Science.gov

Sample records for activation loop tyrosine

  1. Small Molecule Receptor Protein Tyrosine Phosphatase γ (RPTPγ) Ligands That Inhibit Phosphatase Activity via Perturbation of the Tryptophan-Proline-Aspartate (WPD) Loop

    SciTech Connect

    Sheriff, Steven; Beno, Brett R; Zhai, Weixu; Kostich, Walter A; McDonnell, Patricia A; Kish, Kevin; Goldfarb, Valentina; Gao, Mian; Kiefer, Susan E; Yanchunas, Joseph; Huang, Yanling; Shi, Shuhao; Zhu, Shirong; Dzierba, Carolyn; Bronson, Joanne; Macor, John E; Appiah, Kingsley K; Westphal, Ryan S; O’Connell, Jonathan; Gerritz, Samuel W

    2012-11-09

    Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of tyrosine residues, a process that involves a conserved tryptophan-proline-aspartate (WPD) loop in catalysis. In previously determined structures of PTPs, the WPD-loop has been observed in either an 'open' conformation or a 'closed' conformation. In the current work, X-ray structures of the catalytic domain of receptor-like protein tyrosine phosphatase γ (RPTPγ) revealed a ligand-induced 'superopen' conformation not previously reported for PTPs. In the superopen conformation, the ligand acts as an apparent competitive inhibitor and binds in a small hydrophobic pocket adjacent to, but distinct from, the active site. In the open and closed WPD-loop conformations of RPTPγ, the side chain of Trp1026 partially occupies this pocket. In the superopen conformation, Trp1026 is displaced allowing a 3,4-dichlorobenzyl substituent to occupy this site. The bound ligand prevents closure of the WPD-loop over the active site and disrupts the catalytic cycle of the enzyme.

  2. Conformation-selective inhibitors reveal differences in the activation and phosphate-binding loops of the tyrosine kinases Abl and Src.

    PubMed

    Hari, Sanjay B; Perera, B Gayani K; Ranjitkar, Pratistha; Seeliger, Markus A; Maly, Dustin J

    2013-12-20

    Over the past decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely related tyrosine kinases, such as Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abl's activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abl's activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases.

  3. Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

    PubMed Central

    Moise, Gwendolyn; Gallup, Nathan M.; Alexandrova, Anastassia N.; Hengge, Alvan C.; Johnson, Sean J.

    2016-01-01

    Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

  4. Conservative tryptophan mutants of the protein tyrosine phosphatase YopH exhibit impaired WPD-loop function and crystallize with divanadate esters in their active sites.

    PubMed

    Moise, Gwendolyn; Gallup, Nathan M; Alexandrova, Anastassia N; Hengge, Alvan C; Johnson, Sean J

    2015-10-27

    Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

  5. Structural mimicry of a-loop tyrosine phosphorylation by a pathogenic FGF receptor 3 mutation.

    PubMed

    Huang, Zhifeng; Chen, Huaibin; Blais, Steven; Neubert, Thomas A; Li, Xiaokun; Mohammadi, Moosa

    2013-10-01

    The K650E gain-of-function mutation in the tyrosine kinase domain of FGF receptor 3 (FGFR3) causes Thanatophoric Dysplasia type II, a neonatal lethal congenital dwarfism syndrome, and when acquired somatically, it contributes to carcinogenesis. In this report, we determine the crystal structure of the FGFR3 kinase domain harboring this pathogenic mutation and show that the mutation introduces a network of intramolecular hydrogen bonds to stabilize the active-state conformation. In the crystal, the mutant FGFR3 kinases are caught in the act of trans-phosphorylation on a kinase insert autophosphorylation site, emphasizing the fact that the K650E mutation circumvents the requirement for A-loop tyrosine phosphorylation in kinase activation. Analysis of this trans-phosphorylation complex sheds light onto the determinants of tyrosine trans-phosphorylation specificity. We propose that the targeted inhibition of this pathogenic FGFR3 kinase may be achievable by small molecule kinase inhibitors that selectively bind the active-state conformation of FGFR3 kinase.

  6. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

    PubMed

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril

    2014-03-28

    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  7. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases.

    PubMed

    Chen, Sen; Jiang, Xinnong; Gewinner, Christina A; Asara, John M; Simon, Nicholas I; Cai, Changmeng; Cantley, Lewis C; Balk, Steven P

    2013-05-28

    The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr⁵⁷⁷ subsequent to its Src-mediated phosphorylation at Tyr⁵⁷⁶. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, as well as Tyr¹¹⁸⁵, and downstream phosphorylation of the kinase AKT at Thr³⁰⁸ were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser⁴⁷³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases. PMID:23716717

  8. Tyrosine aminotransferase activity in the benzene intoxicated rat

    SciTech Connect

    Bong, M.; Michalska, A.; Laskowska-Klita, T.; Szymczyk, T.

    1985-01-01

    The toxic effect of hydrocarbon solvents on hepatic metabolism manifests itself by changes in the enzymatic pattern of blood serum. Changes in the activity of phosphatases as well as leucine aminopeptidase, glutamine aminotransferase, sorbitol dehydrogenase and ..gamma..-glutamyltransferase were observed in rats intoxicated with different fractions of benzene. Therefore it seemed reasonable to investigate the effect of benzene fraction of petroleum on cellular metabolism. The results of the present work concern the activity of tyrosine aminotransferase, the enzyme involved in catabolism of aromatic amino acid which is known to be under both hormonal and stress dependent control. Changes in tyrosine aminotransferase activity effect the level of tyrosine oxidation as well as the metabolic conversion of this amino acid into tyramine, tyroxin, adrenaline and noradrenaline.

