of the endogenous calpain inhibitor, calpastatin, itself a known calpain substrate. Activation of the tau kinases, glycogen-kinase synthase-3 and cyclin-dependent kinase 5 were also found to occur in Braak stage II-III brain, and these preceded global elevations in tau phosphorylation and the loss of post-synaptic markers. In addition, we identified transient increases in total amyloid precursor protein and pre-synaptic markers in Braak stage II-III brain, that were lost by end stage Alzheimer's disease, that may be indicative of endogenous compensatory responses to the initial stages of neurodegeneration. These findings provide insight into the molecular events that underpin the progression of Alzheimer's disease, and further highlight the rationale for investigating novel treatment

  18. Resistance of Cancer Cells to Targeted Therapies Through the Activation of Compensating Signaling Loops.

    Science.gov (United States)

    von Manstein, Viktoria; Yang, Chul Min; Richter, Diane; Delis, Natalia; Vafaizadeh, Vida; Groner, Bernd

    2013-12-01

    The emergence of low molecular weight kinase inhibitors as "targeted" drugs has led to remarkable advances in the treatment of cancer patients. The clinical benefits of these tumor therapies, however, vary widely in patient populations and with duration of treatment. Intrinsic and acquired resistance against such drugs limits their efficacy. In addition to the well studied mechanisms of resistance based upon drug transport and metabolism, genetic alterations in drug target structures and the activation of compensatory cell signaling have received recent attention. Adaptive responses can be triggered which counteract the initial dependence of tumor cells upon a particular signaling molecule and allow only a transient inhibition of tumor cell growth. These compensating signaling mechanisms are often based upon the relief of repression of regulatory feedback loops. They might involve cell autonomous, intracellular events or they can be mediated via the secretion of growth factor receptor ligands into the tumor microenvironment and signal induction in an auto- or paracrine fashion. The transcription factors Stat3 and Stat5 mediate the biological functions of cytokines, interleukins and growth factors and can be considered as endpoints of multiple signaling pathways. In normal cells this activation is transient and the Stat molecules return to their non-phosphorylated state within a short time period. In tumor cells the balance between activating and de-activating signals is disturbed resulting in the persistent activation of Stat3 or Stat5. The constant activation of Stat3 induces the expression of target genes, which cause the proliferation and survival of cancer cells, as well as their migration and invasive behavior. Activating components of the Jak-Stat pathway have been recognized as potentially valuable drug targets and important principles of compensatory signaling circuit induction during targeted drug treatment have been discovered in the context of kinase

  19. miR-34 activity is modulated through 5'-end phosphorylation in response to DNA damage.

    Science.gov (United States)

    Salzman, David W; Nakamura, Kotoka; Nallur, Sunitha; Dookwah, Michelle T; Metheetrairut, Chanatip; Slack, Frank J; Weidhaas, Joanne B

    2016-01-01

    MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5'-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5'-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level. PMID:26996824

  20. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    Directory of Open Access Journals (Sweden)

    Anshula eSamarajeewa

    2014-11-01

    Full Text Available The serotonin (5-HT type 7 receptor is expressed throughout the CNS including cortical neurons. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA-induced toxicity. The tropomyosin-related kinase B (TrkB receptor is one of the receptors for brain-derived neurotrophic factor (BDNF and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins towards the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.

  1. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    Science.gov (United States)

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  2. The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

    Science.gov (United States)

    McCully, Kilmer S

    2015-01-01

    The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. PMID:25887881

  3. Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells.

    Science.gov (United States)

    Partovian, Chohreh; Simons, Michael

    2004-08-01

    Protein kinase Balpha (PKBalpha/Akt-1) is a key mediator of multiple signaling pathways involved in angiogenesis, cell proliferation and apoptosis among others. The unphosphorylated form of Akt-1 is virtually inactive and its full activation requires two phosphatidylinositol-3,4,5-triphosphate-dependent phosphorylation events, Thr308 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser473 by an undefined kinase that has been termed PDK2. Recent studies have suggested that the Ser473 kinase is a plasma membrane raft-associated kinase. In this study we show that protein kinase Calpha (PKCalpha) translocates to the membrane rafts in response to insulin growth factor-1 (IGF-1) stimulation. Overexpression of PKCalpha increases Ser473 phosphorylation and Akt-1 activity, while inhibition of its activity or expression decreases IGF-1-dependent activation of Akt-1. Furthermore, in vitro, in the presence of phospholipids and calcium, PKCalpha directly phosphorylates Akt-1 at the Ser473 site. We conclude, therefore, that PKCalpha regulates Akt-1 activity via Ser473 phosphorylation and may function as PDK2 in endothelial cells. PMID:15157674

  4. Adrenergic regulation of HSL serine phosphorylation and activity in human skeletal muscle during the onset of exercise.

    Science.gov (United States)

    Talanian, Jason L; Tunstall, Rebecca J; Watt, Matthew J; Duong, Mylinh; Perry, Christopher G R; Steinberg, Gregory R; Kemp, Bruce E; Heigenhauser, George J F; Spriet, Lawrence L

    2006-10-01

    Skeletal muscle hormone-sensitive lipase (HSL) activity is increased by contractions and increases in blood epinephrine (EPI) concentrations and cyclic AMP activation of the adrenergic pathway during prolonged exercise. To determine the importance of hormonal stimulation of HSL activity during the onset of moderate- and high-intensity exercise, nine men [age 24.3 +/- 1.2 yr, 80.8 +/- 5.0 kg, peak oxygen consumption (VO2 peak) 43.9 +/- 3.6 ml x kg(-1) x min(-1)] cycled for 1 min at approximately 65% VO2 peak, rested for 60 min, and cycled at approximately 90% VO2 peak for 1 min. Skeletal muscle biopsies were taken pre- and postexercise, and arterial blood was sampled throughout exercise. Arterial EPI increased (P HSL activity increased (P HSL Ser660 phosphorylation (approximately 55% increase) and ERK1/2 phosphorylation ( approximately 33% increase) were augmented following exercise at both intensities, whereas HSL Ser563 and Ser565 phosphorylation were not different from rest. The results indicate that increases in arterial EPI concentration during the onset of moderate- and high-intensity exercise increase cyclic AMP content, which results in the phosphorylation of HSL Ser660. This adrenergic stimulation contributes to the increase in HSL activity that occurs in human skeletal muscle in the first minute of exercise at 65% and 90% VO2 peak.

  5. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

    KAUST Repository

    Menegazzi, Marta

    2013-12-10

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (-)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568ASTAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation. © Copyright 2014 Federation of European Biochemical Societies. All rights reserved.

  6. Use of an open-loop system to increase physical activity

    Science.gov (United States)

    This study evaluated the effectiveness of an open-loop system that reinforces physical activity with TV watching to increase children’s physical activity. Non-overweight, sedentary boys and girls (8-12 y) were randomized to a group that received feedback of activity counts + reinforcement for physic...

  7. Convoluted dislocation loops induced by helium irradiation in reduced-activation martensitic steel and their impact on mechanical properties

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Fengfeng [Key Laboratory of Artificial Micro- and Nano-structures of Ministry of Education, Hubei Nuclear Solid Physics Key Laboratory, School of Physics and Technology, Wuhan University, Wuhan 430072 (China); Yao, Z. [Department of Mechanical and Materials Engineering, Queen' s University, Kingston, ON, Canada K7L 3N6 (Canada); Guo, Liping, E-mail: guolp@whu.edu.cn [Key Laboratory of Artificial Micro- and Nano-structures of Ministry of Education, Hubei Nuclear Solid Physics Key Laboratory, School of Physics and Technology, Wuhan University, Wuhan 430072 (China); Suo, Jinping [State Key Laboratory of Mould Technology, Institute of Materials Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074 (China); Wen, Yongming [Key Laboratory of Artificial Micro- and Nano-structures of Ministry of Education, Hubei Nuclear Solid Physics Key Laboratory, School of Physics and Technology, Wuhan University, Wuhan 430072 (China)

    2014-06-01

    Helium irradiation induced dislocation loops in reduced-activation martensitic steels were investigated using transmission electron microscopy. The specimens were irradiated with 100 keV helium ions to 0.8 dpa at 350 °C. Unexpectedly, very large dislocation loops were found, significantly larger than that induced by other types of irradiations under the same dose. Moreover, the large loops were convoluted and formed interesting flower-like shape. The large loops were determined as interstitial type. Loops with the Burgers vectors of b=〈100〉 were only observed. Furthermore, irradiation induced hardening caused by these large loops was observed using the nano-indentation technique.

  8. Convoluted dislocation loops induced by helium irradiation in reduced-activation martensitic steel and their impact on mechanical properties

    International Nuclear Information System (INIS)

    Helium irradiation induced dislocation loops in reduced-activation martensitic steels were investigated using transmission electron microscopy. The specimens were irradiated with 100 keV helium ions to 0.8 dpa at 350 °C. Unexpectedly, very large dislocation loops were found, significantly larger than that induced by other types of irradiations under the same dose. Moreover, the large loops were convoluted and formed interesting flower-like shape. The large loops were determined as interstitial type. Loops with the Burgers vectors of b=〈100〉 were only observed. Furthermore, irradiation induced hardening caused by these large loops was observed using the nano-indentation technique

  9. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    Science.gov (United States)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  10. Dyrk1A phosphorylates parkin at Ser-131 and negatively regulates its ubiquitin E3 ligase activity.

    Science.gov (United States)

    Im, Eunju; Chung, Kwang Chul

    2015-08-01

    Mutations of parkin are associated with the occurrence of autosomal recessive familial Parkinson's disease (PD). Parkin acts an E3 ubiquitin ligase, which ubiquitinates target proteins and subsequently regulates either their steady-state levels through the ubiquitin-proteasome system or biochemical properties. In this study, we identify a novel regulatory mechanism of parkin by searching for new regulatory factors. After screening human fetal brain using a yeast two hybrid assay, we found dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) as a novel binding partner of parkin. We also observed that parkin interacts and co-localizes with Dyrk1A in mammalian cells. In addition, Dyrk1A directly phosphorylated parkin at Ser-131, causing the inhibition of its E3 ubiquitin ligase activity. Moreover, Dyrk1A-mediated phosphorylation reduced the binding affinity of parkin to its ubiquitin-conjugating E2 enzyme and substrate, which could be the underlying inhibitory mechanism of parkin activity. Furthermore, Dyrk1A-mediated phosphorylation inhibited the neuroprotective action of parkin against 6-hydroxydopamine toxicity in dopaminergic SH-SY5Y cells. These findings suggest that Dyrk1A acts as a novel functional modulator of parkin. Parkin phosphorylation by Dyrk1A suppresses its E3 ubiquitin ligase activity potentially contributing to the pathogenesis of PD under PD-inducing pathological conditions. Mutations of parkin are linked to autosomal recessive forms of familial Parkinson's disease (PD). According to its functional relevance in abnormal protein aggregation and neuronal cell death, a number of post-translational modifications regulate the ubiquitin E3 ligase activity of parkin. Here we propose a novel inhibitory mechanism of parkin E3 ubiquitin ligase through dual-specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A)-mediated phosphorylation as well as its neuroprotective action against 6-hydroxydopamine (6-OHDA)-induced cell death

  11. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  12. Activation of PI3K/AKT and MAPK pathway through a PDGFRβ-dependent feedback loop is involved in rapamycin resistance in hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Quan-Lin Li

    Full Text Available BACKGROUND: Rapamycin is an attractive approach for the treatment and prevention of HCC recurrence after liver transplantation. However, the objective response rates of rapamycin achieved with single-agent therapy were modest, supporting that rapamycin resistance is a frequently observed characteristic of many cancers. Some studies have been devoted to understanding the mechanisms of rapamycin resistance, however, the mechanisms are cell-type-dependent and studies on rapamycin resistance in HCC are extremely limited. METHODOLOGY/PRINCIPAL FINDINGS: The anti-tumor sensitivity of rapamycin was modest in vitro and in vivo. In both human and rat HCC cells, rapamycin up-regulated the expression and phosphorylation of PDGFRβ in a time and dose-dependent manner as assessed by RT-PCR and western blot analysis. Using siRNA mediated knockdown of PDGFRβ, we confirmed that subsequent activation of AKT and ERK was PDGFRβ-dependent and compromised the anti-tumor activity of rapamycin. Then, blockade of this PDGFRβ-dependent feedback loop by sorafenib enhanced the anti-tumor sensitivity of rapamycin in vitro and in an immunocompetent orthotopic rat model of HCC. CONCLUSIONS: Activation of PI3K/AKT and MAPK pathway through a PDGFRβ-dependent feedback loop compromises the anti-tumor activity of rapamycin in HCC, and blockade of this feedback loop by sorafenib is an attractive approach to improve the anti-tumor effect of rapamycin, particularly in preventing or treating HCC recurrence after liver transplantation.

  13. Flarelike brightenings of active region loops observed with SUMER

    CERN Document Server

    Wang, T J; Solanki, S K; Curdt, W

    2015-01-01

    Coronal loops on the east limb of the Sun were observed by SUMER on SOHO for several days. Small flare-like brightenings are detected very frequently in the hot flare line Fe~{\\small XIX}. We find that the relatively intense events are in good coincidence with the transient brightenings seen by Yohkoh/SXT. A statistical analysis shows that these brightenings have durations of 5-84 min and extensions along the slit of 2-67 Mm. The integrated energy observed in Fe~{\\small XIX} for each event is in the range of $3\\times10^{18}-5\\times10^{23}$ ergs, and the estimated thermal energy ranges from $10^{26}-10^{29}$ ergs. Application of the statistical method proposed by Parnell \\& Jupp (2000) yields a value of 1.5 to 1.8 for the index of a power law relation between the frequency of the events and the radiated energy in Fe~{\\small XIX}, and a value of 1.7 to 1.8 for the index of the frequency distribution of the thermal energy in the energy range $>10^{27}$ ergs. We examine the possibility that these small bright...

  14. p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38

    Science.gov (United States)

    Hori, Takeshi; Moore, Rick

    2016-01-01

    Nuclear receptor constitutive androstane receptor (CAR, NR1I3), which regulates hepatic drug and energy metabolisms as well as cell growth and death, is sequestered in the cytoplasm as its inactive form phosphorylated at threonine 38. CAR activators elicit dephosphorylation, and nonphosphorylated CAR translocates into the nucleus to activate its target genes. CAR was previously found to require p38 mitogen-activated protein kinase (MAPK) to transactivate the cytochrome P450 2B (CYP2B) genes. Here we have demonstrated that p38 MAPK forms a complex with CAR, enables it to bind to the response sequence, phenobarbital-responsive enhancer module (PBREM), within the CYP2B promoter, and thus recruits RNA polymerase II to activate transcription. Subsequently, p38 MAPK elicited rephosphorylation of threonine 38 to inactivate CAR and exclude it from the nucleus. Thus, nuclear p38 MAPK exerted dual regulation by sequentially activating and inactivating CAR-mediated transcription through phosphorylation of threonine 38. PMID:27074912

  15. Preparation, characterization and antibacterial activity of chitosan and phosphorylated chitosan from cuttlebone of Sepia kobiensis (Hoyle, 1885

    Directory of Open Access Journals (Sweden)

    Annaian Shanmugam

    2016-03-01

    Full Text Available Chitosan is a commercially available derivative of chitin that has been extensively studied for its antimicrobial properties. In order to improve the water solubility and its biological activity, the chemical modification or derivatisation is attempted. In the present investigation, the chitosan prepared from the cuttlebone of Sepia kobiensis was being chemically modified by reacting it with orthophosphoric acid so as to obtain phosphorylated chitosan. Then the chitosan and phosphorylated chitosan were structurally characterized through FT-IR spectroscopy. Further the antibacterial activity of chitosan and phosphorylated chitosan was tested against clinically isolated human pathogens (Gram-positive: Streptococcus sp., Streptococcus pneumoniae and Staphylococcus aureus and Gram-negative: Escherichia coli, Vibrio cholerae, V. alginolyticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella sp. and Proteus vulgaris by well diffusion method and the Minimum Inhibitory Concentration (MIC was also calculated. The results of the present study suggests that the chitosan and phosphorylated chitosan has concentration dependent antibacterial activity with variation against several pathogenic human pathogenic bacterial strains which indicates their possible use as antibacterial agents.

  16. Three-dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

    OpenAIRE

    Alamo, Lorenzo; Wriggers, Willy; Pinto, Antonio; Bártoli, Fulvia; Salazar, Leiría; Zhao, Fa-Qing; Craig, Roger; Padrón, Raúl

    2008-01-01

    Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLC). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that my...

  17. Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Taylor, Eric B.; Witczak, Carol A.;

    2010-01-01

    . The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5 AMP activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation......TBC1D4 (also known as AS160) regulates GLUT4 translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of Serine (S)/Threonine (T) residues by upstream kinases resulting in inactivation of Rab-GAP activity leading to GLUT4 mobilization...... that S711 would be recognized by AMPK. Using a phospho-specific antibody against S711, we found that AICAR and contraction increased S711 phosphorylation in mouse skeletal muscle and this increase was abolished in muscle-specific AMPKalpha2 kinase dead transgenic mice. Exercise in human vastus lateralis...

  18. Phosphorylation status of 72 kDa MMP-2 determines its structure and activity in response to peroxynitrite.

    Directory of Open Access Journals (Sweden)

    Anna Laura Jacob-Ferreira

    Full Text Available Matrix metalloproteinase-2 (MMP-2 is a key intra- and extra-cellular protease which contributes to several oxidative stress related pathologies. A molecular understanding of 72 kDa MMP-2 activity, directly mediated by S-glutathiolation of its cysteine residues in the presence of peroxynitrite (ONOO(- and by phosphorylation of its serine and threonine residues, is essential to develop new generation inhibitors of intracellular MMP-2. Within its propeptide and collagen binding domains there is an interesting juxtaposition of predicted phosphorylation sites with nearby cysteine residues which form disulfide bonds. However, the combined effect of these two post-translational modifications on MMP-2 activity has not been studied. The activity of human recombinant 72 kDa MMP-2 (hrMMP-2 following in vitro treatments was measured by troponin I proteolysis assay and a kinetic activity assay using a fluorogenic peptide substrate. ONOO(- treatment in the presence of 30 µM glutathione resulted in concentration-dependent changes in MMP-2 activity, with 0.1-1 µM increasing up to twofold and 100 µM attenuating its activity. Dephosphorylation of MMP-2 with alkaline phosphatase markedly increased its activity by sevenfold, either with or without ONOO(-. Dephosphorylation of MMP-2 also affected the conformational structure of the enzyme as revealed by circular dichroism studies, suggesting an increase in the proportion of α-helices and a decrease in β-strands compared to the phosphorylated form of MMP-2. These results suggest that ONOO(- activation (at low µM and inactivation (at high µM of 72 kDa MMP-2, in the presence or absence of glutathione, is also influenced by its phosphorylation status. These insights into the role of post-translational modifications in the structure and activity of 72 kDa MMP-2 will aid in the development of inhibitors specifically targeting intracellular MMP-2.

  19. Phosphorylation Status of 72 kDa MMP-2 Determines Its Structure and Activity in Response to Peroxynitrite

    Science.gov (United States)

    Jacob-Ferreira, Anna Laura; Kondo, Marcia Yuri; Baral, Pravas Kumar; James, Michael N. G.; Holt, Andrew; Fan, Xiaohu; Schulz, Richard

    2013-01-01

    Matrix metalloproteinase-2 (MMP-2) is a key intra- and extra-cellular protease which contributes to several oxidative stress related pathologies. A molecular understanding of 72 kDa MMP-2 activity, directly mediated by S-glutathiolation of its cysteine residues in the presence of peroxynitrite (ONOO−) and by phosphorylation of its serine and threonine residues, is essential to develop new generation inhibitors of intracellular MMP-2. Within its propeptide and collagen binding domains there is an interesting juxtaposition of predicted phosphorylation sites with nearby cysteine residues which form disulfide bonds. However, the combined effect of these two post-translational modifications on MMP-2 activity has not been studied. The activity of human recombinant 72 kDa MMP-2 (hrMMP-2) following in vitro treatments was measured by troponin I proteolysis assay and a kinetic activity assay using a fluorogenic peptide substrate. ONOO− treatment in the presence of 30 µM glutathione resulted in concentration-dependent changes in MMP-2 activity, with 0.1–1 µM increasing up to twofold and 100 µM attenuating its activity. Dephosphorylation of MMP-2 with alkaline phosphatase markedly increased its activity by sevenfold, either with or without ONOO−. Dephosphorylation of MMP-2 also affected the conformational structure of the enzyme as revealed by circular dichroism studies, suggesting an increase in the proportion of α-helices and a decrease in β-strands compared to the phosphorylated form of MMP-2. These results suggest that ONOO− activation (at low µM) and inactivation (at high µM) of 72 kDa MMP-2, in the presence or absence of glutathione, is also influenced by its phosphorylation status. These insights into the role of post-translational modifications in the structure and activity of 72 kDa MMP-2 will aid in the development of inhibitors specifically targeting intracellular MMP-2. PMID:24013357

  20. Neurotensin Phosphorylates GSK-3α/β through the Activation of PKC in Human Colon Cancer Cells

    Directory of Open Access Journals (Sweden)

    Qingding Wang

    2006-09-01

    Full Text Available Neurotensin (NT, a gastrointestinal hormone, binds its receptor [neurotensin receptor (NTR] to regulate the growth of normal and neoplastic intestinal cells; molecular mechanisms remain largely undefined. Glycogen synthase kinase-3 (GSK-3 regulates diverse cellular processes, including cell growth and apoptosis. Here, we show that NT induces the phosphorylation of GSK-3α/β in the human colon cancer cell line HT29, HCT116, or SW480, which possesses high-affinity NTR. The effect of NT was blocked by inhibitors of protein kinase C (PKC, but not by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK1 or phosphatidylinositol-3 kinase, suggesting a predominant role for PKC in GSK-3β phosphorylation by NT. Pretreatment with Gö6976 (which inhibits PKCα and PKCβ1 or downregulation of endogenous PKCα or PKCβ1 blocked NT-mediated GSK-3β (but not GSK-3α phosphorylation. Moreover, a selective PKCβ inhibitor, LY379196, reduced NT-mediated GSK-3β (but not GSK-3α phosphorylation, suggesting a role for PKCbβ in the NT-mediated phosphorylation of GSK-3β and an undefined kinase in the NT-mediated phosphorylation of GSK-3α. Treatment with NT or the GSK-3 inhibitor SB216763 increased the expression of cyclin D1, a downstream effector protein of GSK-3 and a critical protein for the proliferation of various cells. Our results indicate that NT uses PKC-dependent pathways to modulate GSK-3, which may play a role in the NT regulation of intestinal cell growth.

  1. HIPK1 interacts with c-Myb and modulates its activity through phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Matre, Vilborg; Nordgard, Oddmund; Alm-Kristiansen, Anne Hege; Ledsaak, Marit [Department of Molecular Biosciences, University of Oslo, N-0316 Oslo (Norway); Gabrielsen, Odd Stokke, E-mail: o.s.gabrielsen@imbv.uio.no [Department of Molecular Biosciences, University of Oslo, N-0316 Oslo (Norway)

    2009-10-09

    The transcription factor v-Myb is a potent inducer of myeloid leukaemias, and its cellular homologue c-Myb plays a crucial role in the regulation of haematopoiesis. In a yeast two-hybrid (Y2H) screening we identified the nuclear kinase HIPK1 as an interaction partner for human c-Myb. The interaction involves a C-terminal region of HIPK1, while a bipartite interaction surface was identified in c-Myb, including regions in its N-terminal DNA-binding domain as well as in its C-terminal region. HIPK1 and c-Myb co-localize in distinct nuclear foci upon co-transfection. c-Myb appears to be phosphorylated by HIPK1 in its negative regulatory domain as supported by both in vivo and in vitro data. A functional assay revealed that HIPK1 repressed the ability of c-Myb to activate a chromatin embedded target gene, mim-1, in haematopoetic cells. Our findings point to a novel link between an important kinase and a key regulator of haematopoiesis.

  2. Role of the NC-loop in catalytic activity and stability in lipase from Fervidobacterium changbaicum.

    Directory of Open Access Journals (Sweden)

    Binchun Li

    Full Text Available Flexible NC-loops between the catalytic domain and the cap domain of the α/β hydrolase fold enzymes show remarkable diversity in length, sequence, and configuration. Recent investigations have suggested that the NC-loop might be involved in catalysis and substrate recognition in many enzymes from the α/β hydrolase fold superfamily. To foster a deep understanding of its role in catalysis, stability, and divergent evolution, we here systemically investigated the function of the NC-loop (residues 131-151 in a lipase (FClip1 from thermophilic bacterium Fervidobacterium changbaicum by loop deletion, alanine-scanning mutagenesis and site-directed mutagenesis. We found that the upper part of the NC-loop (residues 131-138 was of great importance to enzyme catalysis. Single substitutions in this region could fine-tune the activity of FClip1 as much as 41-fold, and any deletions from this region rendered the enzyme completely inactive. The lower part of the NC-loop (residues 139-151 was capable of enduring extensive deletions without loss of activity. The shortened mutants in this region were found to show both improved activity and increased stability simultaneously. We therefore speculated that the NC-loop, especially the lower part, would be a perfect target for enzyme engineering to optimize the enzymatic properties, and might present a hot zone for the divergent evolution of α/β hydrolases. Our findings may provide an opportunity for better understanding of the mechanism of divergent evolution in the α/β hydrolase fold superfamily, and may also guide the design of novel biocatalysts for industrial applications.

  3. The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

    Science.gov (United States)

    Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

    2014-09-01

    Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

  4. Disruption of spectrin-like cytoskeleton in differentiating keratinocytes by PKCδ activation is associated with phosphorylated adducin.

    Directory of Open Access Journals (Sweden)

    Kong-Nan Zhao

    Full Text Available Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505 activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505 phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex.

  5. Scaling laws of coronal loops compared to a 3D MHD model of an Active Region

    CERN Document Server

    Bourdin, Philippe-A; Peter, Hardi

    2016-01-01

    Context. The structure and heating of coronal loops are investigated since decades. Established scaling laws relate fundamental quantities like the loop apex temperature, pressure, length, and the coronal heating. Aims. We test such scaling laws against a large-scale 3D MHD model of the Solar corona, which became feasible with nowadays high-performance computing. Methods. We drive an active region simulation a with photospheric observations and found strong similarities to the observed coronal loops in X-rays and EUV wavelength. A 3D reconstruction of stereoscopic observations showed that our model loops have a realistic spatial structure. We compare scaling laws to our model data extracted along an ensemble of field lines. Finally, we fit a new scaling law that represents well hot loops and also cooler structures, which was not possible before only based on observations. Results. Our model data gives some support for scaling laws that were established for hot and EUV-emissive coronal loops. For the RTV scali...

  6. PDGF-induced phosphorylation of Tyr28 in the N-terminus of Fyn affects Fyn activation

    DEFF Research Database (Denmark)

    Hansen, Klaus; Alonso, G; Courtneidge, S A;

    1997-01-01

    Binding of platelet-derived growth factor (PDGF) to its receptors leads to the activation of members of the Src family of protein tyrosine kinases. We show here that Fyn, a member of the Src family, is phosphorylated on Tyr28 in the unique N-terminal part of the molecule after interaction...... with the intracellular domain of the PDGF beta-receptor. Activated Fyn furthermore undergoes autophosphorylation on Tyr30, Tyr39 and Tyr420. When Fyn mutants with Tyr28, Tyr30 or Tyr39 replaced with phenylalanine residues were transfected into NIH3T3 cells a decreased activation after PDGF stimulation was seen......, suggesting a functional importance of the N-terminal tyrosine phosphorylation of Fyn....

  7. Combination of PKCε Activation and PTP1B Inhibition Effectively Suppresses Aβ-Induced GSK-3β Activation and Tau Phosphorylation.

    Science.gov (United States)

    Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

    2016-09-01

    Glycogen synthase kinase-3β (GSK-3β) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer's disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3β by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3β. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3β than each independent treatment, to suppress amyloid β (Aβ)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy. PMID:26328540

  8. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    Science.gov (United States)

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

  9. Epithelial cell ADAM17 activation by Helicobacter pylori: role of ADAM17 C-terminus and Threonine-735 phosphorylation.

    Science.gov (United States)

    McClurg, Urszula L; Danjo, Kazuma; King, Harry O; Scott, Gina B; Robinson, Philip A; Crabtree, Jean E

    2015-03-01

    Helicobacter pylori transactivates the epidermal growth factor receptor (EGFR) on gastric epithelial cells via a signalling cascade involving a disintegrin and metalloprotease 17 (ADAM17) cleavage of membrane bound heparin binding-epidermal growth factor (HB-EGF). The effects of H. pylori on ADAM17 C-terminus in epithelial cells have been examined. Total cellular ADAM17 and surface expression of ADAM17 were significantly increased by H. pylori in AGS gastric epithelial cells. These changes were associated with ADAM17 C-terminal phosphorylation at T375 and S791. AGS cells lacking the ADAM17 C-terminal domain induced significantly attenuated cleavage of HB-EGF and were also unable to upregulate HB-EGF and EGFR transcripts to the same extent as cells expressing full length ADAM17. In mitotic unstimulated AGS and ADAM17 over-expressing AGS cells, ADAM17 was highly T735 phosphorylated indicating ADAM17 T735 phosphorylation is modified during the cell cycle. In conclusion, H. pylori induced ADAM17 C-terminal T735 and/or S791 phosphorylation in gastric epithelial cells are likely to be an important trigger inducing ADAM17 activation and shedding of HB-EGF leading to EGFR transactivation. ADAM17 over-expression in gastric cancer represents a potential target for therapeutic intervention.

  10. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

    Science.gov (United States)

    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  11. Pathogenesis of mucosal injury in the blind loop syndrome. Brush border enzyme activity and glycoprotein degradation.

    Science.gov (United States)

    Jonas, A; Flanagan, P R; Forstner, G G

    1977-12-01

    The effect of intestinal bacterial over-growth on brush border hydrolases and brush border glycoproteins was studied in nonoperated control rats, control rats with surgically introduced jejunal self-emptying blind loops, and rats with surgically introduced jejunal self-filling blind loops. Data were analyzed from blind loop segments, segments above and below the blind loops, and three corresponding segments in the nonoperated controls. Rats with self-filling blind loops had significantly greater fat excretion than controls and exhibited significantly lower conjugated:free bile salt ratios in all three segments. Maltase, sucrase, and lactase activities were significantly reduced in homogenates and isolated brush borders from the self-filling blind loop, but alkaline phosphatase was not affected. The relative degradation rate of homogenate and brush border glycoproteins was assessed by a double-isotope technique involving the injection of d-[6-(3)H]glucosamine 3 h and d-[U-(14)C]glucosamine 19 h before sacrifice, and recorded as a (3)H:(14)C ratio. The relative degradation rate in both homogenate and brush border fractions was significantly greater in most segments from rats with self-filling blind loops. In the upper and blind loop segments from rats with self-filling blind loops, the (3)H:(14)C ratios were higher in the brush border membrane than in the corresponding homogenates, indicating that the increased rates of degradation primarily involve membrane glycoproteins. Incorporation of d-[6-(3)H]glucosamine by brush border glycoproteins was not reduced in rats with self-filling blind loops, suggesting that glycoprotein synthesis was not affected. Polyacrylamide gel electrophoresis of brush border glycoproteins from the contaminated segments indicated that the large molecular weight glycoproteins, which include many of the surface hydrolases, were degraded most rapidly. Brush border maltase, isolated by immunoprecipitation, had (3)H:(14)C ratios characteristic of

  12. Regulation of Gβγi-dependent PLC-β3 activity in smooth muscle: inhibitory phosphorylation of PLC-β3 by PKA and PKG and stimulatory phosphorylation of Gαi-GTPase-activating protein RGS2 by PKG.

    Science.gov (United States)

    Nalli, Ancy D; Kumar, Divya P; Al-Shboul, Othman; Mahavadi, Sunila; Kuemmerle, John F; Grider, John R; Murthy, Karnam S

    2014-11-01

    In gastrointestinal smooth muscle, agonists that bind to Gi-coupled receptors activate preferentially PLC-β3 via Gβγ to stimulate phosphoinositide (PI) hydrolysis and generate inositol 1,4,5-trisphosphate (IP3) leading to IP3-dependent Ca(2+) release and muscle contraction. In the present study, we identified the mechanism of inhibition of PLC-β3-dependent PI hydrolysis by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). Cyclopentyl adenosine (CPA), an adenosine A1 receptor agonist, caused an increase in PI hydrolysis in a concentration-dependent fashion; stimulation was blocked by expression of the carboxyl-terminal sequence of GRK2(495-689), a Gβγ-scavenging peptide, or Gαi minigene but not Gαq minigene. Isoproterenol and S-nitrosoglutathione (GSNO) induced phosphorylation of PLC-β3 and inhibited CPA-induced PI hydrolysis, Ca(2+) release, and muscle contraction. The effect of isoproterenol on all three responses was inhibited by PKA inhibitor, myristoylated PKI, or AKAP inhibitor, Ht-31, whereas the effect of GSNO was selectively inhibited by PKG inhibitor, Rp-cGMPS. GSNO, but not isoproterenol, also phosphorylated Gαi-GTPase-activating protein, RGS2, and enhanced association of Gαi3-GTP and RGS2. The effect of GSNO on PI hydrolysis was partly reversed in cells (i) expressing constitutively active GTPase-resistant Gαi mutant (Q204L), (ii) phosphorylation-site-deficient RGS2 mutant (S46A/S64A), or (iii) siRNA for RGS2. We conclude that PKA and PKG inhibit Gβγi-dependent PLC-β3 activity by direct phosphorylation of PLC-β3. PKG, but not PKA, also inhibits PI hydrolysis indirectly by a mechanism involving phosphorylation of RGS2 and its association with Gαi-GTP. This allows RGS2 to accelerate Gαi-GTPase activity, enhance Gαβγi trimer formation, and inhibit Gβγi-dependent PLC-β3 activity.

  13. Peptides complementary to the active loop of porin P2 from Haemophilus influenzae modulate its activity

    Directory of Open Access Journals (Sweden)

    Galdiero S

    2012-05-01

    Full Text Available Marco Cantisani,1 Mariateresa Vitiello,2 Annarita Falanga,1 Emiliana Finamore,2 Marilena Galdiero,2 Stefania Galdiero11Department of Biological Sciences, CIRPeB and IBB CNR, University of Naples "Federico II," Napoli, Italy; 2Department of Experimental Medicine, II University of Naples, Napoli, ItalyAbstract: Haemophilus influenzae type b (Hib is one of the leading causes of invasive bacterial infection in young children. It is characterized by inflammation that is mainly mediated by cytokines and chemokines. One of the most abundant components of the Hib outer membrane is the P2 porin, which has been shown to induce the release of several inflammatory cytokines. A synthetic peptide corresponding to loop L7 of the porin activates JNK and p38 mitogen-activated protein kinase (MAPK pathways. We report a novel use of the complementary peptide approach to design a peptide that is able to bind selectively to the protein P2, thereby reducing its activity. This work provides insights into essential molecular details of P2 that may affect the pathogenesis of Hib infections where interruption of the signaling cascade could represent an attractive therapeutic strategy.Keywords: complementary-peptide, rational design, porin

  14. Tyrosine 601 of Bacillus subtilis DnaK Undergoes Phosphorylation and Is Crucial for Chaperone Activity and Heat Shock Survival.

    Science.gov (United States)

    Shi, Lei; Ravikumar, Vaishnavi; Derouiche, Abderahmane; Macek, Boris; Mijakovic, Ivan

    2016-01-01

    In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple Stable Isotope Labeling by Amino acids in Cell culture-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type (WT), were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the WT dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis. PMID:27148221

  15. Tyrosine 601 of Bacillus subtilis DnaK undergoes phosphorylation and is crucial for chaperone activity and heat shock survival

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2016-04-01

    Full Text Available In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple SILAC-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type, were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the wild type dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

  16. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C;

    2011-01-01

    PA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems......Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types......, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found...

  17. Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

    Directory of Open Access Journals (Sweden)

    Gomes Ronald R

    2010-11-01

    Full Text Available Abstract Background Immobilized recombinant perlecan domain I (PlnDI binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF165 enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro. Results In solution, PlnDI binds VEGF165 in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF165 mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF165 alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF165 reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951, and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF165 but not VEGF121 significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2. Conclusions Our observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF165 can enhance VEGFR-2 signaling and angiogenic events, in vitro. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, in vivo.

  18. Evidence of Impulsive Heating in Active Region Core Loops

    CERN Document Server

    Tripathi, Durgesh; Klimchuk, James A

    2010-01-01

    Using a full spectral scan of an active region from the Extreme-Ultraviolet Imaging Spectrometer (EIS) we have obtained Emission Measure EM$(T)$ distributions in two different moss regions within the same active region. We have compared these with theoretical transition region EMs derived for three limiting cases, namely \\textit{static equilibrium}, \\textit{strong condensation} and \\textit{strong evaporation} from \\cite{ebtel}. The EM distributions in both the moss regions are strikingly similar and show a monotonically increasing trend from $\\log T[\\mathrm{K}]=5.15 -6.3$. Using photospheric abundances we obtain a consistent EM distribution for all ions. Comparing the observed and theoretical EM distributions, we find that the observed EM distribution is best explained by the \\textit{strong condensation} case (EM$_{con}$), suggesting that a downward enthalpy flux plays an important and possibly dominant role in powering the transition region moss emission. The downflows could be due to unresolved coronal plas...

  19. Cholecystokinin (CCK) stimulates S6 phosphorylation and induced activation of S6 protein kinase in rat pancreatic acini

    Energy Technology Data Exchange (ETDEWEB)

    Sung, C.; Okabayashi, Y.; Williams, J.

    1987-05-01

    CCK and insulin stimulate pancreatic protein synthesis at a post transcriptional step. To better understand this regulation the authors evaluated the phosphorylation state of ribosomal protein S6 and the presence of a specific S6 protein kinase in pancreatic acini from diabetic rats. Both CCK and insulin increased S6 phosphorylation by up to 400% in intact TSP-labelled acini. The phorbol ester 12-0-tetradecanoylphorbol 13-acetate also stimulated both protein synthesis and S6 phosphorlyation suggesting a role for protein kinase C in mediating the effect of CCK. By contrast, the CaS ionophore ionomycin had no effect on either parameter. Recently, insulin has been shown to activate a unique S6 kinase in various cells. To test for its presence, cytosolic extracts were prepared from acini stimulated with CCK and insulin by homogenization in US -glycerophosphate buffer and assayed for the kinase using el-TSP ATP and rat pancreatic ribosomes followed by SDS-polyacrylamide gel electrophoresis. CCK and insulin both increased S6 kinase activity which required neither CaS or phospholipid. The dose response for CCk was similar to S6 phosphorlyation in the intact acini. TPA did not stimulate the S6 kinase. Thus, CCK may induce S6 phosphorylation both via C kinase and by activation of a unique S6 kinase.

  20. ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

    DEFF Research Database (Denmark)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper;

    2016-01-01

    substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (OSR1, HDGF and ccdc82) as ATM...

  1. Behaviour of oscillations in loop structures above active regions

    CERN Document Server

    Kolobov, D Y; Chelpanov, A A; Kochanov, A A; Anfinogentov, S A; Chupin, S A; Myshyakov, I I; Tomin, V E

    2015-01-01

    In this study we combine the multiwavelength ultraviolet -- optical (Solar Dynamics Observatory, SDO) and radio (Nobeyama Radioheliograph, NoRH) observations to get further insight into space-frequency distribution of oscillations at different atmospheric levels of the Sun. We processed the observational data on NOAA 11711 active region and found oscillations propagating from the photospheric level through the transition region upward into the corona. The power maps of low-frequency (1--2 mHz) oscillations reproduce well the fan-like coronal structures visible in the Fe ix 171A line. High frequency oscillations (5--7 mHz) propagate along the vertical magnetic field lines and concentrate inside small-scale elements in the umbra and at the umbra-penumbra boundary. We investigated the dependence of the dominant oscillation frequency upon the distance from the sunspot barycentre to estimate inclination of magnetic tubes in higher levels of sunspots where it cannot be measured directly, and found that this angle i...

  2. Mitogen-activated Protein Kinase (MAPK) Activated by Prostaglandin E2 Phosphorylates Connexin 43 and Closes Osteocytic Hemichannels in Response to Continuous Flow Shear Stress.