  9. L-tyrosine administration increases acetylcholinesterase activity in rats.

    PubMed

    Ferreira, Gabriela K; Carvalho-Silva, Milena; Gonçalves, Cinara L; Vieira, Júlia S; Scaini, Giselli; Ghedim, Fernando V; Deroza, Pedro F; Zugno, Alexandra I; Pereira, Talita C B; Oliveira, Giovanna M T; Kist, Luiza W; Bogo, Maurício R; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

    2012-12-01

    Tyrosinemia is a rare genetic disease caused by mutations on genes that codify enzymes responsible for tyrosine metabolism. Considering that tyrosinemics patients usually present symptoms associated with central nervous system alterations that ranges from slight decreases in intelligence to severe mental retardation, we decided to investigate whether acute and chronic administration of L-tyrosine in rats would affect acetylcholinesterase mRNA expression and enzymatic activity during their development. In our acute protocol, Wistar rats (10 and 30 days old) were killed one hour after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old) and rats were killed 12 h after last injection. Acetylcholinesterase activity was measured by Ellman's method and acetylcholinesterase expression was carried out by a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay. We observed that acute (10 and 30 days old rats) and chronic L-tyrosine administration increased acetylcholinesterase activity in serum and all tested brain areas (hippocampus, striatum and cerebral cortex) when compared to control group. Moreover, there was a significant decrease in mRNA levels of acetylcholinesterase in hippocampus was observed after acute protocol (10 and 30 days old rats) and in striatum after chronic protocol. In case these alterations also occur in the brain of the patients, our results may explain, at least in part, the neurological sequelae associated with high plasma concentrations of tyrosine seen in patients affected by tyrosinemia type II. PMID:23046746

  10. L-tyrosine administration increases acetylcholinesterase activity in rats.

    PubMed

    Ferreira, Gabriela K; Carvalho-Silva, Milena; Gonçalves, Cinara L; Vieira, Júlia S; Scaini, Giselli; Ghedim, Fernando V; Deroza, Pedro F; Zugno, Alexandra I; Pereira, Talita C B; Oliveira, Giovanna M T; Kist, Luiza W; Bogo, Maurício R; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

    2012-12-01

    Tyrosinemia is a rare genetic disease caused by mutations on genes that codify enzymes responsible for tyrosine metabolism. Considering that tyrosinemics patients usually present symptoms associated with central nervous system alterations that ranges from slight decreases in intelligence to severe mental retardation, we decided to investigate whether acute and chronic administration of L-tyrosine in rats would affect acetylcholinesterase mRNA expression and enzymatic activity during their development. In our acute protocol, Wistar rats (10 and 30 days old) were killed one hour after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old) and rats were killed 12 h after last injection. Acetylcholinesterase activity was measured by Ellman's method and acetylcholinesterase expression was carried out by a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay. We observed that acute (10 and 30 days old rats) and chronic L-tyrosine administration increased acetylcholinesterase activity in serum and all tested brain areas (hippocampus, striatum and cerebral cortex) when compared to control group. Moreover, there was a significant decrease in mRNA levels of acetylcholinesterase in hippocampus was observed after acute protocol (10 and 30 days old rats) and in striatum after chronic protocol. In case these alterations also occur in the brain of the patients, our results may explain, at least in part, the neurological sequelae associated with high plasma concentrations of tyrosine seen in patients affected by tyrosinemia type II.

  11. Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety

    SciTech Connect

    Ramos-Santana, Brenda J.; Lopez-Garriga, Juan

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer H-bonding network loop by PheB10Tyr mutation is proposed. Black-Right-Pointing-Pointer The propionate group H-bonding network restricted the flexibility of the heme. Black-Right-Pointing-Pointer The hydrogen bonding interaction modulates the electron density of the iron. Black-Right-Pointing-Pointer Propionate H-bonding network loop explains the heme-ligand stabilization. -- Abstract: Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. {sup 1}H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OH{eta} at 31.00 ppm, GlnE7 N{sub {epsilon}1}H/N{sub {epsilon}2}H at 10.66 ppm/-3.27 ppm, and PheE11 C{sub {delta}}H at 11.75 ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus

  12. Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

    PubMed Central

    1994-01-01

    Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti- Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12- myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins

  13. Asymmetric Tyrosine Kinase Arrangements in Activation or Autophosphorylation of Receptor Tyrosine Kinases

    SciTech Connect

    J Bae; J Schlessinger

    2011-12-31

    Receptor tyrosine kinases (RTKs) play important roles in the control of many cellular processes including cell proliferation, cell adhesion, angiogenesis, and apoptosis. Ligand-induced dimerization of RTKs leads to autophosphorylation and activation of RTKs. Structural studies have shown that while isolated ectodomains of several RTKs form symmetric dimers the isolated cytoplasmic kinase domains of epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) form asymmetric dimers during their activation. Binding of one kinase molecule of EGFR to a second kinase molecule asymmetrically leads to stimulation of kinase activity and enhanced autophosphorylation. Furthermore, the structures of the kinase domain of FGFR1 and FGFR2 reveal the formation of asymmetric interfaces in the processes of autophosphorylation at their specific phosphotyrosine (pY) sites. Disruption of asymmetric dimer interface of EGFR leads to reduction in enzymatic activity and drastic reduction of autophosphorylation of FGFRs in ligandstimulated live cells. These studies demonstrate that asymmetric dimer formation is as a common phenomenon critical for activation and autophosphorylation of RTKs.

  14. Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors

    PubMed Central

    Wahl, Matthew I.; Fluckiger, Anne-Catherine; Kato, Roberta M.; Park, Hyunsun; Witte, Owen N.; Rawlings, David J.

    1997-01-01

    Mutation of Bruton’s tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcɛ receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (≤5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation. PMID:9326643

  15. Protein-tyrosine phosphatase activity of CD45 is activated by sequential phosphorylation by two kinases.

    PubMed Central

    Stover, D R; Walsh, K A

    1994-01-01

    We describe a potential regulatory mechanism for the transmembrane protein-tyrosine phosphatase CD45. Phosphorylation on both tyrosine and serine residues in vitro results in an activation of CD45 specifically toward one artificial substrate but not another. The activation of these kinases appears to be order dependent, as it is enhanced when phosphorylation of tyrosine precedes that of serine but phosphorylation in the reverse order yields no activation. Any of four protein-tyrosine kinases tested, in combination with the protein-serine/threonine kinase, casein kinase II, was capable of mediating this activation in vitro. The time course of phosphorylation of CD45 in response to T-cell activation is consistent with the possibility that this regulatory mechanism is utilized in vivo. Images PMID:7518565

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

    PubMed Central

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

    2016-01-01

    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. PMID:27063862

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2016-04-11

    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.

  19. The Cytoplasmic Adaptor Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization

    SciTech Connect

    Bergamin, E.; Hallock, P; Burden, S; Hubbard, S

    2010-01-01

    Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.

  20. Mitotic chromosome compaction via active loop extrusion

    NASA Astrophysics Data System (ADS)

    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. Tryptophan prenyltransferases showing higher catalytic activities for Friedel-Crafts alkylation of o- and m-tyrosines than tyrosine prenyltransferases.

    PubMed

    Fan, Aili; Xie, Xiulan; Li, Shu-Ming

    2015-07-21

    Tryptophan prenyltransferases FgaPT2, 5-DMATS, 6-DMATSSv and 7-DMATS catalyse regiospecific C-prenylations on the indole ring, while tyrosine prenyltransferases SirD and TyrPT catalyse the O-prenylation of the phenolic hydroxyl group. In this study, we report the Friedel-Crafts alkylation of L-o-tyrosine by these enzymes. Surprisingly, no conversion was detected with SirD and three tryptophan prenyltransferases showed significantly higher activity than another tyrosine prenyltransferase TyrPT. C5-prenylated L-o-tyrosine was identified as a unique product of these enzymes. Using L-m-tyrosine as the prenylation substrate, product formation was only observed with the tryptophan prenyltransferases FgaPT2 and 7-DMATS. C4- and C6-prenylated derivatives were identified in the reaction mixture of FgaPT2. These results provided additional evidence for the similarities and differences between these two subgroups within the DMATS superfamily in their catalytic behaviours.

  2. Tryptophan prenyltransferases showing higher catalytic activities for Friedel-Crafts alkylation of o- and m-tyrosines than tyrosine prenyltransferases.

    PubMed

    Fan, Aili; Xie, Xiulan; Li, Shu-Ming

    2015-07-21

    Tryptophan prenyltransferases FgaPT2, 5-DMATS, 6-DMATSSv and 7-DMATS catalyse regiospecific C-prenylations on the indole ring, while tyrosine prenyltransferases SirD and TyrPT catalyse the O-prenylation of the phenolic hydroxyl group. In this study, we report the Friedel-Crafts alkylation of L-o-tyrosine by these enzymes. Surprisingly, no conversion was detected with SirD and three tryptophan prenyltransferases showed significantly higher activity than another tyrosine prenyltransferase TyrPT. C5-prenylated L-o-tyrosine was identified as a unique product of these enzymes. Using L-m-tyrosine as the prenylation substrate, product formation was only observed with the tryptophan prenyltransferases FgaPT2 and 7-DMATS. C4- and C6-prenylated derivatives were identified in the reaction mixture of FgaPT2. These results provided additional evidence for the similarities and differences between these two subgroups within the DMATS superfamily in their catalytic behaviours. PMID:26077893

  3. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    PubMed Central

    Mezquita, Belén; Mezquita, Pau; Pau, Montserrat; Mezquita, Jovita; Mezquita, Cristóbal

    2014-01-01

    One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer. PMID:24709904

  4. The roles of the conserved tyrosine in the β2-α2 loop of the prion protein.

    PubMed

    Huang, Danzhi; Caflisch, Amedeo

    2015-01-01

    Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP(C)) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP(Sc)) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP(C) proteins have provided evidence of a conformational equilibrium between a 3(10)-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165-175). In vitro cell-free experiments of the seeded conversion of PrP(C) indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3(10)-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3(10)-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species. PMID:26689486

  5. The roles of the conserved tyrosine in the β2-α2 loop of the prion protein.

    PubMed

    Huang, Danzhi; Caflisch, Amedeo

    2015-01-01

    Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP(C)) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP(Sc)) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP(C) proteins have provided evidence of a conformational equilibrium between a 3(10)-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165-175). In vitro cell-free experiments of the seeded conversion of PrP(C) indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3(10)-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3(10)-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.

  6. Diurnal variations in response of rat liver tyrosine aminotransferase activity to food intake.

    PubMed

    Kato, H; Saito, M

    1980-01-01

    Effects of fasting and refeeding on the hepatic tyrosine aminotransferase activity were examined in rats that had been fed during the night. The tyrosine aminotransferase activity showed clear diurnal variations, with a maximal activity after the feeding time. The tyrosine aminotransferase rhythm persisted even under starvation, though the amplitude decreased remarkably. When the starved rats were refed at night, the tyrosine aminotransferase activity increased rapidly to a high level, but it increased slowly to a rather lower level when they were refed in daytime.

  7. Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

    PubMed Central

    Barton, William A.; Dalton, Annamarie C.; Seegar, Tom C.M.; Himanen, Juha P.

    2014-01-01

    The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition. PMID:24478383

  8. Centrosomal targeting of tyrosine kinase activity does not enhance oncogenicity in chronic myeloproliferative disorders.