    Science.gov (United States)

    Riquelme, Manuel A; Burra, Sirisha; Kar, Rekha; Lampe, Paul D; Jiang, Jean X

    2015-11-20

    Cx43 hemichannels serve as a portal for the release of prostaglandins, a critical process in mediating biological responses of mechanical loading on bone formation and remodeling. We have previously observed that fluid flow shear stress (FFSS) opens hemichannels; however, sustained FFSS results in hemichannel closure, as continuous opening of hemichannels is detrimental to cell viability and bone remodeling. However, the mechanism that regulates the closure of the hemichannels is unknown. Here, we show that activation of p44/42 ERK upon continuous FFSS leads to Cx43 phosphorylation at Ser(279)-Ser(282), sites known to be phosphorylated sites by p44/42 MAPK. Incubation of osteocytic MLO-Y4 cells with conditioned media (CM) collected after continuous FFSS increased MAPK-dependent phosphorylation of Cx43. CM treatment inhibited hemichannel opening and this inhibition was reversed when cells were pretreated with the MAPK pathway inhibitor. We found that prostaglandin E2 (PGE2) accumulates in the CM in a time-dependent manner. Treatment with PGE2 increased phospho-p44/42 ERK levels and also Cx43 phosphorylation at Ser(279)-Ser(282) sites. Depletion of PGE2 from CM, and pre-treatment with a p44/42 ERK pathway-specific inhibitor, resulted in a complete inhibition of ERK-dependent Cx43 phosphorylation and attenuated the inhibition of hemichannels by CM and PGE2. Consistently, the opening of hemichannels by FFSS was blocked by PGE2 and CM and this blockage was reversed by U0126 and the CM depleted of PGE2. A similar observation was also obtained in isolated primary osteocytes. Together, results from this study suggest that extracellular PGE2 accumulated after continuous FFSS is responsible for activation of p44/42 ERK signaling and subsequently, direct Cx43 phosphorylation by activated ERK leads to hemichannel closure. PMID:26442583

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

    Science.gov (United States)

    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

  4. R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

    Science.gov (United States)

    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu

    2015-01-01

    R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling. PMID:27029009

  5. LRRK2 Kinase Activity and Biology are Not Uniformly Predicted by its Autophosphorylation and Cellular Phosphorylation Site Status

    Directory of Open Access Journals (Sweden)

    April eReynolds

    2014-06-01

    Full Text Available Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2 gene are the most common genetic predisposition to develop Parkinson’s disease (PD LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.

  6. A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization

    OpenAIRE

    Wilczynska, Malgorzata; Lobov, Sergei; Ohlsson, Per-Ingvar; Ny, Tor

    2003-01-01

    Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize....

  7. Determination of 16N and 19O activities in loop water of swimming pool reactor

    International Nuclear Information System (INIS)

    Measurements of activities for 16N and 19O nuclei in the loop water of swimming pool reactor at China Institute of Atomic Energy were carried out. In order to verify the experiment results, a calculation for same purpose was also performed. The results show their coincidence is well in uncertainty range. The evaluated recommendation data for 18O(n, γ)19O reaction cross sections are also given in the paper. (authors)

  8. TCR-Induced Akt Serine 473 Phosphorylation Is Regulated by Protein Kinase C-Alpha

    OpenAIRE

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

    2010-01-01

    Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser473 in the hydrophobic motif, along with Thr308 in its activation loop, is considered necessary for Akt function. It is widely accepted that Phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr308, but the kinase(s) responsible for phosphorylating Akt at Ser473 (PDK-2) remains elusive. The existence of PDK-2 is considered to be spe...

  9. Closed Loop Control of Active Damped Small DC-link Capacitor Based Drive

    DEFF Research Database (Denmark)

    Maheshwari, Ram Krishan; Munk-Nielsen, Stig

    2010-01-01

    A new method of active damping for small DC-link capacitor based drive system is implemented in stator flux oriented control for an induction machine. The active damping technique is based on a detailed model of the drive system which leads to a very simple implementation. The active damping can...... be achieved either by modifying the machine current reference or by modifying the machine voltage. The correlation between these two methods is shown by using simple analysis and it is verified by experimental results in a three phase induction machine drive. The effect of current control loop bandwidth...

  10. A closed-loop model of the respiratory system: focus on hypercapnia and active expiration.

    Directory of Open Access Journals (Sweden)

    Yaroslav I Molkov

    Full Text Available Breathing is a vital process providing the exchange of gases between the lungs and atmosphere. During quiet breathing, pumping air from the lungs is mostly performed by contraction of the diaphragm during inspiration, and muscle contraction during expiration does not play a significant role in ventilation. In contrast, during intense exercise or severe hypercapnia forced or active expiration occurs in which the abdominal "expiratory" muscles become actively involved in breathing. The mechanisms of this transition remain unknown. To study these mechanisms, we developed a computational model of the closed-loop respiratory system that describes the brainstem respiratory network controlling the pulmonary subsystem representing lung biomechanics and gas (O2 and CO2 exchange and transport. The lung subsystem provides two types of feedback to the neural subsystem: a mechanical one from pulmonary stretch receptors and a chemical one from central chemoreceptors. The neural component of the model simulates the respiratory network that includes several interacting respiratory neuron types within the Bötzinger and pre-Bötzinger complexes, as well as the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG representing the central chemoreception module targeted by chemical feedback. The RTN/pFRG compartment contains an independent neural generator that is activated at an increased CO2 level and controls the abdominal motor output. The lung volume is controlled by two pumps, a major one driven by the diaphragm and an additional one activated by abdominal muscles and involved in active expiration. The model represents the first attempt to model the transition from quiet breathing to breathing with active expiration. The model suggests that the closed-loop respiratory control system switches to active expiration via a quantal acceleration of expiratory activity, when increases in breathing rate and phrenic amplitude no longer provide sufficient

  11. Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts.

    Science.gov (United States)

    Klein, Janet D; Wang, Yanhua; Blount, Mitsi A; Molina, Patrick A; LaRocque, Lauren M; Ruiz, Joseph A; Sands, Jeff M

    2016-05-15

    Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations. PMID:26962099

  12. Gene expression profiles and phosphorylation patterns of AMP-activated protein kinase subunits in various mesenchymal cell types

    Institute of Scientific and Technical Information of China (English)

    Wang Yugang; Fan Qiming; Ma Rui; Lin Wentao; Tang Tingting

    2014-01-01

    Background Recent studies on bone have shown an endocrine role of the skeleton,which could be impaired in various human diseases,including osteoporosis,obesity,and diabetes-associated bone diseases.As a sensor and regulator of energy metabolism,AMP-activated protein kinase (AMPK) may also play an important role in the regulation of bone metabolism.The current study aimed to establish the expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types.Methods Reverse transcription-polymerase chain reaction (PCR) for relative quantification,real-time PCR for absolute quantification,and Western blotting were used to investigate the gene expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types,including primary human mesenchymal stem cells (hMSCs) and hFOB,Saos-2,C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells.Results AMPKα1 and AMPKβ1 mRNAs were abundantly expressed in all cell types.AMPKY1 mRNA was abundantly expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 but not detected in human-derived cell types.AMPKY2 mRNA was mildly expressed in all cell types.AMPKα1 protein was highly expressed in all cell types and AMPKα2 protein was highly expressed only in hFOB and Saos-2 cells.AMPKβ1 protein was abundantly expressed in all cell types except for Saos-2,in which AMPKβ2 protein overwhelmed AMPKβ1 expression.AMPKy1 and AMPKY2 proteins were expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells and only AMPKY2 protein was expressed in hMSCs,hFOB and Saos2 cells.AMPKα was phosphorylated at Thr172 and Ser485 and AMPKβ1 was phosphorylated at Ser108 and Ser182 in all cell types with a specific pattern in each cell type.Conclusion The combination of AMPK α,β,and Y subunits and phosphorylation of AMPKα (Thr172 and Ser485) and AMPKβ1 (Ser108 and Ser182) showed a specific pattern in each cell type.

  13. p38 mitogen-activated protein kinase up-regulates NF-{kappa}B transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Guoping [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China); Liu, Dongxu [Department of Orthodontics, College of Stomatology, Shandong University, Jinan, Shandong Province 250012 (China); Liu, Jing [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Gao, Hui [Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041 (China); Yuan, Xiao, E-mail: yuanxiaoqd@163.com [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Shen, Gang, E-mail: ganshen2007@163.com [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China)

    2010-01-01

    p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-{kappa}B in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-{kappa}B activation during myogenesis, not through down-regulation of degradation of I{kappa}B-{alpha}, and consequent translocation of the p65 subunit of NF-{kappa}B to the nucleus. It is likely that stretch-induced NF-{kappa}B activation by phosphorylation of p65 NF-{kappa}B. Moreover, depletion of p38{alpha} using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-{kappa}B activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-{kappa}B signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The {alpha} isoform of p38MAP kinase regulates the transcriptional activation of NF-{kappa}B following stimulation with cyclic stretch.

  14. p38 mitogen-activated protein kinase up-regulates NF-κB transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

    International Nuclear Information System (INIS)

    p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-κB activation during myogenesis, not through down-regulation of degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that stretch-induced NF-κB activation by phosphorylation of p65 NF-κB. Moreover, depletion of p38α using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-κB activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-κB signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The α isoform of p38MAP kinase regulates the transcriptional activation of NF-κB following stimulation with cyclic stretch.

  15. SATB1 packages densely-looped, transciptionally-active chromatinfor coordinated expression of cytokine genes

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Shutao; Lee, Charles C.; Kohwi-Shigematsu, Terumi

    2006-05-23

    SATB1 is an important regulator of nuclear architecture that anchors specialized DNA sequences onto its cage-like network and recruits chromatin remodeling/modifying factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4, and Il13 from the 200kb cytokine gene cluster region of mouse chromosome 11 during T-helper 2 (Th2)-cell activation. We show that upon cell activation, SATB1 is rapidly induced to form a unique transcriptionally-active chromatin structure that includes the cytokine gene region. Chromatin is folded into numerous small loops all anchored by SATB1, is histone H3 acetylated at lysine 9/14, and associated with Th2-specific factors, GATA3, STAT6, c-Maf, the chromatin-remodeling enzyme Brg-1, and RNA polymerase II across the 200kb region. Before activation, the chromatin displays some of these features, such as association with GATA3 and STAT6, but these were insufficient for cytokine gene expression. Using RNA interference (RNAi), we show that upon cell activation, SATB1 is not only required for chromatin folding into dense loops, but also for c-Maf induction and subsequently for Il4, Il5, and Il13 transcription. Our results show that SATB1 is an important determinant for chromatin architecture that constitutes a novel higher-order, transcriptionally-active chromatin structure upon Th2-cell activation.

  16. Sex differences in hormone-sensitive lipase expression, activity, and phosphorylation in skeletal muscle at rest and during exercise

    DEFF Research Database (Denmark)

    Roepstorff, Carsten; Donsmark, Morten; Thiele, Maja;

    2006-01-01

    Women have been shown to use more intramuscular triacylglycerol (IMTG) during exercise than men. To investigate whether this could be due to sex-specific regulation of hormone-sensitive lipase (HSL) and to use sex comparison as a model to gain further insight into HSL regulation, nine women...... and eight men performed bicycle exercise (90 min, 60% Vo(2peak)), and skeletal muscle HSL expression, phosphorylation, and activity were determined. Supporting previous findings, basal IMTG content (P ... significantly (r = 0.72, P = 0.001). Muscle HSL mRNA (80%, P = 0.11) and protein content (50%, P HSL total activity increased during exercise (47%, P HSL specific activity (HSL activity per HSL protein content...

  17. A novel photoelectrochemical biosensor for protein kinase activity assay based on phosphorylated graphite-like carbon nitride.

    Science.gov (United States)

    Li, Xue; Zhou, Yunlei; Xu, Yan; Xu, Huijie; Wang, Minghui; Yin, Huanshun; Ai, Shiyun

    2016-08-31

    Protein kinases are general and significant regulators in the cell signaling pathway, and it is still greatly desired to achieve simple and quick kinase detection. Herein, we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride (P-g-C3N4) conjugates triggered by Zr(4+) ion coordination. Under optimal conditions, the increased photocurrent is proportional to the protein kinase A (PKA) concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL. Moreover, this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition. The results indicated that the IC50 value (inhibitor concentration producing 50% inhibitor) for ellagic acid was 9.1 μM. Moreover, the developed method is further applied to detect PKA activity in real samples, which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients. Therefore, the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity.

  18. Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle.

    Science.gov (United States)

    Patel, Anant B; Lai, James C K; Chowdhury, Golam M I; Hyder, Fahmeed; Rothman, Douglas L; Shulman, Robert G; Behar, Kevin L

    2014-04-01

    Previous (13)C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-D-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG6P), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions.

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

    Science.gov (United States)

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

    2012-10-01

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

  20. 'Up with the LRRK': a phosphorylated Rab10 assay for evaluation of LRRK2 activity and inhibitor engagement.

    Science.gov (United States)

    Eyers, Patrick A

    2016-09-15

    Protein kinases catalyse the addition of phosphate groups to Ser/Thr and Tyr residues in cognate substrates and are mutated or hyperactive in a variety of diseases, making them important targets for rationally designed drugs. A good example is the Parkinson's disease-associated kinase, leucine-rich repeat kinase 2 (LRRK2), which is mutated (and probably hyperactive) in a small, but significant, subset of patients. An exciting new approach for personalised therapy is the development of central nervous system (CNS)-active small-molecule kinase inhibitors, which could be employed to 'normalise' LRRK2 signalling in affected cell types. However, the development of such drugs requires validated assays for the analysis of target engagement and the assembly of a set of tools for interrogating LRRK2, and its substrates, both in vitro and in vivo A new study published in the Biochemical Journal by Ito et al. establishes that a 'Phos-tag'™-binding assay can be exploited to measure phosphorylation of a recently identified LRRK2 substrate (Ras-related protein in brain 10 (Rab10)), and to compare and contrast relative catalytic output from disease-associated LRRK2 mutants. Powerful in vivo chemical genetic approaches are also disclosed, in which the catalytic activity of LRRK2 is unequivocally linked to the extent of Rab10 phosphorylation and the effects of chemically distinct LRRK2 inhibitors are matched with on-target inhibition mechanisms mediated through LRRK2 and its substrate Rab10. These important findings should simplify the generic analysis of Rab10 phosphorylation in model biological systems and are likely to be applicable to other substrates of LRRK2 (or indeed other kinases) for which phospho-specific antibodies are either absent or unsatisfactory. PMID:27621483

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

    Science.gov (United States)

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

    2015-05-10

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expression of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinase(T172) [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  2. Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.

    Directory of Open Access Journals (Sweden)

    Carla Manuela Abreu

    2013-04-01

    Full Text Available The mediators of the DNA damage response (DDR are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR-specific protein kinases.

  3. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Yong-Whan [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Yoon, Seung-Yong, E-mail: ysy@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Choi, Jung-Eun [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Sang-Min [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Hui-Sun; Choe, Han [Department of Physiology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Seung-Chul [CrystalGenomics, Seoul (Korea, Republic of); Kim, Dong-Hou, E-mail: dhkim@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  4. Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression

    OpenAIRE

    Tawfeek, Hesham A.; Abou-Samra, Abdul B.

    2012-01-01

    Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, β-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Sh...

  5. The plastid casein kinase 2 phosphorylates Rubisco activase at the Thr-78 site but is not essential for regulation of Rubisco activation state

    Directory of Open Access Journals (Sweden)

    Sang Yeol eKim

    2016-03-01

    Full Text Available Rubisco activase (RCA is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730. The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78 has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2 and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

  6. Diphenylarsinic Acid Induced Activation of Cultured Rat Cerebellar Astrocytes: Phosphorylation of Mitogen-Activated Protein Kinases, Upregulation of Transcription Factors, and Release of Brain-Active Cytokines.

    Science.gov (United States)

    Negishi, Takayuki; Matsumoto, Mami; Kojima, Mikiya; Asai, Ryota; Kanehira, Tomoko; Sakaguchi, Fumika; Takahata, Kazuaki; Arakaki, Rina; Aoyama, Yohei; Yoshida, Hikari; Yoshida, Kenji; Yukawa, Kazunori; Tashiro, Tomoko; Hirano, Seishiro

    2016-03-01

    Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow.

  7. Diphenylarsinic Acid Induced Activation of Cultured Rat Cerebellar Astrocytes: Phosphorylation of Mitogen-Activated Protein Kinases, Upregulation of Transcription Factors, and Release of Brain-Active Cytokines.

    Science.gov (United States)

    Negishi, Takayuki; Matsumoto, Mami; Kojima, Mikiya; Asai, Ryota; Kanehira, Tomoko; Sakaguchi, Fumika; Takahata, Kazuaki; Arakaki, Rina; Aoyama, Yohei; Yoshida, Hikari; Yoshida, Kenji; Yukawa, Kazunori; Tashiro, Tomoko; Hirano, Seishiro

    2016-03-01

    Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow. PMID:26645585

  8. Analyzing phosphorylation-dependent regulation of subcellular localization and transcriptional activity of transcriptional coactivator NT-PGC-1α.

    Science.gov (United States)

    Chang, Ji Suk; Gettys, Thomas W

    2013-01-01

    Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is a nuclear transcriptional coactivator that regulates the genes involved in energy metabolism. Recent evidence has been provided that alternative splicing of PPARGC1A gene produces a functional but predominantly cytosolic isoform of PGC-1α (NT-PGC-1α). We have demonstrated that transcriptional coactivation capacity of NT-PGC-1α is directly correlated with its nuclear localization in a PKA phosphorylation-dependent manner. In this chapter, we describe quantitative imaging analysis methods that are developed to measure the relative fluorescence intensity of the protein of interest in the nucleus and cytoplasm in a single cell and the frequency distribution of nuclear/cytoplasmic intensity ratios in the population of cells, respectively. This chapter also describes transient cotransfection and dual-luciferase reporter gene assay that examine the ability of coactivators to activate the transcriptional activity of transcription factors.

  9. c-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Diana M. Dunn

    2015-08-01

    Full Text Available The ability of Heat Shock Protein 90 (Hsp90 to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1, promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo.

  10. Eriocalyxin B Inhibits STAT3 Signaling by Covalently Targeting STAT3 and Blocking Phosphorylation and Activation of STAT3.

    Directory of Open Access Journals (Sweden)

    Xiaokui Yu

    Full Text Available Activated STAT3 plays an important role in oncogenesis by stimulating cell proliferation and resisting apoptosis. STAT3 therefore is an attractive target for cancer therapy. We have screened a traditional Chinese herb medicine compound library and found Eriocalyxin B (EB, a diterpenoid from Isodon eriocalyx, as a specific inhibitor of STAT3. EB selectively inhibited constitutive as well as IL-6-induced phosphorylation of STAT3 and induced apoptosis of STAT3-dependent tumor cells. EB did not affect the upstream protein tyrosine kinases or the phosphatase (PTPase of STAT3, but rather interacted directly with STAT3. The effects of EB could be abolished by DTT or GSH, suggesting a thiol-mediated covalent linkage between EB and STAT3. Site mutagenesis of cysteine in and near the SH2 domain of STAT3 identified Cys712 to be the critical amino acid for the EB-induced inactivation of STAT3. Furthermore, LC/MS/MS analyses demonstrated that an α, β-unsaturated carbonyl of EB covalently interacted with the Cys712 of STAT3. Computational modeling analyses also supported a direct interaction between EB and the Cys712 of STAT3. These data strongly suggest that EB directly targets STAT3 through a covalent linkage to inhibit the phosphorylation and activation of STAT3 and induces apoptosis of STAT3-dependent tumor cells.

  11. Trichothecin induces cell death in NF-κB constitutively activated human cancer cells via inhibition of IKKβ phosphorylation.

    Directory of Open Access Journals (Sweden)

    Jia Su

    Full Text Available Constitutive activation of the transcription factor nuclear factor-κB (NF-κB is involved in tumorigenesis and chemo-resistance. As the key regulator of NF-κB, IKKβ is a major therapeutic target for various cancers. Trichothecin (TCN is a metabolite isolated from an endophytic fungus of the herbal plant Maytenus hookeri Loes. In this study, we evaluated the anti-tumor activity of TCN and found that TCN markedly inhibits the growth of cancer cells with constitutively activated NF-κB. TCN induces G0/G1 cell cycle arrest and apoptosis in cancer cells, activating pro-apoptotic proteins, including caspase-3, -8 and PARP-1, and decreasing the expression of anti-apoptotic proteins Bcl-2, Bcl-xL, and survivin. Reporter activity assay and target genes expression analysis illustrated that TCN works as a potent inhibitor of the NF-κB signaling pathway. TCN inhibits the phosphorylation and degradation of IκBα and blocks the nuclear translocation of p65, and thus inhibits the expression of NF-κB target genes XIAP, cyclin D1, and Bcl-xL. Though TCN does not directly interfere with IKKβ kinase, it suppresses the phosphorylation of IKKβ. Overexpression of constitutively activated IKKβ aborted TCN induced cancer cell apoptosis, whereas knockdown of endogenous IKKβ with siRNA sensitized cancer cells toward apoptosis induced by TCN. Moreover, TCN showed a markedly weaker effect on normal cells. These findings suggest that TCN may be a potential therapeutic candidate for cancer treatment, targeting NF-κB signaling.

  12. Suppressive effect of CORM-2 on LPS-induced platelet activation by glycoprotein mediated HS1 phosphorylation interference.

    Directory of Open Access Journals (Sweden)

    Dadong Liu

    Full Text Available In recent years, it has been discovered that septic patients display coagulation abnormalities. Platelets play a major role in the coagulation system. Studies have confirmed that carbon monoxide (CO has important cytoprotective and anti-inflammatory function. However, whether CO could alter abnormal activation of platelets and coagulation and thereby reduce the incidence of mortality during sepsis has not been defined. In this report, we have used CO-releasing molecules (CORM-2 to determine whether CO inhibits LPS-induced abnormal activation of platelets and have explored the potential mechanisms. LPS was used to induce activation of platelets in vitro, which were purified from the peripheral venous blood of healthy adult donors. CORM-2 was applied as a potential therapeutic agent. CORM-2 preconditioning and delayed treatment were also studied. We found that in the LPS groups, the function of platelets such as spreading, aggregation, and release were enhanced abnormally. By contrast, the platelets in the CORM-2 group were gently activated. Further studies showed that the expression of platelet membrane glycoproteins increased in the LPS group. Coincidently, both hematopoietic lineage cell-specific protein 1 and its phosphorylated form also increased dramatically. These phenomena were less dramatically seen in the CORM-2 groups. Taken together, we conclude that during LPS stimulation, platelets were abnormally activated, and this functional state may be associated with the signal that is transmitted between membrane glycoproteins and HS1. CORM-released CO suppresses the abnormal activation of platelets by interfering with glycoprotein-mediated HS1 phosphorylation.

  13. DOUBLE LOOP ACTIVE VIBRATION CONTROL OF PNEUMATIC ISOLATOR WITH TWO SEPARATE CHAMBERS

    Institute of Scientific and Technical Information of China (English)

    YANG Qingjun; LI Jun; WANG Zuwen

    2006-01-01

    A newly designed pneumatic spring with two separate chambers is promoted and double-loop active control is introduced to overcome the following drawbacks of passive pneumatic level with different force load. The design of two separate chambers is for the purpose of tuning support frequency and force independently and each chamber is controlled by a different valve. The inner one of double-loop structure is pressure control, and in order to obtain good performance,nonlinearities compensation and motion flow rate compensation (MFRC) are added besides the basic cascade compensation, and the influence of tube length is studied. The outer loop has two functions:one is to eliminate the resonance caused by isolation support and to broaden the isolation frequency band by payload velocity feedback and base velocity feed forward, and the other is to rune support force and support stiffness simultaneously and independently, which means the support force will have no effect on support stiffness. Theoretical analysis and experiment results show that the three drawbacks are overcome simultaneously.

  14. Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion.

    OpenAIRE

    Kawakami, Y; Nishimoto, H; Kitaura, J.; Maeda-Yamamoto, M.; Kato, R.; Littman, D; Leitges, M.; Rawlings, D; Kawakami, T

    2004-01-01

    Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phospho...

  15. Cyclin H binding to the RARα activation function (AF)-2 domain directs phosphorylation of the AF-1 domain by cyclin-dependent kinase 7

    OpenAIRE

    Bour, Gaétan; Gaillard, Emilie; Bruck, Nathalie; Lalevée, Sébastien; Plassat, Jean-Luc; Busso, Didier; Samama, Jean-Pierre; Rochette-Egly, Cécile

    2005-01-01

    The transcriptional activity of nuclear retinoic acid receptors (RARs), which act as RAR/retinoid X receptor (RXR) heterodimers, depends on two activation functions, AF-1 and AF-2, which are targets for phosphorylations and synergize for the activation of retinoic acid target genes. The N-terminal AF-1 domain of RARα is phosphorylated at S77 by the cyclin-dependent kinase (cdk)-activating kinase (CAK) subcomplex (cdk7/cyclin H/MAT1) of the general transcription factor TFIIH. Here, we show tha...

  16. CK1δ activity is modulated by CDK2/E- and CDK5/p35-mediated phosphorylation.

    Science.gov (United States)

    Ianes, Chiara; Xu, Pengfei; Werz, Natalie; Meng, Zhigang; Henne-Bruns, Doris; Bischof, Joachim; Knippschild, Uwe

    2016-02-01

    CK1 protein kinases form a family of serine/threonine kinases which are highly conserved through different species and ubiquitously expressed. CK1 family members can phosphorylate numerous substrates thereby regulating different biological processes including membrane trafficking, cell cycle regulation, circadian rhythm, apoptosis, and signal transduction. Deregulation of CK1 activity and/or expression contributes to the development of neurological diseases and cancer. Therefore, CK1 became an interesting target for drug development and it is relevant to further understand the mechanisms of its regulation. In the present study, Cyclin-dependent kinase 2/Cyclin E (CDK2/E) and Cyclin-dependent kinase 5/p35 (CDK5/p35) were identified as cellular kinases able to modulate CK1δ activity through site-specific phosphorylation of its C-terminal domain. Furthermore, pre-incubation of CK1δ with CDK2/E or CDK5/p35 reduces CK1δ activity in vitro, indicating a functional impact of the interaction between CK1δ and CDK/cyclin complexes. Interestingly, inhibition of Cyclin-dependent kinases by Dinaciclib increases CK1δ activity in pancreatic cancer cells. In summary, these results suggest that CK1δ activity can be modulated by the interplay between CK1δ and CDK2/E or CDK5/p35. These findings extend our knowledge about CK1δ regulation and may be of use for future development of CK1-related therapeutic strategies in the treatment of neurological diseases or cancer. PMID:26464264

  17. Adiponectin Upregulates MiR-133a in Cardiac Hypertrophy through AMPK Activation and Reduced ERK1/2 Phosphorylation.

    Directory of Open Access Journals (Sweden)

    Ying Li

    Full Text Available Adiponectin and miR-133a are key regulators in cardiac hypertrophy. However, whether APN has a potential effect on miR-133a remains unclear. In this study, we aimed to investigate whether APN could regulate miR-133a expression in Angiotensin II (Ang II induced cardiac hypertrophy in vivo and in vitro. Lentiviral-mediated adiponectin treatment attenuated cardiac hypertrophy induced by Ang II infusion in male wistar rats as determined by reduced cell surface area and mRNA levels of atrial natriuretic peptide (ANF and brain natriuretic peptide (BNP, also the reduced left ventricular end-diastolic posterior wall thickness (LVPWd and end-diastolic interventricular septal thickness (IVSd. Meanwhile, APN elevated miR-133a level which was downregulated by Ang II. To further investigate the underlying molecular mechanisms, we treated neonatal rat ventricular myocytes (NRVMs with recombinant rat APN before Ang II stimulation. Pretreating cells with recombinant APN promoted AMP-activated protein kinase (AMPK phosphorylation and inhibited ERK activation. By using the inhibitor of AMPK or a lentiviral vector expressing AMPK short hairpin RNA (shRNA cancelled the positive effect of APN on miR-133a. The ERK inhibitor PD98059 reversed the downregulation of miR-133a induced by Ang II. These results indicated that the AMPK activation and ERK inhibition were responsible for the positive effect of APN on miR-133a. Furthermore, adiponectin receptor 1 (AdipoR1 mRNA expression was inhibited by Ang II stimulation. The positive effects of APN on AMPK activation and miR-133a, and the inhibitory effect on ERK phosphorylation were inhibited in NRVMs transfected with lentiviral AdipoR1shRNA. In addition, APN depressed the elevated expression of connective tissue growth factor (CTGF, a direct target of miR-133a, through the AMPK pathway. Taken together, our data indicated that APN reversed miR-133a levels through AMPK activation, reduced ERK1/2 phosphorylation in

  18. The role of mitochondrial fusion and StAR phosphorylation in the regulation of StAR activity and steroidogenesis.

    Science.gov (United States)

    Castillo, Ana F; Orlando, Ulises; Helfenberger, Katia E; Poderoso, Cecilia; Podesta, Ernesto J

    2015-06-15

    The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroidogenesis, i.e. the delivery of cholesterol from the outer (OMM) to the inner (IMM) mitochondrial membrane. StAR is a 37-kDa protein with an N-terminal mitochondrial targeting sequence that is cleaved off during mitochondrial import to yield 30-kDa intramitochondrial StAR. StAR acts exclusively on the OMM and its activity is proportional to how long it remains on the OMM. However, the precise fashion and the molecular mechanism in which StAR remains on the OMM have not been elucidated yet. In this work we will discuss the role of mitochondrial fusion and StAR phosphorylation by the extracellular signal-regulated kinases 1/2 (ERK1/2) as part of the mechanism that regulates StAR retention on the OMM and activity.

  19. Active roll control: system design and hardware-in-the-loop test bench

    OpenAIRE

    A. Sorniotti; Morgando, A; Velardocchia, M

    2006-01-01

    The first part of the article describes the targets related to the design of an Active Roll Control (ARC) system, based on the hydraulic actuation of the anti-roll bars of an automobile. Then the basic static and dynamic design principles of the system are commented upon in detail. The second part of the article presents the hardware-in-the-loop test bench implemented to evaluate the designed system. In the end, the main experimental results are summarized and discussed from the point of view...

  20. Pin1-mediated Sp1 phosphorylation by CDK1 increases Sp1 stability and decreases its DNA-binding activity during mitosis.

    Science.gov (United States)

    Yang, Hang-Che; Chuang, Jian-Ying; Jeng, Wen-Yih; Liu, Chia-I; Wang, Andrew H-J; Lu, Pei-Jung; Chang, Wen-Chang; Hung, Jan-Jong

    2014-12-16

    We have shown that Sp1 phosphorylation at Thr739 decreases its DNA-binding activity. In this study, we found that phosphorylation of Sp1 at Thr739 alone is necessary, but not sufficient for the inhibition of its DNA-binding activity during mitosis. We demonstrated that Pin1 could be recruited to the Thr739(p)-Pro motif of Sp1 to modulate the interaction between phospho-Sp1 and CDK1, thereby facilitating CDK1-mediated phosphorylation of Sp1 at Ser720, Thr723 and Thr737 during mitosis. Loss of the C-terminal end of Sp1 (amino acids 741-785) significantly increased Sp1 phosphorylation, implying that the C-terminus inhibits CDK1-mediated Sp1 phosphorylation. Binding analysis of Sp1 peptides to Pin1 by isothermal titration calorimetry indicated that Pin1 interacts with Thr739(p)-Sp1 peptide but not with Thr739-Sp1 peptide. X-ray crystallography data showed that the Thr739(p)-Sp1 peptide occupies the active site of Pin1. Increased Sp1 phosphorylation by CDK1 during mitosis not only stabilized Sp1 levels by decreasing interaction with ubiquitin E3-ligase RNF4 but also caused Sp1 to move out of the chromosomes completely by decreasing its DNA-binding activity, thereby facilitating cell cycle progression. Thus, Pin1-mediated conformational changes in the C-terminal region of Sp1 are critical for increased CDK1-mediated Sp1 phosphorylation to facilitate cell cycle progression during mitosis.

  1. Elevation of cortical serotonin transporter activity upon peripheral immune challenge is regulated independently of p38 mitogen-activated protein kinase activation and transporter phosphorylation.

    Science.gov (United States)

    Schwamborn, Robert; Brown, Eric; Haase, Jana

    2016-05-01

    The serotonin transporter (SERT) is responsible for high-affinity serotonin (5-HT) uptake from extracellular fluid and is a prominent pharmacological target in the treatment of depression. In recent years, depression has also been linked to immune system activation. Inflammatory conditions can cause sickness behaviour and depression-like symptoms in both animals and humans. Since SERT has been proposed as one of the molecular targets in inflammation-induced depression, we applied the widely used lipopolysaccharides (LPS) model to study the effects of peripheral inflammation on SERT activity in the brain. We show that 24 h after intraperitoneal LPS administration, SERT-mediated 5-HT uptake is significantly enhanced in the frontal cortex. Analysis of uptake kinetics revealed that the transport capacity (Vmax ) of cortical SERT was increased in LPS-injected animals, while the Km value remained unchanged. The increase in Vmax was neither due to increased SERT protein expression nor increased synaptic surface exposure. The suppression of SERT activity upon inhibition of p38 MAPK was not selective for LPS-induced enhancement of SERT function. In addition, SERT activity changes in LPS-treated rats are unaffected by nitric oxide synthase and protein kinase G inhibitors. Using the Phos-Tag method, we identified five SERT-specific protein bands representing distinct phosphorylation states of SERT. However, the enhancement of SERT activity in LPS-treated rats was not correlated with altered transporter phosphorylation. Together with previous studies by others, our results suggest that SERT is regulated by multiple mechanisms in response to peripheral immune system activation. Peripheral injection of lipopolysaccharide (LPS) induces characteristic sickness and depression-like behaviour in rats over a period of at least 24 h. We show here that the activity of the serotonin transporter (SERT), a prominent antidepressant target, is up-regulated 24 h following LPS

  2. Novel STAT3 phosphorylation inhibitors exhibit potent growth-suppressive activity in pancreatic and breast cancer cells.

    Science.gov (United States)

    Lin, Li; Hutzen, Brian; Zuo, Mingxin; Ball, Sarah; Deangelis, Stephanie; Foust, Elizabeth; Pandit, Bulbul; Ihnat, Michael A; Shenoy, Satyendra S; Kulp, Samuel; Li, Pui-Kai; Li, Chenglong; Fuchs, James; Lin, Jiayuh

    2010-03-15

    The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently detected in most types of human cancer where it plays important roles in survival, drug resistance, angiogenesis, and other functions. Targeting constitutive STAT3 signaling is thus an attractive therapeutic approach for these cancers. We have recently developed novel small-molecule STAT3 inhibitors, known as FLLL31 and FLLL32, which are derived from curcumin (the primary bioactive compound of turmeric). These compounds are designed to bind selectively to Janus kinase 2 and the STAT3 Src homology-2 domain, which serve crucial roles in STAT3 dimerization and signal transduction. Here we show that FLLL31 and FLLL32 are effective inhibitors of STAT3 phosphorylation, DNA-binding activity, and transactivation in vitro, leading to the impediment of multiple oncogenic processes and the induction of apoptosis in pancreatic and breast cancer cell lines. FLLL31 and FLLL32 also inhibit colony formation in soft agar and cell invasion and exhibit synergy with the anticancer drug doxorubicin against breast cancer cells. In addition, we show that FLLL32 can inhibit the induction of STAT3 phosphorylation by IFNalpha and interleukin-6 in breast cancer cells. We also show that administration of FLLL32 can inhibit tumor growth and vascularity in chicken embryo xenografts as well as substantially reduce tumor volumes in mouse xenografts. Our findings highlight the potential of these new compounds and their efficacy in targeting pancreatic and breast cancers that exhibit constitutive STAT3 signaling. PMID:20215512

  3. Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation

    Institute of Scientific and Technical Information of China (English)

    Jian LIU; Si-tu YANG; Lang BU; Jing-ping OU-YANG; Jing-fang ZHANG; Jin-zhi LU; De-ling ZHANG; Ke LI; Ke SU; Jing WANG; Ye-min ZHANG; Nian WANG

    2013-01-01

    Aim:To establish the mechanism responsible for the stimulation of glucose uptake by Astragalus polysaccharide (APS),extracted from Astragalus membranaceus Bunge,in L6 myotubes in vitro.Methods:APS-stimulated glucose uptake in L6 myotubes was measured using the 2-deoxy-[3H]-D-glucose method.The adenine nucleotide contents in the cells were measured by HPLC.The phosphorylation of AMP-activated protein kinase (AMPK) and Akt substrate of 160 kDa (AS160) was examined using Western blot analysis.The cells transfected with 4P mutant AS160 (AS160-4P) were constructed using gene transfer approach.Results:Treatment of L6 myotubes with APS (100-1600 μg/mL) significantly increased glucose uptake in time-and concentration-dependent manners.The maximal glucose uptake was reached in the cells treated with APS (400 μg/mL) for 36 h.The APS-stimulated glucose uptake was significantly attenuated by pretreatment with Compound C,a selective AMPK inhibitor or in the cells overexpressing AS160-4P.Treatment of L6 myotubes with APS strongly promoted the activation of AMPK.We further demonstrated that either Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) or liver kinase B1 (LKB1) mediated APS-induced activation of AMPK in L6 myotubes,and the increased cellular AMP:ATP ratio was also involved.Treatment of L6 myotubes with APS robustly enhanced the phosphorylation of AS160,which was significantly attenuated by pretreatment with Compound C.Conclusion:Our results demonstrate that APS stimulates glucose uptake in L6 myotubes through the AMP-AMPK-AS160 pathway,which may contribute to its hypoglycemic effect.

  4. Asymmetry of the Active Site Loop Conformation between Subunits of Glutamate-1-semialdehyde Aminomutase in Solution

    Directory of Open Access Journals (Sweden)

    Barbara Campanini

    2013-01-01

    Full Text Available Glutamate-1-semialdehyde aminomutase (GSAM is a dimeric, pyridoxal 5′-phosphate (PLP- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5′-phosphate (PMP to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

  5. COX-2 activation is associated with Akt phosphorylation and poor survival in ER-negative, HER2-positive breast cancer

    Directory of Open Access Journals (Sweden)

    Goodman Julie E

    2010-11-01

    Full Text Available Abstract Background Inducible cyclooxgenase-2 (COX-2 is commonly overexpressed in breast tumors and is a target for cancer therapy. Here, we studied the association of COX-2 with breast cancer survival and how this association is influenced by tumor estrogen and HER2 receptor status and Akt pathway activation. Methods Tumor COX-2, HER2 and estrogen receptor α (ER expression and phosphorylation of Akt, BAD, and caspase-9 were analyzed immunohistochemically in 248 cases of breast cancer. Spearman's correlation and multivariable logistic regression analyses were used to examine the relationship between COX-2 and tumor characteristics. Kaplan-Meier survival and multivariable Cox proportional hazards regression analyses were used to examine the relationship between COX-2 and disease-specific survival. Results COX-2 was significantly associated with breast cancer outcome in ER-negative [Hazard ratio (HR = 2.72; 95% confidence interval (CI, 1.36-5.41; comparing high versus low COX-2] and HER2 overexpressing breast cancer (HR = 2.84; 95% CI, 1.07-7.52. However, the hazard of poor survival associated with increased COX-2 was highest among patients who were both ER-negative and HER2-positive (HR = 5.95; 95% CI, 1.01-34.9. Notably, COX-2 expression in the ER-negative and HER2-positive tumors correlated significantly with increased phosphorylation of Akt and of the two Akt targets, BAD at Ser136 and caspase-9 at Ser196. Conclusions Up-regulation of COX-2 in ER-negative and HER2-positive breast tumors is associated with Akt pathway activation and is a marker of poor outcome. The findings suggest that COX-2-specific inhibitors and inhibitors of the Akt pathway may act synergistically as anticancer drugs in the ER-negative and HER2-positive breast cancer subtype.