    PubMed

    Bochtler, T; Kirsch, M; Maier, B; Bachmann, J; Klingmüller, U; Anderhub, S; Ho, A D; Krämer, A

    2012-04-01

    Constitutive tyrosine kinase activation by reciprocal chromosomal translocation is a common pathogenetic mechanism in chronic myeloproliferative disorders. Since centrosomal proteins have been recurrently identified as translocation partners of tyrosine kinases FGFR1, JAK2, PDGFRα and PDGFRβ in these diseases, a role for the centrosome in oncogenic transformation has been hypothesized. In this study, we addressed the functional role of centrosomally targeted tyrosine kinase activity. First, centrosomal localization was not routinely found for all chimeric fusion proteins tested. Second, targeting of tyrosine kinases to the centrosome by creating artificial chimeric fusion kinases with the centrosomal targeting domain of AKAP450 failed to enhance the oncogenic transforming potential in both Ba/F3 and U2OS cells, although phospho-tyrosine-mediated signal transduction pathways were initiated at the centrosome. We conclude that the centrosomal localization of constitutively activated tyrosine kinases does not contribute to disease pathogenesis in chronic myeloproliferative disorders. PMID:22015771

  9. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    NASA Astrophysics Data System (ADS)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  10. Hierarchical curiosity loops and active sensing.

    PubMed

    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

  11. Small-Molecule Inhibition and Activation-Loop Trans-Phosphorylation of the IGF1 Receptor

    SciTech Connect

    Wu,J.; Li, W.; Craddock, B.; Foreman, K.; Mulvihill, M.; Ji, Q.; Miller, W.; Hubbard, S.

    2008-01-01

    The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1, 5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.

  12. Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

    PubMed Central

    Song, Gyun Jee; Kim, Jaehong; Kim, Jong-Heon; Song, Seungeun; Park, Hana; Zhang, Zhong-Yin

    2016-01-01

    Protein tyrosine phosphatases (PTPs) are key regulatory factors in inflammatory signaling pathways. Although PTPs have been extensively studied, little is known about their role in neuroinflammation. In the present study, we examined the expression of 6 different PTPs (PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ) and their role in glial activation and neuroinflammation. All PTPs were expressed in brain and glia. The expression of PTP1B, SHP2, and LYP was enhanced in the inflamed brain. The expression of PTP1B, TC-PTP, and LYP was increased after treating microglia cells with lipopolysaccharide (LPS). To examine the role of PTPs in microglial activation and neuroinflammation, we used specific pharmacological inhibitors of PTPs. Inhibition of PTP1B, TC-PTP, SHP2, LYP, and RPTPβ suppressed nitric oxide production in LPS-treated microglial cells in a dose-dependent manner. Furthermore, intracerebroventricular injection of PTP1B, TC-PTP, SHP2, and RPTPβ inhibitors downregulated microglial activation in an LPS-induced neuroinflammation model. Our results indicate that multiple PTPs are involved in regulating microglial activation and neuroinflammation, with different expression patterns and specific functions. Thus, PTP inhibitors can be exploited for therapeutic modulation of microglial activation in neuroinflammatory diseases. PMID:27790059

  13. A cytosolic activator of DNA replication is tyrosine phosphorylated in its active form.

    PubMed

    Fresa, K L; Autieri, M V; Coffman, F D; Georgoff, I; Cohen, S

    1993-04-01

    Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a > 90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from non-proliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself. PMID:7683270

  14. Tyrosine Phosphorylation of SGEF Regulates RhoG Activity and Cell Migration

    PubMed Central

    Okuyama, Yusuke; Umeda, Kentaro; Negishi, Manabu; Katoh, Hironori

    2016-01-01

    SGEF and Ephexin4 are members of the Ephexin subfamily of RhoGEFs that specifically activate the small GTPase RhoG. It is reported that Ephexin1 and Ephexin5, two well-characterized Ephexin subfamily RhoGEFs, are tyrosine-phosphorylated by Src, and that their phosphorylation affect their activities and functions. In this study, we show that SGEF, but not Ephexin4, is tyrosine-phosphorylated by Src. Tyrosine phosphorylation of SGEF suppresses its interaction with RhoG, the elevation of RhoG activity, and SGEF-mediated promotion of cell migration. We identified tyrosine 530 (Y530), which is located within the Dbl homology domain, as a major phosphorylation site of SGEF by Src, and Y530F mutation blocked the inhibitory effect of Src on SGEF. Taken together, these results suggest that the activity of SGEF is negatively regulated by tyrosine phosphorylation of the DH domain. PMID:27437949

  15. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    PubMed Central

    Maruyama, Ichiro N.

    2014-01-01

    Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights. PMID:24758840

  16. 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.

    PubMed

    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

  17. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  18. Tyrosine inhibits creatine kinase activity in cerebral cortex of young rats.

    PubMed

    de Andrade, Rodrigo Binkowski; Gemelli, Tanise; Rojas, Denise Bertin; Funchal, Cláudia; Dutra-Filho, Carlos Severo; Wannmacher, Clovis Milton Duval

    2011-09-01

    Tyrosine accumulates in inborn errors of tyrosine catabolism, especially in tyrosinemia type II, where tyrosine levels are highly elevated in tissues and physiological fluids of affected patients. Tyrosinemia type II is a disorder of autosomal recessive inheritance characterized by neurological symptoms similar to those observed in patients with creatine deficiency syndromes. Considering that the mechanisms of brain damage in these disorders are poorly known, in the present study our main objective was to investigate the in vivo and in vitro effects of different concentrations and preincubation times of tyrosine on cytosolic and mitochondrial creatine kinase activities of the cerebral cortex from 14-day-old Wistar rats. The cytosolic CK was reduced by 15% at 1 mM and 32% at 2 mM tyrosine. Similarly, the mitochondrial CK was inhibited by 15% at 1 mM and 22% at 2 mM tyrosine. We observed that the inhibition caused by tyrosine was concentration-dependent and was prevented by reduced glutathione. Results also indicated that mitochondrial, but not cytosolic creatine kinase activity was inhibited by tyrosine in a time-dependent way. Finally, a single injection of L-Tyrosine methyl ester administered i.p. decreased cytosolic (31%) and mitochondrial (18%) creatine kinase activities of brain cortex from rats. Considering that creatine kinase is an enzyme dependent of thiol residues for its function and tyrosine induces oxidative stress, the results suggest that the inhibition caused by tyrosine might occur by oxidation of essential sulfhydryl groups of the enzyme. In case this also occurs in patients with tyrosinemia, it is possible that creatine kinase inhibition may contribute to the neurological dysfunction characteristic of tyrosinemia.

  19. Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

    PubMed Central

    van Oers, N S; Tao, W; Watts, J D; Johnson, P; Aebersold, R; Teh, H S

    1993-01-01

    The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta. Images PMID:7689151

  20. Partial purification and characterization of an enzyme from pea nuclei with protein tyrosine phosphatase activity.

    PubMed

    Guo, Y L; Roux, S J

    1995-01-01

    A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate.

  1. Novel post-translational incorporation of tyrosine in PMA-activated polymorphonuclear leukocytes (PMN)

    SciTech Connect

    Nath, J.; Oliver, C.; Ohno, Y.; Gallin, J.I.

    1986-03-05

    During studies undertaken to determine whether stimulation of tubulin tyrosinolation occurs in PMA-activated PMN, a distinctly different and novel post-translational incorporation of tyrosine into multiple PMN proteins was observed. The reaction also occurred in organelle-depleted neutrophil cytoplasts and was highly exaggerated in organelle-enriched karyogranuloplasts. The incorporation was specific for tyrosine, did not require extracellular Ca/sup 2 +/ and was inhibited in the presence of a variety of reducing agents, intracellular scavengers of oxygen radicals and inhibitors of peroxidase-mediated reactions. The PMA-induced incorporation of tyrosine was completely absent in PMN from patients with chronic granulomatous disease, but occurred normally in PMN of a patient with myeloperoxidase deficiency. Moreover, the incorporation of tyrosine was blocked by N-acetyl-L-tyrosine but not by phenylalanine suggesting a requirement for the phenolic group. A two-fold increase in stable protein carbonyl derivatives was demonstrated suggesting an increased oxidative modification of the proteins. SDS urea PAGE and reversed phase HPLC did not reveal any detectable changes in the extent of protein cross-linking. The PMN tyrosine pool was approximately 900 ..mu..M and yet only 1 ..mu..M tyrosine was added in these experiments. The functional significance of this reaction is not yet clear.

  2. JAK tyrosine kinases promote hierarchical activation of Rho and Rap modules of integrin activation.

    PubMed

    Montresor, Alessio; Bolomini-Vittori, Matteo; Toffali, Lara; Rossi, Barbara; Constantin, Gabriela; Laudanna, Carlo

    2013-12-23

    Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1- and VLA-4-mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Gαi-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation.

  3. DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster abelson proto-oncogene homolog

    SciTech Connect

    Henkemeyer, M.J.; Bennett, R.L.; Gertler, F.B.; Hoffmann, F.M.

    1988-02-01

    The authors report their molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type 1b 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.

  4. Activation loop 3 and the 170 loop interact in the active conformation of coagulation factor VIIa.

    PubMed

    Persson, Egon; Olsen, Ole H

    2009-06-01

    The initiation of blood coagulation involves tissue factor (TF)-induced allosteric activation of factor VIIa (FVIIa), which circulates in a zymogen-like state. In addition, the (most) active conformation of FVIIa presumably relies on a number of intramolecular interactions. We have characterized the role of Gly372(223) in FVIIa, which is the sole residue in activation loop 3 that is capable of forming backbone hydrogen bonds with the unusually long 170 loop and with activation loop 2, by studying the effects of replacement with Ala [G372(223)A]. G372A-FVIIa, both in the free and TF-bound form, exhibited reduced cleavage of factor X (FX) and of peptidyl substrates, and had increased K(m) values compared with wild-type FVIIa. Inhibition of G372A-FVIIa.sTF by p-aminobenzamidine was characterized by a seven-fold higher K(i) than obtained with FVIIa.sTF. Crystallographic and modelling data suggest that the most active conformation of FVIIa depends on the backbone hydrogen bond between Gly372(223) and Arg315(170C) in the 170 loop. Despite the reduced activity and inhibitor susceptibility, native and active site-inhibited G372A-FVIIa bound sTF with the same affinity as the corresponding forms of FVIIa, and burial of the N-terminus of the protease domain increased similarly upon sTF binding to G372A-FVIIa and FVIIa. Thus Gly372(223) in FVIIa appears to play a critical role in maturation of the S1 pocket and adjacent subsites, but does not appear to be of importance for TF binding and the ensuing allostery. PMID:19490111

  5. Tetrodotoxin-insensitive Na+ channel activator palytoxin inhibits tyrosine uptake into cultured bovine adrenal chromaffin cells

    SciTech Connect

    Morita, K.; Teraoka, K.; Azuma, M.; Oka, M.; Hamano, S. )

    1991-07-01

    The effects of the tetrodotoxin-insensitive Na+ channel activator palytoxin on both the secretion of endogenous catecholamines and the formation of 14C-catecholamines from (14C)tyrosine were examined using cultured bovine adrenal chromaffin cells. Palytoxin was shown to cause the stimulation of catecholamine secretion in a concentration-dependent manner. However, this toxin caused the reduction rather than the stimulation of 14C-catecholamine formation at the same concentrations. Palytoxin failed to cause any alteration in the activity of tyrosine hydroxylase prepared from bovine adrenal medulla. Furthermore, the uptake of (14C)tyrosine into the cells was shown to be inhibited by this toxin under the conditions in which the suppression of 14C-catecholamine formation was observed, and this inhibitory action on tyrosine uptake was closely correlated with that on catecholamine formation. The inhibitory action of palytoxin on tyrosine uptake into the cells was observed to be noncompetitive, and this effect was not altered by the removal of Na+ from the incubation mixture. These results suggest that palytoxin may be able to inhibit the uptake of (14C)tyrosine into the cells, resulting in the suppression of 14C-catecholamine formation, probably through its direct action on the plasma membranes of bovine adrenal chromaffin cells.