  6. AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of β-catenin at Ser 552

    International Nuclear Information System (INIS)

    AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; its activity is regulated by a plethora of physiological conditions, exercises and many anti-diabetic drugs. Recent studies show that AMPK involves in cell differentiation but the underlying mechanism remains undefined. Wingless Int-1 (Wnt)/β-catenin signaling pathway regulates the differentiation of mesenchymal stem cells through enhancing β-catenin/T-cell transcription factor 1 (TCF) mediated transcription. The objective of this study was to determine whether AMPK cross-talks with Wnt/β-catenin signaling through phosphorylation of β-catenin. C3H10T1/2 mesenchymal cells were used. Chemical inhibition of AMPK and the expression of a dominant negative AMPK decreased phosphorylation of β-catenin at Ser 552. The β-catenin/TCF mediated transcription was correlated with AMPK activity. In vitro, pure AMPK phosphorylated β-catenin at Ser 552 and the mutation of Ser 552 to Ala prevented such phosphorylation, which was further confirmed using [γ-32P]ATP autoradiography. In conclusion, AMPK phosphorylates β-catenin at Ser 552, which stabilizes β-catenin, enhances β-catenin/TCF mediated transcription, expanding AMPK from regulation of energy metabolism to cell differentiation and development via cross-talking with the Wnt/β-catenin signaling pathway.

  7. Mutations in the catalytic loop HRD motif alter the activity and function of Drosophila Src64.

    Directory of Open Access Journals (Sweden)

    Taylor C Strong

    Full Text Available The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele.

  8. Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation

    OpenAIRE

    Mahajan, Nupam P.; Liu, Yuanbo; Majumder, Samarpan; Warren, Maria R.; Parker, Carol E.; Mohler, James L.; Earp, H. Shelton; Whang, Young E.

    2007-01-01

    Activation of the androgen receptor (AR) may play a role in androgen-independent progression of prostate cancer. Multiple mechanisms of AR activation, including stimulation by tyrosine kinases, have been postulated. We and others have recently shown involvement of activated Cdc42-associated tyrosine kinase Ack1 in advanced human prostate cancer. Here we provide the molecular basis for interplay between Ack1 and AR in prostate cancer cells. Activated Ack1 promoted androgen-independent growth o...

  9. Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus.

    Science.gov (United States)

    Kim, Yea Woon; Lee, Sungkung; Yun, Jangmi; Kim, AeRi

    2015-03-18

    Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer-promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them.

  10. cAMP-dependent Protein Kinase Phosphorylation Produces Interdomain Movement in SUR2B Leading to Activation of the Vascular KATP Channel*S⃞

    OpenAIRE

    Shi, Yun; Chen, Xianfeng; Wu, Zhongying; Shi, Weiwei; Yang, Yang; Cui, Ningren; Jiang, Chun; Harrison, Robert W.

    2008-01-01

    Vascular ATP-sensitive K+ channels are activated by multiple vasodilating hormones and neurotransmitters via PKA. A critical PKA phosphorylation site (Ser-1387) is found in the second nucleotide-binding domain (NBD2) of the SUR2B subunit. To understand how phosphorylation at Ser-1387 leads to changes in channel activity, we modeled the SUR2B using a newly crystallized ABC protein SAV1866. The model showed that Ser-1387 was located on the interface of NBD2 with TMD1 and...

  11. Platelet-activating factor (PAF)-dependent biochemical, morphologic, and physiologic responses of human platelets: Demonstration of translocation of protein kinase C associated with protein phosphorylation

    International Nuclear Information System (INIS)

    Platelet-activating factor (PAF) is a potent stimulus for platelet aggregation and secretion. PAF has been shown to stimulate the phosphatidylinositol (PI) pathway in platelets, which implies that PAF should activate protein kinase C. In this study, measurements of PI metabolites, the elevation of intracellular free calcium concentration, (Ca2+)i, the activation of protein kinase C, and the phosphorylation of platelet proteins (using a two-dimensional gel electrophoretic technique) were performed before and after the addition of 10(-8) M PAF to human platelets. These findings were correlated with morphologic changes in the platelets as determined by immunoelectron microscopic studies on the cytoskeleton and by X-ray analysis of dense bodies. The results show that PAF stimulates the production of PI metabolites and causes an increase in the membrane-associated activity of protein kinase C. These changes are accompanied by a rise in the (Ca2+)i and protein phosphorylation. The increase in protein kinase C activity reaches a maximum at approximately 60 s, a time frame that is consistent with the protein phosphorylation and the subsequent morphologic and secretory events. X-ray analysis revealed two types of dense bodies containing various amounts of calcium which appeared to be released sequentially after PAF activation. These results suggest that the protein phosphorylation that controls the physiologic events resulting from PAF activation of human platelets is catalyzed by protein kinase C

  12. 17β-estradiol rapidly activates calcium release from intracellular stores via the GPR30 pathway and MAPK phosphorylation in osteocyte-like MLO-Y4 cells

    KAUST Repository

    Ren, Jian

    2012-03-06

    Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E 2) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E 2 elevated [Ca 2+] i and increased Ca 2+ oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E 2 mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E 2 activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E 2 induces the non-genomic responses Ca 2+ release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E 2 responses. © 2012 Springer Science+Business Media, LLC.

  13. 17β-estradiol rapidly activates calcium release from intracellular stores via the GPR30 pathway and MAPK phosphorylation in osteocyte-like MLO-Y4 cells.

    Science.gov (United States)

    Ren, Jian; Wu, Jun Hua

    2012-05-01

    Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E(2)) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E(2) elevated [Ca(2+)]( i ) and increased Ca(2+) oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E(2) mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E(2) activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E(2) induces the non-genomic responses Ca(2+) release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E(2) responses. PMID:22392527

  14. Rac1/p21-activated kinase pathway controls retinoblastoma protein phosphorylation and E2F transcription factor activation in B lymphocytes.

    Science.gov (United States)

    Zaldua, Natalia; Llavero, Francisco; Artaso, Alain; Gálvez, Patricia; Lacerda, Hadriano M; Parada, Luis A; Zugaza, José L

    2016-02-01

    Small GTPases of the Ras superfamily are capable of activating E2F-dependent transcription leading to cell proliferation, but the molecular mechanisms are poorly understood. In this study, using immortalized chicken DT40 B cell lines to investigate the role of the Vav/Rac signalling cascade on B cell proliferation, it is shown that the proliferative response triggered by B cell receptor activation is dramatically reduced in the absence of Vav3 expression. Analysis of this proliferative defect shows that in the absence of Vav3 expression, retinoblastoma protein (RB) phosphorylation and the subsequent E2F activation do not take place. By combining pharmacological and genetic approaches, phosphatidylinositol-3-kinase and phospholipase Cγ2 (PLCγ2) were identified as the key regulatory signalling molecules upstream of the Vav3/Rac pathway leading to RB phosphorylation and E2F transcription factor activation. Additionally, vav3(-/-) and plcγ2(-/-) DT40 B cells were not able to activate the RB-E2F complex wild-type phenotype when these genetically modified cells were transfected with constitutively active forms of RhoA or Cdc42. However, when these knockout cells were transfected with different constitutively active versions of PLCγ, Vav or Rac1, not only activation of the RB-E2F complex wild-type phenotype was recovered but also the cellular proliferation. Furthermore, by evaluating the effect of two known effector mutants of Rac1 (Rac1(Q61L/F37A) and Rac1(Q61L/Y40C) ), the RB-E2F complex activation dependency on p21-activated kinase (PAK) and protein kinase Cε (PKCε) activities was established, being independent of both actin cytoskeleton reorganization and Ras activity. These results suggest that PAK1 and PKCε may be potential therapeutic targets to stop uncontrolled B cell proliferation mediated by the Vav/Rac pathway.

  15. Non-hypoxic activation of the negative regulatory feedback loop of prolyl-hydroxylase oxygen sensors.

    Science.gov (United States)

    Tug, Suzan; Delos Reyes, Buena; Fandrey, Joachim; Berchner-Pfannschmidt, Utta

    2009-07-10

    Hypoxia inducible factors (HIF) coordinate cellular responses towards hypoxia. HIFs are mainly regulated by a group of prolyl-hydroxylases (PHDs) that in the presence of oxygen, target the HIFalpha subunit for degradation. Herein, we studied the role of nitric oxide (NO) in regulating PHD activities under normoxic conditions. In the present study we show that different NO-donors initially inhibited endogenous PHD2 activity which led to accumulation of HIF-1alpha subsequently to enhance HIF-1 dependent increased PHD2 promoter activity. Consequently PHD2 abundance and activity were strongly induced which caused downregulation of HIF-1alpha. Interestingly, upregulation of endogenous PHD2 activity by NO was not found in cells that lack an intact pVHL dependent degradation pathway. Recovery of PHD activity required intact cells and was not observed in cell extracts or recombinant PHD2. In conclusion induction of endogenous PHD2 activity by NO is dependent on a feedback loop initiated despite normoxic conditions. PMID:19427832

  16. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.

    OpenAIRE

    Bunone, G; Briand, P A; Miksicek, R J; Picard, D.

    1996-01-01

    The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of 'steroid-independent activation' of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N-terminal transcriptional activation function AF-1, but not the C-terminal AF-2, is necessary...

  17. Inhibition of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses proliferation and STAT3 phosphorylation independently of Jak3

    DEFF Research Database (Denmark)

    Marzec, Michal; Kasprzycka, Monika; Ptasznik, Andrzej;

    2005-01-01

    Aberrant expression of the ALK tyrosine kinase as a chimeric protein with nucleophosmin (NPM) and other partners plays a key role in malignant cell transformation of T-lymphocytes and other cells. Here we report that two small-molecule, structurally related, quinazoline-type compounds, WHI-131...... and WHI-154, directly inhibit enzymatic activity of NPM/ALK as demonstrated by in vitro kinase assays using a synthetic tyrosine-rich oligopeptide and the kinase itself as the substrates. The inhibition of NPM/ALK activity resulted in malignant T cells in suppression of their growth, induction...... of apoptosis and inhibition of tyrosine phosphorylation of STAT3, the key effector of the NPM/ALK-induced oncogenesis. We also show that the STAT3 tyrosine phosphorylation is mediated in the malignant T cells by NPM/ALK independently of Jak3 kinase as evidenced by the presence of STAT3 phosphorylation...

  18. Loss of p53-regulatory protein IFI16 induces NBS1 leading to activation of p53-mediated checkpoint by phosphorylation of p53 SER37.

    Science.gov (United States)

    Tawara, Hideyuki; Fujiuchi, Nobuko; Sironi, Juan; Martin, Sarah; Aglipay, Jason; Ouchi, Mutsuko; Taga, Makoto; Chen, Phang-Lang; Ouchi, Toru

    2008-01-01

    Our previous results that IFI16 is involved in p53 transcription activity under conditions of ionizing radiation (IR), and that the protein is frequently lost in human breast cancer cell lines and breast adenocarcinoma tissues suggesting that IFI16 plays a crucial role in controlling cell growth. Here, we show that loss of IFI16 by RNA interference in cell culture causes elevated phosphorylation of p53 Ser37 and accumulated NBS1 (nibrin) and p21WAF1, leading to growth retardation. Consistent with these observations, doxycyclin-induced NBS1 caused accumulation of p21WAF1 and increased phosphorylation of p53 Ser37, leading to cell cycle arrest in G1 phase. Wortmannin treatment was found to decrease p53 Ser37 phosphorylation in NBS-induced cells. These results suggest that loss of IFI16 activates p53 checkpoint through NBS1-DNA-PKcs pathway. PMID:17981542

  19. Laboratory Scale Study of Activated Sludge Process in Jet Loop Reactor for Waste WaterTreatment

    Directory of Open Access Journals (Sweden)

    M. S. Patil

    2014-03-01

    Full Text Available The present study was undertaken to evaluate the feasibility of Activated Sludge Process (ASP for the treatment of synthetic wastewater and to develop a simple design criteria under local conditions.A laboratory scale Compact jet loop reactor model comprising of an aeration tank and final clarifier was used for this purpose.Settled synthetic wastewater was used as influent to the aeration tank. The Chemical Oxygen Demand (COD of the influent and effluent was measured to find process efficiency at various mixed liquorvolatile suspended solids (MLVSS and hydraulic retention time (θ. The results of the studydemonstrated that an efficiency of above 95% could be obtained for COD if the ASP is operated atan MLVSS concentration of 3000 mg/L keeping an aeration time of 1 hour.In the present investigation the preliminary studies were carried out in a lab scale Jet loop reactor made of glass. Synthetic waste water having a composition of 1000 mg/L mixed with other nutrients such as Urea, Primary and secondary Potassium phosphates, Magnesium sulfate, Iron chloriderequired for the bacteria was prepared in the laboratory and reduction in COD and the increase inSuspended Solids (SSand the Sludge Volume Index (SVI were determined.

  20. Shutoff and agonist-triggered internalization of protease-activated receptor 1 can be separated by mutation of putative phosphorylation sites in the cytoplasmic tail.

    Science.gov (United States)

    Hammes, S R; Shapiro, M J; Coughlin, S R

    1999-07-20

    The thrombin receptor PAR1 becomes rapidly phosphorylated upon activation by either thrombin or exogenous SFLLRN agonist peptide. Substitution of alanine for all serine and threonine residues in the receptor's cytoplasmic carboxyl-terminal tail ablated phosphorylation and yielded a receptor defective in both shutoff and agonist-triggered internalization. These observations suggested that activation-dependent phosphorylation of PAR1's cytoplasmic tail is required for both shutoff and agonist-triggered internalization. To identify the phosphorylation site(s) that are necessary for these functions, we generated three mutant receptors in which alanine was substituted for serine and threonine residues in the amino-terminal, middle, and carboxyl-terminal thirds of PAR1's cytoplasmic tail. When stably expressed in fibroblasts, all three mutated receptors were rapidly phosphorylated in response to agonist, while a mutant in which all serines and threonines in the cytoplasmic tail were converted to alanines was not. This result suggests that phosphorylation can occur at multiple sites in PAR1's cytoplasmic tail. Alanine substitutions in the N-terminal and C-terminal portions of the tail had no effect on either receptor shutoff or agonist-triggered internalization. By contrast, alanine substitutions in the "middle" serine cluster between Ser(391) and Ser(406) yielded a receptor with considerably slower shutoff of signaling after thrombin activation than the wild type. Surprisingly, this same mutant was indistinguishable from the wild type in agonist-triggered internalization and degradation. Overexpression of G protein-coupled receptor kinase 2 (GRK2) and GRK3 "suppressed" the shutoff defect of the S --> A (391-406) mutant, consistent with this defect being due to altered receptor phosphorylation. These results suggest that specific phosphorylation sites are required for rapid receptor shutoff, but phosphorylation at multiple alternative sites is sufficient for agonist

  1. Large loop conformation sampling using the activation relaxation technique, ART-nouveau method.

    Science.gov (United States)

    St-Pierre, Jean-François; Mousseau, Normand

    2012-07-01

    We present an adaptation of the ART-nouveau energy surface sampling method to the problem of loop structure prediction. This method, previously used to study protein folding pathways and peptide aggregation, is well suited to the problem of sampling the conformation space of large loops by targeting probable folding pathways instead of sampling exhaustively that space. The number of sampled conformations needed by ART nouveau to find the global energy minimum for a loop was found to scale linearly with the sequence length of the loop for loops between 8 and about 20 amino acids. Considering the linear scaling dependence of the computation cost on the loop sequence length for sampling new conformations, we estimate the total computational cost of sampling larger loops to scale quadratically compared to the exponential scaling of exhaustive search methods.

  2. Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman.

    Science.gov (United States)

    Hafver, Tandekile Lubelwana; Hodne, Kjetil; Wanichawan, Pimthanya; Aronsen, Jan Magnus; Dalhus, Bjørn; Lunde, Per Kristian; Lunde, Marianne; Martinsen, Marita; Enger, Ulla Helene; Fuller, William; Sjaastad, Ivar; Louch, William Edward; Sejersted, Ole Mathias; Carlson, Cathrine Rein

    2016-02-26

    The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

  3. Effect of nitric oxide-induced tyrosine phosphorylation of calcium-activated potassium channel α subunit on vascular hyporesponsiveness in rats

    Institute of Scientific and Technical Information of China (English)

    ZHOU Rong; LIU Liang-ming; HU De-yao

    2005-01-01

    Objective: To study the effect of nitric oxide-induced tyrosine phosphorylation of large-conductance calcium-activated potassium (BKCa) channel α subunit on vascular hyporesponsiveness in rats. Methods: A total of 46 Wistar rats of either sex, weighing 250 g±20 g, were used in this study. Models of vascular hyporesponsiveness induced by hemorrhagic shock (30 mm Hg for 2 hours) in vivo and by L-arginine in vitro were established respectively. The vascular responsiveness of isolated superior mesenteric arteries to norepinephrine was observed. Tyrosine phosphorylation of BKCa α subunit was evaluated with methods of immunoprecipitation and Western blotting. Results: In the smooth muscle cells of the superior mesenteric arteries, the expression of BKCa α subunit tyrosine phosphorylation increased following hemorrhagic shock, and L-arginine could induce BKCa channel α subunit tyrosine phosphorylation in a time- and dose-dependent manner. L-NAME (Nω-nitro-L-arginine-methyl-ester), a nitric oxide synthetase inhibitor, could partly restore the decreased vasoresponsiveness of the superior mesenteric arteries after hemorrhagic shock in rats. Down-regulating the protein tyrosine phosphorylation with genistein, a widely-used special protein tyrosine kinase inhibitor, could partly improve the decreased vasoresponsiveness of the superior mesenteric arteries induced by L-arginine in vitro, while up-regulating the protein tyrosine phosphorylation with Na3VO4, a protein tyrosine phosphatase inhibitor, could further decrease the nitric oxide-induced vascular hyporesponsiveness, which could be partly ameliorated by 0.1 mmol/L tetrabutylammonium chloride (TEA), a selective BKCa inhibitor at this concentration. Conclusions: Nitric oxide can induce the tyrosine phosphorylation of BKCa α subunit, which influences the vascular hyporesponsiveness in hemorrhagic shock rats or induced by L-arginine in vitro.

  4. Increased iPLA2 activity and levels of phosphorylated GSK3B in platelets are associated with donepezil treatment in Alzheimer's disease patients.

    Science.gov (United States)

    Talib, L L; Hototian, S R; Joaquim, H P G; Forlenza, O V; Gattaz, W F

    2015-12-01

    Reduced phospholipase A2 (PLA2) activity and increased phosphorylation of glycogen synthase kinase 3B (GSK3B) participate in the production of beta-amyloid plaques and of neurofibrillary tangles, which are two neuropathological hallmarks of Alzheimer's disease (AD). Experimental evidences suggest a neuroprotective effect of the cholinesterase inhibitor donepezil in the treatment the disease. The aims of the present study were to evaluate in AD patients the effects of treatment with donepezil on PLA2 activity and GSK3B level. Thirty patients with AD were treated during 6 months with 10 mg daily of donepezil. Radio-enzymatic assays were used to measure PLA2 activity and Elisa assays for GSK3B level, both in platelets. Before treatment and after 3 and 6 months on donepezil, AD patients underwent a cognitive assessment and platelet samples were collected. Values were compared to a healthy control group of 42 sex- and age-matched elderly individuals. Before treatment, iPLA2 activity was lower in patients with AD as compared to controls (p cognitive improvement during treatment (p = 0.037). After 6 months, we found an increase in phosphorylated GSK3B (p = 0.02). The present findings suggest two possible mechanisms by which donepezil delays the progression of AD. The increment of iPLA2 activity may reduce the production of beta-amyloid plaques, whereas the phosphorylation of GSK3B inactivates the enzyme, reducing thus the phosphorylation of tau protein. PMID:25920742

  5. The role of β18-β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    XIA LiQiu; WANG FaXiang; DING XueZhi; ZHAO XinMin; FU ZuJiao; QUAN MeiFang; YU ZiNiu

    2008-01-01

    The β18-β19 loop in domain Ⅲ of Cry1Ac toxin is unique among Bacillus thuringiensis Cry proteins. In this study, the role of the loop structure in insecticidal activity of Cry1Ac toxin was investigated. Alanine scanning mutations within the loop were initially generated and most mutants were over-expressed and reduced toxicity at different degrees, except mutant N546A that showed almost 2 times enhanced toxicity against Helicoverpa armigera larvae. Further mutagenic analysis of N546 re-vealed that a charged amino acid in this position would cause very unfavorable influence on insecti-cidal activity. In addition, the deletion of N546 led to protein instability because of destruction of the loop integrity. Besides, mutant W544F was much more toxic than W544Y, indicating that hydrophobic nature of the position was important for maintaining the stability and activity of Cry1Ac protein. These findings are the first biological evidence for a structural function of β18-β19 loop in insecticidal activity of the Cry1Ac toxin.

  6. From Concept-to-Flight: An Active Active Fluid Loop Based Thermal Control System for Mars Science Laboratory Rover

    Science.gov (United States)

    Birur, Gajanana C.; Bhandari, Pradeep; Bame, David; Karlmann, Paul; Mastropietro, A. J.; Liu, Yuanming; Miller, Jennifer; Pauken, Michael; Lyra, Jacqueline

    2012-01-01

    The Mars Science Laboratory (MSL) rover, Curiosity, which was launched on November 26, 2011, incorporates a novel active thermal control system to keep the sensitive electronics and science instruments at safe operating and survival temperatures. While the diurnal temperature variations on the Mars surface range from -120 C to +30 C, the sensitive equipment are kept within -40 C to +50 C. The active thermal control system is based on a single-phase mechanically pumped fluid loop (MPFL) system which removes or recovers excess waste heat and manages it to maintain the sensitive equipment inside the rover at safe temperatures. This paper will describe the entire process of developing this active thermal control system for the MSL rover from concept to flight implementation. The development of the rover thermal control system during its architecture, design, fabrication, integration, testing, and launch is described.

  7. Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop

    DEFF Research Database (Denmark)

    Mokrosinski, Jacek; Frimurer, Thomas M; Sivertsen, Bjoern;

    2012-01-01

    . Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal-ion site stabilizing an a-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment...

  8. Density functional theory studies of MTSL nitroxide side chain conformations attached to an activation loop

    CERN Document Server

    Concilio, Maria Grazia; Bayliss, Richard; Burgess, Selena G

    2016-01-01

    A quantum-mechanical (QM) method rooted on density functional theory (DFT) has been employed to determine conformations of the methane-thiosulfonate spin label (MTSL) attached to a fragment extracted from the activation loop of Aurora-A kinase. The features of the calculated energy surface revealed low energy barriers between isoenergetic minima and the system could be described in a population of 76 rotamers that can be also considered for other systems since it was found that the X3, X4 and X5 do not depend on the previous two dihedral angles. Conformational states obtained were seen to comparable to those obtained in the {\\alpha}-helix systems studied previously, indicating that the protein backbone does not affect the torsional profiles significantly and suggesting the possibility to use determined conformations for other protein systems for further modelling studies.

  9. Activation of phosphorylating-p38 mitogen-activated protein kinase and its relationship with localization of intestinal stem cells in rats after ischemia-reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Xiao-Bing Fu; Feng Xing; Yin-Hui Yang; Tong-Zhu Sun; Bao-Chen Guo

    2003-01-01

    AIM: To investigate the expression of phosphorylating p38mitogen-activated protein kinase (MAPK) in rat small intestine after ischemia-reperfusion (I/R) insult and its relationship with the localization of intestinal stem cells.METHODS: Forty-eight Wistar rats were divided randomly into three groups, namely intestinal ischemia-reperfusion group (R), intestinal ischemia group (Ⅰ) and sham-operated control group (C). In group I, the animals were killed 45minutes after superior mesenteric artery (SMA) occlusion,while in group R the rats sustained SMA occlusion for 45 minutes and reperfusion for 2, 6, 12 or 24 hours respectively. In shamoperated control group, SMA was separated, but without occlusion. The activity of plasma diamine oxidase (DAO)was determined. Intestinal tissue samples were also taken for histological analysis and immunohistochemical analysis of MAPK p38 detection and intestinal stem cell localization.RESULTS: The changes in histological structure and plasma DAO levels indicated that the intestinal barrier was damaged after intestinal I/R injury. In group C and I, each crypt contained 5-6 p38 MAPK positive cells, which were mainly located in the lower region of the crypts. This was consistent with the distribution of intestinal stem cells. The presence of positive cells in crypts increased with the time of reperfusion and reached its peak at 12 hours after reperfusion (35.6%).CONCLUSION: After intestinal T/R injury, the expression of phosphorylating-p38 MAPK in small intestine increased with the duration of reperfusion, and its distribution coincided with that of intestinal stem cells and their daughter cells,indicating that phosphorylating-p38 might be a possible marker of intestinal stem cells.

  10. BRF1 Protein Turnover and mRNA Decay Activity Are Regulated by Protein Kinase B at the Same Phosphorylation Sites▿

    OpenAIRE

    Benjamin, Don; Schmidlin, Martin; Min, Lu; Gross, Brigitte; Moroni, Christoph

    2006-01-01

    BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to al...

  11. The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;

    1993-01-01

    Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise...... to reconstituted CK2 holoenzymes displaying basal catalytic activity, thermostability and responsiveness to polylysine, identical to those of wild-type holoenzyme, whose reconstitution, moreover, is not affected by previous phosphorylation of the beta-subunit at either its N-terminal or C-terminal sites. Unlike...

  12. Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

    Directory of Open Access Journals (Sweden)

    Zhang Xue

    2006-07-01

    Full Text Available Abstract Background Aberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17 tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated. Results Phosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells. Conclusion Tumor suppressor PTEN can alleviate the phosporylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity

  13. Phosphorylation of synaptosomal cytoplasmic proteins: Inhibition of calcium-activated, phospholipid-dependent protein kinase (protein kinase c) by bay k 8644.

    Science.gov (United States)

    Robinson, P J; Lovenberg, W

    1988-01-01

    The phosphorylation of specific substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was examined in striatal synaptosomal cytoplasm. The phosphoprotein substrata were termed group C phosphoprotems and were divided into two subgroups: group C(1) phosphoproteins (P83, P45A, P21 and P18) were found in both cytoplasm and synaptosomal membranes and, although stimulated by phosphatidylserine, only required exogamous calcium for their labeling; group C(2) phosphoproteins (P120, P96, P21.5, P18.5 and P16) were found predominantly in the cytoplasm and were absolutely dependent upon exogenous calcium and phosphatidylserme for their labeling. Several criteria were used to identify these proteins as specific protein kinase C substrates: (a) their phosphorylation was stimulated to a greater extent by Ca(2+) /phosphatidylserine/diolein than by Ca(2+) alone or Cal(2+) /calmodulin (group C(1)) or was completely dependent upon Ca(2+) /phosphatdylserine/diolein (group C(2)); (b) supermaximal concentrations of the cAMP-dependent protein kinase inhibitor were without effect; (c) their phosphorylation was stimulated by oleic acid, which selectively activates protein kinase C in the absence of Ca(2+); (d) NaCl, which inhibited cAMP- and Ca(2+)/calmodulindependent phosphorylation, slightly increased phosphorylation of group C(1) and slightly decreased phosphorylation of group C(2) phosphoproteins. Maximal phosphorylation of P96 and other group C phosphoproteins occurred within 60 s and was followed by a slow decay rate while substrata of calmodulin-dependent protein kinase were maximally labeled within 20-30 s and rapidly dephosphorylated. The phosphorylation of all group C phosphoproteins was inhibited by the calcium channel agomst BAY K 8644, however, group C(2) phosphoproteins were considerably more sensitive. The IC(50) for inhibition of P96 labeling was 19 ?M. but for P83 was 190 ?M. Group B phosphoproteins were also slightly inhibited, and the

  14. The role of GH receptor tyrosine phosphorylation in Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, J A; Hansen, L H; Wang, X;

    1997-01-01

    . Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA...

  15. Complete Sucrose Metabolism Requires Fructose Phosphotransferase Activity in Corynebacterium glutamicum To Ensure Phosphorylation of Liberated Fructose

    OpenAIRE

    Dominguez, H.; Lindley, N. D.

    1996-01-01

    Sucrose uptake by Corynebacterium glutamicum involves a phosphoenolpyruvate-dependent sucrose phosphotransferase (PTS), but in the absence of fructokinase, further metabolism of the liberated fructose requires efflux of the fructose and reassimilation via the fructose PTS. Mutant strains lacking detectable fructose-transporting PTS activity accumulated fructose extracellularly but consumed sucrose at rates comparable to those of the wild-type strain.

  16. Biological activities of the homologous loop regions in the laminin α chain LG modules.

    Science.gov (United States)

    Katagiri, Fumihiko; Hara, Toshihiro; Yamada, Yuji; Urushibata, Shunsuke; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi

    2014-06-10

    Each laminin α chain (α1-α5 chains) has chain-specific diverse biological functions. The C-terminal globular domain of the α chain consists of five laminin-like globular (LG1-5) modules and plays a critical role in biological activities. The LG modules consist of a 14-stranded β-sheet (A-N) sandwich structure. Previously, we described the chain-specific biological activities of the loop regions between the E and F strands in the LG4 modules using five homologous peptides (G4EF1-G4EF5). Here, we further analyze the biological activities of the E-F strands loop regions in the rest of LG modules. We designed 20 homologous peptides (approximately 20 amino acid length), and 17 soluble peptides were used for the cell attachment assay. Thirteen peptides promoted cell attachment activity with different cell morphologies. Cell attachment to peptides G1EF1, G1EF2, G2EF1, G3EF4, and G5EF4 was inhibited by heparin, and peptides G1EF1, G1EF2, and G2EF1 specifically bound to syndecan-overexpressing cells. Cell attachment to peptides G2EF3, G3EF1, G3EF3, G5EF1, G5EF3, and G5EF5 was inhibited EDTA. Further, cell attachment to peptides G3EF3, G5EF1, and G5EF5 was inhibited by both anti-integrin α2 and β1 antibodies, whereas cell attachment to peptide G5EF3 was inhibited by only anti-integrin β1 antibody. Cell attachment to peptides G1EF4, G3EF4, and G5EF4 was inhibited by both heparin and EDTA and was not inhibited by anti-integrin antibodies. The active peptide sequence alignments suggest that the syndecan-binding peptides contain a "basic amino acid (BAA)-Gly-BAA" motif in the middle of the molecule and that the integrin-binding peptides contain an "acidic amino acid (AAA)"-Gly-BAA motif. Core-switched peptide analyses suggested that the "BAA-Gly-BAA" motif is critical for binding to syndecans and that the "AAA-Gly-BAA" motif has potential to recognize integrins. These findings are useful for understanding chain-specific biological activities of laminins and to evaluate

  17. Tyrosine phosphorylation of the E3 ubiquitin ligase TRIM21 positively regulates interaction with IRF3 and hence TRIM21 activity.

    Directory of Open Access Journals (Sweden)

    Kevin B Stacey

    Full Text Available Patients suffering from Systemic Lupus Erythematous (SLE have elevated type I interferon (IFN levels which correlate with disease activity and severity. TRIM21, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that targets the transcription factor IRF3 in order to turn off and limit type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs. However, how the activity of TRIM21 is regulated downstream of TLRs is unknown. In this study we demonstrate that TRIM21 is tyrosine phosphorylated following TLR3 and TLR4 stimulation, suggesting that its activity is potentially regulated by tyrosine phosphorylation. Using Netphos, we have identified three key tyrosines that are strongly predicted to be phosphorylated, two of which are conserved between the human and murine forms of TRIM21, at residues 343, 388, and 393, all of which have been mutated from tyrosine to phenylalanine (Y343F, Y388F, and Y393F. We have observed that tyrosine phosphorylation of TRIM21 only occurs in the substrate binding PRY/SPRY domain, and that Y393, and to a lesser extent, Y388 are required for TRIM21 to function as a negative regulator of IFN-β promoter activity. Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-β promoter. Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4. Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics.

  18. Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2+2a) phosphorylation

    DEFF Research Database (Denmark)

    Friedrichsen, Martin; Birk, Jesper Bratz; Richter, Erik;

    2013-01-01

    was positively associated with pAkt-T308 (P=0.01) and Akt2 activity (P=0.04), but not pAkt-S473 or IRS-1-PI3K activity. Furthermore, pAkt-T308 and Akt2 activity were negatively associated with NH(2)-terminal GS phosphorylation (P=0.001 for both), which in turn was negatively associated with insulin-stimulated GS...

  19. CaMKII, but not protein kinase A, regulates Rpt6 phosphorylation and proteasome activity during the formation of long-term memories

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    2013-08-01

    Full Text Available CaMKII and Protein Kinase A (PKA are thought to be critical for synaptic plasticity and memory formation through their regulation of protein synthesis. Consistent with this, numerous studies have reported that CaMKII, PKA and protein synthesis are critical for long-term memory formation. Recently, we found that protein degradation through the ubiquitin-proteasome system is also critical for long-term memory formation in the amygdala. However, the mechanism by which ubiquitin-proteasome activity is regulated during memory formation and how protein degradation interacts with known intracellular signaling pathways important for learning remain unknown. Recently, evidence has emerged suggesting that both CaMKII and PKA are capable of regulating proteasome activity in vitro through the phosphorylation of proteasome regulatory subunit Rpt6 at Serine-120, though whether they regulate Rpt6 phosphorylation and proteasome function in vivo remains unknown. In the present study we demonstrate for the first time that fear conditioning transiently modifies a proteasome regulatory subunit and proteasome catalytic activity in the mammalian brain in a CaMKII-dependent manner. We found increases in the phosphorylation of proteasome ATPase subunit Rpt6 at Serine-120 and an enhancement in proteasome activity in the amygdala following fear conditioning. Pharmacological manipulation of CaMKII, but not PKA, in vivo significantly reduced both the learning-induced increase in Rpt6 Serine-120 phosphorylation and the increase in proteasome activity without directly affecting protein polyubiquitination levels. These results indicate a novel role for CaMKII in memory formation through its regulation of protein degradation and suggest that CaMKII regulates Rpt6 phosphorylation and proteasome function both in vitro and in vivo.

  20. Type 2 diabetes is associated with altered NF-¿B DNA binding activity, JNK phosphorylation, and AMPK phosphorylation in skeletal muscle after LPS

    DEFF Research Database (Denmark)

    Andreasen, Anne Sofie; Kelly, Meghan; Berg, Ronan Martin Griffin;

    2011-01-01

    Systemic inflammation is often associated with impaired glucose metabolism. We therefore studied the activation of inflammatory pathway intermediates that interfere with glucose uptake during systemic inflammation by applying a standardised inflammatory stimulus in vivo. After ethical approval, i...

  1. Lipopolysaccharide-induced activation of NF-{kappa}B non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharyya, Sumit; Borthakur, Alip; Dudeja, Pradeep K. [Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Chicago, IL 60612-7227 (United States); Tobacman, Joanne K., E-mail: jkt@uic.edu [Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Chicago, IL 60612-7227 (United States)

    2010-11-15

    Background and aims: B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-{kappa}B (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods: Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-{kappa}B activation, including nuclear RelB and p52 and phospho- and total NF-{kappa}B inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results: By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-I{kappa}B{alpha} of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions: LPS induces activation of both canonical and non-canonical pathways of NF-{kappa}B in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.

  2. (S)-Lacosamide Binding to Collapsin Response Mediator Protein 2 (CRMP2) Regulates CaV2.2 Activity by Subverting Its Phosphorylation by Cdk5.

    Science.gov (United States)

    Moutal, Aubin; François-Moutal, Liberty; Perez-Miller, Samantha; Cottier, Karissa; Chew, Lindsey Anne; Yeon, Seul Ki; Dai, Jixun; Park, Ki Duk; Khanna, May; Khanna, Rajesh

    2016-04-01

    The neuronal circuit remodels during development as well as in human neuropathologies such as epilepsy. Neurite outgrowth is an obligatory step in these events. We recently reported that alterations in the phosphorylation state of an axon specification/guidance protein, the collapsin response mediator protein 2 (CRMP2), play a major role in the activity-dependent regulation of neurite outgrowth. We also identified (S)-LCM, an inactive stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Here, we investigated the mechanism by which (S)-LCM affects CRMP2 phosphorylation by two key kinases, cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β (GSK-3β). (S)-LCM application to embryonic cortical neurons resulted in reduced levels of Cdk5- and GSK-3β-phosphorylated CRMP2. Mechanistically, (S)-LCM increased CRMP2 binding to both Cdk5- and GSK-3β without affecting binding of CRMP2 to its canonical partner tubulin. Saturation transfer difference nuclear magnetic resonance (STD NMR) and differential scanning fluorimetry (DSF) experiments demonstrated direct binding of (S)-LCM to CRMP2. Using an in vitro luminescent kinase assay, we observed that (S)-LCM specifically inhibited Cdk5-mediated phosphorylation of CRMP2. Cross-linking experiments and analytical ultracentrifugation showed no effect of (S)-LCM on the oligomerization state of CRMP2. The increased association between Cdk5-phosphorylated CRMP2 and CaV2.2 was reduced by (S)-LCM in vitro and in vivo. This reduction translated into a decrease of calcium influx via CaV2.2 in (S)-LCM-treated neurons compared to controls. (S)-LCM, to our knowledge, is the first molecule described to directly inhibit CRMP2 phosphorylation and may be useful for delineating CRMP2-facilitated functions.

  3. [Effects of acute hypobaric hypoxia and exhaustive exercise on AMP-activated protein kinase phosphorylation in rat skeletal muscle].

    Science.gov (United States)

    Yang, Tao; Huang, Qing-Yuan; Shan, Fa-Bo; Guan, Li-Bin; Cai, Ming-Chun

    2012-04-25

    The present study was aimed to explore the changes of phosphorylated AMP-activated protein kinase (pAMPK) level in skeletal muscle after exposure to acute hypobaric hypoxia and exhaustive exercise. Thirty-two male Sprague-Dawley (SD) rats were randomly divided into sea level and high altitude groups. The rats in high altitude group were submitted to simulated 5 000 m of high altitude in a hypobaric chamber for 24 h, and sea level group was maintained at normal conditions. All the rats were subjected to exhaustive swimming exercise. The exhaustion time was recorded. Before and after the exercise, blood lactate and glycogen content in skeletal muscle were determined; AMPK and pAMPK levels in skeletal muscle were detected by Western blot. The results showed that the exhaustion time was significantly decreased after exposure to high altitude. At the moment of exhaustion, high altitude group had lower blood lactate concentration and higher surplus glycogen content in gastrocnemius compared with sea level group. Exhaustive exercise significantly increased the pAMPK/AMPK ratio in rat skeletal muscles from both sea level and high altitude groups. However, high altitude group showed lower pAMPK/AMPK ratio after exhaustion compared to sea level group. These results suggest that, after exposure to acute hypobaric hypoxia, the decrement in exercise capacity may not be due to running out of glycogen, accumulation of lactate or disturbance in energy status in skeletal muscle. PMID:22513470

  4. Activation of the plasma membrane Na/H antiporter salt-overly-sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain

    KAUST Repository

    Quintero, Francisco J.

    2011-01-24

    The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2-SOS3 calcium-dependent protein kinase complex upregulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2-SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2-SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

  5. Dietary agent, benzyl isothiocyanate inhibits signal transducer and activator of transcription 3 phosphorylation and collaborates with sulforaphane in the growth suppression of PANC-1 cancer cells

    Directory of Open Access Journals (Sweden)

    Deangelis Stephanie

    2009-08-01

    Full Text Available Abstract The Signal Transducer and Activator of Transcription (STAT proteins comprise a family of latent transcription factors with diverse functions. STAT3 has well established roles in cell proliferation, growth and survival, and its persistent activation has been detected with high frequency in many human cancers. As constitutive activation of STAT3 appears to be vital for the continued survival of these cancerous cells, it has emerged as an attractive target for chemotherapeutics. We examined whether the inhibitory activities of bioactive compounds from cruciferous vegetables, such as Benzyl isothiocyanate (BITC and sulforaphane, extended to STAT3 activation in PANC-1 human pancreatic cancer cells. BITC and sulforaphane were both capable of inhibiting cell viability and inducing apoptosis in PANC-1. Sulforaphane had minimal effect on the direct inhibition of STAT3 tyrosine phosphorylation, however, suggesting its inhibitory activities are most likely STAT3-independent. Conversely, BITC was shown to inhibit the tyrosine phosphorylation of STAT3, but not the phosphorylation of ERK1/2, MAPK and p70S6 kinase. These results suggest that STAT3 may be one of the targets of BITC-mediated inhibition of cell viability in PANC-1 cancer cells. In addition, we show that BITC can prevent the induction of STAT3 activation by Interleukin-6 in MDA-MB-453 breast cancer cells. Furthermore, combinations of BITC and sulforaphane inhibited cell viability and STAT3 phosphorylation more dramatically than either agent alone. These findings suggest that the combination of the dietary agents BITC and sulforaphane has potent inhibitory activity in pancreatic cancer cells and that they may have translational potential as chemopreventative or therapeutic agents.

  6. Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation.

    OpenAIRE

    Nicholson, S. E.; Oates, A. C.; Harpur, A G; Ziemiecki, A; Wilks, A F; Layton, J E

    1994-01-01

    Granulocyte-colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of cells of the neutrophil lineage by interaction with a specific receptor. Early signal transduction events following G-CSF receptor activation were studied. We detected tyrosine phosphorylation of both the G-CSF receptor and the protein tyrosine kinase JAK1 following G-CSF binding to the human G-CSF receptor. In vitro, the kinase activity of JAK1 was increased by G-CSF stimulation. Coimmunoprecipi...

  7. Active-Flux-Based, V/f-with-Stabilizing-Loops Versus Sensorless Vector Control of IPMSM Drives

    DEFF Research Database (Denmark)

    Moldovan, Ana; Blaabjerg, Frede; Boldea, Ion

    2011-01-01

    . By this control strategy, a fast dynamic speed response, without steady state error and without speed or current regulators, for all AC machines is obtained. The second control method is a sensorless vector control strategy which also has been implemented and tested, just for comparison.......This paper proposes two control methods for Interior Permanent Magnet Synchronous Motor (IPMSM) Drives. The first one is a V/f control with two stabilizing loops: one loop based on active flux balance for voltage magnitude correction and a second, based on speed error, with voltage phase correction...

  8. Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation.

    Science.gov (United States)

    Schaarschmidt, Joerg; Nagel, Marcus B M; Huth, Sandra; Jaeschke, Holger; Moretti, Rocco; Hintze, Vera; von Bergen, Martin; Kalkhof, Stefan; Meiler, Jens; Paschke, Ralf

    2016-07-01

    The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor. PMID:27129207

  9. Lyn- and PLC-beta3-dependent regulation of SHP-1 phosphorylation controls Stat5 activity and myelomonocytic leukemia-like disease.

    Science.gov (United States)

    Xiao, Wenbin; Ando, Tomoaki; Wang, Huan-You; Kawakami, Yuko; Kawakami, Toshiaki

    2010-12-23

    Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-β3-dependent manner. Thus, PLC-β3-deficient mice develop myeloproliferative neoplasm, like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-β3 doubly deficient lyn(-/-);PLC-β3(-/-) mice develop a Stat5-dependent, fatal myelodysplastic/myeloproliferative neoplasm, similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-β3(-/-) mice that cause the CMML-like disease, phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated, resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore, SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand, Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore, we identify a novel Lyn/PLC-β3-mediated regulatory mechanism of SHP-1 and Stat5 activities.

  10. Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients.

    Science.gov (United States)

    Chiker, Sara; Pennaneach, Vincent; Loew, Damarys; Dingli, Florent; Biard, Denis; Cordelières, Fabrice P; Gemble, Simon; Vacher, Sophie; Bieche, Ivan; Hall, Janet; Fernet, Marie

    2015-01-01

    Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing. PMID:26237679

  11. Serine 83 in DosR, a response regulator from Mycobacterium tuberculosis, promotes its transition from an activated, phosphorylated state to an inactive, unphosphorylated state.

    Science.gov (United States)

    Cho, Ha Yeon; Kang, Beom Sik

    2014-02-21

    A sensor kinase, DosS, and its corresponding response regulator, DosR, constitute a two component system for regulating gene expression under hypoxic conditions in Mycobacterium tuberculosis. Among response regulators in M. tuberculosis, NarL has high sequence similarity to DosR, and autophosphorylated DosS transfers its phosphate group not only to DosR but also to NarL. Phosphorylated DosR is more rapidly dephosphorylated than phosphorylated NarL. DosR and NarL differ with respect to the amino acids at positions T+1 and T+2 around the phosphorylation sites in the N-terminal phosphoacceptor domain; NarL has S83 and Y84, whereas DosR has A90 and H91. A DosR S83A mutant shows prolonged phosphorylation. Structural comparison with a histidinol phosphate phosphatase suggests that the hydroxyl group of DosR S83 could play a role in activating the water molecule involved in the triggering of autodephosphorylation.

  12. MicroRNAs Constitute a Negative Feedback Loop in Streptococcus pneumoniae-Induced Macrophage Activation.

    Science.gov (United States)

    Griss, Kathrin; Bertrams, Wilhelm; Sittka-Stark, Alexandra; Seidel, Kerstin; Stielow, Christina; Hippenstiel, Stefan; Suttorp, Norbert; Eberhardt, Martin; Wilhelm, Jochen; Vera, Julio; Schmeck, Bernd

    2016-07-15

    Streptococcus pneumoniae causes high mortality as a major pneumonia-inducing pathogen. In pneumonia, control of innate immunity is necessary to prevent organ damage. We assessed the role of microRNAs (miRNAs) as regulators in pneumococcal infection of human macrophages. Exposure of primary blood-derived human macrophages with pneumococci resulted in transcriptional changes in several gene clusters and a significant deregulation of 10 microRNAs. Computational network analysis retrieved miRNA-146a as one putatively important regulator of pneumococci-induced host cell activation. Its induction depended on bacterial structural integrity and was completely inhibited by blocking Toll-like receptor 2 (TLR-2) or depleting its mediator MyD88. Furthermore, induction of miRNA-146a release did not require the autocrine feedback of interleukin 1β and tumor necrosis factor α released from infected macrophages, and it repressed the TLR-2 downstream mediators IRAK-1 and TRAF-6, as well as the inflammatory factors cyclooxygenase 2 and interleukin 1β. In summary, pneumococci recognition induces a negative feedback loop, preventing excessive inflammation via miR-146a and potentially other miRNAs. PMID:26984146

  13. The cell-specific activity of the estrogen receptor α may be fine-tuned by phosphorylation-induced structural gymnastics

    Science.gov (United States)

    Gburcik, Valentina; Picard, Didier

    2006-01-01

    The estrogen receptor α (ERα) regulates the transcription of target genes by recruiting coregulator proteins through several domains including the two activation functions AF1 and AF2. The contribution of the N-terminally located AF1 activity is particularly important in differentiated cells, and for ERα to integrate inputs from other signaling pathways. However, how the phosphorylation of key residues influences AF1 activity has long remained mysterious, in part because the naturally disordered AF1 domain has resisted a structural characterization. The recent discovery of two coregulators that are specific for a phosphorylated form of AF1 suggests that phosphorylation, possibly in conjunction with the subsequent binding of these coregulators, may enforce a stable structure. The binding of the "pioneer" coregulators might facilitate the subsequent recruitment of yet other coregulators. Different AF1 folds may be enabled by the combinatorial action of posttranslational modifications and coregulator binding thereby fine-tuning ERα activities in a cell- and promoter-specific fashion. PMID:16604168

  14. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

    Energy Technology Data Exchange (ETDEWEB)

    Smet-Nocca, Caroline, E-mail: caroline.smet@univ-lille1.fr; Launay, Helene; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle, E-mail: isabelle.landrieu@univ-lille1.fr [Universite de Lille-Nord de France, Institut Federatif de Recherches 147, CNRS UMR 8576 (France)

    2013-04-15

    The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer's disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the {sup 1}H,{sup 15}N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

  15. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

    International Nuclear Information System (INIS)

    The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1H,15N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

  16. Truncation and Activation of Dual Specificity Tyrosine Phosphorylation-regulated Kinase 1A by Calpain I: A MOLECULAR MECHANISM LINKED TO TAU PATHOLOGY IN ALZHEIMER DISEASE.

    Science.gov (United States)

    Jin, Nana; Yin, Xiaomin; Gu, Jianlan; Zhang, Xinhua; Shi, Jianhua; Qian, Wei; Ji, Yuhua; Cao, Maohong; Gu, Xiaosong; Ding, Fei; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei

    2015-06-12

    Hyperphosphorylation and dysregulation of exon 10 splicing of Tau are pivotally involved in pathogenesis of Alzheimer disease (AD) and/or other tauopathies. Alternative splicing of Tau exon 10, which encodes the second microtubule-binding repeat, generates Tau isoforms containing three and four microtubule-binding repeats, termed 3R-Taus and 4R-Taus, respectively. Dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A) lies at the Down syndrome critical region of chromosome 21. Overexpression of this kinase may contribute to the early Tau pathology in Down syndrome via phosphorylation of Tau and dysregulation of Tau exon 10. Here, we report that Dyrk1A was truncated at the C terminus and was associated with overactivation of calpain I in AD brain. Calpain I proteolyzed Dyrk1A in vitro first at the C terminus and further at the N terminus and enhanced its kinase activity toward Tau via increased Vmax but not Km. C-terminal truncation of Dyrk1A resulted in stronger activity than its full-length protein in promotion of exon 10 exclusion and phosphorylation of Tau. Dyrk1A was truncated in kainic acid-induced excitotoxic mouse brains and coincided with an increase in 3R-Tau expression and phosphorylation of Tau via calpain activation. Moreover, truncation of Dyrk1A was correlated with an increase in the ratio of 3R-Tau/4R-Tau and Tau hyperphosphorylation in AD brain. Collectively, these findings suggest that truncation/activation of Dyrk1A by Ca(2+)/calpain I might contribute to Tau pathology via promotion of exon 10 exclusion and hyperphosphorylation of Tau in AD brain. PMID:25918155

  17. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    Science.gov (United States)

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

  18. Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

    Directory of Open Access Journals (Sweden)

    Raabe Timothy D

    2003-04-01

    Full Text Available Abstract Background Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97 have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. Results Using confocal microscopy and western analysis techniques, we determined 1 the immunolabeled structures are truly within the nucleus; 2 the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs and it can be identified on other intermediate filament proteins (IFs in other cell types; and 3 there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. Conclusion We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

  19. Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

    NARCIS (Netherlands)

    Parikh, Kaushal; Diks, Sander H.; Tuynman, Jurriaan H. B.; Verhaar, Auke; Lowenberg, Mark; Hommes, Daan W.; Joore, Jos; Pandey, Akhilesh; Peppelenbosch, Maikel P.

    2009-01-01

    Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to pred

  20. A Requirement for SOCS-1 and SOCS-3 Phosphorylation in Bcr-Abl-Induced Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Xiaoxue Qiu

    2012-06-01

    Full Text Available Suppressors of cytokine signaling 1 and 3 (SOCS-1 and SOCS-3 are inhibitors of the Janus tyrosine kinase (JAK/signal transducers and activators of transcription (STAT pathway and function in a negative feedback loop during cytokine signaling. Abl transformation is associated with constitutive activation of JAK/STAT-dependent signaling. However, the mechanism by which Abl oncoproteins bypass SOCS inhibitory regulation remains poorly defined. Here, we demonstrate that coexpression of Bcr-Abl with SOCS-1 or SOCS-3 results in tyrosine phosphorylation of these SOCS proteins. Interestingly, SOCS-1 is highly tyrosine phosphorylated in one of five primary chronic myelogenous leukemia samples. Bcr-Abl-dependent tyrosine phosphorylation of SOCS-1 and SOCS-3 occurs mainly on Tyr 155 and Tyr 204 residues of SOCS-1 and on Tyr 221 residue of SOCS-3. We observed that phosphorylation of these SOCS proteins was associated with their binding to Bcr-Abl. Bcr-Abl-dependent phosphorylation of SOCS-1 and SOCS-3 diminished their inhibitory effects on the activation of JAK and STAT5 and thereby enhanced JAK/STAT5 signaling. Strikingly, disrupting the tyrosine phosphorylation of SOCS-1 or SOCS-3 impaired the expression of Bcl-XL protein and sensitized K562 leukemic cells to undergo apoptosis. Moreover, selective mutation of tyrosine phosphorylation sites of SOCS-1 or SOCS-3 significantly blocked Bcr-Abl-mediated tumorigenesis in nude mice and inhibited Bcr-Abl-mediated murine bone marrow transformation. Together, these results reveal a mechanism of how Bcr-Abl may overcome SOCS-1 and SOCS-3 inhibition to constitutively activate the JAK/STAT-dependent signaling, and suggest that Bcr-Abl may critically requires tyrosine phosphorylation of SOCS-1 and SOCS-3 to mediate tumorigenesis when these SOCS proteins are present in cells.

  1. Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription

    Directory of Open Access Journals (Sweden)

    Harrich David

    2005-02-01

    Full Text Available Abstract Background The interferon (IFN-induced, dsRNA-dependent serine/threonine protein kinase, PKR, plays a key regulatory role in the IFN-mediated anti-viral response by blocking translation in the infected cell by phosphorylating the alpha subunit of elongation factor 2 (eIF2. The human immunodeficiency virus type 1 (HIV-1 evades the anti-viral IFN response through the binding of one of its major transcriptional regulatory proteins, Tat, to PKR. HIV-1 Tat acts as a substrate homologue for the enzyme, competing with eIF2α, and inhibiting the translational block. It has been shown that during the interaction with PKR, Tat becomes phosphorylated at three residues: serine 62, threonine 64 and serine 68. We have investigated the effect of this phosphorylation on the function of Tat in viral transcription. HIV-1 Tat activates transcription elongation by first binding to TAR RNA, a stem-loop structure found at the 5' end of all viral transcripts. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR. Results We have investigated the effect of phosphorylation on Tat-mediated transactivation. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR. In vitro phosphorylation experiments with a series of bacterial expression constructs carrying the wild-type tat gene or mutants of the gene with alanine substitutions at one, two, or all three of the serine/threonine PKR phosphorylation sites, showed that these were subject to different levels of phosphorylation by PKR and displayed distinct kinetic behaviour. These results also suggested a cooperative role for the phosphorylation of S68 in conjunction with S62 and T64. We examined the effect of phosphorylation on Tat-mediated transactivation of the HIV-1 LTR in vivo with a series of analogous mammalian expression constructs. Co-transfection experiments showed a gradual reduction in transactivation as the

  2. Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

    Science.gov (United States)

    Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

    2016-04-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

  3. RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients

    International Nuclear Information System (INIS)

    A major obstacle in treating colorectal cancer (CRC) is the acquired resistance to chemotherapeutic agents. An important protein in the regulation of cancer cell death and clinical outcome is Raf kinase inhibitor protein (RKIP). In contrast, activated signal transducer and activator of transcription 3 (STAT3) is a protein that promotes tumor cell survival by inhibiting apoptosis and has an important role in cancer progression in many of cancer types. The aim of this study was to evaluate the regulation of RKIP and STAT3 after treatment with clinically relevant chemotherapeutic agents (camptothecin (CPT) and oxaliplatin (OXP)) and the cytokine interleukin-6 (IL-6) in HCT116 colon cancer cells as well as evaluate the association between RKIP and STAT3 with clinical outcome of Stage II colon cancer patients. HCT-116 colon cancer cells were treated with CPT, OXP, and IL-6 separately or in combination in a time and dose-dependent manner and examined for phosphorylated and non-phosphorylated RKIP and STAT3 via Western blot analysis. STAT3 transcriptional activity was measured via a luciferase reporter assay in HCT116 cells treated with CPT, IL-6 or transfected with JAK 1, 2 separately or in combination. We extended these observations and determined STAT3 and RKIP/ pRKIP in tumor microarrays (TMA) in stage II colon cancer patients. We demonstrate IL-6-mediated activation of STAT3 occurs in conjunction with the phosphorylation of RKIP in vitro in human colon cancer cells. OXP and CPT block IL-6 mediated STAT3 activation and RKIP phosphorylation via the inhibition of the interaction of STAT3 with gp130. We determined that STAT3 and nuclear pRKIP are significantly associated with poor patient prognosis in stage II colon cancer patients. In the analysis of tumor samples from stage II colon cancer patients and the human colon carcinoma cell line HCT116, pRKIP and STAT3, 2 proteins potentially involved in the resistance to conventional treatments were detected. The

  4. Active harmonic filtering using current-controlled, grid-connected DG units with closed-loop power control

    DEFF Research Database (Denmark)

    He, Jinwei; Li, Yun Wei; Blaabjerg, Frede;

    2014-01-01

    The increasing application of nonlinear loads may cause distribution system power quality issues. In order to utilize distributed generation (DG) unit interfacing converters to actively compensate harmonics, this paper proposes an enhanced current control approach, which seamlessly integrates...... voltage detection are not necessary for the proposed harmonic compensation method. Moreover, a closed-loop power control scheme is employed to directly derive the fundamental current reference without using any phase-locked loops (PLL). The proposed power control scheme effectively eliminates the impacts...... of steady-state fundamental current tracking errors in the DG units. Thus, an accurate power control is realized even when the harmonic compensation functions are activated. In addition, this paper also briefly discusses the performance of the proposed method when DG unit is connected to a grid...

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

    Science.gov (United States)

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

    2016-09-01

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

  6. Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites.

    Science.gov (United States)

    Martin, D W; Sachs, J R

    1999-06-01

    The phosphorylation capacity of Na+,K+-ATPase preparations in common use is much less than expected on the basis of the molecular weight of the enzyme deduced from cDNA sequences. This has led to the popularity of half-of-the-sites or flip-flop models for the enzyme reaction mechanism. We have prepared Na+,K+-ATPase from nasal salt glands of salt-adapted ducks which has a phosphorylation capacity and specific activity near the theoretical maxima. Preparations with specific activities of >60 micromol (mg of protein)-1 min-1 at 37 degrees C had phosphorylation capacities of >60 nmol/mg of protein, and the rate of turnover of the enzyme was 9690 min-1, within the range reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the protein on SDS-PAGE which was alpha and beta chains, especially at the highest specific activity which indicates that all of the alphabeta protomers are active. The gels of the most reactive preparations contained only alpha and beta chains, but less active preparations contained a number of extraneous proteins. The major contaminant was actin. The preparation did not contain any protein which migrated in the molecular weight range of the gamma subunit. The subunit composition of the enzyme was alpha1 and beta1 only. This is the first report of a pure, homogeneous, fully active preparation of the protein. Reaction models which incorporate a half-of-the-sites or flip-flop mechanism do not apply to this enzyme.

  7. Cyclin-dependent kinase 5 modulates STAT3 and androgen receptor activation through phosphorylation of Ser⁷²⁷ on STAT3 in prostate cancer cells.

    Science.gov (United States)

    Hsu, Fu-Ning; Chen, Mei-Chih; Lin, Kuan-Chia; Peng, Yu-Ting; Li, Pei-Chi; Lin, Eugene; Chiang, Ming-Ching; Hsieh, Jer-Tsong; Lin, Ho

    2013-10-15

    Cyclin-dependent kinase 5 (Cdk5) is known to regulate prostate cancer metastasis. Our previous results indicated that Cdk5 activates androgen receptor (AR) and supports prostate cancer growth. We also found that STAT3 is a target of Cdk5 in promoting thyroid cancer cell growth, whereas STAT3 may play a role as a regulator to AR activation under cytokine control. In this study, we investigated the regulation of Cdk5 and its activator p35 on STAT3/AR signaling in prostate cancer cells. Our results show that Cdk5 biochemically interacts with STAT3 and that this interaction depends on Cdk5 activation in prostate cancer cells. The phosphorylation of STAT3 at Ser⁷²⁷ (p-Ser⁷²⁷-STAT3) is regulated by Cdk5 in cells and xenograft tumors. The mutant of STAT3 S727A reduces its interaction with Cdk5. We further show that the nuclear distribution of p-Ser⁷²⁷-STAT3 and the expression of STAT3-regulated genes (junB, c-fos, c-myc, and survivin) are regulated by Cdk5 activation. STAT3 mutant does not further decrease cell proliferation upon Cdk5 inhibition, which implies that the role of STAT3 regulated by Cdk5 correlates to cell proliferation control. Interestingly, Cdk5 may regulate the interaction between STAT3 and AR through phosphorylation of Ser⁷²⁷-STAT3 and therefore upregulate AR protein stability and transactivation. Correspondingly, clinical evidence shows that the level of p-Ser⁷²⁷-STAT3 is significantly correlated with Gleason score and the levels of upstream regulators (Cdk5 and p35) as well as downstream protein (AR). In conclusion, this study demonstrates that Cdk5 regulates STAT3 activation through Ser⁷²⁷ phosphorylation and further promotes AR activation by protein-protein interaction in prostate cancer cells.

  8. Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Yogi, Alvaro; Callera, Glaucia E. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Mecawi, André S. [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Batalhão, Marcelo E.; Carnio, Evelin C. [Department of General and Specialized Nursing, College of Nursing of Ribeirão Preto, USP, São Paulo (Brazil); Antunes-Rodrigues, José [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Queiroz, Regina H. [Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences, USP, São Paulo (Brazil); Touyz, Rhian M. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Tirapelli, Carlos R., E-mail: crtirapelli@eerp.usp.br [Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP (Brazil)

    2012-11-01

    Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT{sub 1} receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT{sub 1}-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT{sub 1} receptor activation.

  9. Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

    International Nuclear Information System (INIS)

    Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT1-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT1 receptor activation. ► Translocation of p47

  10. HDAC3 as a molecular chaperone for shuttling phosphorylated TR2 to PML: a novel deacetylase activity-independent function of HDAC3.

    Directory of Open Access Journals (Sweden)

    Pawan Gupta

    Full Text Available TR2 is an orphan nuclear receptor specifically expressed in early embryos (Wei and Hsu, 1994, and a transcription factor for transcriptional regulation of important genes in stem cells including the gate keeper Oct4 (Park et al. 2007. TR2 is known to function as an activator (Wei et al. 2000, or a repressor (Chinpaisal et al., 1998, Gupta et al. 2007. Due to the lack of specific ligands, mechanisms triggering its activator or repressor function have remained puzzling for decades. Recently, we found that all-trans retinoic acid (atRA triggers the activation of extracellular-signal-regulated kinase 2 (ERK2, which phosphorylates TR2 and stimulates its partitioning to promyelocytic leukemia (PML nuclear bodies, thereby converting the activator function of TR2 into repression (Gupta et al. 2008; Park et al. 2007. Recruitment of TR2 to PML is a crucial step in the conversion of TR2 from an activator to a repressor. However, it is unclear how phosphorylated TR2 is recruited to PML, an essential step in converting TR2 from an activator to a repressor. In the present study, we use both in vitro and in vivo systems to address the problem of recruiting TR2 to PML nuclear bodies. First, we identify histone deacetylase 3 (HDAC3 as an effector molecule. HDAC3 is known to interact with TR2 (Franco et al. 2001 and this interaction is enhanced by the atRA-stimulated phosphorylation of TR2 at Thr-210 (Gupta et al. 2008. Secondly, in this study, we also find that the carrier function of HDAC3 is independent of its deacetylase activity. Thirdly, we find another novel activity of atRA that stimulates nuclear enrichment of HDAC3 to form nuclear complex with PML, which is ERK2 independent. This is the first report identifying a deacetylase-independent function for HDAC3, which serves as a specific carrier molecule that targets a specifically phosphorylated protein to PML NBs. This is also the first study delineating how protein recruitment to PML nuclear bodies occurs

  11. Phosphatidylinositol-3-kinase-dependent phosphorylation of SLP-76 by the lymphoma-associated ITK-SYK fusion-protein

    International Nuclear Information System (INIS)

    Recurrent chromosomal translocations have long been implicated in various types of lymphomas and other malignancies. Novel recurrent t(5;9)(q33;q22) has been recently discovered in un-specified peripheral T-cell lymphoma. To elucidate the role of this translocation, the corresponding fusion construct encoding the N-terminal portion of the ITK kinase and the C-terminal catalytic region of the SYK kinase was generated. We herein show that the ITK-SYK fusion-protein is constitutively active. Moreover, we demonstrate that ITK-SYK is phosphorylated on key tyrosine residues and is capable of potently phosphorylating the related adapter proteins BLNK and SLP-76. In transiently transfected cells, SYK was phosphorylated at Y352 but not detectably at the activation-loop tyrosines Y525/Y526. In contrast, ITK-SYK was phosphorylated both at Y212 and the activation-loop tyrosines Y385/Y386, corresponding to Y352 and Y525/Y526 in SYK, respectively. In resting primary lymphocytes, ITK-SYK predominantly localizes to the cell surface. In addition, we demonstrate that following stimulation, the ITK-SYK fusion-protein in cell lines translocates to the cell membrane and, moreover, that this phenomenon as well as SLP-76 phosphorylation are blocked upon phosphatidylinositol-3-kinase (PI3-kinase) inhibition.

  12. Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

    Science.gov (United States)

    Han, Cho Rong; Jun, Do Youn; Kim, Yoon Hee; Lee, Ji Young; Kim, Young Ho

    2013-10-01

    In Jurkat T cell clone (JT/Neo), G2/M arrest, apoptotic sub-G1 peak, mitochondrial membrane potential (Δψm) loss, and TUNEL-positive DNA fragmentation were induced following exposure to 17α-estradiol (17α-E2), whereas none of these events (except for G2/M arrest) were induced in Jurkat cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, phosphorylation at Thr161 and dephosphorylation at Tyr15 of Cdk1, upregulation of cyclin B1 level, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation, and Bim phosphorylation were detected in the presence of Bcl-2 overexpression. However, the 17α-E2-induced upregulation of Bak levels, activation of Bak, activation of caspase-3, and PARP degradation were abrogated by Bcl-2 overexpression. In the presence of the G1/S blocking agent hydroxyurea, 17α-E2 failed to induce G2/M arrest and all apoptotic events including Cdk1 activation and phosphorylation of Bcl-2, Mcl-1 and Bim. The 17α-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor but not by aurora A and aurora B kinase inhibitors. Immunofluorescence microscopic analysis showed that an aberrant bipolar microtubule array, incomplete chromosome congression at the metaphase plate, and prometaphase arrest, which was reversible, were the underlying factors for 17α-E2-induced mitotic arrest. The in vitro microtubule polymerization assay showed that 17α-E2 could directly inhibit microtubule formation. These results show that the apoptogenic activity of 17α-E2 was due to the impaired mitotic spindle assembly causing prometaphase arrest and prolonged Cdk1 activation, the phosphorylation of Bcl-2, Mcl-1 and Bim, and the activation of Bak and mitochondria-dependent caspase cascade. PMID:23707954

  13. Benchmarking and Hardware-In-The-Loop Operation of a 2014 MAZDA SkyActiv

    Science.gov (United States)

    Engine Performance evaluation in support of LD MTE. EPA used elements of its ALPHA model to apply hardware-in-the-loop (HIL) controls to the SKYACTIV engine test setup to better understand how the engine would operate in a chassis test after combined with future leading edge tech...

  14. Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Pegan, Scott D. [Univ. of Denver, CO (United States); Rukseree, Kamolchanok [National Center for Genetic Engineering and Biotechnology (BIOTEC), Tha Khlong (Thailand); Capodagli, Glenn C. [Univ. of Denver, CO (United States); Baker, Erica A. [Univ. of Denver, CO (United States); Krasnykh, Olga [Univ. of Illinois, Chicago, IL (United States); Franzblau, Scott G. [Univ. of Illinois, Chicago, IL (United States); Mesecar, Andrew D. [Purdue Univ., West Lafayette, IN (United States)

    2013-01-08

    The class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprises one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation–deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA–PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation–protonation step of the MtFBA reaction mechanism. Furthermore, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.

  15. Homology models of dipeptidyl peptidases 8 and 9 with a focus on loop predictions near the active site.

    Science.gov (United States)

    Rummey, Christian; Metz, Günther

    2007-01-01

    Dipeptidyl peptidase 4 (DP4) inhibitors are currently under intensive investigation in late-stage clinical trials as a treatment for type II diabetes. Lack of selectivity toward the related enzymes DP8 and DP9 has recently emerged as a possible source of drug-induced toxicity. Unlike DP4, X-ray structures of DP8 and DP9 are not yet available. As an aid to understanding the structural basis for selectivity, the authors have constructed homology models of DP8 and DP9 based on the X-ray coordinates of DP4. Accurate sequence alignment reveals common structural features indicative for a well-preserved overall fold comprising two domains, namely, a hydrolase domain and a so-called beta-propeller, which together form the active site deeply buried within the protein. The conformation of two loops inside this deep cavity is particularly relevant for the active sites. The authors used a published protocol for loop prediction based on conformational sampling and energy analysis to generate plausible solutions for these two loops. The predictive power of the approach was successfully evaluated for the template protein DP4 and two additional known structures from the same protein family, namely, FAP and DPX. The authors also show that inclusion of the covalent ligand NVP-728 greatly enhances the refinement. Based on the established evaluation protocol, the corresponding loops of DP8 and DP9 were predicted and the resulting active sites were compared with DP4. In particular, the authors conclude that differences in the P2-pocket are relevant for the design of selective DP4 inhibitors. The loss of key interactions in DP8 and DP9 as predicted from their models is consistent with the selectivity profile of the DP4 clinical candidate MK-431.

  16. Cell type-specific anti-cancer properties of valproic acid: independent effects on HDAC activity and Erk1/2 phosphorylation

    DEFF Research Database (Denmark)

    Gotfryd, Kamil; Skladchikova, Galina; Lepekhin, Eugene E;

    2010-01-01

    ABSTRACT: BACKGROUND: The anti-epileptic drug valproic acid (VPA) has attracted attention as an anti-cancer agent. Methods: The present study investigated effects of VPA exposure on histone deacetylase (HDAC) inhibition, cell growth, cell speed, and the degree of Erk1/2 phosphorylation in 10 cell...... lines (BT4C, BT4Cn, U87MG, N2a, PC12-E2, CSML0, CSML100, HeLa, L929, Swiss 3T3). Results: VPA induced significant histone deacetylase (HDAC) inhibition in most of the cell lines, but the degree of inhibition was highly cell type-specific. Moreover, cell growth, motility and the degree of Erk1/2...... phosphorylation were inhibited, activated, or unaffected by VPA in a cell type-specific manner. Importantly, no relationship was found between the effects of VPA on HDAC inhibition and changes in the degree of Erk1/2 phosphorylation, cell growth, or motility. In contrast, VPA-induced modulation of the MAPK...

  17. Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

    Energy Technology Data Exchange (ETDEWEB)

    Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

    2015-07-15

    Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

  18. Transcriptional activation of p21(WAF¹/CIP¹) is mediated by increased DNA binding activity and increased interaction between p53 and Sp1 via phosphorylation during replicative senescence of human embryonic fibroblasts.

    Science.gov (United States)

    Kim, Hyun-Seok; Heo, Jee-In; Park, Seong-Hoon; Shin, Jong-Yeon; Kang, Hong-Jun; Kim, Min-Ju; Kim, Sung Chan; Kim, Jaebong; Park, Jae-Bong; Lee, Jae-Yong

    2014-01-01

    Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation.

  19. Phosphorylation of CDK9 at Ser175 enhances HIV transcription and is a marker of activated P-TEFb in CD4(+ T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Uri R Mbonye

    Full Text Available The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification-tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative

  20. The Mek1 phosphorylation cascade plays a role in meiotic recombination of Schizosaccharomyces pombe.

    Science.gov (United States)

    Tougan, Takahiro; Kasama, Takashi; Ohtaka, Ayami; Okuzaki, Daisuke; Saito, Takamune T; Russell, Paul; Nojima, Hiroshi

    2010-12-01

    Mek1 is a Chk2/Rad53/Cds1-related protein kinase that is required for proper meiotic progression of Schizosaccharomyces pombe. However, the molecular mechanisms of Mek1 regulation and Mek1 phosphorylation targets are unclear. Here, we report that Mek1 is phosphorylated at serine-12 (S12), S14 and threonine-15 (T15) by Rad3 (ATR) and/or Tel1 (ATM) kinases that are activated by meiotic programmed double-strand breaks (DSBs). Mutations of these sites by alanine replacement caused abnormal meiotic progression and recombination rates. Phosphorylation of these sites triggers autophosphorylation of Mek1; indeed, alanine replacement mutations of Mek1-T318 and -T322 residues in the activation loop of Mek1 reduced Mek1 kinase activity and meiotic recombination rates. Substrates of Mek1 include Mus81-T275, Rdh54-T6 and Rdh54-T673. Mus81-T275 is known to regulate the Mus81 function in DNA cleavage, whereas Rdh54-T6A/T673A mutant cells showed abnormal meiotic recombination. Taken together, we conclude that the phosphorylation of Mek1 by Rad3 or Tel1, Mek1 autophosphorylation and Mus81 or Rdh54 phosphorylation by Mek1 regulate meiotic progression in S. pombe. PMID:21084840

  1. Dynamin Reduces Pyk2 Y402 Phosphorylation and Src Binding in Osteoclasts ▿ †

    Science.gov (United States)

    Bruzzaniti, Angela; Neff, Lynn; Sandoval, Amanda; Du, Liping; Horne, William C.; Baron, Roland

    2009-01-01

    Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin's GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2. PMID:19380485

  2. Dynamin reduces Pyk2 Y402 phosphorylation and SRC binding in osteoclasts.

    Science.gov (United States)

    Bruzzaniti, Angela; Neff, Lynn; Sandoval, Amanda; Du, Liping; Horne, William C; Baron, Roland

    2009-07-01

    Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin's GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2. PMID:19380485

  3. Properties of phosphorylated thymidylate synthase.

    Science.gov (United States)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  4. Activities in the SBL-FPL loops for LMFBR safety studies, (2)

    International Nuclear Information System (INIS)

    As the first step for the safety study of liquid metal-cooled fast breeder reactors, it is necessary to understand the initial events of potential accidents in LMFBRs. Local faults and sodium boiling are conceivable. The sodium boiling test loop (SBL) and the fuel failure propagation test loop (FPL) were constructed to investigate such phenomena experimentally. The two loops share one sodium dump tank and the purification system, and the whole facility is called ''Sodium installation for experiment of nuclear reactor safety analysis (SIENA)''. In almost all experiments, electrically heated fuel pin simulators were used in the form of pin bundles. The experimental studies using SBL-FPL have been carried out by the ''Core safety sodium behavior experiment group (CSSBEG)''. In addition, the group has operated two water test rigs for the experiments on large bubble behavior during hypothetical core disruptive accidents. A local event may successively cause the failure of adjacent fuel pins by the local worsening of cooling capability. The studies on fuel pin contact, FP gas release, local blockage and anomaly detection were carried out. The cooling of pin bundles by boiling in low heat flux and low flow rate conditions was tested. The behavior of bubbles in HCDA and the behavior of melted claddings were also investigated. (Kako, I.)

  5. Increased activity of the Vesicular Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor TI-VAMP/VAMP7 by Tyrosine Phosphorylation in the Longin Domain*

    Science.gov (United States)

    Burgo, Andrea; Casano, Alessandra M.; Kuster, Aurelia; Arold, Stefan T.; Wang, Guan; Nola, Sébastien; Verraes, Agathe; Dingli, Florent; Loew, Damarys; Galli, Thierry

    2013-01-01

    Vesicular (v)- and target (t)-SNAREs play essential roles in intracellular membrane fusion through the formation of cytoplasmic α-helical bundles. Several v-SNAREs have a Longin N-terminal extension that, by promoting a closed conformation, plays an autoinhibitory function and decreases SNARE complex formation and membrane fusion efficiency. The molecular mechanism leading to Longin v-SNARE activation is largely unknown. Here we find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise. In search of a potential downstream mechanism, we found that TI-VAMP is phosphorylated in vitro by c-Src kinase on tyrosine 45 of the Longin domain. Accordingly, a mutation of tyrosine 45 into glutamate, but not phenylalanine, activates both t-SNARE binding and exocytosis. Activation of TI-VAMP-mediated exocytosis thus relies on tyrosine phosphorylation. PMID:23471971

  6. Increased activity of the vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor TI-VAMP/VAMP7 by tyrosine phosphorylation in the Longin domain.

    Science.gov (United States)

    Burgo, Andrea; Casano, Alessandra M; Kuster, Aurelia; Arold, Stefan T; Wang, Guan; Nola, Sébastien; Verraes, Agathe; Dingli, Florent; Loew, Damarys; Galli, Thierry

    2013-04-26

    Vesicular (v)- and target (t)-SNAREs play essential roles in intracellular membrane fusion through the formation of cytoplasmic α-helical bundles. Several v-SNAREs have a Longin N-terminal extension that, by promoting a closed conformation, plays an autoinhibitory function and decreases SNARE complex formation and membrane fusion efficiency. The molecular mechanism leading to Longin v-SNARE activation is largely unknown. Here we find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise. In search of a potential downstream mechanism, we found that TI-VAMP is phosphorylated in vitro by c-Src kinase on tyrosine 45 of the Longin domain. Accordingly, a mutation of tyrosine 45 into glutamate, but not phenylalanine, activates both t-SNARE binding and exocytosis. Activation of TI-VAMP-mediated exocytosis thus relies on tyrosine phosphorylation.

  7. Physical Activity Capture Technology With Potential for Incorporation Into Closed-Loop Control for Type 1 Diabetes.

    Science.gov (United States)

    Dadlani, Vikash; Levine, James A; McCrady-Spitzer, Shelly K; Dassau, Eyal; Kudva, Yogish C

    2015-11-01

    Physical activity is an important determinant of glucose variability in type 1 diabetes (T1D). It has been incorporated as a nonglucose input into closed-loop control (CLC) protocols for T1D during the last 4 years mainly by 3 research groups in single center based controlled clinical trials involving a maximum of 18 subjects in any 1 study. Although physical activity data capture may have clinical benefit in patients with T1D by impacting cardiovascular fitness and optimal body weight achievement and maintenance, limited number of such studies have been conducted to date. Clinical trial registries provide information about a single small sample size 2 center prospective study incorporating physical activity data input to modulate closed-loop control in T1D that are seeking to build on prior studies. We expect an increase in such studies especially since the NIH has expanded support of this type of research with additional grants starting in the second half of 2015. Studies (1) involving patients with other disorders that have lasted 12 weeks or longer and tracked physical activity and (2) including both aerobic and resistance activity may offer insights about the user experience and device optimization even as single input CLC heads into real-world clinical trials over the next few years and nonglucose input is introduced as the next advance. PMID:26481641

  8. Physical Activity Capture Technology With Potential for Incorporation Into Closed-Loop Control for Type 1 Diabetes.

    Science.gov (United States)

    Dadlani, Vikash; Levine, James A; McCrady-Spitzer, Shelly K; Dassau, Eyal; Kudva, Yogish C

    2015-10-18

    Physical activity is an important determinant of glucose variability in type 1 diabetes (T1D). It has been incorporated as a nonglucose input into closed-loop control (CLC) protocols for T1D during the last 4 years mainly by 3 research groups in single center based controlled clinical trials involving a maximum of 18 subjects in any 1 study. Although physical activity data capture may have clinical benefit in patients with T1D by impacting cardiovascular fitness and optimal body weight achievement and maintenance, limited number of such studies have been conducted to date. Clinical trial registries provide information about a single small sample size 2 center prospective study incorporating physical activity data input to modulate closed-loop control in T1D that are seeking to build on prior studies. We expect an increase in such studies especially since the NIH has expanded support of this type of research with additional grants starting in the second half of 2015. Studies (1) involving patients with other disorders that have lasted 12 weeks or longer and tracked physical activity and (2) including both aerobic and resistance activity may offer insights about the user experience and device optimization even as single input CLC heads into real-world clinical trials over the next few years and nonglucose input is introduced as the next advance.

  9. Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

    Energy Technology Data Exchange (ETDEWEB)

    Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie; Coppola, Thierry [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France); Mazella, Jean, E-mail: mazella@ipmc.cnrs.fr [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)

    2011-10-14

    Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

  10. Exercise maintains euglycemia in association with decreased activation of c-Jun NH2-terminal kinase and serine phosphorylation of IRS-1 in the liver of ZDF rats.