  6. Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1.

    PubMed Central

    Reiland, J; Ott, V L; Lebakken, C S; Yeaman, C; McCarthy, J; Rapraeger, A C

    1996-01-01

    Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane

  7. DMBA induces tyrosine phosphorylation of PLC-[gamma]1 and activates the tyrosine kinases lck and fyn in the HPB-ALL human T-cell line

    SciTech Connect

    Archuleta, M.M.; Schieven, G.L.; Ledbetter, J.A.; Burchiel, S.W. . Coll. of Pharmacy)

    1993-01-01

    Previous studies in this laboratory have demonstrated that DMBA alters biochemical events associated with lymphocyte activation including formation of the second messenger IP[sub 3] and the release of intracellular Ca[sup 2+]. The purpose of the present studies was to evaluate the mechanisms by which DMBA induces IP[sub 3] formation and Ca[sup 2+] release by examining phosphorylation of membrane associated proteins and activation of protein tyrosine kinases lck and fyn. These studies demonstrated that exposure of HPB-ALL cells to 10[mu]M DMBA resulted in a time- and dose-dependent increase in tyrosine phosphorylation of PLC-[gamma]1 that correlated with our earlier findings of IP[sub 3] formation and Ca[sup 2+] release. These results indicate that the effects of DMBA on the PI-PLC signaling pathway are in part, the result of DMBA-induced tyrosine phosphorylation of the PLC-[gamma]1 enzyme. The mechanism of DMBA- induced tyrosine phosphorylation of PLC-[gamma]1 may be due to activation of fyn or lck kinase activity, since it was found that DMBA increased the activity of these PTKs by more than 2-fold. Therefore, these studies demonstrate that DMBA may disrupt T cell activation by stimulating PTK activation with concomitant tyrosine phosphorylation of PLC-[gamma]1, release of IP[sub 3], and mobilization of intracellular Ca[sup 2+].

  8. DMBA induces tyrosine phosphorylation of PLC-{gamma}1 and activates the tyrosine kinases lck and fyn in the HPB-ALL human T-cell line

    SciTech Connect

    Archuleta, M.M.; Schieven, G.L.; Ledbetter, J.A.; Burchiel, S.W.

    1993-02-01

    Previous studies in this laboratory have demonstrated that DMBA alters biochemical events associated with lymphocyte activation including formation of the second messenger IP{sub 3} and the release of intracellular Ca{sup 2+}. The purpose of the present studies was to evaluate the mechanisms by which DMBA induces IP{sub 3} formation and Ca{sup 2+} release by examining phosphorylation of membrane associated proteins and activation of protein tyrosine kinases lck and fyn. These studies demonstrated that exposure of HPB-ALL cells to 10{mu}M DMBA resulted in a time- and dose-dependent increase in tyrosine phosphorylation of PLC-{gamma}1 that correlated with our earlier findings of IP{sub 3} formation and Ca{sup 2+} release. These results indicate that the effects of DMBA on the PI-PLC signaling pathway are in part, the result of DMBA-induced tyrosine phosphorylation of the PLC-{gamma}1 enzyme. The mechanism of DMBA- induced tyrosine phosphorylation of PLC-{gamma}1 may be due to activation of fyn or lck kinase activity, since it was found that DMBA increased the activity of these PTKs by more than 2-fold. Therefore, these studies demonstrate that DMBA may disrupt T cell activation by stimulating PTK activation with concomitant tyrosine phosphorylation of PLC-{gamma}1, release of IP{sub 3}, and mobilization of intracellular Ca{sup 2+}.

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

    PubMed Central

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

    2014-01-01

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

  10. Redundant kinase activation and resistance of EGFR-tyrosine kinase inhibitors

    PubMed Central

    Luo, Min; Fu, Li-Wu

    2014-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic effects against that tumors harboring EGFR activating mutations in the EGFR intracytoplasmic tyrosine kinase domain and resulted in cell apoptosis. Unfortunately, a number of patients ultimately developed resistance by multiple mechanisms. Thus, elucidation of the mechanism of resistance to EGFR-TKIs can provide strategies for blocking or reversing the situation. Recent studies suggested that redundant kinase activation plays pivotal roles in escaping from the effects of EGFR-TKIs. Herein, we aimed to characterize several molecular events involved in the resistance to EGFR-TKIs mediated by redundant kinase activation. PMID:25520855

  11. Activation of spleen tyrosine kinase (Syk) at fertilization in Rhinella arenarum eggs.

    PubMed

    Mouguelar, Valeria S; Coux, Gabriela

    2014-01-01

    Recently, we have provided evidence for the involvement of a cytosolic tyrosine-phosphorylatable 70 kDa oocyte protein in Rhinella arenarum (Anura: Bufonidae) fertilization. The aim of the present work was to characterize its phosphorylation, determine the identity of this protein and establish its biological role during the fertilization process. Tyrosine phosphorylation of the 70 kDa protein was not observed in eggs activated with the calcium ionophore A23187. Pretreatment of oocytes with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation of the 70 kDa protein. In order to identify this protein, we examined the presence in amphibian oocytes of non-receptor 70 kDa tyrosine kinase members of the Syk/Zap70 and Tec families by RT-PCR using degenerate primers. We found that R. arenarum oocytes contain the transcripts coding for Syk and Tec kinases. Western blot analysis confirmed the presence of Syk protein in unfertilized oocytes and eggs. Studies using phospho-Syk specific antibodies showed that fertilization rapidly (less than 10 minutes) induces phosphorylation on Syk tyrosine residues (352 and 525/526) that are necessary for the activation of the enzyme. Finally, specific inhibition of Syk with the R406 compound provoked a diminished fertilization score, thereby confirming a functional role of the Syk protein during R. arenarum fertilization. To our knowledge this is the first time that Syk is described as a player in the signaling cascade activated after fertilization.