    Science.gov (United States)

    Király, Michael A; Campbell, Jon; Park, Edward; Bates, Holly E; Yue, Jessica T Y; Rao, Venket; Matthews, Stephen G; Bikopoulos, George; Rozakis-Adcock, Maria; Giacca, Adria; Vranic, Mladen; Riddell, Michael C

    2010-03-01

    Stress-activated systems and oxidative stress are involved in insulin resistance, which, along with beta-cell failure, contribute to the development of type 2 diabetes mellitus (T2DM). Exercise improves insulin resistance and glucose tolerance, and these adaptations may, in part, be related to reductions in inflammation and oxidative stress. We investigated circulating and tissue-specific markers of inflammation and oxidative stress and insulin-signaling pathways in a rodent model of T2DM, the Zucker diabetic fatty rat, with and without voluntary exercise. At 5 wk of age, Zucker diabetic fatty rats (n = 8-9/group) were divided into basal (B), voluntary exercise (E), and sedentary control (S) groups. B rats were euthanized at 6 wk of age, and S and E rats were euthanized 10 wk later. E rats ran approximately 5 km/day, which improved insulin sensitivity and maintained fed and fasted glucose levels and glucose tolerance. Ten weeks of exercise also decreased whole body markers of inflammation and oxidative stress in plasma and liver, including lowered circulating IL-6, haptoglobin, and malondialdehyde levels, hepatic protein oxidation, and phosphorylated JNK, the latter indicating decreased JNK activity. Hepatic phosphoenolpyruvate carboxykinase levels and Ser(307)-phosphorylated insulin receptor substrate-1 were also reduced in E compared with S rats. In summary, we show that, in a rodent model of T2DM, voluntary exercise decreases circulating markers of inflammation and oxidative stress and lowers hepatic JNK activation and Ser(307)-phosphorylated insulin receptor substrate-1. These changes in oxidative stress markers and inflammation are associated with decreased hyperglycemia and insulin resistance and reduced expression of the main gluconeogenic enzyme phosphoenolpyruvate carboxykinase. PMID:19996384

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

    Science.gov (United States)

    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

  12. Translation of the radioresistance kinase TLK1B is induced by γ-irradiation through activation of mTOR and phosphorylation of 4E-BP1

    Directory of Open Access Journals (Sweden)

    De Benedetti Arrigo

    2004-04-01

    Full Text Available Abstract Background The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila, or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and chromosome missegregation probably due to alterations in chromatin remodeling capacity. Overexpression of TLK1B, a spliced variant of the TLK1 mRNA, in a model mouse cell line increases their resistance to ionizing radiation, also likely through changes in chromatin remodeling. The TLK1B mRNA is translationally repressed by its 5'UTR and is regulated by the availability of eIF4E. We now report that radiation or doxorubicin result in an increase in the translation of TLK1B, and we have uncovered the likely mechanism for this effect. Results Radiation causes a shift in the polysomal distribution of TLK1B mRNA, from the untranslated region and small polysomes to the large polysomes, concomitant with an increase in the expression of TLK1B protein. This change is preceded by an increase in phosphorylation of the eIF4E inhibitory protein 4E-BP1, which releases eIF4E when it is phosphorylated. The phosphorylation of 4E-BP1 depends on mTOR, since rapamycin blocked the increase in phosphorylation induced by radiation, and prevented the increase in TLK1B protein expression. The activation of mTOR was likely due to the rapid activation of Akt following radiation. The activation of Akt could be inhibited with wortmannin, an inhibitor of PI3 kinase, hence placing PI3 kinase upstream of Akt as a very early event following radiation. Wortmannin also inhibited translation of TLK1B mRNA following activation by IR. This was shown both by western blot and by measuring the initiation capacity of the mRNA, as indicated by its distribution on polysomes. Conclusions The translational upregulation of TLK1B elicited by DNA double strand breaks

  13. Theoretical Study on the Activity of α-COOH and β-COOH of N-Phosphoryl Aspartic Acids

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The bio-mimic reactions of N-phosphoryl amino acids are very important in the study of many biochemical processes. The difference of reactivity between α-COOH and β-COOH in phosphoryl aspartic acid was studied by theoretical study (Hartree-Fock and Density Functional methods) in this paper. The intermediates Ⅱ containing five-membered ring were more stable than Ⅲ with six-membered ring. While for intermediates Ⅲ, the isomers with six-membered ring in apical-equatorial spanning arrangement were more stable than those with di-equatorial spanning arrangement. At B3LYP/6-31G** level, it was shown that transition states Ⅳ and V involving α-COOH or β-COOH group had energy barriers of ΔE = 58.67 kJ·mol-1 and 103.94 kJ·mol-1, respectively. These results were in agreement with the experimental data. So the α-COOH group was involved in form of the intramolecular penta-coordinate phosphoric-carboxylic mixed anhydride intermediates, but not β-COOH group.

  14. When phosphorylated at Thr148, the β2-subunit of AMP-activated kinase does not associate with glycogen in skeletal muscle.

    Science.gov (United States)

    Xu, Hongyang; Frankenberg, Noni T; Lamb, Graham D; Gooley, Paul R; Stapleton, David I; Murphy, Robyn M

    2016-07-01

    The 5'-AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of α- and β-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, ∼60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an ∼80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total β2-AMPK that was phosphorylated in whole muscle homogenates measured by immunoprecipitation. These findings suggest that, in rat skeletal muscle, β2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate β2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of β2-AMPK in skeletal muscle. PMID:27099349

  15. The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F.

    Science.gov (United States)

    Gorges, Laura L; Lents, Nathan H; Baldassare, Joseph J

    2008-11-01

    The retinoblastoma protein pRb plays a pivotal role in G(1)- to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and COOH-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the NH(2)-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using "reverse mutational analysis" of Rb(DeltaCDK) (a dominantly active repressive allele of Rb), we have previously shown that restoration of Thr-373 is sufficient to render Rb(DeltaCDK) sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the NH(2)-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we have expanded this analysis to include additional residues in the NH(2)-terminal region of pRb and further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further have found that removal of the COOH-terminal domain of either RbDeltaCDK(+T373) or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Our data demonstrate a novel function for the NH(2)-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.

  16. Distribution of phosphorylated Elk-1 in rat brain after Y-maze active avoidance training in a temporal manner

    Institute of Scientific and Technical Information of China (English)

    Xuhong Chen; Siyun Shu; Zhenjiang Liang; Xinmin Bao; Lixue Chen; Yongming Wu

    2006-01-01

    BACKGROUND: Elk-1 mRNA distributes extensively in the neurons of mice, rat and human brains, and the Elk-1 expression may be correlated with the synaptic plasticity, learning and memory.OBJECTIVE: To observe the distribution of phosphorylated Elk-1 (pElk-1) in whole brain of rats received Y-maze active avoidance training and the changes of pElk-1 expression at different time points after training.DESIGN: A randomized controlled study.SETTING: Research Room of Neurobiology, the Second Affiliated Hospital of Southern Medical University.MATERIALS: Fifty-five male clean-degree SD rats of 3-4 months old, weighing 200-250 g, were provided by the Experimental Animal Center of Southern Medical University. The rabbit anti-monoclonal pElk-1 antibody was purchased from Cell Signal Transduction Company, and ABC kit from Vector Company.METHODS: The experiments were carried out in the Research Room of Neurobiology, Second Affiliated Hospital of Southern Medical University from September 2004 to February 2005. ① Grouping: The rats were randomly divided into training group (n = 25), sham-training group (n = 25) and norm al control group (n = 5), and the training and sham-training groups were observed at 0, 1, 3, 6 and 24 hours after training, which represented the five phases in the process of learning and memory. ② Y-maze training: The rats were preconditioned in the electrical Y-maze apparatus, 20 minutes a day for 3 days continuously, and training began from the 4th day. In the training group, the rats were trained with the combination of light and electricity. Each rat repeated for 60 times in each training, and the correct times were recorded, those correct for less than 25 times were taken as unqualified, and excluded from the training group, and supplemented by other rats in time. In the sham-training group, there was no fixed correlation between the application of light and electricity. The rats in the normal control group were given not any training. ③Detection of p

  17. Evidence against a role for the JIL-1 kinase in H3S28 phosphorylation and 14-3-3 recruitment to active genes in Drosophila.

    Directory of Open Access Journals (Sweden)

    Chao Wang

    Full Text Available JIL-1 is the major kinase controlling phosphorylation of histone H3S10 and has been demonstrated to function to counteract heterochromatization and gene silencing. However, an alternative model has been proposed in which JIL-1 is required for transcription to occur, additionally phosphorylates H3S28, and recruits 14-3-3 to active genes. Since these findings are incompatible with our previous demonstration that there are robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at developmental or heat shock-induced polytene chromosome puffs, we have reexamined JIL-1's possible role in H3S28 phosphorylation and 14-3-3 recruitment. Using two different H3S28ph antibodies we show by immunocytochemistry and immunoblotting that in Drosophila the H3S28ph mark is not present at detectable levels above background on polytene chromosomes at interphase but only on chromosomes at pro-, meta-, and anaphase during cell division in S2 cells and third instar larval neuroblasts. Moreover, this mitotic H3S28ph signal is also present in a JIL-1 null mutant background at undiminished levels suggesting that JIL-1 is not the mitotic H3S28ph kinase. We also demonstrate that H3S28ph is not enriched at heat shock puffs. Using two different pan-specific 14-3-3 antibodies as well as an enhancer trap 14-3-3ε-GFP line we show that 14-3-3, while present in salivary gland nuclei, does not localize to chromosomes but only to the nuclear matrix surrounding the chromosomes. In our hands 14-3-3 is not recruited to developmental or heat shock puffs. Furthermore, using a lacO repeat tethering system to target LacI-JIL-1 to ectopic sites on polytene chromosomes we show that only H3S10ph is present and upregulated at such sites, not H3S28ph or 14-3-3. Thus, our results argue strongly against a model where JIL-1 is required for H3S28 phosphorylation and 14-3-3 recruitment at active genes.

  18. Ras-activated RSK1 phosphorylates EBP50 to regulate its nuclear localization and promote cell proliferation.

    Science.gov (United States)

    Lim, Hooi Cheng; Jou, Tzuu-Shuh

    2016-03-01

    Differential subcellular localization of EBP50 leads to its controversial role in cancer biology either as a tumor suppressor when it resides at the membrane periphery, or a tumor facilitator at the nucleus. However, the mechanism behind nuclear localization of EBP50 remains unclear. A RNA interference screening identified the downstream effector of the Ras-ERK cascade, RSK1, as the molecule unique for nuclear transport of EBP50. RSK1 binds to EBP50 and phosphorylates it at a conserved threonine residue at position 156 (T156) under the regulation of growth factor. Mutagenesis experiments confirmed the significance of T156 residue in nuclear localization of EBP50, cellular proliferation, and oncogenic transformation. Our study sheds light on a possible therapeutic strategy targeting at this aberrant nuclear expression of EBP50 without affecting the normal physiological function of EBP50 at other subcellular localization.

  19. BAFF induces spleen CD4+ T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    International Nuclear Information System (INIS)

    Highlights: ► Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4+ T cells. ► Carrying out siRNA technology to study FOXO3A protein function. ► Helpful to understand the T cell especially CD4+ T cell‘s role in immunological reaction. -- Abstract: The TNF ligand family member “B cell-activating factor belonging to the TNF family” (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4+ spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4+ T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4+ spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4+ T cell proliferation.

  20. Antidepressant-like activity of resveratrol treatment in the forced swim test and tail suspension test in mice: the HPA axis, BDNF expression and phosphorylation of ERK.

    Science.gov (United States)

    Wang, Zhen; Gu, Jianhua; Wang, Xueer; Xie, Kai; Luan, Qinsong; Wan, Nianqing; Zhang, Qun; Jiang, Hong; Liu, Dexiang

    2013-11-01

    Resveratrol is a natural polyphenol enriched in Polygonum cuspidatum and has diverse biological activities. There is only limited information about the antidepressant-like effect of resveratrol. The present study assessed whether resveratrol treatment (20, 40 and 80mg/kg, i.p., 21days) has an antidepressant-like effect on the forced swim test (FST) and tail suspension test (TST) in mice and examined what its molecular targets might be. The results showed that resveratrol administration produced antidepressant-like effects in mice, evidenced by the reduced immobility time in the FST and TST, while it had no effect on the locomotor activity in the open field test. Resveratrol treatment significantly reduced serum corticosterone levels, which had been elevated by the FST and TST. Moreover, resveratrol increased brain-derived neurotrophic factor (BDNF) protein and extracellular signal-regulated kinase (ERK) phosphorylation levels in the prefrontal cortex and hippocampus. All of these antidepressant-like effects of resveratrol were essentially similar to those observed with the clinical antidepressant, fluoxetine. These results suggest that the antidepressant-like effects of resveratrol in the FST and TST are mediated, at least in part, by modulating hypothalamic-pituitary-adrenal axis, BDNF and ERK phosphorylation expression in the brain region of mice.

  1. Proteomic analysis of the oil palm fruit mesocarp reveals elevated oxidative phosphorylation activity is critical for increased storage oil production.

    Science.gov (United States)

    Loei, Hendrick; Lim, Justin; Tan, Melvin; Lim, Teck Kwang; Lin, Qing Song; Chew, Fook Tim; Kulaveerasingam, Harikrishna; Chung, Maxey C M

    2013-11-01

    Palm oil is a highly versatile commodity with wide applications in the food, cosmetics, and biofuel industries. Storage oil in the oil palm mesocarp can make up a remarkable 80% of its dry mass, making it the oil crop with the richest oil content in the world. As such, there has been an ongoing interest in understanding the mechanism of oil production in oil palm fruits. To identify the proteome changes during oil palm fruit maturation and factors affecting oil yield in oil palm fruits, we examined the proteomic profiles of oil palm mesocarps at four developing stages--12, 16, 18, and 22 weeks after pollination--by 8-plex iTRAQ labeling coupled to 2D-LC and MALDI-TOF/TOF MS. It was found that proteins from several important metabolic processes, including starch and sucrose metabolism, glycolysis, pentose phosphate shunt, fatty acid biosynthesis, and oxidative phosphorylation, were differentially expressed in a concerted manner. These increases led to an increase in carbon flux and a diversion of resources such as ATP and NADH that are required for lipid biosynthesis. The temporal proteome profiles between the high-oil-yielding (HY) and low-oil-yielding (LY) fruits also showed significant differences in the levels of proteins involved in the regulation of the TCA cycle and oxidative phosphorylation. In particular, the expression level of the β subunit of the ATP synthase complex (complex IV of the electron transport chain) was found to be increased during fruit maturation in HY but decreased in the LY during the fruit maturation. These results suggested that increased energy supply is necessary for augmented oil yield in the HY oil palm trees. PMID:24083564

  2. OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation.

    Science.gov (United States)

    Zhang, Baowen; Wang, Xiaolong; Zhao, Zhiying; Wang, Ruiju; Huang, Xiahe; Zhu, Yali; Yuan, Li; Wang, Yingchun; Xu, Xiaodong; Burlingame, Alma L; Gao, Yingjie; Sun, Yu; Tang, Wenqiang

    2016-02-01

    Many plant receptor kinases transduce signals through receptor-like cytoplasmic kinases (RLCKs); however, the molecular mechanisms that create an effective on-off switch are unknown. The receptor kinase BR INSENSITIVE1 (BRI1) transduces brassinosteroid (BR) signal by phosphorylating members of the BR-signaling kinase (BSK) family of RLCKs, which contain a kinase domain and a C-terminal tetratricopeptide repeat (TPR) domain. Here, we show that the BR signaling function of BSKs is conserved in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) and that the TPR domain of BSKs functions as a "phospho-switchable" autoregulatory domain to control BSKs' activity. Genetic studies revealed that OsBSK3 is a positive regulator of BR signaling in rice, while in vivo and in vitro assays demonstrated that OsBRI1 interacts directly with and phosphorylates OsBSK3. The TPR domain of OsBSK3, which interacts directly with the protein's kinase domain, serves as an autoinhibitory domain to prevent OsBSK3 from interacting with bri1-SUPPRESSOR1 (BSU1). Phosphorylation of OsBSK3 by OsBRI1 disrupts the interaction between its TPR and kinase domains, thereby increasing the binding between OsBSK3's kinase domain and BSU1. Our results not only demonstrate that OsBSK3 plays a conserved role in regulating BR signaling in rice, but also provide insight into the molecular mechanism by which BSK family proteins are inhibited under basal conditions but switched on by the upstream receptor kinase BRI1.

  3. Decreased hepatic phosphorylated p38 mitogen-activated protein kinase contributes to attenuation of thioacetamide-induced hepatic necrosis in diet-induced obese mice.

    Science.gov (United States)

    Shirai, Makoto; Arakawa, Shingo; Teranishi, Munehiro; Kai, Kiyonori

    2016-04-01

    We previously reported that thioacetamide (TA)-induced hepatocellular necrosis was attenuated in mice fed a high-fat diet (HFD mice) compared with mice fed a normal rodent diet (ND mice). In this study, we investigated whether p38 mitogen-activated protein kinase (p38 MAPK) was involved in this attenuation. Western blot analysis revealed that hepatic phosphorylated p38 MAPK protein decreased at 8 and 24 hours (hr) after TA dosing in the HFD mice, while it decreased only at 24 hr in the ND mice in comparison to the time- and diet-matched, vehicle-treated mice. p38 MAPK regulates various biological functions including inflammation, therefore, hepatic metabolomics analysis focusing on pro-inflammatory lipid mediators was performed. At 24 hr after TA dosing, only one pro-inflammatory mediator, 12-hydroxyeicosatetraenoic acid (HETE), was higher in the HFD mice. On the other hand, in addition to 12-HETE, 15-HETE and 12-hydroxyeicosapentaenoic acid (HEPE) were higher and omega-3/omega-6 polyunsaturated fatty acids ratios were lower in the ND mice at 24 hr. These results of metabolomics indicated that less pro-inflammatory state was seen in HFD mice than in ND mice at 24 hr. Finally, to confirm whether the observed decrease in phosphorylated p38 MAPK could attenuate TA-induced hepatocellular necrosis, we showed that SB203580 hydrochloride, an inhibitor of p38 MAPK, partially attenuated TA-induced hepatic necrosis in ND mice. Collectively, these results suggest that a prompt decrease in phosphorylation of p38 MAPK after TA administration is one of the factors that attenuate TA-induced hepatic necrosis in HFD mice. PMID:26961609

  4. Structural studies of conformational changes of proteins upon phosphorylation: Structures of activated CheY, CheY-N16-FliM complex, and AAA {sup +} ATPase domain of NtrC1 in both inactive and active states

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seok-Yong

    2003-04-10

    Protein phosphorylation is a general mechanism for signal transduction as well as regulation of cellular function. Unlike phosphorylation in eukaryotic systems that uses Ser/Thr for the sites of modification, two-component signal transduction systems, which are prevalent in bacteria, archea, and lower eukaryotes, use an aspartate as the site of phosphorylation. Two-component systems comprise a histidine kinase and a receiver domain. The conformational change of the receiver domain upon phosphorylation leads to signal transfer to the downstream target, a process that had not been understood well at the molecular level. The transient nature of the phospho-Asp bond had made structural studies difficult. The discovery of an excellent analogue for acylphosphate, BeF{sub 3}{sup -}, enabled structural study of activated receiver domains. The structure of activated Chemotaxis protein Y (CheY) was determined both by NMR spectroscopy and X-ray crystallography. These structures revealed the molecular basis of the conformational change that is coupled to phosphorylation. Phosphorylation of the conserved Asp residue in the active site allows hydrogen bonding of the T87 O{gamma} to phospho-aspartate, which in turn leads to the rotation of Y106 into the ''in'' position (termed Y-T coupling). The structure of activated CheY complexed with the 16 N-terminal residues of FliM (N16-FliM), its target, was also determined by X-ray crystallography and confirmed the proposed mechanism of activation (Y-T coupling). First, N16-FliM binds to the region on CheY that undergoes a significant conformational change. Second, the ''in'' position of Y106 presents a better binding surface for FliM because the sidechain of Y106 in the inactive form of CheY (''out'' position) sterically interferes with binding of N16-FliM. In addition to confirmation of Y-T coupling, the structure of the activated CheY-N16-FliM complex suggested that the N16

  5. The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

    Science.gov (United States)

    Knight, Jonathan D.; Li, Rong; Botchan, Michael

    1991-04-01

    The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

  6. Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane.

    Science.gov (United States)

    Chiu, C-F; Ho, M-Y; Peng, J-M; Hung, S-W; Lee, W-H; Liang, C-M; Liang, S-M

    2013-02-01

    Prohibitin (PHB) is indispensable for Ras-induced Raf-1 activation, cell migration and growth; however, the exact role of PHB in the molecular pathogenesis of cancer metastasis remains largely unexamined. Here, we found a positive correlation between plasma membrane-associated PHB and the clinical stages of cancer. The level of PHB phosphorylated at threonine 258 (T258) and tyrosine 259 (Y259) in human cancer-cell membranes correlated with the invasiveness of cancer cells. Overexpression of phosphorylated PHB (phospho-PHB) in the lipid-raft domain of the cell membrane enhanced cell migration/invasion through PI3K/Akt and Raf-1/ERK activation. It also enhanced epithelial-mesenchymal transition, matrix metalloproteinase-2 activity and invasiveness of cancer cells in vitro. Immunoprecipitation analysis demonstrated that phospho-PHB associated with Raf-1, Akt and Ras in the membrane and was essential for the activation of Raf-1 signaling by Ras. Mice implanted with cancer cells stably overexpressing PHB in the plasma membrane showed enlarged cervical tumors, enhanced metastasis and shorter survival time compared with mice implanted with cancer cells without PHB overexpression. Dephosphorylation of PHB at T258 by site-directed mutagenesis diminished the in vitro and in vivo effects of PHB. These results suggest that increase in phospho-PHB T258 in the raft domain of the plasma membrane has a role in the Ras-driven activation of PI3K/Akt and Raf-1/ERK-signaling cascades and results in the promotion of cancer metastasis.

  7. Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.

    Science.gov (United States)

    Hutson, N J; Kerbey, A L; Randle, P J; Sugden, P H

    1978-08-01

    1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with

  8. Using Coronal Loops to Reconstruct the Magnetic Field of an Active Region Before and After a Major Flare

    CERN Document Server

    Malanushenko, A; DeRosa, M L; Wheatland, M S

    2013-01-01

    The shapes of solar coronal loops are sensitive to the presence of electrical currents that are the carriers of the nonpotential energy available for impulsive activity. We use this information in a new method for modeling the coronal magnetic field of AR 11158 as a nonlinear force-free field (NLFFF). The observations used are coronal images around time of major flare activity on 2011/02/15, together with the surface line-of-sight magnetic field measurements. The data are from the Helioseismic and Magnetic Imager and Atmospheric Imaging Assembly (HMI and AIA, respectively) onboard the Solar Dynamics Observatory (SDO). The model fields are constrained to approximate the coronal loop configurations as closely as possible, while also subject to the force-free constraints. The method does not use transverse photospheric magnetic field components as input, and is thereby distinct from methods for modeling NLFFFs based on photospheric vector magnetograms. We validate the method using observations of AR 11158 at a t...

  9. Intrinsic monitoring of learning success facilitates memory encoding via the activation of the SN/VTA-Hippocampal loop.

    Science.gov (United States)

    Ripollés, Pablo; Marco-Pallarés, Josep; Alicart, Helena; Tempelmann, Claus; Rodríguez-Fornells, Antoni; Noesselt, Toemme

    2016-01-01

    Humans constantly learn in the absence of explicit rewards. However, the neurobiological mechanisms supporting this type of internally-guided learning (without explicit feedback) are still unclear. Here, participants who completed a task in which no external reward/feedback was provided, exhibited enhanced fMRI-signals within the dopaminergic midbrain, hippocampus, and ventral striatum (the SN/VTA-Hippocampal loop) when successfully grasping the meaning of new-words. Importantly, new-words that were better remembered showed increased activation and enhanced functional connectivity between the midbrain, hippocampus, and ventral striatum. Moreover, enhanced emotion-related physiological measures and subjective pleasantness ratings during encoding were associated with remembered new-words after 24 hr. Furthermore, increased subjective pleasantness ratings were also related to new-words remembered after seven days. These results suggest that intrinsic-potentially reward-related-signals, triggered by self-monitoring of correct performance, can promote the storage of new information into long-term memory through the activation of the SN/VTA-Hippocampal loop, possibly via dopaminergic modulation of the midbrain. PMID:27644419

  10. Intrinsic monitoring of learning success facilitates memory encoding via the activation of the SN/VTA-Hippocampal loop

    Science.gov (United States)

    Ripollés, Pablo; Marco-Pallarés, Josep; Alicart, Helena; Tempelmann, Claus; Rodríguez-Fornells, Antoni; Noesselt, Toemme

    2016-01-01

    Humans constantly learn in the absence of explicit rewards. However, the neurobiological mechanisms supporting this type of internally-guided learning (without explicit feedback) are still unclear. Here, participants who completed a task in which no external reward/feedback was provided, exhibited enhanced fMRI-signals within the dopaminergic midbrain, hippocampus, and ventral striatum (the SN/VTA-Hippocampal loop) when successfully grasping the meaning of new-words. Importantly, new-words that were better remembered showed increased activation and enhanced functional connectivity between the midbrain, hippocampus, and ventral striatum. Moreover, enhanced emotion-related physiological measures and subjective pleasantness ratings during encoding were associated with remembered new-words after 24 hr. Furthermore, increased subjective pleasantness ratings were also related to new-words remembered after seven days. These results suggest that intrinsic—potentially reward-related—signals, triggered by self-monitoring of correct performance, can promote the storage of new information into long-term memory through the activation of the SN/VTA-Hippocampal loop, possibly via dopaminergic modulation of the midbrain. DOI: http://dx.doi.org/10.7554/eLife.17441.001 PMID:27644419

  11. Propofol directly increases tau phosphorylation.

    Directory of Open Access Journals (Sweden)

    Robert A Whittington

    Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

  12. Differential roles of PDK1- and PDK2-phosphorylation sites in the yeast AGC kinases Ypk1, Pkc1 and Sch9.

    Science.gov (United States)

    Roelants, Françoise M; Torrance, Pamela D; Thorner, Jeremy

    2004-10-01

    Saccharomyces cerevisiae Pkh1 and Pkh2 (orthologues of mammalian protein kinase, PDK1) are functionally redundant. These kinases activate three AGC family kinases involved in the maintenance of cell wall integrity: Ypk1 and Ypk2, two closely related, functionally redundant enzymes (orthologues of mammalian protein kinase SGK), and Pkc1 (orthologue of mammalian protein kinase PRK2). Pkh1 and Pkh2 activate Ypk1, Ypk2 and Pkc1 by phosphorylating a Thr in a conserved sequence motif (PDK1 site) within the activation loop of these proteins. A fourth protein kinase involved in growth control and stress response, Sch9 (orthologue of mammalian protein kinase c-Akt/PKB), also carries the conserved activation loop motif. Like other AGC family kinases, Ypk1, Ypk2, Pkc1 and Sch9 also carry a second conserved sequence motif situated in a region C-terminal to the catalytic domain, called the hydrophobic motif (PDK2 site). Currently, there is still controversy surrounding the identity of the enzyme responsible for phosphorylating this second site and the necessity for phosphorylation at this site for in vivo function. Here, genetic and biochemical methods have been used to investigate the physiological consequences of phosphorylation at the PDK1 and PDK2 sites of Ypk1, Pkc1 and Sch9. It was found that phosphorylation at the PDK1 site in the activation loop is indispensable for the essential functions of all three kinases in vivo, whereas phosphorylation at the PDK2 motif plays a non-essential and much more subtle role in modulating the ability of these kinases to regulate the downstream processes in which they participate. PMID:15470109

  13. p21-Activated kinase 6 (PAK6) inhibits prostate cancer growth via phosphorylation of androgen receptor and tumorigenic E3 ligase murine double minute-2 (Mdm2).

    Science.gov (United States)

    Liu, Tong; Li, Yang; Gu, Hui; Zhu, Ge; Li, Jiabin; Cao, Liu; Li, Feng

    2013-02-01

    The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.

  14. CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    Directory of Open Access Journals (Sweden)

    Breuer Denitra

    2012-11-01

    Full Text Available Abstract Background HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity. Results The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94 that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation. Conclusion Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for

  15. Suppressing Akt phosphorylation and activating Fas by safrole oxide inhibited angiogenesis and induced vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 and serum.

    Science.gov (United States)

    Zhao, Jing; Miao, Junying; Zhao, Baoxiang; Zhang, Shangli; Yin, Deling

    2006-01-01

    At present, vascular endothelial cell (VEC) apoptosis induced by deprivation of fibroblast growth factor-2 (FGF-2) and serum has been well studied. But how to trigger VEC apoptosis in the presence of FGF-2 and serum is not well known. To address this question, in this study, the effects of safrole oxide on angiogenesis and VEC growth stimulated by FGF-2 were investigated. The results showed that safrole oxide inhibited angiogenesis and induced VEC apoptosis in the presence of FGF-2 and serum. To understand the possible mechanism of safrole oxide acting, we first examined the phosphorylation of Akt and the activity of nitric oxide synthase (NOS); secondly, we analyzed the expressions and distributions of Fas and P53; then we measured the activity of phosphatidylcholine specific phospholipase C (PC-PLC) in the VECs treated with and without safrole oxide. The results showed that this small molecule obviously suppressed Akt phosphorylation and the activity of NOS, and promoted the expressions of Fas and P53 markedly. Simultaneously, Fas protein clumped on cell membrane, instead of homogenously distributed. The activity of PC-PLC was not changed obviously. The data suggested that safrole oxide effectively inhibited angiogenesis and triggered VEC apoptosis in the presence of FGF-2 and serum, and it might perform its functions by suppressing Akt/NOS signal pathway, upregulating the expressions of Fas and P53 and modifying the distributing pattern of Fas in VEC. This finding provided a powerful chemical probe for promoting VEC apoptosis during angiogenesis stimulated by FGF-2.

  16. A flexible loop controlling the enzymatic activity and specificity in a glycosyl hydrolase family 19 endochitinase from barley seeds

    DEFF Research Database (Denmark)

    Fukamizo, Tamo; Miyake, Ryoh; Tamura, Atsushi;

    2009-01-01

    predominantly and the dimer and tetramer in lesser amounts. When the mutated enzymes were used instead of the wild type, the enzyme cleavage sites in the hexamer substrate were clearly shifted, and the ß-anomer selectivity was eliminated. The mutation effects on the enzymatic activity and stability were much....... Thermal stability and specific activities toward glycol chitin and chitin hexasaccharide were significantly affected by the individual mutations. When N-acetylglucosamine hexamer was hydrolyzed by the wild type, the ß-anomer of the substrate was preferentially hydrolyzed, producing the trimer...... mutation and the flexibility increases in the order of W72A, W72A/W82A and W82A. We conclude that Trp72 interacts with the sugar residue but Trp82 modulates the loop flexibility, which controls the protein stability and enzymatic properties. These tryptophan residues are likely to interact with each other...

  17. Phosphorylation of DegU is essential for activation of amyE expression in Bacillus subtilis

    Indian Academy of Sciences (India)

    Monica Gupta; K Krishnamurthy Rao

    2014-12-01

    Alpha ()-amylase (amyE) is one of the major exo-enzymes secreted by Bacillus subtilis during the post-exponential phase. The DegS-DegU two-component system regulates expression of majority of post-exponentially expressed genes in B. subtilis. It has been demonstrated that varying levels of the phosphorylated form of DegU (DegU-P) control different cellular processes. Exo-protease production is observed when effective concentration of DegU-P rises in the cell, whereas swarming motility is favoured at very low amounts of DegU-P. In this study we show that like other exo-proteases, expression of amyE is positively regulated by increase in DegU-P levels in the cell. We also demonstrate that residues at the DNA-binding helix-turn-helix (HTH) motif of DegU are necessary for the amyE expression. This observation is further reinforced by demonstrating the direct interaction of DegU on amyE promoter.

  18. The protein C omega-loop substitution Asn2Ile is associated with reduced protein C anticoagulant activity.

    LENUS (Irish Health Repository)

    Preston, Roger J S

    2012-02-01

    We report a kindred with heritable protein C (PC) deficiency in which two siblings with severe thrombosis showed a composite type I and IIb PC deficiency phenotype, identified using commercial PC assays (proband: PC antigen 42 u\\/dl, amidolytic activity 40 u\\/dl, anticoagulant activity 9 u\\/dl). The independent PROC nucleotide variations c.669C>A (predictive of Ser181Arg) and c.131C>T (predictive of Asn2Ile) segregated with the type I and type IIb PC deficiency phenotypes respectively, but co-segregated in the siblings with severe thrombosis. Soluble thrombomodulin (sTM)-mediated inhibition of plasma thrombin generation from an individual with PC-Asn2Ile was lower (endogenous thrombin potential (ETP) 56 +\\/- 1% that of ETP determined without sTM) than control plasma (ETP 15 +\\/- 2%) indicating reduced PC anticoagulant activity. Recombinant APC-Asn2Ile exhibited normal amidolytic activity but impaired anticoagulant activity. Protein S (PS)-dependent anticoagulant activity of recombinant APC-Asn2Ile and binding of recombinant APC-Asn2Ile to endothelial protein C receptor (EPCR) were reduced compared to recombinant wild-type APC. Asn2 lies within the omega-loop of the PC\\/APC Gla domain and this region is critical for calcium-induced folding and subsequent interactions with anionic phospholipids, EPCR and PS. The disruption of these interactions in this naturally-occurring PC variant highlights their collective importance in mediating APC anticoagulant activity in vivo.

  19. SARM modulates MyD88-mediated TLR activation through BB-loop dependent TIR-TIR interactions.

    Science.gov (United States)

    Carlsson, Emil; Ding, Jeak Ling; Byrne, Bernadette

    2016-02-01

    Toll-like receptors (TLRs) recognise invading pathogens and initiate an innate immune response by recruiting intracellular adaptor proteins via heterotypic Toll/interleukin-1 receptor (TIR) domain interactions. Of the five TIR domain-containing adaptor proteins identified, Sterile α- and armadillo-motif-containing protein (SARM) is functionally unique; suppressing immune signalling instead of promoting it. Here we demonstrate that the recombinantly expressed and purified SARM TIR domain interacts with both the major human TLR adaptors, MyD88 and TRIF. A single glycine residue located in the BB-loop of the SARM TIR domain, G601, was identified as essential for interaction. A short peptide derived from this motif was also found to interact with MyD88 in vitro. SARM expression in HEK293 cells was found to significantly suppress lipopolysaccharide (LPS)-mediated upregulation of inflammatory cytokines, IL-8 and TNF-α, an effect lost in the G601A mutant. The same result was observed with cytokine activation initiated by MyD88 expression and stimulation of TLR2 with lipoteichoic acid (LTA), suggesting that SARM is capable of suppressing both TRIF- and MyD88- dependent TLR signalling. Our findings indicate that SARM acts on a broader set of target proteins than previously thought, and that the BB-loop motif is functionally important, giving further insight into the endogenous mechanisms used to suppress inflammation in immune cells. PMID:26592460

  20. A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase

    DEFF Research Database (Denmark)

    Henriksen, Annette; Aghajari, Nushin; Jensen, Kaj Frank;

    1996-01-01

    is thereafter converted to uridine 5‘-monophosphate by OMP decarboxylase. We have determined the 2.4 Å structure of Escherichia coli OPRTase, ligated with sulfate, by molecular replacement and refined the structure to an R-factor of 18.3% for all data. In the structure of the E. coli enzyme we have determined...... the fold of a flexible loop region with a highly conserved amino acid sequence among OPRTases, a region known to take part in catalysis. The structure of this region was not determined in the model used for molecular replacement, and it involves interactions at the dimer interface through a bound sulfate...... ion. Crystalline E. coli OPRTase is a homodimer, with sulfate ions inhibiting enzyme activity bound in the dimer interface close to the flexible loop region. Although this loop is very close in space to the sulfate binding site, and sulfate is found in both interfaces of the homodimer, the loop...

  1. Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

    Science.gov (United States)

    Holt, Andrew W; Martin, Danielle N; Shaver, Patti R; Adderley, Shaquria P; Stone, Joshua D; Joshi, Chintamani N; Francisco, Jake T; Lust, Robert M; Weidner, Douglas A; Shewchuk, Brian M; Tulis, David A

    2016-09-01

    Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls

  2. Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

    Science.gov (United States)

    Holt, Andrew W; Martin, Danielle N; Shaver, Patti R; Adderley, Shaquria P; Stone, Joshua D; Joshi, Chintamani N; Francisco, Jake T; Lust, Robert M; Weidner, Douglas A; Shewchuk, Brian M; Tulis, David A

    2016-09-01

    Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls

  3. hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells

    OpenAIRE

    Chang, J T; Lu, Y-C; Chen, Y-J; Tseng, C-P; Chen, Y-L; Fang, C-W; Cheng, A-J

    2006-01-01

    Telomerase activity is suppressed in normal somatic tissues but is activated in most cancer cells. We have previously found that all six telomerase subunit proteins, including hTERT and hsp90 are needed for full enzyme activity. Telomerase activity has been reported to be upregulated by protein kinase C (PKC), but the mechanism is not clear. In this study, we examined how PKC regulates telomerase activity in head and neck cancer cells. PKC inhibitor, bisindolylmaleimide I (BIS), inhibited tel...

  4. RKIP phosphorylation-dependent ERK1 activation stimulates adipogenic lipid accumulation in 3T3-L1 preadipocytes overexpressing LC3.

    Science.gov (United States)

    Hahm, Jong Ryeal; Ahmed, Mahmoud; Kim, Deok Ryong

    2016-09-01

    3T3-L1 preadipocytes undergo adipogenesis in response to treatment with dexamethaxone, 1-methyl-3-isobutylxanthine, and insulin (DMI) through activation of several adipogenic transcription factors. Many autophagy-related proteins are also highly activated in the earlier stages of adipogenesis, and the LC3 conjugation system is required for formation of lipid droplets. Here, we investigated the effect of overexpression of green fluorescent protein (GFP)-LC3 fusion protein on adipogenesis. Overexpression of GFP-LC3 in 3T3-L1 preadipocytes using poly-l-lysine-assisted adenoviral GFP-LC3 transduction was sufficient to produce intracellular lipid droplets. Indeed, GFP-LC3 overexpression stimulated expression of some adipogenic transcription factors (e.g., C/EBPα or β, PPARγ, SREBP2). In particular, SREBP2 was highly activated in preadipocytes transfected with adenoviral GFP-LC3. Also, phosphorylation of Raf kinase inhibitory protein (RKIP) at serine 153, consequently stimulating extracellular-signal regulated kinase (ERK)1 activity, was significantly increased during adipogenesis induced by either poly-l-lysine-assisted adenoviral GFP-LC3 transduction or culture in the presence of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Furthermore, RKIP knockdown promoted ERK1 and PPARγ activation, and significantly increased the intracellular accumulation of triacylglycerides in DMI-induced adipogenesis. In conclusion, GFP-LC3 overexpression in 3T3-L1 preadipocytes stimulates adipocyte differentiation via direct modulation of RKIP-dependent ERK1 activity.

  5. Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

    Energy Technology Data Exchange (ETDEWEB)

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J. (Virginia Tech); (UMC)

    2012-11-01

    Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 {angstrom} movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k{sub cat}. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

  6. The importance of hinge sequence for loop function and catalytic activity in the reaction catalyzed by triosephosphate isomerase.

    Science.gov (United States)

    Xiang, J; Sun, J; Sampson, N S

    2001-04-01

    We have determined the sequence requirements for the N-terminal protein hinge of the active-site lid of triosephosphate isomerase. The codons for the hinge (PVW) were replaced with a genetic library of all possible 8000 amino acid combinations. The most active of these 8000 mutants were selected using in vivo complementation of a triosephosphate isomerase-deficient strain of Escherichia coli, DF502. Approximately 0.3 % of the mutants complement DF502 with an activity that is between 10 and 70 % of wild-type activity. They all contain Pro at the first position. Furthermore, the sequences of these hinge mutants reveal that hydrophobic packing is very important for efficient formation of the enediol intermediate. However, the reduced catalytic activities observed are not due to increased rates of loop opening. To explore the relationship between the N-terminal and C-terminal hinges, three semi-active mutants from the N-terminal hinge selection experiment (PLH, PHS and PTF), and six active C-terminal hinge mutants from previous work (NSS, LWA, YSL, KTK, NPN, KVA) were combined to form 18 "double-hinge" mutants. The activities of these mutants suggest that the N-terminal and C-terminal hinge structures affect one another. It appears that specific side-chain interactions are important for forming a catalytically active enzyme, but not for preventing release of the unstable enediol intermediate from the active site of the enzyme. The independence of intermediate release on amino acid sequence is consistent with the absence of a "universal" hinge sequence in structurally related enzymes.

  7. Neuregulin1–β decreases interleukin–1β–induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability

    Science.gov (United States)

    Wu, Limin; Ramirez, Servio H.; Andrews, Allison M.; Leung, Wendy; Itoh, Kanako; Wu, Jiang; Arai, Ken; Lo, Eng H.; Lok, Josephine

    2016-01-01

    Neuregulin-1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type-specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL-1β-induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1-β isoform acts on IL-1β-induced endothelial permeability. Our data show that NRG1-β increases barrier function, measured by transendothelial electrical resistance, and decreases IL-1β-induced hyperpermeability, measured by dextran-40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1-β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2-associated tyrosine kinase, reduces the effect of NRG1-β on IL-1β-induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1-β signaling affects changes in the brain microvasculature in the setting of neuroinflammation. PMID:26438054

  8. BAFF induces spleen CD4{sup +} T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Fang; Chen, Rongjing [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Liu, Baojun [Laboratory of Lung, Inflammation and Cancers, Huashan Hospital, Fudan University, Shanghai (China); Zhang, Xiaoping [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Han, Junli; Wang, Haining [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Shen, Gang [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Tao, Jiang, E-mail: taojiang2012@yahoo.cn [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4{sup +} T cells. Black-Right-Pointing-Pointer Carrying out siRNA technology to study FOXO3A protein function. Black-Right-Pointing-Pointer Helpful to understand the T cell especially CD4{sup +} T cell's role in immunological reaction. -- Abstract: The TNF ligand family member 'B cell-activating factor belonging to the TNF family' (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4{sup +} spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4{sup +} T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4{sup +} spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4{sup +} T cell proliferation.

  9. Phosphorylation and activation of nuclear Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) by Ca{sup 2+}/calmodulin-dependent protein kinase I (CaMKI)

    Energy Technology Data Exchange (ETDEWEB)

    Onouchi, Takashi [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan); Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan); Ishida, Atsuhiko [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521 (Japan); Kameshita, Isamu [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer CaMKP-N/PPM1E underwent proteolytic processing and translocated to cytosol. Black-Right-Pointing-Pointer The proteolysis was effectively inhibited by the proteasome inhibitors. Black-Right-Pointing-Pointer Ser-480 of zebrafish CaMKP-N was phosphorylated by cytosolic CaMKI. Black-Right-Pointing-Pointer Phosphorylation-mimic mutants of CaMKP-N showed enhanced activity. Black-Right-Pointing-Pointer These results suggest that CaMKP-N is regulated by CaMKI. -- Abstract: Nuclear Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) is an enzyme that dephosphorylates and downregulates multifunctional Ca{sup 2+}/calmodulin-dependent protein kinases (CaMKs) as well as AMP-dependent protein kinase. In our previous study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing and translocated to cytosol in a proteasome inhibitor-sensitive manner. In the present study, we found that zCaMKP-N is regulated by phosphorylation at Ser-480. When zCaMKP-N was incubated with the activated CaMKI, time-dependent phosphorylation of the enzyme was observed. This phosphorylation was significantly reduced when Ser-480 was replaced by Ala, suggesting that CaMKI phosphorylates Ser-480 of zCaMKP-N. Phosphorylation-mimic mutants, S480D and S480E, showed higher phosphatase activities than those of wild type and S480A mutant in solution-based phosphatase assay using various substrates. Furthermore, autophosphorylation of CaMKII after ionomycin treatment was more severely attenuated in Neuro2a cells when CaMKII was cotransfected with the phosphorylation-mimic mutant of zCaMKP-N than with the wild-type or non-phosphorylatable zCaMKP-N. These results strongly suggest that phosphorylation of zCaMKP-N at Ser-480 by CaMKI activates CaMKP-N catalytic activity and thereby downregulates multifunctional CaMKs in the cytosol.

  10. Molecular dynamics simulation of phosphorylation-induced conformational transitions in the mycobacterium tuberculosis response regulator PrrA

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Guo [Los Alamos National Laboratory; Mcmahon, Benjamin H [Los Alamos National Laboratory; Tung, Chang - Shung [Los Alamos National Laboratory

    2008-01-01

    Phosphorylation-activated modulation of response regulators (RR) is predominantly used by bacteria as a strategy in regulating their two-component signaling (TCS) systems, the underlying molecular mechanisms are however far from fully understood. In this work we have conducted a molecular dynamics (MD) simulation of the phosphorylation-induced conformational transitions of RRs with the Mycobacterium Tuberculosis PrrA as a particular example. Starting from the full-length inactive structure of PrrA we introduced a local disturbance by phosphorylating the conserved aspartic acid residue, Asp-58, in the regulatory domain. A Go-model-type algorithm packaged with AMBER force fields was then applied to simulate the dynamics upon phosphorylation. The MD simulation shows that the phosphorylation of Asp-58 facilitates PrrA, whose inactive state has a compact conformation with a closed interdomain interface, to open up with its interdomain separation being increased by an average of about 1.5 {angstrom} for a simulation of 20 ns. The trans-activation loop, which is completely buried within the interdomain interface in the inactive PrrA, is found to become more exposed with the phosphorylated structure as well. These results provide more structural details of how the phosphorylation of a local aspartate activates PrrA to undergo a global conformational rearrangement toward its extended active state. This work also indicates that MD simulations can serve as a fast tool to unravel the regulation mechanisms of all RRs, which is especially valuable when the structures of full-length active RRs are currently unavailable.

  11. Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

    Directory of Open Access Journals (Sweden)

    Thiel Gerald

    2009-05-01

    Full Text Available Abstract Background The serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts. Results Stimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK. Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M

  12. Nonlinear Mr Model Inversion for Semi-Active Control Enhancement With Open-Loop Force Compensation

    OpenAIRE

    Reader, Daniel Martin

    2009-01-01

    The increased prevalence of semi-active control systems is largely due to the emergence of cost effective commercially available controllable damper technology such as Magneto-Rheological (MR) devices. Unfortunately, MR dampers exhibit highly nonlinear behavior, thus presenting an often over-looked complexity to the control system designer. With regards to controlling dampers, the well-known Skyhook Damping control algorithm has enjoyed great success for both fully active and semi-active co...

  13. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seon Sook; Lee, Eun Hye [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Lee, Kooyeon [Department of Bio-Health Technology, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Jo, Su-Hyun, E-mail: suhyunjo@kangwon.ac.kr [Department of Physiology, BK21 Plus Graduate Program, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Seo, Su Ryeon, E-mail: suryeonseo@kangwon.ac.kr [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

    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.

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

    Science.gov (United States)

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

    2005-10-01

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

  15. Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1.

    OpenAIRE

    Weiss, A; Koretzky, G; Schatzman, R C; Kadlecek, T

    1991-01-01

    Stimulation of the T-cell antigen receptor (TCR), which itself is not a protein-tyrosine kinase (PTK), activates a PTK and phospholipase C (PLC). Using the human T-cell leukemic line Jurkat and normal peripheral blood lymphocytes, we demonstrate that stimulation of the TCR specifically induces the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Stimulation of the human muscarinic receptor, subtype 1, when expressed in Jurkat activates PLC through a guanine nu...

  16. Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain

    OpenAIRE

    Kurbatskaya, Ksenia; Phillips, Emma Claire; Croft, Cara Louise; Dentoni, Giacomo; Hughes, Martina; Wade, Matthew Austen James; Al-Sarraj, Safa; Troakes, Claire; O'Neill, Michael; Gomez Perez-Nievas, Beatriz; Hanger, Diane Pamela; Noble, Wendy Jane

    2016-01-01

    Alterations in calcium homeostasis are widely reported to contribute to synaptic degeneration and neuronal loss in Alzheimer’s disease. Elevated cytosolic calcium concentrations lead to activation of the calcium-sensitive cysteine protease, calpain, which has a number of substrates known to be abnormally regulated in disease. Analysis of human brain has shown that calpain activity is elevated in AD compared to controls, and that calpain-mediated proteolysis regulates the activity of important...

  17. Phosphorylation influences the binding of the yeast RAP1 protein to the upstream activating sequence of the PGK gene.

    OpenAIRE

    Tsang, J S; Henry, Y A; Chambers, A.; Kingsman, A J; Kingsman, S M

    1990-01-01

    Yeast repressor activator protein 1 (RAP1) binds in vitro to specific DNA sequences that are found in diverse genetic elements. Expression of the yeast phosphoglycerate kinase gene (PGK) requires the binding of RAP1 to the activator core sequence within the upstream activating sequence (UAS) of PGK. A DNA fragment Z+ which contains the activator core sequence of the PGK(UAS) has been shown to bind RAP1. Here we report that phosphatase treatment of RAP1 affected its binding to the PGK(UAS) but...

  18. Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C*

    OpenAIRE

    Lum, Michelle A.; Balaburski, Gregor M; Murphy, Maureen E.; Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    Background: Mechanisms of activation-induced PKC down-regulation are poorly understood. A characterized pathway involves priming site dephosphorylation and degradation of the dephosphorylated species.

  19. Identification of Phosphorylation Consensus Sequences and Endogenous Neuronal Substrates of the Psychiatric Risk Kinase TNIK.

    Science.gov (United States)

    Wang, Qi; Amato, Stephen P; Rubitski, David M; Hayward, Matthew M; Kormos, Bethany L; Verhoest, Patrick R; Xu, Lan; Brandon, Nicholas J; Ehlers, Michael D

    2016-02-01

    Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase highly expressed in the brain and enriched in the postsynaptic density of glutamatergic synapses in the mammalian brain. Accumulating genetic evidence and functional data have implicated TNIK as a risk factor for psychiatric disorders. However, the endogenous substrates of TNIK in neurons are unknown. Here, we describe a novel selective small molecule inhibitor of the TNIK kinase family. Using this inhibitor, we report the identification of endogenous neuronal TNIK substrates by immunoprecipitation with a phosphomotif antibody followed by mass spectrometry. Phosphorylation consensus sequences were defined by phosphopeptide sequence analysis. Among the identified substrates were members of the delta-catenin family including p120-catenin, δ-catenin, and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), each of which is linked to psychiatric or neurologic disorders. Using p120-catenin as a representative substrate, we show TNIK-induced p120-catenin phosphorylation in cells requires intact kinase activity and phosphorylation of TNIK at T181 and T187 in the activation loop. Addition of the small molecule TNIK inhibitor or knocking down TNIK by two shRNAs reduced endogenous p120-catenin phosphorylation in cells. Together, using a TNIK inhibitor and phosphomotif antibody, we identify endogenous substrates of TNIK in neurons, define consensus sequences for TNIK, and suggest signaling pathways by which TNIK influences synaptic development and function linked to psychiatric and neurologic disorders. PMID:26645429

  20. Duration of streptozotocin-induced diabetes differentially affects p38-mitogen-activated protein kinase (MAPK phosphorylation in renal and vascular dysfunction

    Directory of Open Access Journals (Sweden)

    Gupta Akanksha

    2005-03-01

    Full Text Available Abstract Background In the present study we tested the hypothesis that progression of streptozotocin (STZ-induced diabetes (14-days to 28-days would produce renal and vascular dysfunction that correlate with altered p38- mitogen-activated protein kinase (p38-MAPK phosphorylation in kidneys and thoracic aorta. Methods Male Sprague Dawley rats (350–400 g were randomized into three groups: sham (N = 6, 14-days diabetic (N = 6 and 28-days diabetic rats (N = 6. Diabetes was induced using a single tail vein injection of STZ (60 mg/kg, I.V. on the first day. Rats were monitored for 28 days and food, water intake and plasma glucose levels were noted. At both 14-days and 28-days post diabetes blood samples were collected and kidney cortex, medulla and aorta were harvested from each rat. Results The diabetic rats lost body weight at both 14-days (-10% and 28-days (-13% more significantly as compared to sham (+10% group. Glucose levels were significantly elevated in the diabetic rats at both 14-days and 28-days post-STZ administration. Renal dysfunction as evidenced by renal hypertrophy, increased plasma creatinine concentration and reduced renal blood flow was observed in 14-days and 28-days diabetes. Vascular dysfunction as evidenced by decreased carotid blood flow was observed in 14-days and 28-days diabetes. We observed an up-regulation of inducible nitric oxide synthase (iNOS, prepro endothelin-1 (preproET-1 and phosphorylated p38-MAPK in thoracic aorta and kidney cortex but not in kidney medulla in 28-days diabetes group. Conclusion The study provides evidence that diabetes produces vascular and renal dysfunction with a profound effect on signaling mechanisms at later stage of diabetes.

  1. A Closed-Loop Model of the Respiratory System: Focus on Hypercapnia and Active Expiration

    OpenAIRE

    Molkov, Yaroslav I.; Shevtsova, Natalia A.; Park, Choongseok; Ben-Tal, Alona; Smith, Jeffrey C.; Rubin, Jonathan E.; Rybak, Ilya A.

    2014-01-01

    Breathing is a vital process providing the exchange of gases between the lungs and atmosphere. During quiet breathing, pumping air from the lungs is mostly performed by contraction of the diaphragm during inspiration, and muscle contraction during expiration does not play a significant role in ventilation. In contrast, during intense exercise or severe hypercapnia forced or active expiration occurs in which the abdominal “expiratory” muscles become actively involved in breathing. The mechanis...

  2. Activation of Bacillus subtilis Ugd by the BY-Kinase PtkA Proceeds via Phosphorylation of Its Residue Tyrosine 70

    DEFF Research Database (Denmark)

    Petranovic, Dina; Grangeasse, C.; Macek, B.;

    2009-01-01

    -specific phosphoproteomic study indicated that tyrosine 70 is phosphorylated in the Bacillus subtilis UDP-glucose dehydrogenase Ugd. In this study we confirm that this tyrosine 70 is indeed the main residue phosphorylated by the cognate BY-kinase PtkA. Homology-based modeling of the Ugd structure using structures from UDP...

  3. Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria.

    Science.gov (United States)

    Kavanagh, N I; Ainscow, E K; Brand, M D

    2000-02-24

    Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.

  4. Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Ya-Hsin, E-mail: yhcheng@mail.cmu.edu.tw [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China); Li, Lih-Ann; Lin, Pinpin; Cheng, Li-Chuan [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan, ROC (China); Hung, Chein-Hui [Graduate Institute of Clinical Medicine Sciences, Chang Gung University, Puizi City, Chiayi 613, Taiwan, ROC (China); Chang, Nai Wen [Department of Biochemistry, School of Medicine, China Medical University, Taichung, Taiwan, ROC (China); Lin, Chingju [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China)

    2012-09-15

    Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phase and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3β, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation. -- Highlights: ► Baicalein causes the G1 phase arrest by decreasing Rb phosphorylation. ► Baicalein modulates AhR-mediated cell proliferation. ► Both AhR activation and cyclin D1 degradation results in hypophosphorylation of Rb. ► Baicalein facilitates cyclin D1 degradation by signalling the GSK-3β pathway.

  5. Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation

    International Nuclear Information System (INIS)

    Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phase and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3β, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation. -- Highlights: ► Baicalein causes the G1 phase arrest by decreasing Rb phosphorylation. ► Baicalein modulates AhR-mediated cell proliferation. ► Both AhR activation and cyclin D1 degradation results in hypophosphorylation of Rb. ► Baicalein facilitates cyclin D1 degradation by signalling the GSK-3β pathway.

  6. Ras-mutant cancer cells display B-Raf binding to Ras that activates extracellular signal-regulated kinase and is inhibited by protein kinase A phosphorylation.

    Science.gov (United States)

    Li, Yanping; Takahashi, Maho; Stork, Philip J S

    2013-09-20

    The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.

  7. Leukemia Mediated Endothelial Cell Activation Modulates Leukemia Cell Susceptibility to Chemotherapy through a Positive Feedback Loop Mechanism.

    Directory of Open Access Journals (Sweden)

    Bahareh Pezeshkian

    Full Text Available In acute myeloid leukemia (AML, the chances of achieving disease-free survival are low. Studies have demonstrated a supportive role of endothelial cells (ECs in normal hematopoiesis. Here we show that similar intercellular relationships exist in leukemia. We demonstrate that leukemia cells themselves initiate these interactions by directly modulating the behavior of resting ECs through the induction of EC activation. In this inflammatory state, activated ECs induce the adhesion of a sub-set of leukemia cells through the cell adhesion molecule E-selectin. These adherent leukemia cells are sequestered in a quiescent state and are unaffected by chemotherapy. The ability of adherent cells to later detach and again become proliferative following exposure to chemotherapy suggests a role of this process in relapse. Interestingly, differing leukemia subtypes modulate this process to varying degrees, which may explain the varied response of AML patients to chemotherapy and relapse rates. Finally, because leukemia cells themselves induce EC activation, we postulate a positive-feedback loop in leukemia that exists to support the growth and relapse of the disease. Together, the data defines a new mechanism describing how ECs and leukemia cells interact during leukemogenesis, which could be used to develop novel treatments for those with AML.

  8. Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53

    OpenAIRE

    Osborn, Alexander J.; Elledge, Stephen J.

    2003-01-01

    When DNA replication is stalled, a signal transduction pathway is activated that promotes the stability of stalled forks and resumption of DNA synthesis. In budding yeast, this pathway includes the kinases Mec1 and Rad53. Here we report that the Mediator protein Mrc1, which is required for normal DNA replication and for activation of Rad53, is present at replication forks. Mrc1 initially binds early-replicating sequences and moves along chromatin with the replication f...

  9. Triptolide (PG-490) induces apoptosis of dendritic cells through sequential p38 MAP kinase phosphorylation and caspase 3 activation

    Institute of Scientific and Technical Information of China (English)

    Liu Q; Chen T; Chen H; Zhang M; Li N; Lu Z; Ma P; Cao X

    2004-01-01

    Dendritic cells (DCs) are the most potent antigen-presenting cells that play crucial roles in the regulation of immune response. Triptolide, an active component purified from the medicinal plant Tripterygium wilfordii Hook F. , has been demonstrated to act as a potent immunosuppressive drug capable of inhibiting T cell activation and proliferation. However, little is known about the effects of triptolide on DCs. The present study shows that triptolide does not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserine exposure, mitochondria potential decrease, and nuclear DNA condensation. Triptolide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that the anti-inflammatory and immunosuppressive activities of triptolide may be due, in part,to its apoptosis-inducing effects on DCs.

  10. Cellular regulation by protein phosphorylation.

    Science.gov (United States)

    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.

  11. FIRST THREE-DIMENSIONAL RECONSTRUCTIONS OF CORONAL LOOPS WITH THE STEREO A+B SPACECRAFT. III. INSTANT STEREOSCOPIC TOMOGRAPHY OF ACTIVE REGIONS

    International Nuclear Information System (INIS)

    Here we develop a novel three-dimensional (3D) reconstruction method of the coronal plasma of an active region by combining stereoscopic triangulation of loops with density and temperature modeling of coronal loops with a filling factor equivalent to tomographic volume rendering. Because this method requires only a stereoscopic image pair in multiple temperature filters, which are sampled within ∼1 minute with the recent STEREO/EUVI instrument, this method is about four orders of magnitude faster than conventional solar rotation-based tomography. We reconstruct the 3D density and temperature distribution of active region NOAA 10955 by stereoscopic triangulation of 70 loops, which are used as a skeleton for a 3D field interpolation of some 7000 loop components, leading to a 3D model that reproduces the observed fluxes in each stereoscopic image pair with an accuracy of a few percents (of the average flux) in each pixel. With the stereoscopic tomography we infer also a differential emission measure distribution over the entire temperature range of T ∼ 104-107, with predictions for the transition region and hotter corona in soft X-rays. The tomographic 3D model provides also large statistics of physical parameters. We find that the extreme-ultraviolet loops with apex temperatures of Tm ∼m ∼ 4-7 MK are near-hydrostatic. The new 3D reconstruction model is fully independent of any magnetic field data and is promising for future tests of theoretical magnetic field models and coronal heating models.

  12. Structure of TSA2 reveals novel features of the active-site loop of peroxiredoxins

    DEFF Research Database (Denmark)

    Nielsen, Maja Holch; Kidmose, Rune Thomas; Jenner, Lasse Bohl

    2016-01-01

    Saccharomyces cerevisiae TSA2 belongs to the family of typical 2-Cys peroxiredoxins, a ubiquitously expressed family of redox-active enzymes that utilize a conserved peroxidatic cysteine to reduce peroxides. Typical 2-Cys peroxiredoxins have been shown to be involved in protection against oxidative...... stress and in hydrogen peroxide signalling. Furthermore, several 2-Cys peroxiredoxins, including S. cerevisiae TSA1 and TSA2, are able to switch to chaperone activity upon hyperoxidation of their peroxidatic cysteine. This makes the sensitivity to hyperoxidation of the peroxidatic cysteine a very....... This requires a local unfolding of the active site and the C-terminus. The balance between the fully folded and locally unfolded conformations is of key importance for the reactivity and sensitivity to hyperoxidation of the different peroxiredoxins. Here, the structure of a C48S mutant of TSA2 from S...

  13. Measurement of fission product activity in the Peach Bottom Reactor primary coolant loop

    International Nuclear Information System (INIS)

    The distribution of gamma-emitting radionuclides deposited in the primary circuit of the Peach Bottom High-Temperature Gas-Cooled Reactor (HTGR) at end-of-life has been determined by in situ gamma scanning. The work was part of the Peach Bottom End-of-Life Program and was performed by the IRT Corporation under subcontract to General Atomic Company. The measurements were made to support a design method verification exercise. The specific activity on the ducts was measured by external scans at local points with a Ge(Li) detector and by internal scans with a travelling intrinsic germanium detector (after destructive removal of trepan samples); the activity on the steam generator tube bundle was determined by traversing selected tubes with travelling CdTe detectors from the water side. Calibration measurements on mockups allowed reduction of the spectra to specific activity

  14. Biphasic Regulation of Yes-associated Protein (YAP) Cellular Localization, Phosphorylation, and Activity by G Protein-coupled Receptor Agonists in Intestinal Epithelial Cells: A NOVEL ROLE FOR PROTEIN KINASE D (PKD).

    Science.gov (United States)

    Wang, Jia; Sinnett-Smith, James; Stevens, Jan V; Young, Steven H; Rozengurt, Enrique

    2016-08-19

    We examined the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in intestinal epithelial cells. Our results show that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II, a potent mitogen for these cells, induced rapid translocation of YAP from the nucleus to the cytoplasm (within 15 min) and a concomitant increase in YAP phosphorylation at Ser(127) and Ser(397) Angiotensin II elicited YAP phosphorylation and cytoplasmic accumulation in a dose-dependent manner (ED50 = 0.3 nm). Similar YAP responses were provoked by stimulation with vasopressin or serum. Treatment of the cells with the protein kinase D (PKD) family inhibitors CRT0066101 and kb NB 142-70 prevented the increase in YAP phosphorylation on Ser(127) and Ser(397) via Lats2, YAP cytoplasmic accumulation, and increase in the mRNA levels of YAP/TEAD-regulated genes (Ctgf and Areg). Furthermore, siRNA-mediated knockdown of PKD1, PKD2, and PKD3 markedly attenuated YAP nuclear-cytoplasmic shuttling, phosphorylation at Ser(127), and induction of Ctgf and Areg expression in response to GPCR activation. These results identify a novel role for the PKD family in the control of biphasic localization, phosphorylation, and transcriptional activity of YAP in intestinal epithelial cells. In turn, YAP and TAZ are necessary for the stimulation of the proliferative response of intestinal epithelial cells to GPCR agonists that act via PKD. The discovery of interaction between YAP and PKD pathways identifies a novel cross-talk in signal transduction and demonstrates, for the first time, that the PKDs feed into the YAP pathway. PMID:27369082

  15. Quantitative phosphoproteomics unravels biased phosphorylation of serotonin 2A receptor at Ser280 by hallucinogenic versus nonhallucinogenic agonists.

    Science.gov (United States)

    Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

    2014-05-01

    The serotonin 5-HT(2A) receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT(2A) receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT(2A) receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT(2A) agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser(280)) located in the third intracellular loop of the 5-HT(2A) receptor, a region important for its desensitization. The specific phosphorylation of Ser(280) by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT(2A) receptors at Ser(280) in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser(280) to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased

  16. Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists*

    Science.gov (United States)

    Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J.; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

    2014-01-01

    The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of

  17. A novel mechanism involved in the coupling of mitochondrial biogenesis to oxidative phosphorylation

    Directory of Open Access Journals (Sweden)

    Jelena Ostojić

    2014-01-01

    Full Text Available Mitochondria are essential organelles that are central to a multitude of cellular processes, including oxidative phosphorylation (OXPHOS, which produces most of the ATP in animal cells. Thus it is important to understand not only the mechanisms and biogenesis of this energy production machinery but also how it is regulated in both physiological and pathological contexts. A recent study by Ostojić et al. [Cell Metabolism (2013 18, 567-577] has uncovered a regulatory loop by which the biogenesis of a major enzyme of the OXPHOS pathway, the respiratory complex III, is coupled to the energy producing activity of the mitochondria.

  18. N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias.

    Science.gov (United States)

    Soto, Antonio G; Smith, Thomas H; Chen, Buxin; Bhattacharya, Supriyo; Cordova, Isabel Canto; Kenakin, Terry; Vaidehi, Nagarajan; Trejo, JoAnn

    2015-07-01

    Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias. PMID:26100877

  19. Active magnetic bearing control loop modeling for a finite element rotordynamics code

    Science.gov (United States)

    Genta, Giancarlo; Delprete, Cristiana; Carabelli, Stefano

    1994-01-01

    A mathematical model of an active electromagnetic bearing which includes the actuator, the sensor and the control system is developed and implemented in a specialized finite element code for rotordynamic analysis. The element formulation and its incorporation in the model of the machine are described in detail. A solution procedure, based on a modal approach in which the number of retained modes is controlled by the user, is then shown together with other procedures for computing the steady-state response to both static and unbalance forces. An example of application shows the numerical results obtained on a model of an electric motor suspended on a five active-axis magnetic suspension. The comparison of some of these results with the experimental characteristics of the actual system shows the ability of the present model to predict its performance.

  20. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    International Nuclear Information System (INIS)

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  1. The Adaptogens Rhodiola and Schizandra Modify the Response to Immobilization Stress in Rabbits by Suppressing the Increase of Phosphorylated Stress-activated Protein Kinase, Nitric Oxide and Cortisol

    Directory of Open Access Journals (Sweden)

    Alexander Panossian

    2007-01-01

    Full Text Available Adaptogens possess anti-fatigue and anti-stress activities that can increase mental and physical working performance against a background of fatigue or stress. The aim of the present study was to ascertain which mediators of stress response are significantly involved in the mechanisms of action of adaptogens, and to determine their relevance as biochemical markers for evaluating anti-stress effects in rabbits subjected to restraint stress. Blood levels of stress-activated protein kinase (SAPK/JNK, the phosphorylated kinase p-SAPK/p-JNK, nitric oxide (NO, cortisol, testosterone, prostaglandin E2, leukotriene B4 and thromboxane B2 were determined in groups of animals prior to daily oral administration of placebo, rhodioloside or extracts of Eleutherococcus senticosus, Schizandra chinensis, Rhodiola rosea, Bryonia alba and Panax ginseng over a 7 day period. Ten minutes after the fi nal treatment, animals were immobilized for 2 hours and blood levels of the markers re-determined. In the placebo group, only p-SAPK/p-JNK, NO and cortisol were increased significantly (by 200–300% cf basal levels following restraint stress, whilst in animals that had received multiple doses of adaptogens/stress-protectors, the levels of NO and cortisol remained practically unchanged after acute stress. Rhodioloside and extracts of S. chinensis and R. rosea were the most active inhibitors of stress-induced p-SAPK/p-JNK. E. senticosus, B. alba and P. ginseng exerted little effect on p-SAPK/p-JNK levels. It is suggested that the inhibitory effects of R. rosea and S. chinensis on p-SAPK/p-JNK activation may be associated with their anti-depressant activity as well as their positive effects on mental performance under stress.

  2. DYRK1A-mediated phosphorylation of GluN2A at Ser1048 regulates the surface expression and channel activity of GluN1/GluN2A receptors

    Directory of Open Access Journals (Sweden)

    Cristina eGrau

    2014-10-01

    Full Text Available N-methyl-D-aspartate glutamate receptors (NMDARs play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A, suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs was modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser1048 of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser1048 hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser1048 increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons.

  3. DYRK1A-mediated phosphorylation of GluN2A at Ser(1048) regulates the surface expression and channel activity of GluN1/GluN2A receptors.

    Science.gov (United States)

    Grau, Cristina; Arató, Krisztina; Fernández-Fernández, José M; Valderrama, Aitana; Sindreu, Carlos; Fillat, Cristina; Ferrer, Isidre; de la Luna, Susana; Altafaj, Xavier

    2014-01-01

    N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser(1048) of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser(1048) hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser(1048) increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons. PMID:25368549

  4. Oxidative phosphorylation revisited

    DEFF Research Database (Denmark)

    Nath, Sunil; Villadsen, John

    2015-01-01

    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...... 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...

  5. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    International Nuclear Information System (INIS)

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer

  6. First Three-Dimensional Reconstructions of Coronal Loops with the STEREO A+B Spacecraft. III. Instant Stereoscopic Tomography of Active Regions

    Science.gov (United States)

    Aschwanden, Markus J.; Wuelser, Jean-Pierre; Nitta, Nariaki V.; Lemen, James R.; Sandman, Anne

    2009-04-01

    Here we develop a novel three-dimensional (3D) reconstruction method of the coronal plasma of an active region by combining stereoscopic triangulation of loops with density and temperature modeling of coronal loops with a filling factor equivalent to tomographic volume rendering. Because this method requires only a stereoscopic image pair in multiple temperature filters, which are sampled within ≈1 minute with the recent STEREO/EUVI instrument, this method is about four orders of magnitude faster than conventional solar rotation-based tomography. We reconstruct the 3D density and temperature distribution of active region NOAA 10955 by stereoscopic triangulation of 70 loops, which are used as a skeleton for a 3D field interpolation of some 7000 loop components, leading to a 3D model that reproduces the observed fluxes in each stereoscopic image pair with an accuracy of a few percents (of the average flux) in each pixel. With the stereoscopic tomography we infer also a differential emission measure distribution over the entire temperature range of T ≈ 104-107, with predictions for the transition region and hotter corona in soft X-rays. The tomographic 3D model provides also large statistics of physical parameters. We find that the extreme-ultraviolet loops with apex temperatures of Tm lsim 3.0 MK tend to be super-hydrostatic, while hotter loops with Tm ≈ 4-7 MK are near-hydrostatic. The new 3D reconstruction model is fully independent of any magnetic field data and is promising for future tests of theoretical magnetic field models and coronal heating models.

  7. Loop-to-loop coupling.

    Energy Technology Data Exchange (ETDEWEB)

    Warne, Larry Kevin; Lucero, Larry Martin; Langston, William L.; Salazar, Robert Austin; Coleman, Phillip Dale; Basilio, Lorena I.; Bacon, Larry Donald

    2012-05-01

    This report estimates inductively-coupled energy to a low-impedance load in a loop-to-loop arrangement. Both analytical models and full-wave numerical simulations are used and the resulting fields, coupled powers and energies are compared. The energies are simply estimated from the coupled powers through approximations to the energy theorem. The transmitter loop is taken to be either a circular geometry or a rectangular-loop (stripline-type) geometry that was used in an experimental setup. Simple magnetic field models are constructed and used to estimate the mutual inductance to the receiving loop, which is taken to be circular with one or several turns. Circuit elements are estimated and used to determine the coupled current and power (an equivalent antenna picture is also given). These results are compared to an electromagnetic simulation of the transmitter geometry. Simple approximate relations are also given to estimate coupled energy from the power. The effect of additional loads in the form of attached leads, forming transmission lines, are considered. The results are summarized in a set of susceptibility-type curves. Finally, we also consider drives to the cables themselves and the resulting common-to-differential mode currents in the load.

  8. Constitutive phosphorylation of Shc proteins in human tumors

    DEFF Research Database (Denmark)

    Pelicci, G; Lanfrancone, L; Salcini, A E;

    1995-01-01

    cells. In tumor cells with known TK gene alterations Shc proteins were constitutively phosphorylated and complexed with the activated TK. No constitutive Shc phosphorylation was found in primary cell cultures and normal tissues. In 14 of 27 tumor cell lines with no reported TK alterations, Shc proteins...... activated TKs and that the analysis of Shc phosphorylation allow the identification of tumors with constitutive TK activation....

  9. Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

    Science.gov (United States)

    Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

    2016-04-01

    R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low

  10. QM/MD studies of the dynamics of the MTSL spin label in Aurora-A kinase protein activation loop

    CERN Document Server

    Concilio, Maria Grazia; Bayliss, Richard; Burgess, Selena

    2015-01-01

    Molecular dynamics(MD)simulations using a graphics processing unit (GPU) has been employed in order to determine the conformational space of the methane-thiosulfonate spin label (MTSL) attached to the activation loop of the Aurora-A kinase protein and compared with quantum mechanical (QM) methods rooted on density functional theory (DFT). MD provided a wealth of information about interactions between the MTSL and the residues of the protein and on the different motional contributions to the overall dynamics of the MTSL. Data obtained from MD were seen to be in good agreement with those obtained from QM but the dynamics of the system revealed more interactions than those observed from QM methods. A strong correlation between the tumbling of the protein and the transitions of the X4 and X5 dihedral angles of the MTSL, was observed with a consequent effect also the distribution of the nitroxide(NO)group in the space. Theoretical EPR spectra calculated from opportunely selected MD frames showing interactions betw...

  11. MicroRNA biogenesis factor DRB1 is a phosphorylation target of mitogen activated protein kinase MPK3 in both rice and Arabidopsis.

    Science.gov (United States)

    Raghuram, Badmi; Sheikh, Arsheed H; Rustagi, Yashika; Sinha, Alok K

    2015-02-01

    MicroRNA (miRNA) biogenesis requires AtDRB1 (double-stranded RNA binding protein)/HYL1 (Hyponastic Leaves1) protein for processing and maturation of miRNA precursors. The AtDRB1/HYL1 protein associates with AtDCL1 (Dicer-Like1) and accurately processes primary-miRNAs (pri-mRNAs) first to precursor-miRNAs (pre-miRNAs) and finally to mature miRNAs. The dephosphorylation of AtDRB1/HYL1 protein is very important for the precise processing of miRNA precursors. The monocot model crop plant Oryza sativa encodes four orthologues of AtDRB1/HYL1 protein, the only one encoded by Arabidopsis thaliana. The present study focuses on the functionality of the O. sativa DRBs as the orthologues of AtDRB1/HYL1 by using RNA binding assays and in planta protein-protein interaction analysis. Further, mitogen-activated protein kinase MPK3 is established as the kinase phosphorylating DRB1 protein in both the model plants, O. sativa and Arabidopsis. MicroRNA microarray analysis in atmpk3 and atmpk6 mutants indicate the importance of AtMPK3 in maintaining the level of miRNAs in the plant.

  12. Ghrelin promotes intestinal epithelial cell proliferation through PI3K/Akt pathway and EGFR trans-activation both converging to ERK 1/2 phosphorylation.

    Science.gov (United States)

    Waseem, Talat; Duxbury, Mark; Ashley, Stanley W; Robinson, Malcolm K

    2014-02-01

    Little is known about ghrelin's effects on intestinal epithelial cells even though it is known to be a mitogen for a variety of other cell types. Because ghrelin is released in close proximity to the proliferative compartment of the intestinal tract, we hypothesized that ghrelin may have potent pro-proliferative effect on intestinal epithelial cells as well. To test this hypothesis, we characterized the effects of ghrelin on FHs74Int and Caco-2 intestinal epithelial cell lines in vitro. We found that ghrelin has potent dose dependent proliferative effects in both cell lines through a yet to be characterized G protein coupled growth hormone secretagogue receptor (GHS-R) subtype. Consistent with above findings, cell cycle flowcytometric analyses demonstrated that ghrelin shifts cells from the G1 to S phase and thereby promotes cell cycle progression. Further characterization of subcellular events, suggested that ghrelin mediates its pro-proliferative effect through Adenylate cyclase (AC)-independent epidermal growth factor receptor (EGFR) trans-activation and PI3K-Akt phosphorylation. Both these pathways converge to stimulate MAPK, ERK 1/2 downstream. The role of ghrelin in states where intestinal mucosal injury and rapid mucosal repair occur warrants further investigation.

  13. Protein phosphorylation during coconut zygotic embryo development

    International Nuclear Information System (INIS)

    Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species

  14. Antcin K, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects via Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in Muscles

    Directory of Open Access Journals (Sweden)

    Yueh-Hsiung Kuo

    2016-01-01

    Full Text Available The purpose of this study was to screen firstly the potential effects of antcin K (AnK, the main constituent of the fruiting body of Antrodia camphorata, in vitro and further evaluate the activities and mechanisms in high-fat-diet- (HFD- induced mice. Following 8-week HFD-induction, mice were treated with AnK, fenofibrate (Feno, metformin (Metf, or vehicle for 4 weeks afterward. In C2C12 myotube cells, the membrane GLUT4 and phospho-Akt expressions were higher in insulin and AnK-treated groups than in the control group. It was observed that AnK-treated mice significantly lowered blood glucose, triglyceride, total cholesterol, and leptin levels in AnK-treated groups. Of interest, AnK at 40 mg/kg/day dosage displayed both antihyperglycemic effect comparable to Metf (300 mg/kg/day and antihypertriglyceridemic effect comparable to Feno (250 mg/kg/day. The combination of significantly increased skeletal muscular membrane expression levels of glucose transporter 4 (GLUT4 but decreased hepatic glucose-6-phosphatase (G6 Pase mRNA levels by AnK thus contributed to a decrease in blood glucose levels. Furthermore, AnK enhanced phosphorylation of AMP-activated protein kinase (phospho-AMPK expressions in the muscle and liver. Moreover, AnK treatment exhibited inhibition of hepatic fatty acid synthase (FAS but enhancement of fatty acid oxidation peroxisome proliferator-activated receptor α (PPARα expression coincident with reduced sterol response element binding protein-1c (SREBP-1c mRNA levels in the liver may contribute to decreased plasma triglycerides, hepatic steatosis, and total cholesterol levels. The present findings indicate that AnK displays an advantageous therapeutic potential for the management of type 2 diabetes and hyperlipidemia.

  15. Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages.

    Science.gov (United States)

    Clark, Kristopher; MacKenzie, Kirsty F; Petkevicius, Kasparas; Kristariyanto, Yosua; Zhang, Jiazhen; Choi, Hwan Geun; Peggie, Mark; Plater, Lorna; Pedrioli, Patrick G A; McIver, Ed; Gray, Nathanael S; Arthur, J Simon C; Cohen, Philip

    2012-10-16

    Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation, and resolution of inflammation. Classically activated (M1) macrophages produce proinflammatory mediators to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages (M2b) produce high levels of anti-inflammatory molecules, such as IL-10, and low levels of proinflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Here, we demonstrate that the salt-inducible kinases (SIKs) restrict the formation of regulatory macrophages and that their inhibition induces striking increases in many of the characteristic markers of regulatory macrophages, greatly stimulating the production of IL-10 and other anti-inflammatory molecules. We show that SIK inhibitors elevate IL-10 production by inducing the dephosphorylation of cAMP response element-binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3, its dissociation from 14-3-3 proteins and its translocation to the nucleus where it enhances a gene transcription program controlled by CREB. Importantly, the effects of SIK inhibitors on IL-10 production are lost in macrophages that express a drug-resistant mutant of SIK2. These findings identify SIKs as a key molecular switch whose inhibition reprograms macrophages to an anti-inflammatory phenotype. The remarkable effects of SIK inhibitors on macrophage function suggest that drugs that target these protein kinases may have therapeutic potential for the treatment of inflammatory and autoimmune diseases.

  16. Closed-loop feedback control and bifurcation analysis of epileptiform activity via optogenetic stimulation in a mathematical model of human cortex

    Science.gov (United States)

    Selvaraj, Prashanth; Sleigh, Jamie W.; Kirsch, Heidi E.; Szeri, Andrew J.

    2016-01-01

    Optogenetics provides a method of neuron stimulation that has high spatial, temporal, and cell-type specificity. Here we present a model of optogenetic feedback control that targets the inhibitory population, which expresses light-sensitive channelrhodopsin-2 channels, in a mean-field model of undifferentiated cortex that is driven to seizures. The inhibitory population is illuminated with an intensity that is a function of electrode measurements obtained via the cortical model. We test the efficacy of this control method on seizurelike activity observed in two parameter spaces of the cortical model that most closely correspond to seizures observed in patients. We also compare the effect of closed-loop and open-loop control on seizurelike activity using a less-complicated ordinary differential equation model of the undifferentiated cortex in parameter space. Seizurelike activity is successfully suppressed in both parameter planes using optimal illumination intensities less likely to have adverse effects on cortical tissue.