  12. Assays to measure the activation of membrane tyrosine kinase receptors: focus on cellular methods.

    PubMed

    Minor, Lisa K

    2003-09-01

    Many methods have been explored as means to measure the activation and inhibition of tyrosine kinase receptors, in vitro using the isolated kinase domain, and in living cells. Kinase activity has been measured in enzyme assays using a peptide substrate, but with different detection systems. These include the radioactive FlashPlate assay, the fluorescent resonance energy transfer (FRET) assay, the dissociation-enhance lanthanide fluorescence immunoassay (DELFIA) and other formats. These methods have successfully identified inhibitors of receptor activity. Cell-based assays have recently emerged to measure receptor activation and inhibition. When membrane tyrosine kinase receptors become activated, they increase their state of phosphorylation. This phosphorylation may lead to an increase in tyrosine kinase-specific activity. Methods have been developed that take advantage of these properties. These include measuring the ligand-stimulated total tyrosine phosphorylation of the receptor using a DELFIA or an ELISA assay, measuring ligand-stimulated enzyme activation of the receptor by quantifying enzyme activity, and dimerization of the activated receptor using bioluminescence resonance energy transfer (BRET). Although cell-based assays are still in their infancy, these techniques may prove a valuable addition to the receptor screening strategy.

  13. Inactivation of tyrosine hydroxylase activity by ascorbate in vitro and in rat PC12 cells.

    PubMed

    Wilgus, H; Roskoski, R

    1988-10-01

    Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC50 of 5.9 microM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC50, 11 microM) and dehydroascorbate (EC50, 970 microM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant proteolysis of the purified enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25-50%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2901463

  14. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    SciTech Connect

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  15. Morin inhibits STAT3 tyrosine 705 phosphorylation in tumor cells through activation of protein tyrosine phosphatase SHP1.

    PubMed

    Gupta, Subash C; Phromnoi, Kanokkarn; Aggarwal, Bharat B

    2013-04-01

    The major goal of cancer drug discovery is to find an agent that is safe and affordable, yet effective against cancer. Here we show that morin (3,5,7,2',4'-pentahydroxyflavone) has potential against cancer cells through suppression of the signal transducer and activator of transcription 3 (STAT3) pathway, which is closely linked to the transformation, survival, proliferation, and metastasis of cancer. We found that morin completely suppressed inducible and constitutively activated STAT3 and blocked the nuclear translocation of STAT3 and its DNA binding in multiple myeloma and head and neck squamous carcinoma cells. Morin inhibited activated Src, JAK-1, and JAK-2, all of which are linked to STAT3 activation, while up-regulating a protein inhibitor of activated STAT3, PIAS3. Pervanadate reversed the effects of morin on STAT3 phosphorylation, indicating the role of a protein tyrosine phosphatase. Furthermore, morin induced SHP1 expression at both the mRNA and protein levels, and silencing of SHP1 abrogated the effect of morin on STAT3 phosphorylation, indicating that morin mediates its effects on STAT3 through SHP1. Suppression of STAT3 correlated with the down-regulation of various gene products linked to tumor survival, proliferation, and angiogenesis and led to sensitization of tumor cells to thalidomide and bortezomib. Comparing the activities of morin with those of four structurally related flavonols demonstrated the importance of hydroxyl groups in the B ring in inhibiting STAT3 activation. These findings suggest that morin suppresses the STAT3 pathway, leading to the down-regulation of STAT3-dependent gene expression and chemosensitization of tumor cells.

  16. Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine.

    PubMed

    Blomberg, Margareta R A

    2016-01-26

    Cytochrome c oxidase, the terminal enzyme in the respiratory chain, reduces molecular oxygen to water and stores the released energy through electrogenic chemistry and proton pumping across the membrane. Apart from the heme-copper binuclear center, there is a conserved tyrosine residue in the active site (BNC). The tyrosine delivers both an electron and a proton during the O-O bond cleavage step, forming a tyrosyl radical. The catalytic cycle then occurs in four reduction steps, each taking up one proton for the chemistry (water formation) and one proton to be pumped. It is here suggested that in three of the reduction steps the chemical proton enters the center of the BNC, leaving the tyrosine unprotonated with radical character. The reproprotonation of the tyrosine occurs first in the final reduction step before binding the next oxygen molecule. It is also suggested that this reduction mechanism and the presence of the tyrosine are essential for the proton pumping. Density functional theory calculations on large cluster models of the active site show that only the intermediates with the proton in the center of the BNC and with an unprotonated tyrosyl radical have a high electron affinity of similar size as the electron donor, which is essential for the ability to take up two protons per electron and thus for the proton pumping. This type of reduction mechanism is also the only one that gives a free energy profile in accordance with experimental observations for the amount of proton pumping in the working enzyme.