  17. Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji [Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Lee, Hui-Young [Department of Internal Medicine, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Hong, Suntaek [Laboratory of Cancer Cell Biology, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon 406-840 (Korea, Republic of); Kim, Seong-Jin [CHA Cancer Institute, CHA University of Medicine and Science, Seoul 135-081 (Korea, Republic of); Kim, Byung-Chul, E-mail: bckim@kangwon.ac.kr [Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

    2013-06-14

    Highlights: •H{sub 2}O{sub 2} inhibits TGF-β1-induced cell cycle arrest. •H{sub 2}O{sub 2} induces Smad3 linker phosphorylation through Akt-ERK1/2 pathway. •H{sub 2}O{sub 2}-mediated suppression of TGF-β signal requires Smad3 linker phosphorylation. •This is a first report about interplay between H{sub 2}O{sub 2} and growth inhibition pathway. -- Abstract: Hydrogen peroxide (H{sub 2}O{sub 2}) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H{sub 2}O{sub 2} are less understood. Here we report an important mechanism for antagonistic effects of H{sub 2}O{sub 2} on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H{sub 2}O{sub 2} (0.05–0.2 mM) completely blocked TGF-β1-mediated induction of p15{sup INK4B} expression and increase of its promoter activity. Interestingly, H{sub 2}O{sub 2} selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H{sub 2}O{sub 2} increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H{sub 2}O{sub 2} on TGF-β1-induced increase of p15{sup INK4B}-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H{sub 2}O{sub 2} as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing

  18. Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway.

    Science.gov (United States)

    Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Hui-Young; Hong, Suntaek; Kim, Seong-Jin; Kim, Byung-Chul

    2013-06-14

    Hydrogen peroxide (H2O2) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H2O2 are less understood. Here we report an important mechanism for antagonistic effects of H2O2 on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H2O2 (0.05-0.2 mM) completely blocked TGF-β1-mediated induction of p15(INK4B) expression and increase of its promoter activity. Interestingly, H2O2 selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H2O2 increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H2O2 on TGF-β1-induced increase of p15(INK4B)-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H2O2 as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing a potential mechanism whereby H2O2 antagonizes the cytostatic function of TGF-β1.

  19. Genistein inhibits phorbol ester-induced NF-κB transcriptional activity and COX-2 expression by blocking the phosphorylation of p65/RelA in human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Myung-Hoon; Kim, Do-Hee [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Na, Hye-Kyung [Department of Food and Nutrition, Sungshin Women' s University, Seoul (Korea, Republic of); Kim, Jung-Hwan; Kim, Ha-Na [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Haegeman, Guy [LEGEST, University of Gent (Belgium); Surh, Young-Joon, E-mail: surh@snu.ac.kr [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul (Korea, Republic of); Cancer Research Institute, Seoul National University, Seoul (Korea, Republic of)

    2014-10-15

    Genistein, an isoflavone present in soy products, has chemopreventive effects on mammary carcinogenesis. In the present study, we have investigated the effects of genistein on phorbol ester-induced expression of cyclooxygenase-2 (COX-2) that plays an important role in the pathophysiology of inflammation-associated carcinogenesis. Pretreatment of cultured human breast epithelial (MCF10A) cells with genistein reduced COX-2 expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). There are multiple lines of evidence supporting that the induction of COX-2 is regulated by the eukaryotic transcription factor NF-κB. Genistein failed to inhibit TPA-induced nuclear translocation and DNA binding of NF-κB as well as degradation of IκB. However, genistein abrogated the TPA-induced transcriptional activity of NF-κB as determined by the luciferase reporter gene assay. Genistein inhibited phosphorylation of the p65 subunit of NF-κB and its interaction with cAMP regulatory element-binding protein-binding protein (CBP)/p300 and TATA-binding protein (TBP). TPA-induced NF-κB phosphorylation was abolished by pharmacological inhibition of extracellular signal-regulated kinase (ERK). Likewise, pharmacologic inhibition or dominant negative mutation of ERK suppressed phosphorylation of p65. The above findings, taken together, suggest that genistein inhibits TPA-induced COX-2 expression in MCF10A cells by blocking ERK-mediated phosphorylation of p65 and its subsequent interaction with CBP and TBP.

  20. Protein kinase C-α signals P115RhoGEF phosphorylation and RhoA activation in TNF-α-induced mouse brain microvascular endothelial cell barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Deng Xiaolu

    2011-04-01

    Full Text Available Abstract Background Tumor necrosis factor-α (TNF-α, a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC barrier dysfunction. Methods Bend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL. RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER. p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability. Results We observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER. Conclusions Taken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF

  1. The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher [MUSC; (UNC)

    2013-04-22

    Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5–α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5–α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.

  2. Resveratrol inhibits phosphorylation within the signal transduction and activator of transcription 3 signaling pathway by activating sirtuin 1 in SW1353 chondrosarcoma cells.

    Science.gov (United States)

    Jin, Haidong; Chen, Hui; Yu, Kehe; Zhang, Jingdong; Li, Bin; Cai, Ningyu; Pan, Jun

    2016-09-01

    The present study assessed the mechanism by which resveratrol (Res) inhibits the growth of SW1353 chondrosarcoma cells and examined whether sirtuin 1 (Sirt1) activation affects phosphorylation within the signal transduction and activator of transcription 3 (STAT3) signaling pathway. The present study used SW1353 chondrosarcoma cells in the logarithmic phase of growth (control and treatment groups). The latter group was treated with Res at 25 and 50 µmol/l for 24 h, and cell viability, proliferation and apoptosis were analyzed using the cell counting kit‑8 assay, colony counting and Hoechst staining, respectively. The expression levels of caspase‑3, cleaved caspase‑3, B‑cell lymphoma‑2 (BCL‑2), BCL-2 associated X protein (Bax), STAT3 and phosphorylated (p‑)STAT3) were measured by Western blotting. SW1353 cells were transfected with small interfering (si)RNA targeting Sirt1 and the expression levels of Sirt1, STAT3 and p-STAT3 were assessed. Exposure of SW1353 cells to Res reduced cell viability in a dose‑dependent manner (P<0.01). Additionally, cell proliferation was significantly inhibited and the cell nuclei exhibited apoptotic characteristics. Cleaved caspase‑3, Sirt1 and Bax levels were upregulated. The expression levels of BCL‑2 and p‑STAT3 were downregulated. Additionally, the BCL‑2/Bax ratio was reduced compared with the control group. The total STAT3 level was unaffected. Res treatment activated Sirt1, however, in cells transfected with Sirt1‑siRNA, the ability of resveratrol to suppress p‑STAT3 expression was compromised. Overall, it was revealed that Res treatment induced apoptosis, inhibited proliferation and affected phosphorylation within the STAT3 signaling pathway by activating Sirt1 in SW1353 chondrosarcoma cells.

  3. Imbalanced expression of mitogen-activated protein kinase phosphatase-1 and phosphorylated extracellular signal-regulated kinases in lung squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Kai WANG; Min ZHANG; Ying-ying QIAN; Zhe-yuan DING; Jun-huiLV; Hua-hao SHEN

    2011-01-01

    Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2),as the key regulatory mechanism of the MAPKs,in lung squamous cell carcinoma (SCC).Methods:We assessed the expressions of MKP-1 and p-ERK1/2in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis.Results:Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was negatively correlated with tumor differentiation (P<0.01).However,the expression of p-ERK1/2 or ERK1/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was positively correlated with tumor differentiation (P<0.01).Conclusions:Our data indicates the relevance of MKP-1 and p-ERK1/2 in SCC as a potential positive and negative prognostic factor.The imbalanced expression of MKP-1 and p-ERK1/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.

  4. Amphetamine elevates phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the rat forebrain via activating dopamine D1 and D2 receptors.

    Science.gov (United States)

    Xue, Bing; Fitzgerald, Cole A; Jin, Dao-Zhong; Mao, Li-Min; Wang, John Q

    2016-09-01

    Psychostimulants have an impact on protein synthesis, although underlying molecular mechanisms are unclear. Eukaryotic initiation factor 2α-subunit (eIF2α) is a key player in initiation of protein translation and is regulated by phosphorylation. While this factor is sensitive to changing synaptic input and is critical for synaptic plasticity, its sensitivity to stimulants is poorly understood. Here we systematically characterized responses of eIF2α to a systemic administration of the stimulant amphetamine (AMPH) in dopamine responsive regions of adult rat brains. Intraperitoneal injection of AMPH at 5mg/kg increased eIF2α phosphorylation at serine 51 in the striatum. This increase was transient. In the medial prefrontal cortex (mPFC), AMPH induced a relatively delayed phosphorylation of the factor. Pretreatment with a dopamine D1 receptor antagonist SCH23390 blocked the AMPH-stimulated eIF2α phosphorylation in both the striatum and mPFC. Similarly, a dopamine D2 receptor antagonist eticlopride reduced the effect of AMPH in the two regions. Two antagonists alone did not alter basal eIF2α phosphorylation. AMPH and two antagonists did not change the amount of total eIF2α proteins in both regions. These results demonstrate the sensitivity of eIF2α to stimulant exposure. AMPH possesses the ability to stimulate eIF2α phosphorylation in striatal and mPFC neurons in vivo in a D1 and D2 receptor-dependent manner. PMID:27338925

  5. Role of Unusual P Loop Ejection and Autophosphorylation in HipA-Mediated Persistence and Multidrug Tolerance

    Directory of Open Access Journals (Sweden)

    Maria A. Schumacher

    2012-09-01

    Full Text Available HipA is a bacterial serine/threonine protein kinase that phosphorylates targets, bringing about persistence and multidrug tolerance. Autophosphorylation of residue Ser150 is a critical regulatory mechanism of HipA function. Intriguingly, Ser150 is not located on the activation loop, as are other kinases; instead, it is in the protein core, where it forms part of the ATP-binding “P loop motif.” How this buried residue is phosphorylated and regulates kinase activity is unclear. Here, we report multiple structures that reveal the P loop motif's exhibition of a remarkable “in-out” conformational equilibrium, which allows access to Ser150 and its intermolecular autophosphorylation. Phosphorylated Ser150 stabilizes the “out state,” which inactivates the kinase by disrupting the ATP-binding pocket. Thus, our data reveal a mechanism of protein kinase regulation that is vital for multidrug tolerance and persistence, as kinase inactivation provides the critical first step in allowing dormant cells to revert to the growth phenotype and to reinfect the host.

  6. Compartment-Specific Phosphorylation of Squid Neurofilaments.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  8. Signal Transducer and Activator of Transcription (Stat)-Induced Stat Inhibitor 1 (Ssi-1)/Suppressor of Cytokine Signaling 1 (Socs1) Inhibits Insulin Signal Transduction Pathway through Modulating Insulin Receptor Substrate 1 (Irs-1) Phosphorylation

    OpenAIRE

    Kawazoe, Yoshinori; Naka, Tetsuji; Fujimoto, Minoru; Kohzaki, Hidetsugu; Morita, Yoshiaki; Narazaki, Masashi; Okumura, Kohichi; Saitoh, Hiroshi; Nakagawa, Reiko; Uchiyama, Yasuo; Akira, Shizuo; Kishimoto, Tadamitsu

    2001-01-01

    Signal transducer and activator of transcription (STAT)-induced STAT inhibitor 1 (SSI-1) is known to function as a negative feedback regulator of cytokine signaling, but it is unclear whether it is involved in other biological events. Here, we show that SSI-1 participates and plays an important role in the insulin signal transduction pathway. SSI-1–deficient mice showed a significantly low level of blood sugar. While the forced expression of SSI-1 reduced the phosphorylation level of insulin ...

  9. Benchmarking and Hardware-In-The-Loop Operation of a 2014 MAZDA SkyActiv (SAE 2016-01-1007)

    Science.gov (United States)

    Engine Performance evaluation in support of LD MTE. EPA used elements of its ALPHA model to apply hardware-in-the-loop (HIL) controls to the SKYACTIV engine test setup to better understand how the engine would operate in a chassis test after combined with future leading edge tech...

  10. Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

    DEFF Research Database (Denmark)

    Xiang, B; Yu, G H; Guo, J;

    2001-01-01

    ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

  11. Fibroblast growth factor receptor 3 interacts with and activates TGFβ-activated kinase 1 tyrosine phosphorylation and NFκB signaling in multiple myeloma and bladder cancer.

    Directory of Open Access Journals (Sweden)

    Lisa Salazar

    Full Text Available Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1. Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3 tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.

  12. Antidiabetic and Antihyperlipidemic Effects of Clitocybe nuda on Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    Directory of Open Access Journals (Sweden)

    Mei-Hsing Chen

    2014-01-01

    Full Text Available The objective of this study was to evaluate the antihyperlipidemic and antihyperglycemic effects and mechanism of the extract of Clitocybe nuda (CNE, in high-fat- (HF- fed mice. C57BL/6J was randomly divided into two groups: the control (CON group was fed with a low-fat diet, whereas the experimental group was fed with a HF diet for 8 weeks. Then, the HF group was subdivided into five groups and was given orally CNE (including C1: 0.2, C2: 0.5, and C3: 1.0 g/kg/day extracts or rosiglitazone (Rosi or vehicle for 4 weeks. CNE effectively prevented HF-diet-induced increases in the levels of blood glucose, triglyceride, insulin (P<0.001, P<0.01, P<0.05, resp. and attenuated insulin resistance. By treatment with CNE, body weight gain, weights of white adipose tissue (WAT and hepatic triacylglycerol content were reduced; moreover, adipocytes in the visceral depots showed a reduction in size. By treatment with CNE, the protein contents of glucose transporter 4 (GLUT4 were significantly increased in C3-treated group in the skeletal muscle. Furthermore, CNE reduces the hepatic expression of glucose-6-phosphatase (G6Pase and glucose production. CNE significantly increases protein contents of phospho-AMP-activated protein kinase (AMPK in the skeletal muscle and adipose and liver tissues. Therefore, it is possible that the activation of AMPK by CNE leads to diminished gluconeogenesis in the liver and enhanced glucose uptake in skeletal muscle. It is shown that CNE exhibits hypolipidemic effect in HF-fed mice by increasing ATGL expression, which is known to help triglyceride to hydrolyze. Moreover, antidiabetic properties of CNE occurred as a result of decreased hepatic glucose production via G6Pase downregulation and improved insulin sensitization. Thus, amelioration of diabetic and dyslipidemic states by CNE in HF-fed mice occurred by regulation of GLUT4, G6Pase, ATGL, and AMPK phosphorylation.

  13. Transcription, Signaling Receptor Activity, Oxidative Phosphorylation, and Fatty Acid Metabolism Mediate the Presence of Closely Related Species in Distinct Intertidal and Cold-Seep Habitats.

    Science.gov (United States)

    Van Campenhout, Jelle; Vanreusel, Ann; Van Belleghem, Steven; Derycke, Sofie

    2016-01-01

    Bathyal cold seeps are isolated extreme deep-sea environments characterized by low species diversity while biomass can be high. The Håkon Mosby mud volcano (Barents Sea, 1,280 m) is a rather stable chemosynthetic driven habitat characterized by prominent surface bacterial mats with high sulfide concentrations and low oxygen levels. Here, the nematode Halomonhystera hermesi thrives in high abundances (11,000 individuals 10 cm(-2)). Halomonhystera hermesi is a member of the intertidal Halomonhystera disjuncta species complex that includes five cryptic species (GD1-5). GD1-5's common habitat is characterized by strong environmental fluctuations. Here, we compared the transcriptomes of H. hermesi and GD1, H. hermesi's closest relative. Genes encoding proteins involved in oxidative phosphorylation are more strongly expressed in H. hermesi than in GD1, and many genes were only observed in H. hermesi while being completely absent in GD1. Both observations could in part be attributed to high sulfide concentrations and low oxygen levels. Additionally, fatty acid elongation was also prominent in H. hermesi confirming the importance of highly unsaturated fatty acids in this species. Significant higher amounts of transcription factors and genes involved in signaling receptor activity were observed in GD1 (many of which were completely absent in H. hermesi), allowing fast signaling and transcriptional reprogramming which can mediate survival in dynamic intertidal environments. GC content was approximately 8% higher in H. hermesi coding unigenes resulting in differential codon usage between both species and a higher proportion of amino acids with GC-rich codons in H. hermesi. In general our results showed that most pathways were active in both environments and that only three genes are under natural selection. This indicates that also plasticity should be taken in consideration in the evolutionary history of Halomonhystera species. Such plasticity, as well as possible

  14. Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Giovanna Damia

    2001-01-01

    Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

  15. Protein phosphatase 2a (PP2A binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

    Directory of Open Access Journals (Sweden)

    Jones Candace A

    2011-10-01

    residues lying between striatin's calmodulin-binding and WD-domains and recruits the PP2A A/C heterodimer to its coiled-coil/oligomerization domain. Residues outside the previously reported coiled-coil domain of striatin are necessary for its oligomerization. Striatin-associated PP2A is critical for Mst3 dephosphorylation and inactivation. Upon inhibition of PP2A, Mst3 activation appears to involve autophosphorylation of multiple activation loop phosphorylation sites. Mob3 can associate with striatin sequences C-terminal to the Mst3 binding site but also with sequences proximal to striatin-associated PP2A, consistent with a possible role for Mob 3 in the regulation of Mst3 by PP2A.

  16. Time-resolved emission from bright hot pixels of an active region observed in the EUV band with SDO/AIA and multi-stranded loop modeling

    CERN Document Server

    Tajfirouze, E; Petralia, A; Testa, P

    2015-01-01

    Evidence for small amounts of very hot plasma has been found in active regions and might be the indication of an impulsive heating, released at spatial scales smaller than the cross section of a single loop. We investigate the heating and substructure of coronal loops in the core of one such active region by analyzing the light curves in the smallest resolution elements of solar observations in two EUV channels (94 A and 335 A) from the Atmospheric Imaging Assembly on-board the Solar Dynamics Observatory. We model the evolution of a bundle of strands heated by a storm of nanoflares by means of a hydrodynamic 0D loop model (EBTEL). The light curves obtained from the random combination of those of single strands are compared to the observed light curves either in a single pixel or in a row of pixels, simultaneously in the two channels and using two independent methods: an artificial intelligent system (Probabilistic Neural Network, PNN) and a simple cross-correlation technique. We explore the space of the param...

  17. Phosphorylating enzymes involved in glucose fermentation of Actinomyces naeslundii.

    OpenAIRE

    Takahashi, N.; Kalfas, S; Yamada, T.

    1995-01-01

    Enzymatic activities involved in glucose fermentation of Actinomyces naeslundii were studied with glucose-grown cells from batch cultures. Glucose could be phosphorylated to glucose 6-phosphate by a glucokinase that utilized polyphosphate and GTP instead of ATP as a phosphoryl donor. Glucose 6-phosphate was further metabolized to the end products lactate, formate, acetate, and succinate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase was only PPi. Phos...

  18. A New Intermolecular Phosphoryl Transfer between Serine and Histidine Residues

    Institute of Scientific and Technical Information of China (English)

    SU,Yu-Qian; NIU,Ming-Yu; CAO,Shu-Xia; ZHANG,Jian-Chen; QU,Ling-Bo; LIAO,Xin-Cheng; ZHAO,Yu-Fen

    2004-01-01

    @@ Phosphoryl transfer constitutes one of the most important reactions in functionalized molecules, bioorganic chemistry and biochemistry.[1] The transformations are involved in diverse processes, such as activated state change of phosphorus, DNA/RNA synthesis, energy metabolism and signal transduction. So, phosphoryl transfer reaction which can be performed by either intramolecular or intermolecular phosphorylation and dephosphorylation mechanism has been investigated by many scientists in wide fields.

  19. Activation of protein kinase C or cAMP-dependent protein kinase increases phosphorylation of the c-erbA-encoded thyroid hormone receptor and of the v-erbA-encoded protein

    DEFF Research Database (Denmark)

    Goldberg, Y; Glineur, C; Gesquière, J C;

    1988-01-01

    -v-erbA is enhanced 10-fold following treatment of cells with activators of either protein kinase C or cAMP-dependent protein kinase. Since cAMP-dependent protein kinase phosphorylates both p46c-erbA and P75gag-v-erbA in vitro at the same site as that observed in vivo, at least part of the c......, defined by the limit tryptic phosphopeptide 28SSQCLVK, is retained on the v-erbA-encoded P75gag-v-erbA protein. This site is located in the amino-terminal domain of these molecules, 21 amino acids upstream of the DNA-binding region. Phosphorylation of this site in both p46c-erbA and P75gag...

  20. The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase.

    Science.gov (United States)

    Montagner, Caroline; Nigen, Michaël; Jacquin, Olivier; Willet, Nicolas; Dumoulin, Mireille; Karsisiotis, Andreas Ioannis; Roberts, Gordon C K; Damblon, Christian; Redfield, Christina; Matagne, André

    2016-07-29

    Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus β-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG(0)) of 32 ± 2 kJ·mol(-1) For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG(0) value of 65 ± 1.4 kJ·mol(-1) These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity. PMID:27235401

  1. Direct Optical Ice Sensing and Closed-Loop Controller Design for Active De-icing of Wind Turbines Using Distributed Heating

    Science.gov (United States)

    Shajiee, Shervin

    Ice accumulation on wind turbines operating in cold regions reduces power generation by degrading aerodynamic efficiency and causes mass imbalance and fatigue loads on the blades. Due to blade rotation and variation of the pitch angle, different locations on the blade experience large variations of Reynolds number, Nusselt number, heat loss, and non-uniform ice distribution. Hence, applying different amounts of heat flux in different blade locations can provide more effective de-icing for the same total power consumption. This large variation of required heat flux motivates using distributed resistive heating, with the capability of locally adjusting thermal power as a function of location on the blade. The main contributions of this research are developing the experimental feasibility of direct ice sensing using an optical sensing technique as well as development of a computational framework for implementation of closed-loop localized active de-icing using distributed sensing. A script-base module was developed in a commercial finite-element software (ANSYS) which provides the capability of (i) Closed-loop de-icing simulations for a distributed network of sensors and actuators, (ii) investigating different closed-loop thermal control schemes and their de-icing efficiency (iii) optimizing thermal actuation for a distributed resistive heating, and (iv) analyzing different faulty scenarios for sensors and thermal actuators under known faults in the network. Different surrogate models were used to enhance the computational efficiency of this approach. The results showed that optimal value of control parameters in a distributed network of heaters depends on convective heat transfer characteristics, layout of heaters and type of closed-loop controller scheme used for thermal actuation. Furthermore, It was shown that closed-loop control provides much faster de-icing than the open-loop constant heat flux thermal actuation. It was observed both experimentally and

  2. Localization of active, dually phosphorylated extracellular signal-regulated kinase 1 and 2 in colorectal cancer with or without activating BRAF and KRAS mutations

    DEFF Research Database (Denmark)

    Holck, Susanne; Bonde, Jesper; Pedersen, Helle;

    2016-01-01

    . The same applied to 4 of the 14 KRAS(m) tumors. A phophorylation-insensitive antibody demonstrated that lack of pERK staining did not reflect defect expression of ERK1/2 protein. Thus, increased staining for pERK does not correlate to BRAF or KRAS mutations even with a highly optimized procedure. Further......Colorectal cancers (CRC) often show activating mutations of the KRAS or BRAF genes, which stimulate the extracellular signal-regulated kinase (ERK) pathway, thus increasing cell proliferation and inhibiting apoptosis. However, immunohistochemical results on ERK activation in such tumors differ...... greatly. Recently, using a highly optimized immunohistochemical method, we obtained evidence that high levels of ERK activation in rectal adenocarcinomas were associated with resistance to radiochemotherapy. In order to determine whether KRAS and/or BRAF mutations correlate to immunohistochemically...

  3. Extended frequency compensation of a diamagnetic loop

    International Nuclear Information System (INIS)

    A method of compensation for a diamagnetic loop that is magnetically coupled to a concentric stainless-steel vacuum vessel is presented. This compensation method accounts for imperfect magnetic coupling between the vessel eddy currents, the diamagnetic loop, and the plasma diamagnetic currents, and it also corrects for a finite loading resistance on the diamagnetic loop. A procedure for adjusting and calibrating the active-filter compensation circuit is presented. It can be applied to internal or external diamagnetic loops. (author)

  4. Phosphorylation of DGCR8 Increases Its Intracellular Stability and Induces a Progrowth miRNA Profile

    Directory of Open Access Journals (Sweden)

    Kristina M. Herbert

    2013-11-01

    Full Text Available During miRNA biogenesis, the microprocessor complex (MC, which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8’s ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.

  5. Loop Quantum Gravity

    Directory of Open Access Journals (Sweden)

    Rovelli Carlo

    1998-01-01

    Full Text Available The problem of finding the quantum theory of the gravitational field, and thus understanding what is quantum spacetime, is still open. One of the most active of the current approaches is loop quantum gravity. Loop quantum gravity is a mathematically well-defined, non-perturbative and background independent quantization of general relativity, with its conventional matter couplings. Research in loop quantum gravity today forms a vast area, ranging from mathematical foundations to physical applications. Among the most significant results obtained are: (i The computation of the physical spectra of geometrical quantities such as area and volume, which yields quantitative predictions on Planck-scale physics. (ii A derivation of the Bekenstein-Hawking black hole entropy formula. (iii An intriguing physical picture of the microstructure of quantum physical space, characterized by a polymer-like Planck scale discreteness. This discreteness emerges naturally from the quantum theory and provides a mathematically well-defined realization of Wheeler's intuition of a spacetime ``foam''. Long standing open problems within the approach (lack of a scalar product, over-completeness of the loop basis, implementation of reality conditions have been fully solved. The weak part of the approach is the treatment of the dynamics: at present there exist several proposals, which are intensely debated. Here, I provide a general overview of ideas, techniques, results and open problems of this candidate theory of quantum gravity, and a guide to the relevant literature.

  6. Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy.

    Science.gov (United States)

    Rippe, K; Guthold, M; von Hippel, P H; Bustamante, C

    1997-07-11

    Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase x sigma 54 (RNAP x sigma 54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C). DNA-protein complexes were deposited on mica and images were recorded in air. The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP x sigma 54 glnA promoter. By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP x sigma 54 at the glnA promoter; (3) association between DNA bound RNAP x sigma 54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP x sigma 54. Measurements of the DNA bending angle of RNAP x sigma 54 closed promoter complexes yielded an apparent bending angle of 49(+/-24) degrees. Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50(+/-24) degrees and 114(+/-18) degrees, suggesting that the transition from the RNAP x sigma 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.

  7. Rollercoaster loop shapes

    Science.gov (United States)

    Pendrill, Ann-Marie

    2005-11-01

    Many modern rollercoasters feature loops. Although textbook loops are often circular, real rollercoaster loops are not. In this paper, we look into the mathematical description of various possible loop shapes, as well as their riding properties. We also discuss how a study of loop shapes can be used in physics education.

  8. A Crystallographic Snapshot of Tyrosine Trans-phosphorylation in Action

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Xu, C; Ma, J; Eliseenkova, A; Li, W; Pollock, P; Pitteloud, N; Miller, W; Neubert, T; Mohammadi, M

    2008-01-01

    Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 {angstrom} away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

  9. Phosphorylation of Cdc5 regulates its accumulation

    Directory of Open Access Journals (Sweden)

    Simpson-Lavy Kobi J

    2011-12-01

    Full Text Available Abstract Background Cdc5 (polo kinase/Plk1 is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28 phosphorylation site present. Findings Cdc5T23A introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans and drastically reduces Clb2 levels. Conclusions Phosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1 caps Cdc5 and other CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.

  10. Enhanced activation of human T cell clones specific for virus-like particles expressing the HIV V3 loop in the presence of HIV V3 loop-specific polyclonal antibodies

    Science.gov (United States)

    Peifang, S.; Pira, G. L.; Fenoglio, D.; Harris, S.; Costa, M. G.; Venturino, V.; Dessì, V.; Layton, G.; Laman, J.; Huisman, J. G.; Manca, F.

    1994-01-01

    Recombinant virus-like particles (VLP), formed by the yeast Ty p1 protein, carrying the HIV gp120 V3 loop on their surface (V3-VLP) have been tested in vitro for immunogenicity and antigenicity by using VLP p1-specific human CD4+ T cell lines and clones. VLP-specific human T cell lines and clones were generated from normal individuals, indicating that VLP-specific precursor cells present in the peripheral lymphocyte pool can be induced to expand clonally upon antigen challenge in vitro, in the absence of previous immunization. It was also shown that V3-specific polyclonal antibodies enhance V3-VLP-induced activation of VLP-specific T cell clones. Antibody-dependent potentiation has been shown previously in other antigen systems, and it depends on enhanced uptake of complexed antigen by Fc receptor-positive antigen-presenting cells. Since in this case antigen is internalized by presenting cells as a complex, it can be inferred that a similar event of antibody-mediated antigen uptake can take place with V3-specific B cells, resulting in presentation by the B cells of T helper epitopes derived from processing of the VLP p1 moiety. This suggests that T helper cells specific for the carrier VLP p1 protein can be activated to provide help to V3-specific B cells in the presence of the appropriate antigen construct. PMID:7915974

  11. Phospholipase Cϵ Activates Nuclear Factor-κB Signaling by Causing Cytoplasmic Localization of Ribosomal S6 Kinase and Facilitating Its Phosphorylation of Inhibitor κB in Colon Epithelial Cells.

    Science.gov (United States)

    Wakita, Masahiro; Edamatsu, Hironori; Li, Mingzhen; Emi, Aki; Kitazawa, Sohei; Kataoka, Tohru

    2016-06-10

    Phospholipase Cϵ (PLCϵ), an effector of Ras and Rap small GTPases, plays a crucial role in inflammation by augmenting proinflammatory cytokine expression. This proinflammatory function of PLCϵ is implicated in its facilitative role in tumor promotion and progression during skin and colorectal carcinogenesis, although their direct link remains to be established. Moreover, the molecular mechanism underlying these functions of PLCϵ remains unknown except that PKD works downstream of PLCϵ. Here we show by employing the colitis-induced colorectal carcinogenesis model, where Apc(Min) (/+) mice are administered with dextran sulfate sodium, that PLCϵ knock-out alleviates the colitis and suppresses the following tumorigenesis concomitant with marked attenuation of proinflammatory cytokine expression. In human colon epithelial Caco2 cells, TNF-α induces sustained expression of proinflammatory molecules and sustained activation of nuclear factor-κB (NF-κB) and PKD, the late phases of which are suppressed by not only siRNA-mediated PLCϵ knockdown but also treatment with a lysophosphatidic acid (LPA) receptor antagonist. Also, LPA stimulation induces these events in an early time course, suggesting that LPA mediates TNF-α signaling in an autocrine manner. Moreover, PLCϵ knockdown results in inhibition of phosphorylation of IκB by ribosomal S6 kinase (RSK) but not by IκB kinases. Subcellular fractionation suggests that enhanced phosphorylation of a scaffolding protein, PEA15 (phosphoprotein enriched in astrocytes 15), downstream of the PLCϵ-PKD axis causes sustained cytoplasmic localization of phosphorylated RSK, thereby facilitating IκB phosphorylation in the cytoplasm. These results suggest the crucial role of the TNF-α-LPA-LPA receptor-PLCϵ-PKD-PEA15-RSK-IκB-NF-κB pathway in facilitating inflammation and inflammation-associated carcinogenesis in the colon. PMID:27053111

  12. Direct Demonstration That Loop1 of Scap Binds to Loop7: A CRUCIAL EVENT IN CHOLESTEROL HOMEOSTASIS.

    Science.gov (United States)

    Zhang, Yinxin; Lee, Kwang Min; Kinch, Lisa N; Clark, Lindsay; Grishin, Nick V; Rosenbaum, Daniel M; Brown, Michael S; Goldstein, Joseph L; Radhakrishnan, Arun

    2016-06-10

    Cholesterol homeostasis is mediated by Scap, a polytopic endoplasmic reticulum (ER) protein that transports sterol regulatory element-binding proteins from the ER to Golgi, where they are processed to forms that activate cholesterol synthesis. Scap has eight transmembrane helices and two large luminal loops, designated Loop1 and Loop7. We earlier provided indirect evidence that Loop1 binds to Loop7, allowing Scap to bind COPII proteins for transport in coated vesicles. When ER cholesterol rises, it binds to Loop1. We hypothesized that this causes dissociation from Loop7, abrogating COPII binding. Here we demonstrate direct binding of the two loops when expressed as isolated fragments or as a fusion protein. Expressed alone, Loop1 remained intracellular and membrane-bound. When Loop7 was co-expressed, it bound to Loop1, and the soluble complex was secreted. A Loop1-Loop7 fusion protein was also secreted, and the two loops remained bound when the linker between them was cleaved by a protease. Point mutations that disrupt the Loop1-Loop7 interaction prevented secretion of the Loop1-Loop7 fusion protein. These data provide direct documentation of intramolecular Loop1-Loop7 binding, a central event in cholesterol homeostasis. PMID:27068746

  13. Functional characterization of human RSK4, a new 90-kDa ribosomal S6 kinase, reveals constitutive activation in most cell types

    DEFF Research Database (Denmark)

    Dümmler, Bettina A; Hauge, Camilla; Silber, Joachim;

    2005-01-01

    The 90-kDa ribosomal S6 kinases (RSK1-3) are important mediators of growth factor stimulation of cellular proliferation, survival, and differentiation and are activated via coordinated phosphorylation by ERK and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Here we performed the functional...... for phosphorylation of Ser232, a key regulatory site in the activation loop of the N-terminal kinase domain, that in other RSKs is phosphorylated by PDK1. The unusual regulation and growth factor-independent kinase activity indicate that RSK4 is functionally distinct from other RSKs and may help explain recent...

  14. Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop.

    Science.gov (United States)

    Breig, O; Théoleyre, O; Douablin, A; Baklouti, F

    2010-05-13

    Spi-1/PU.1 oncogene is downregulated as proerythroblasts undergo terminal differentiation. Insertion of the Friend virus upstream of the Spi-1/PU.1 locus leads to the constitutive upregulation of Spi-1/PU.1, and a subsequent block in the differentiation of the affected erythroblasts. We have shown that sustained overexpression of Spi-1/PU.1 also inhibits the erythroid splicing of protein 4.1R exon 16, irrespective of chemical induction of differentiation. Here, we show a positive feedback loop that couples constitutive phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling to high expression of Spi-1/PU.1 in Friend erythroleukemia cells. Inhibition of PI3K/AKT results in Spi-1/PU.1 downregulation in a stepwise manner and induces cell differentiation. Chromatin immunoprecipitation assays further supported the positive autoregulatory effect of Spi-1/PU.1. Mutational analysis indicated that Ser41, but not Ser148, is necessary for Spi-1/PU.1-mediated repression of hemoglobin expression, whereas both Ser residues are required for Spi-1/PU.1 inhibition of the erythroid splicing event. We further show that inhibition of the erythroid transcriptional and splicing events are strictly dependent on distinct Spi-1/PU.1 phosphorylation modifications rather than Spi-1/PU.1 expression level per se. Our data further support the fact that Spi-1/PU.1 inhibits 4.1R erythroid splicing through two different pathways, and bring new insights into the extracellular signal impact triggered by erythropoietin on late erythroid regulatory program, including pre-mRNA splicing.

  15. Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion.

    Science.gov (United States)

    Kawakami, Yuko; Nishimoto, Hajime; Kitaura, Jiro; Maeda-Yamamoto, Mari; Kato, Roberta M; Littman, Dan R; Leitges, Michael; Rawlings, David J; Kawakami, Toshiaki

    2004-11-12

    Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2. PMID:15364915

  16. Tubulin binds to the cytoplasmic loop of TRESK background K⁺ channel in vitro.

    Directory of Open Access Journals (Sweden)

    Péter Enyedi

    Full Text Available The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18. Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell.

  17. Alternative loop rings

    CERN Document Server

    Goodaire, EG; Polcino Milies, C

    1996-01-01

    For the past ten years, alternative loop rings have intrigued mathematicians from a wide cross-section of modern algebra. As a consequence, the theory of alternative loop rings has grown tremendously. One of the main developments is the complete characterization of loops which have an alternative but not associative, loop ring. Furthermore, there is a very close relationship between the algebraic structures of loop rings and of group rings over 2-groups. Another major topic of research is the study of the unit loop of the integral loop ring. Here the interaction between loop rings and group ri

  18. Closed-Loop Neuroscience and Non-Invasive Brain Stimulation: A Tale of Two Loops.

    Science.gov (United States)

    Zrenner, Christoph; Belardinelli, Paolo; Müller-Dahlhaus, Florian; Ziemann, Ulf

    2016-01-01

    Closed-loop neuroscience is receiving increasing attention with recent technological advances that enable complex feedback loops to be implemented with millisecond resolution on commodity hardware. We summarize emerging conceptual and methodological frameworks that are available to experimenters investigating a "brain in the loop" using non-invasive brain stimulation and briefly review the experimental and therapeutic implications. We take the view that closed-loop neuroscience in fact deals with two conceptually quite different loops: a "brain-state dynamics" loop, used to couple with and modulate the trajectory of neuronal activity patterns, and a "task dynamics" loop, that is the bidirectional motor-sensory interaction between brain and (simulated) environment, and which enables goal-directed behavioral tasks to be incorporated. Both loops need to be considered and combined to realize the full experimental and therapeutic potential of closed-loop neuroscience. PMID:27092055

  19. Symposia on Plant (Protein Phosphorylation

    Directory of Open Access Journals (Sweden)

    Sacco C. De Vries

    2012-08-01

    Full Text Available From September 14-16, 2011 the twelfth symposium on Plant Protein Phosphorylation was held in Tübingen, Germany. The topic is as broad as the name suggests and covers all aspects of this important means of protein modification in plants. I have had the pleasure of attending the 2007 and the 2011 symposia. The interesting concept behind these meetings is to hear about the same biochemical mechanism operative in a multitude of experimental systems. The meetings are quite informal and present an excellent mix ranging from technology to biochemical experience and novel findings and tools.The two-and-a-half-day program was divided into five double sessions: biotic interactions, hormone signaling, abiotic interactions, Mitogen Activated Protein Kinase (MAPK and Ca++ pathways and phosphoproteomics. It was hosted by the Zentrum für Molekularbiologie der Pflanzen (ZMBP and the organizing committee chaired by Klaus Harter.

  20. Dynamical behaviour in coronal loops

    Science.gov (United States)

    Haisch, Bernhard M.

    1986-01-01

    Rapid variability has been found in two active region coronal loops observed by the X-ray Polychromator (XRP) and the Hard X-ray Imaging Spectrometer (HXIS) onboard the Solar Maximum Mission (SMM). There appear to be surprisingly few observations of the short-time scale behavior of hot loops, and the evidence presented herein lends support to the hypothesis that coronal heating may be impulsive and driven by flaring.

  1. Ghrelin receptor activity amplifies hippocampal N-methyl-d-aspartate receptor-mediated postsynaptic currents and increases phosphorylation of the GluN1 subunit at Ser896 and Ser897.

    Science.gov (United States)

    Muniz, Brandon G; Isokawa, Masako

    2015-12-01

    Although ghrelin and its cognate receptor growth hormone secretagogue receptor (GHSR1a) are highly localized in the hypothalamic nuclei for the regulation of metabolic states and feeding, GHSR1a is also highly localized in the hippocampus, suggesting its involvement in extra-hypothalamic functions. Indeed, exogenous application of ghrelin has been reported to improve hippocampal learning and memory. However, the underlying mechanism of ghrelin regulation of hippocampal functions is poorly understood. Here, we report ghrelin-promoted phosphorylation of GluN1 and amplified N-methyl-d-aspartate receptor (NMDAR)-mediated excitatory postsynaptic currents in the CA1 pyramidal cells of the hippocampus in slice preparations. The ghrelin-induced responses were sensitive to a GHSR1a antagonist and inverse agonist, and were absent in GHSR1a homozygous knock-out mice. These results indicated that activation of GHSR1a was critical in the ghrelin-induced enhancement of the NMDAR function. Interestingly, heterozygous mouse hippocampi were also insensitive to ghrelin treatment, suggesting that a slight reduction in the availability of GHSR1a may be sufficient to negate the effect of ghrelin on GluN1 phosphorylation and NMDAR channel activities. In addition, NMDAR-mediated spike currents, which are of dendritic origin, were blocked by the GHSR1a antagonist, suggesting the presence of GHSR1a on the pyramidal cell dendrites in physical proximity to NMDAR. Together with our findings on the localization of GHSR1a in the CA1 region of the hippocampus, which was shown by fluorescent ghrelin binding, immunoreactivity, and enhanced green fluorescent protein reporter gene expression, we conclude that the activation of GHSR1a favours rapid modulation of the NMDAR-mediated glutamatergic synaptic transmission by phosphorylating GluN1 in the hippocampus. PMID:26490687

  2. Integrin-linked Kinase Modulates Lipopolysaccharide- and Helicobacter pylori-induced Nuclear Factor κB-activated Tumor Necrosis Factor-α Production via Regulation of p65 Serine 536 Phosphorylation*

    Science.gov (United States)

    Ahmed, Afsar U.; Sarvestani, Soroush T.; Gantier, Michael P.; Williams, Bryan R. G.; Hannigan, Gregory E.

    2014-01-01

    Integrin-linked kinase (ILK) is a ubiquitously expressed and highly conserved serine-threonine protein kinase that regulates cellular responses to a wide variety of extracellular stimuli. ILK is involved in cell-matrix interactions, cytoskeletal organization, and cell signaling. ILK signaling has also been implicated in oncogenesis and progression of cancers. However, its role in the innate immune system remains unknown. Here, we show that ILK mediates pro-inflammatory signaling in response to lipopolysaccharide (LPS). Pharmacological or genetic inhibition of ILK in mouse embryonic fibroblasts and macrophages selectively blocks LPS-induced production of the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). ILK is required for LPS-induced activation of nuclear factor κB (NF-κB) and transcriptional induction of TNF-α. The modulation of LPS-induced TNF-α synthesis by ILK does not involve the classical NF-κB pathway, because IκB-α degradation and p65 nuclear translocation are both unaffected by ILK inhibition. Instead, ILK is involved in an alternative activation of NF-κB signaling by modulating the phosphorylation of p65 at Ser-536. Furthermore, ILK-mediated alternative NF-κB activation through p65 Ser-536 phosphorylation also occurs during Helicobacter pylori infection in macrophages and gastric cancer cells. Moreover, ILK is required for H. pylori-induced TNF-α secretion in macrophages. Although ILK-mediated phosphorylation of p65 at Ser-536 is independent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway during LPS stimulation, upon H. pylori infection this event is dependent on the PI3K/Akt pathway. Our findings implicate ILK as a critical regulatory molecule for the NF-κB-mediated pro-inflammatory signaling pathway, which is essential for innate immune responses against pathogenic microorganisms. PMID:25100717

  3. Integrin-linked kinase modulates lipopolysaccharide- and Helicobacter pylori-induced nuclear factor κB-activated tumor necrosis factor-α production via regulation of p65 serine 536 phosphorylation.

    Science.gov (United States)

    Ahmed, Afsar U; Sarvestani, Soroush T; Gantier, Michael P; Williams, Bryan R G; Hannigan, Gregory E

    2014-10-01

    Integrin-linked kinase (ILK) is a ubiquitously expressed and highly conserved serine-threonine protein kinase that regulates cellular responses to a wide variety of extracellular stimuli. ILK is involved in cell-matrix interactions, cytoskeletal organization, and cell signaling. ILK signaling has also been implicated in oncogenesis and progression of cancers. However, its role in the innate immune system remains unknown. Here, we show that ILK mediates pro-inflammatory signaling in response to lipopolysaccharide (LPS). Pharmacological or genetic inhibition of ILK in mouse embryonic fibroblasts and macrophages selectively blocks LPS-induced production of the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). ILK is required for LPS-induced activation of nuclear factor κB (NF-κB) and transcriptional induction of TNF-α. The modulation of LPS-induced TNF-α synthesis by ILK does not involve the classical NF-κB pathway, because IκB-α degradation and p65 nuclear translocation are both unaffected by ILK inhibition. Instead, ILK is involved in an alternative activation of NF-κB signaling by modulating the phosphorylation of p65 at Ser-536. Furthermore, ILK-mediated alternative NF-κB activation through p65 Ser-536 phosphorylation also occurs during Helicobacter pylori infection in macrophages and gastric cancer cells. Moreover, ILK is required for H. pylori-induced TNF-α secretion in macrophages. Although ILK-mediated phosphorylation of p65 at Ser-536 is independent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway during LPS stimulation, upon H. pylori infection this event is dependent on the PI3K/Akt pathway. Our findings implicate ILK as a critical regulatory molecule for the NF-κB-mediated pro-inflammatory signaling pathway, which is essential for innate immune responses against pathogenic microorganisms. PMID:25100717

  4. Time-resolved Emission from Bright Hot Pixels of an Active Region Observed in the EUV Band with SDO/AIA and Multi-stranded Loop Modeling

    Science.gov (United States)

    Tajfirouze, E.; Reale, F.; Petralia, A.; Testa, P.

    2016-01-01

    Evidence of small amounts of very hot plasma has been found in active regions and might be an indication of impulsive heating released at spatial scales smaller than the cross-section of a single loop. We investigate the heating and substructure of coronal loops in the core of one such active region by analyzing the light curves in the smallest resolution elements of solar observations in two EUV channels (94 and 335 Å) from the Atmospheric Imaging Assembly on board the Solar Dynamics Observatory. We model the evolution of a bundle of strands heated by a storm of nanoflares by means of a hydrodynamic 0D loop model (EBTEL). The light curves obtained from a random combination of those of single strands are compared to the observed light curves either in a single pixel or in a row of pixels, simultaneously in the two channels, and using two independent methods: an artificial intelligent system (Probabilistic Neural Network) and a simple cross-correlation technique. We explore the space of the parameters to constrain the distribution of the heat pulses, their duration, their spatial size, and, as a feedback on the data, their signatures on the light curves. From both methods the best agreement is obtained for a relatively large population of events (1000) with a short duration (less than 1 minute) and a relatively shallow distribution (power law with index 1.5) in a limited energy range (1.5 decades). The feedback on the data indicates that bumps in the light curves, especially in the 94 Å channel, are signatures of a heating excess that occurred a few minutes before.

  5. Inhibition of human immunodeficiency virus type 1 Tat-dependent activation of translation in Xenopus oocytes by the benzodiazepine Ro24-7429 requires trans-activation response element loop sequences.

    Science.gov (United States)

    Braddock, M; Cannon, P; Muckenthaler, M; Kingsman, A J; Kingsman, S M

    1994-01-01

    Two benzodiazepine compounds, [7-chloro-5-(2-pyrryl)-3H-1,4 benzodiazapin-2-(H)-one] (Ro5-3335) and [7-chloro-5-(1H-pyrrol-2-yl)-3H-benzo[e] [1,4] diazepin-2-yl]- methylamine (Ro24-7429), inhibit human immunodeficiency virus type 1 (HIV-1) replication via a specific effect on the function of the transactivator protein, Tat. To gain further insight into the mechanism of action of these compounds, we have tested their effects in an alternative assay for Tat activation in Xenopus oocytes. In this system, translation of trans-activation response element (TAR)-containing RNA is activated by Tat. Both compounds specifically blocked activation of translation in a dose-dependent fashion, with Ro24-7429 showing the greater potency. In the Xenopus oocyte system, as in mammalian cells, mutation of the TAR loop sequences abolishes Tat action. However, it is possible to obtain TAR-specific, Tat-dependent activation of a target RNA with a mutation in the loop provided that this target is in large excess. This result has been interpreted as indicating that a negative factor has been titrated (M. Braddock, R. Powell, A.D. Blanchard, A.J. Kingsman, and S.M. Kingsman, FASEB J. 7:214-222, 1993). Interestingly Ro24-7429 was unable to inhibit the TAR-specific but loop sequence-independent mode of translational activation. This finding suggests that a specific loop-binding cellular factor may mediate the effects of this inhibitor of Tat action. Consistent with this notion, we could not detect any effect of Ro24-7429 on the efficiency of specific Tat binding to TAR in vitro. PMID:8254735

  6. α-MSH Stimulates Glucose Uptake in Mouse Muscle and Phosphorylates Rab-GTPase-Activating Protein TBC1D1 Independently of AMPK

    Science.gov (United States)

    Enriori, Pablo J.; Jensen, Thomas Elbenhardt; Garcia-Rudaz, Cecilia; Litwak, Sara A.; Raun, Kirsten; Wojtaszewski, Jørgen; Wulff, Birgitte Schjellerup; Cowley, Michael A.

    2016-01-01

    The melanocortin system includes five G-protein coupled receptors (family A) defined as MC1R-MC5R, which are stimulated by endogenous agonists derived from proopiomelanocortin (POMC). The melanocortin system has been intensely studied for its central actions in body weight and energy expenditure regulation, which are mainly mediated by MC4R. The pituitary gland is the source of various POMC-derived hormones released to the circulation, which raises the possibility that there may be actions of the melanocortins on peripheral energy homeostasis. In this study, we examined the molecular signaling pathway involved in α-MSH-stimulated glucose uptake in differentiated L6 myotubes and mouse muscle explants. In order to examine the involvement of AMPK, we investigate α-MSH stimulation in both wild type and AMPK deficient mice. We found that α-MSH significantly induces phosphorylation of TBC1 domain (TBC1D) family member 1 (S237 and T596), which is independent of upstream PKA and AMPK. We find no evidence to support that α-MSH-stimulated glucose uptake involves TBC1D4 phosphorylation (T642 and S704) or GLUT4 translocation. PMID:27467141

  7. A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39

    DEFF Research Database (Denmark)

    Storjohann, Laura; Holst, Birgitte; Schwartz, Thue W

    2008-01-01

    . Disruption of the nonconserved disulfide bridge by mutagenesis led to an increase in the Zn (2+) potency. This phenotype, with an approximate 10-fold increase in agonist potency and a slight increase in E max, was mimicked by treatment of the wild-type receptor with TCEP at low concentrations, which had no......A highly conserved feature across all families of 7TM receptors is a disulfide bridge between a Cys residue located at the extracellular end of transmembrane segment III (TM-III) and one in extracellular loop 2 (ECL-2). The zinc sensor GPR39 contains four Cys residues in the extracellular domains....... By using mutagenesis, treatment with the reducing agent TCEP, and a labeling procedure for free sulfhydryl groups, we identify the pairing of these Cys residues in two disulfide bridges: the prototypical bridge between Cys (108) in TM-III and Cys (210) in ECL-2 and a second disulfide bridge...

  8. The stem-loop luciferase assay for polyadenylation (SLAP) method for determining CstF-64-dependent polyadenylation activity.

    Science.gov (United States)

    Hockert, J Andrew; Macdonald, Clinton C

    2014-01-01

    Polyadenylation is an essential cellular process in eukaryotic cells (Edmonds M and Abrams R, J Biol Chem 235, 1142-1149, 1960; Zhao J et al., Microbiol Mol Biol Rev 63, 405-445, 1999; Edmonds M, Progr Nucleic Acid Res Mol Biol 71, 285-389, 2002). For this reason, it has been difficult to examine the functions of specific polyadenylation proteins in vivo. Here, we describe a cell culture assay that allows structure-function experiments on CstF-64, a protein that binds to pre-mRNAs downstream of the cleavage site for accurate and efficient polyadenylation. We also demonstrate that the stem-loop luciferase assay for polyadenylation (SLAP) accurately reflects CstF-64-dependent polyadenylation. This assay could be easily adapted to the study of other important RNA-binding proteins in polyadenylation.

  9. Phosphorylation Regulates Functions of ZEB1 Transcription Factor.

    Science.gov (United States)

    Llorens, M Candelaria; Lorenzatti, Guadalupe; Cavallo, Natalia L; Vaglienti, Maria V; Perrone, Ana P; Carenbauer, Anne L; Darling, Douglas S; Cabanillas, Ana M

    2016-10-01

    ZEB1 transcription factor is important in both development and disease, including many TGFβ-induced responses, and the epithelial-to-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA-binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF-1) can inhibit transcriptional repression by either a ZEB1-ZD2 domain clone, or full-length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. Immunoprecipitation with anti-ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK consensus site at Thr-867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA-binding measured by EMSA, IGF-1-induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP-ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF-1. J. Cell. Physiol. 231: 2205-2217, 2016. © 2016 Wiley Periodicals, Inc. PMID:26868487

  10. Allo-antigen stimulated CD8+ T-cells suppress NF-κB and Ets-1 DNA binding activity, and inhibit phosphorylated NF-κB p65 nuclear localization in CD4+ T-cells.

    Science.gov (United States)

    Nagashima, Ryuichi; Kawakami, Fumitaka; Takahashi, Shinichiro; Obata, Fumiya; Kubo, Makoto

    2014-08-01

    CD8+ T-cells of asymptomatic HIV-1 carriers (AC) suppress human immunodeficiency virus type 1 (HIV-1) replication in a class I major histocompatibility complex (MHC-I)-restricted and -unrestricted manner. In order to investigate the mechanism of MHC-I-unrestricted CD8+ T-cell-mediated HIV-1 suppression, we previously established allo-antigen stimulated CD8+T-cells from HIV-1-uninfected donors. These allo-antigen stimulated CD8+ T-cells suppressed HIV-1 replication in acutely infected autologous CD4+ T-cells when directly co-cultured. To elucidate the mechanism of HIV-1 replication suppression, we analyzed DNA-binding activity and phosphorylation of transcriptional factors associated with HIV-1 replication by electrophoresis mobility shift assay and Western blotting. When CD4+ T-cells were cultured with allo-antigen stimulated CD8+ T-cells, the reduction of NF-κB and Ets-1 DNA-binding activity was observed. Nuclear localization of NF-κB p65 and Ets-1 was suppressed in CD4+ T-cells. Although NF-κB p65 and Ets-1 are known to be regulated by protein kinase A (PKA), no difference was observed in the expression and phosphorylation of the PKA catalytic subunit in CD4+ T-cells cultured with PHA-treated CD8+ T-cells or allo-antigen stimulated CD8+ T-cells. Cyclic AMP is also known to enter through gap junctions, but the suppression of HIV-1 replication mediated by allo-antigen stimulated CD8+ T-cells was not affected by the gap junction inhibitor. The nuclear transport of phosphorylated NF-κB p65 (Ser276) was inhibited only in CD4+ T-cells cultured with allo-antigen stimulated CD8+ T-cells. Our results indicate that allo-antigen stimulated CD8+ T-cells suppress the transcriptional activity of NF-κB p65 or Ets-1 in an antigen-nonspecific manner, and inhibit the nuclear transport of phosphorylated NF-κB p65 (Ser276).

  11. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr;

    2015-01-01

    , 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 m...

  12. Phosphorylation of chicken growth hormone

    Energy Technology Data Exchange (ETDEWEB)

    Aramburo, C.; Montiel, J.L. (Universidad Nacional Autonoma de Mexico (Mexico)); Donoghue, D.; Scanes, C.G. (Rutgers Univ., New Brunswick, NJ (USA)); Berghman, L.R. (Laboratory for Neuroendocrinology and Immunological Biotechnology, Louvain (Belgium))

    1990-01-01

    The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and {gamma}-{sup 32}P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of {sup 32}P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of {sup 32}P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer.

  13. Effect of Phosphorylation on Hydrogen-Bonding Interactions of the Active Site Histidine of the Phosphocarrier Protein HPr of the Phosphoenolpyruvate-Dependent Phosphotransferase System Determined by 15N NMR Spectroscopy

    NARCIS (Netherlands)

    Dijk, Alard A. van; Lange, Liesbeth C.M. de; Bachovchin, William W.; Robillard, George T.

    1990-01-01

    The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this histidi

  14. Toward a Systems-Level View of Dynamic Phosphorylation Networks

    Directory of Open Access Journals (Sweden)

    Robert Howard Newman

    2014-08-01

    Full Text Available To better understand how cells sense and respond to their environment, it is important to understand the organization and regulation of the phosphorylation networks that underlie most cellular signal transduction pathways. These networks, which are composed of protein kinases, protein phosphatases and their respective cellular targets, are highly dynamic. Importantly, to achieve signaling specificity, phosphorylation networks must be regulated at several levels, including at the level of protein expression, substrate recognition, and spatiotemporal modulation of enzymatic activity. Here, we briefly summarize some of the traditional methods used to study the phosphorylation status of cellular proteins before focusing our attention on several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks. Together, these approaches promise to lead to a systems-level view of dynamic phosphorylation networks.

  15. Ion channels, phosphorylation and mammalian sperm capacitation.

    Science.gov (United States)

    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.

  16. Ion channels, phosphorylation and mammalian sperm capacitation

    Institute of Scientific and Technical Information of China (English)

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

    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.

  17. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    Science.gov (United States)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  18. The Synthesis of a Series of Phosphoryl Coumarins

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Different hydroxy substituted coumarins were successfully phosphorylated with diisopropylphophite (DIPPH) by the Atherton-Todd reaction in 76-89% yields. Moreover, the reaction activities of different hydroxys of the coumarins in the Atherton-Todd reaction were studied.

  19. Hypertension alters phosphorylation of VASP in brain endothelial cells.

    Science.gov (United States)

    Arlier, Zulfikar; Basar, Murat; Kocamaz, Erdogan; Kiraz, Kemal; Tanriover, Gamze; Kocer, Gunnur; Arlier, Sefa; Giray, Semih; Nasırcılar, Seher; Gunduz, Filiz; Senturk, Umit K; Demir, Necdet

    2015-04-01

    Hypertension impairs cerebral vascular function. Vasodilator-stimulated phosphoprotein (VASP) mediates active reorganization of the cytoskeleton via membrane ruffling, aggregation and tethering of actin filaments. VASP regulation of endothelial barrier function has been demonstrated by studies using VASP(-/-) animals under conditions associated with tissue hypoxia. We hypothesize that hypertension regulates VASP expression and/or phosphorylation in endothelial cells, thereby contributing to dysfunction in the cerebral vasculature. Because exercise has direct and indirect salutary effects on vascular systems that have been damaged by hypertension, we also investigated the effect of exercise on maintenance of VASP expression and/or phosphorylation. We used immunohistochemistry, Western blotting and immunocytochemistry to examine the effect of hypertension on VASP expression and phosphorylation in brain endothelial cells in normotensive [Wistar-Kyoto (WKY)] and spontaneously hypertensive (SH) rats under normal and exercise conditions. In addition, we analyzed VASP regulation in normoxia- and hypoxia-induced endothelial cells. Brain endothelial cells exhibited significantly lower VASP immunoreactivity and phosphorylation at the Ser157 residue in SHR versus WKY rats. Exercise reversed hypertension-induced alterations in VASP phosphorylation. Western blotting and immunocytochemistry indicated reduction in VASP phosphorylation in hypoxic versus normoxic endothelial cells. These results suggest that diminished VASP expression and/or Ser157 phosphorylation mediates endothelial changes associated with hypertension and exercise may normalize these changes, at least in part, by restoring VASP phosphorylation. PMID:24894047

  20. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Teramura, Takeshi, E-mail: teramura@med.kindai.ac.jp [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Takehara, Toshiyuki; Onodera, Yuta [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Nakagawa, Koichi; Hamanishi, Chiaki [Department of Orthopaedic Surgery, Kinki University, Faculty of Medicine, Osaka (Japan); Fukuda, Kanji [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Department of Orthopaedic Surgery, Kinki University, Faculty of Medicine, Osaka (Japan)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

  1. Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi.

    Science.gov (United States)

    Aizen, Joseph; Chandler, Jennifer C; Fitzgibbon, Quinn P; Sagi, Amir; Battaglene, Stephen C; Elizur, Abigail; Ventura, Tomer

    2016-04-01

    In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date, the biochemical pathways through which IAG exerts its effects are poorly understood and could be elucidated through the production of a functional recombinant IAG (rIAG). We have successfully expressed glycosylated, biologically active IAG using the Pichia pastoris yeast expression system. We co-expressed recombinant single-chain precursor molecules consisting of the B and A chains (the mature hormone) tethered by a flexible linker, producing rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). We then tested the biological activity of each, through the ability to increase phosphorylation in the testis; both Sv and Cq rIAGs significantly elevated phosphorylation specific to their species, and in a dose-dependent manner. Mr rIAG was tested on Macrobrachium australiense (Ma), eliciting a similar response. Moreover, using bioinformatics analyses of the de novo assembled spiny lobster transcriptome, we identified a spiny lobster tyrosine kinase insulin receptor (Sv-TKIR). We validated this discovery with a receptor activation assay in COS-7 cells expressing Sv-TKIR, using a reporter SRE-LUC system designed for RTKs, with each of the rIAG proteins acting as the activation ligand. Using recombinant proteins, we aim to develop specific tools to control sexual development through the administration of IAG within the critical sexual differentiation time window. The biologically active rIAGs generated might facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological mode of action of IAG in crustaceans. PMID:26883686

  2. BRAFV600E mutation, TIMP-1 upregulation, and NF-κB activation: closing the loop on the papillary thyroid cancer trilogy.

    Science.gov (United States)

    Bommarito, Alessandra; Richiusa, Pierina; Carissimi, Elvira; Pizzolanti, Giuseppe; Rodolico, Vito; Zito, Giovanni; Criscimanna, Angela; Di Blasi, Francesco; Pitrone, Maria; Zerilli, Monica; Amato, Marco C; Spinelli, Gaetano; Carina, Valeria; Modica, Giuseppe; Latteri, M Adelfio; Galluzzo, Aldo; Giordano, Carla

    2011-12-01

    BRAF(V600E) is the most common mutation found in papillary thyroid carcinoma (PTC). Tissue inhibitor of metalloproteinases (TIMP-1) and nuclear factor (NF)-κB have been shown to play an important role in thyroid cancer. In particular, TIMP-1 binds its receptor CD63 on cell surface membrane and activates Akt signaling pathway, which is eventually responsible for its anti-apoptotic activity. The aim of our study was to evaluate whether interplay among these three factors exists and exerts a functional role in PTCs. To this purpose, 56 PTC specimens were analyzed for BRAF(V600E) mutation, TIMP-1 expression, and NF-κB activation. We found that BRAF(V600E) mutation occurs selectively in PTC nodules and is associated with hyperactivation of NF-κB and upregulation of both TIMP-1 and its receptor CD63. To assess the functional relationship among these factors, we first silenced BRAF gene in BCPAP cells, harboring BRAF(V600E) mutation. We found that silencing causes a marked decrease in TIMP-1 expression and NF-κB binding activity, as well as decreased invasiveness. After treatment with specific inhibitors of MAPK pathway, we found that only sorafenib was able to increase IκB-α and reduce both TIMP-1 expression and Akt phosphorylation in BCPAP cells, indicating that BRAF(V600E) activates NF-κB and this pathway is MEK-independent. Taken together, our findings demonstrate that BRAF(V600E) causes upregulation of TIMP-1 via NF-κB. TIMP-1 binds then its surface receptor CD63, leading eventually to Akt activation, which in turn confers antiapoptotic behavior and promotion of cell invasion. The recognition of this functional trilogy provides insight on how BRAF(V600E) determines cancer initiation, progression, and invasiveness in PTC, also identifying new therapeutic targets for the treatment of highly aggressive forms.

  3. Mécanismes de régulation de la NADPH Oxydase NOX1 : rôle de la phosphorylation de NOXA1 ( NOX Activator 1 ) et de NOXO1 ( NOX Organizer 1)

    OpenAIRE

    Debbabi, Maya

    2011-01-01

    Les NADPH oxydases constituent une famille d’enzymes dont la fonction est dédiée à la production de formes réactives de l’oxygène. NOX1, un des membres de cette famille, est abondamment exprimée dans le colon et sa dérégulation pourrait être associée aux maladies inflammatoires chroniques de l’intestin. Les mécanismes qui modulent l’activation de NOX1 demeurent mal connus. Au cours de ma thèse je me suis donc intéressée à l’étude de la phosphorylation de NOXA1 et NOXO1, deux sous-unités régul...

  4. Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

    Science.gov (United States)

    Iranshahy, Milad; Tayarani-Najaran, Zahra; Kasaian, Jamal; Ghandadi, Morteza; Emami, Seyed Ahmad; Asili, Javad; Chandran, Jima N; Schneider, Bernd; Iranshahi, Mehrdad

    2016-02-01

    Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation. PMID:26581585

  5. Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

    Science.gov (United States)

    Iranshahy, Milad; Tayarani-Najaran, Zahra; Kasaian, Jamal; Ghandadi, Morteza; Emami, Seyed Ahmad; Asili, Javad; Chandran, Jima N; Schneider, Bernd; Iranshahi, Mehrdad

    2016-02-01

    Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation.

  6. Compensation effects and relation between the activation energy of spin transition and the hysteresis loop width for an iron(ii) complex.

    Science.gov (United States)

    Bushuev, Mark B; Pishchur, Denis P; Nikolaenkova, Elena B; Krivopalov, Viktor P

    2016-06-22

    The enthalpy-entropy compensation was observed for the cooperative → spin transition (the phase is a mononuclear complex [FeL2](BF4)2, L is 4-(3,5-dimethyl-1H-pyrazol-1-yl)-2-(pyridin-2-yl)-6-methylpyrimidine). The physical origin of this effect is the fact that the → spin transition is the first order phase transition accompanied by noticeable variations in the Tonset↑, ΔH and ΔS values. Higher ΔH and ΔS values are correlated with higher Tonset↑ values. The higher the enthalpy and entropy of the spin transition, the wider the hysteresis loop. The kinetic compensation effect, i.e. a linear relationship between ln A and Ea, was observed for the → spin transition. Moreover, an isokinetic relationship was detected in this system: the Arrhenius lines (ln k vs. 1/T) obtained from magnetochemical data for different samples of the phase undergoing the → transition show a common point of intersection (Tiso = 490 ± 2 K, ln kiso = -6.0 ± 0.2). The validity of this conclusion was confirmed by the Exner-Linert statistical method. This means that the isokinetic relationship and the kinetic compensation effect (ln A vs. Ea) in this system are true ones. The existence of a true kinetic compensation effect is supported independently by the fact that the hysteresis loop width for the cooperative spin transition ↔ increases with increasing activation barrier height. Estimating the energy of excitations for the phase with Tiso ∼ 490 K yields wavenumbers of ca. 340 cm(-1) corresponding to the frequencies of the stretching vibrations of the Fe(LS)-N bonds, i.e. the bonds directly involved in the mechanism of the spin transition. This is the first observation of the kinetic compensation effect (ln A vs. Ea) and the isokinetic relationship for a cooperative spin crossover system showing thermal hysteresis. Our results provide the first experimental evidence that the higher the activation barrier for the spin transition, the wider the hysteresis loop for a

  7. Compensation effects and relation between the activation energy of spin transition and the hysteresis loop width for an iron(ii) complex.

    Science.gov (United States)

    Bushuev, Mark B; Pishchur, Denis P; Nikolaenkova, Elena B; Krivopalov, Viktor P

    2016-06-22

    The enthalpy-entropy compensation was observed for the cooperative → spin transition (the phase is a mononuclear complex [FeL2](BF4)2, L is 4-(3,5-dimethyl-1H-pyrazol-1-yl)-2-(pyridin-2-yl)-6-methylpyrimidine). The physical origin of this effect is the fact that the → spin transition is the first order phase transition accompanied by noticeable variations in the Tonset↑, ΔH and ΔS values. Higher ΔH and ΔS values are correlated with higher Tonset↑ values. The higher the enthalpy and entropy of the spin transition, the wider the hysteresis loop. The kinetic compensation effect, i.e. a linear relationship between ln A and Ea, was observed for the → spin transition. Moreover, an isokinetic relationship was detected in this system: the Arrhenius lines (ln k vs. 1/T) obtained from magnetochemical data for different samples of the phase undergoing the → transition show a common point of intersection (Tiso = 490 ± 2 K, ln kiso = -6.0 ± 0.2). The validity of this conclusion was confirmed by the Exner-Linert statistical method. This means that the isokinetic relationship and the kinetic compensation effect (ln A vs. Ea) in this system are true ones. The existence of a true kinetic compensation effect is supported independently by the fact that the hysteresis loop width for the cooperative spin transition ↔ increases with increasing activation barrier height. Estimating the energy of excitations for the phase with Tiso ∼ 490 K yields wavenumbers of ca. 340 cm(-1) corresponding to the frequencies of the stretching vibrations of the Fe(LS)-N bonds, i.e. the bonds directly involved in the mechanism of the spin transition. This is the first observation of the kinetic compensation effect (ln A vs. Ea) and the isokinetic relationship for a cooperative spin crossover system showing thermal hysteresis. Our results provide the first experimental evidence that the higher the activation barrier for the spin transition, the wider the hysteresis loop for a

  8. Loss of the anorexic response to systemic 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside administration despite reducing hypothalamic AMP-activated protein kinase phosphorylation in insulin-deficient rats.

    Directory of Open Access Journals (Sweden)

    Kaio F Vitzel

    Full Text Available This study tested whether chronic systemic administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR could attenuate hyperphagia, reduce lean and fat mass losses, and improve whole-body energy homeostasis in insulin-deficient rats. Male Wistar rats were first rendered diabetic through streptozotocin (STZ administration and then intraperitoneally injected with AICAR for 7 consecutive days. Food and water intake, ambulatory activity, and energy expenditure were assessed at the end of the AICAR-treatment period. Blood was collected for circulating leptin measurement and the hypothalami were extracted for the determination of suppressor of cytokine signaling 3 (SOCS3 content, as well as the content and phosphorylation of AMP-kinase (AMPK, acetyl-CoA carboxylase (ACC, and the signal transducer and activator of transcription 3 (STAT3. Rats were thoroughly dissected for adiposity and lean body mass (LBM determinations. In non-diabetic rats, despite reducing adiposity, AICAR increased (∼1.7-fold circulating leptin and reduced hypothalamic SOCS3 content and food intake by 67% and 25%, respectively. The anorexic effect of AICAR was lost in diabetic rats, even though hypothalamic AMPK and ACC phosphorylation markedly decreased in these animals. Importantly, hypothalamic SOCS3 and STAT3 levels remained elevated and reduced, respectively, after treatment of insulin-deficient rats with AICAR. Diabetic rats were lethargic and displayed marked losses of fat and LBM. AICAR treatment increased ambulatory activity and whole-body energy expenditure while also attenuating diabetes-induced fat and LBM losses. In conclusion, AICAR did not reverse hyperphagia, but it promoted anti-catabolic effects on skeletal muscle and fat, enhanced spontaneous physical activity, and improved the ability of rats to cope with the diabetes-induced dysfunctional alterations in glucose metabolism and whole-body energy homeostasis.

  9. A P-loop Mutation in G[alpha] Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Bosch, Dustin E.; Willard, Francis S.; Ramanujam, Ravikrishna; Kimple, Adam J.; Willard, Melinda D.; Naqvi, Naweed I.; Siderovski, David P. (UNC); (Singapore)

    2012-10-23

    Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active G{alpha}{beta}{gamma} heterotrimer relies on nucleotide cycling by the G{alpha} subunit: exchange of GTP for GDP activates G{alpha}, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting G{alpha} to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of G{alpha} subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that G{alpha}(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon G{alpha}{sub i1}(G42R) binding to GDP {center_dot} AlF{sub 4}{sup -} or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. G{alpha}(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with G{beta}{gamma} and GoLoco motifs in any nucleotide state. The corresponding G{alpha}{sub q}(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the G{alpha} subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two G{alpha} mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.

  10. Bak apoptotic function is not directly regulated by phosphorylation.

    Science.gov (United States)

    Tran, V H; Bartolo, R; Westphal, D; Alsop, A; Dewson, G; Kluck, R M

    2013-01-01

    During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation. On 1D-IEF, Bak extracted from a range of cell types migrated as a single band near the predicted isoelectric point of 5.6 both before and after phosphatase treatment, indicating that Bak is not significantly phosphorylated at any residue. In contrast, three engineered 'phosphotagged' Bak variants showed a second band at lower pI, indicating phosphorylation. Apoptosis induced by several stimuli failed to alter Bak pI, indicating little change in phosphorylation status. In addition, alanine substitution of tyrosine-108 and other putative phosphorylation sites failed to enhance Bak activation or pro-apoptotic function. In summary, Bak is not significantly phosphorylated at any residue, and Bak activation during apoptosis does not require dephosphorylation. PMID:23303126

  11. Epstein-Barr virus-negative aggressive natural killer-cell leukaemia with high P-glycoprotein activity and phosphorylated extracellular signal-regulated protein kinases 1 and 2

    Directory of Open Access Journals (Sweden)

    Sanja Perkovic

    2012-09-01

    Full Text Available Aggressive natural killer-cell leukaemia (ANKL is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV and P-glycoprotein (P-gp expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56+ NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2 MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.

  12. PKC isoforms interact with and phosphorylate DNMT1

    Directory of Open Access Journals (Sweden)

    Pradhan Sriharsa

    2011-05-01

    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  13. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice.

    Science.gov (United States)

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-01-01

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated

  14. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    Directory of Open Access Journals (Sweden)

    Yueh-Hsiung Kuo

    2016-06-01

    Full Text Available This study investigated the potential effects of dehydroeburicoic acid (TT, a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom on membrane glucose transporter 4 (GLUT4 and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4 and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels, fenofibrate (Feno (at 0.25 g/kg body weight, metformin (Metf (at 0.3 g/kg body weight or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%. TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase, an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated

  15. Control mechanisms in mitochondrial oxidative phosphorylation

    Institute of Scientific and Technical Information of China (English)

    Jana Hroudová; Zdeněk Fi(s)ar

    2013-01-01

    Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5'- triphosphate production is regulated by many control mechanism–firstly by oxygen, substrate level, adenosine-5'-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by "second control mechanisms," such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5'-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.

  16. 脂肪细胞AMPK活性对NF-κB活性的影响%Effects of the Phosphorylation of AMPK on NF-κB Activity in Adipocytes

    Institute of Scientific and Technical Information of China (English)

    郑丽英; 张君; 徐文静; 陆环; 李宏; 冯晓朋; 王金宝; 谢建新

    2012-01-01

    为检测AICAR激活及Compound C抑制脂肪细胞AMPK磷酸化后对NF-κB的磷酸化活性的影响,探讨肥胖启动炎症的分子机制.将3T3-L1细胞诱导为成熟脂肪细胞后,实验分3个处理:基础培养液组(对照组)、实验组(基础培养液组分别加入AICAR和Compound C).运用Western blot检测药物干预后AMPK与NF-κB的磷酸化水平.结果显示,AICAR培养1h脂肪细胞内AMPK磷酸化水平增加,Compound C培养1h脂肪细胞内AMPK磷酸化水平降低,差异有统计学意义(P<0.05).AICAR培养1h脂肪细胞内NF-κB磷酸化水平降低,Compound C培养1h脂肪细胞NF-κB磷酸化水平增高,差异有统计学意义(P<0.05).由此可知,AMPK活性与NF-κB活性呈一定的负相关,AMPK可抑制NF-κB信号,肥胖导致炎症可能是AMPK活性降低引发NF-κB信号活性增强有关.%To explore the effects of the phosphorylation of AMPK activated by AICAR and inhibited by Compound C on NF-κB activity in adipocytes, and investigate molecular mechanisms of obesity-related inflammation. 3T3-L1 cells were induced into adipocytes, and three treatments were used in this experiment. AICAR and Compound C were added respectively into the standard culture medium (control), AMPK and NF-kB phosphorylation were examined by Western blot. AICAR activated AMPK and inhibited NF-κB phospho-rylation(P<0.05). Compound C inhibited AMPK and activated NF-κB phosphorylatioa AMPK activities and NF-κB activities have a certain negative correlation, and AMPK can inhibit NF-κB signaling pathway. The decrease of AMPK activities may enhance the activities of NF-kB signaling pathway, and then lead to obesity-related inflammation.

  17. Ergostatrien-3β-ol from Antrodia camphorata inhibits diabetes and hyperlipidemia in high-fat-diet treated mice via regulation of hepatic related genes, glucose transporter 4, and AMP-activated protein kinase phosphorylation.

    Science.gov (United States)

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2015-03-11

    This study was designed to explore the effects and mechanism of ergostatrien-3β-ol (EK100) from the submerged whole broth of Antrodia camphorata on diabetes and dyslipidemia in high fat diet (HFD)-fed mice for 12 weeks. The C57BL/6J mouse fed with a high fat diet (HFD) could induce insulin resistance and hyperlipidemia. After 8 week of induction, mice were receiving EK100 (at three dosages) or fenofibrate (Feno) or rosiglitazone (Rosi) or vehicle by oral gavage 4 weeks afterward. HFD-fed mice display increased blood glucose, glycated hemoglobin (HbA1c), total cholesterol (TC), triglyceride (TG), insulin, and leptin levels. These blood markers were significantly lower in EK100-treated mice, and finally ameliorated insulin resistance. EK100 treatment exhibited reduced hepatic ballooning degeneration and size of visceral adipocytes. Glucose transporter 4 (GLUT4) proteins and phosphorylation of Akt in skeletal muscle were significantly increased in EK100- and Rosi-treated mice. EK100, Feno, and Rosi treatment led to significant increases in phosphorylation of AMP-activated protein kinase (phospho-AMPK) protein in both skeletal muscle and liver. Moreover, EK100 caused a decrease in hepatic expressions of phosphenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6 Pase), and decreased glucose production. EK100 lowered blood TG level by inhibition of hepatic fatty acid synthesis by dampening sterol response element binding protein-1c (SREBP-1c) but increasing expression of peroxisome proliferator activated receptor α (PPARα). Moreover, EK100-treated mice reduced blood TC levels by decreased hepatic expressions of SREBP2, which plays a major role in the regulation of cholesterol synthesis. EK100 increased high-density lipoprotein cholesterol (HDL-C) concentrations by increasing expressions of apolipoprotein A-I (apo A-I) in liver tissue. Our findings manifest that EK100 may have therapeutic potential in treating type 2 diabetes associated with hyperlipidemia

  18. PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Lu, Yan, E-mail: luyan@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Shen, Pingping, E-mail: ppshen@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Model Animal Research Center (MARC), Nanjing University, Nanjing (China)

    2014-03-10

    Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080

  19. The consequences of selective inhibition of signal transducer and activator of transcription 3 (STAT3) tyrosine705 phosphorylation by phosphopeptide mimetic prodrugs targeting the Src homology 2 (SH2) domain.

    Science.gov (United States)

    McMurray, John S; Mandal, Pijus K; Liao, Warren S; Klostergaard, Jim; Robertson, Fredika M

    2012-10-01

    Herein we review our progress on the development of phosphopeptide-based prodrugs targeting the SH2 domain of STAT3 to prevent recruitment to cytokine and growth factor receptors, activation, nuclear translocation and transcription of genes involved in cancer. We developed high affinity phosphopeptides (K I = 46-200 nM). Corresponding prodrugs inhibited constitutive and IL-6 induced Tyr705 phosphorylation at 0.5-1 μM in a variety of human cancer cell lines. They were not cytotoxic at 5 μM in vitro but they inhibited tumor growth in a human xenograft breast cancer model in mice, accompanied by reduced VEGF expression and angiogenesis. PMID:24058783

  20. Crystal Structure of the Redox-Active Cofactor Dibromothymoquinone Bound to Circadian Clock Protein KaiA and Structural Basis for Dibromothymoquinone's Ability to Prevent Stimulation of KaiC Phosphorylation by KaiA

    Energy Technology Data Exchange (ETDEWEB)

    Pattanayek, Rekha; Sidiqi, Said K.; Egli, Martin [Vanderbilt-MED

    2013-09-19

    KaiA protein that stimulates KaiC phosphorylation in the cyanobacterial circadian clock was recently shown to be destabilized by dibromothymoquinone (DBMIB), thus revealing KaiA as a sensor of the plastoquinone (PQ) redox state and suggesting an indirect control of the clock by light through PQ redox changes. Here we show using X-ray crystallography that several DBMIBs are bound to KaiA dimer. Some binding modes are consistent with oligomerization of N-terminal KaiA pseudoreceiver domains and/or reduced interdomain flexibility. DBMIB bound to the C-terminal KaiA (C-KaiA) domain and limited stimulation of KaiC kinase activity by C-KaiA in the presence of DBMIB demonstrate that the cofactor may weakly inhibit KaiA-KaiC binding.

  1. Functional map of arrestin binding to phosphorylated opsin, with and without agonist.

    Science.gov (United States)

    Peterhans, Christian; Lally, Ciara C M; Ostermaier, Martin K; Sommer, Martha E; Standfuss, Jörg

    2016-01-01

    Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct. PMID:27350090

  2. Tuning the Phosphoryl Donor Specificity of Dihydroxyacetone Kinase from ATP to Inorganic Polyp