  17. The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity.

    PubMed Central

    Bergman, M; Mustelin, T; Oetken, C; Partanen, J; Flint, N A; Amrein, K E; Autero, M; Burn, P; Alitalo, K

    1992-01-01

    Protein tyrosine kinases participate in the transduction and modulation of signals that regulate proliferation and differentiation of cells. Excessive or deregulated protein tyrosine kinase activity can cause malignant transformation. The catalytic activity of the T cell protein tyrosine kinase p56lck is normally suppressed by phosphorylation of a carboxyl-terminal tyrosine, Tyr-505, by another cellular protein tyrosine kinase. Here we characterize a human cytosolic 50 kDa protein tyrosine kinase, p50csk, which specifically phosphorylates Tyr-505 of p56lck and a synthetic peptide containing this site. Phosphorylation of Tyr-505 suppressed the catalytic activity of p56lck. We suggest that p50csk negatively regulates p56lck, and perhaps other cellular src family kinases. Images PMID:1639064

  18. Phosphorylation of Tip60 Tyrosine 327 by Abl Kinase Inhibits HAT Activity through Association with FE65

    PubMed Central

    Shin, Sung Hwa; Kang, Sang Sun

    2013-01-01

    The transfer of acetyl groups from acetyl coenzyme A to the ε amino group of internal lysine residues is catalyzed by Tip60, which is in the MYST family of nuclear histone acetyltransferases (HATs). The tyrosine phosphorylation of Tip60 seems to be a unique modification. We present evidence that Tip60 is modified on tyrosine 327 by Abl kinase. We show that this causes functional changes in HAT activity and the subcellular localization of TIP60, which forms a complex with Abl kinase. The Tip60 mutation Y327F abolished tyrosine phosphorylation, reduced the inhibition of Tip60 HAT activity, and caused G0-G1 arrest and association with FE65. Thus, our findings for the first time suggested a novel regulation mechanism of Tip60. Regulation was through phosphorylation of tyrosine 327 by Abl tyrosine kinase and depended on environmental conditions, suggesting that the tyrosine residue of Tip60 is important for the activation process. PMID:24044023

  19. Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and vascular integrity.

    PubMed

    Boulaftali, Yacine; Hess, Paul R; Kahn, Mark L; Bergmeier, Wolfgang

    2014-03-28

    Platelets are well-known for their critical role in hemostasis, that is, the prevention of blood loss at sites of mechanical vessel injury. Inappropriate platelet activation and adhesion, however, can lead to thrombotic complications, such as myocardial infarction and stroke. To fulfill its role in hemostasis, the platelet is equipped with various G protein-coupled receptors that mediate the response to soluble agonists such as thrombin, ADP, and thromboxane A2. In addition to G protein-coupled receptors, platelets express 3 glycoproteins that belong to the family of immunoreceptor tyrosine-based activation motif receptors: Fc receptor γ chain, which is noncovalently associated with the glycoprotein VI collagen receptor, C-type lectin 2, the receptor for podoplanin, and Fc receptor γII A, a low-affinity receptor for immune complexes. Although both genetic and chemical approaches have documented a critical role for platelet G protein-coupled receptors in hemostasis, the contribution of immunoreceptor tyrosine-based activation motif receptors to this process is less defined. Studies performed during the past decade, however, have identified new roles for platelet immunoreceptor tyrosine-based activation motif signaling in vascular integrity in utero and at sites of inflammation. The purpose of this review is to summarize recent findings on how platelet immunoreceptor tyrosine-based activation motif signaling controls vascular integrity, both in the presence and absence of mechanical injury. PMID:24677237

  20. Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and vascular integrity.

    PubMed

    Boulaftali, Yacine; Hess, Paul R; Kahn, Mark L; Bergmeier, Wolfgang

    2014-03-28

    Platelets are well-known for their critical role in hemostasis, that is, the prevention of blood loss at sites of mechanical vessel injury. Inappropriate platelet activation and adhesion, however, can lead to thrombotic complications, such as myocardial infarction and stroke. To fulfill its role in hemostasis, the platelet is equipped with various G protein-coupled receptors that mediate the response to soluble agonists such as thrombin, ADP, and thromboxane A2. In addition to G protein-coupled receptors, platelets express 3 glycoproteins that belong to the family of immunoreceptor tyrosine-based activation motif receptors: Fc receptor γ chain, which is noncovalently associated with the glycoprotein VI collagen receptor, C-type lectin 2, the receptor for podoplanin, and Fc receptor γII A, a low-affinity receptor for immune complexes. Although both genetic and chemical approaches have documented a critical role for platelet G protein-coupled receptors in hemostasis, the contribution of immunoreceptor tyrosine-based activation motif receptors to this process is less defined. Studies performed during the past decade, however, have identified new roles for platelet immunoreceptor tyrosine-based activation motif signaling in vascular integrity in utero and at sites of inflammation. The purpose of this review is to summarize recent findings on how platelet immunoreceptor tyrosine-based activation motif signaling controls vascular integrity, both in the presence and absence of mechanical injury.

  1. 3D MHD Models of Active Region Loops

    NASA Technical Reports Server (NTRS)

    Ofman, Leon

    2004-01-01

    Present imaging and spectroscopic observations of active region loops allow to determine many physical parameters of the coronal loops, such as the density, temperature, velocity of flows in loops, and the magnetic field. However, due to projection effects many of these parameters remain ambiguous. Three dimensional imaging in EUV by the STEREO spacecraft will help to resolve the projection ambiguities, and the observations could be used to setup 3D MHD models of active region loops to study the dynamics and stability of active regions. Here the results of 3D MHD models of active region loops are presented, and the progress towards more realistic 3D MHD models of active regions. In particular the effects of impulsive events on the excitation of active region loop oscillations, and the generation, propagations and reflection of EIT waves are shown. It is shown how 3D MHD models together with 3D EUV observations can be used as a diagnostic tool for active region loop physical parameters, and to advance the science of the sources of solar coronal activity.

  2. Models for the activation pathway of epidermal growth factor receptor protein-tyrosine kinase

    SciTech Connect

    Campion, S.R.; Niyogi, S.K. )

    1991-03-15

    Activation of the epidermal growth factor (EGF) receptor's intrinsic protein-tyrosine kinase activity, which occurs upon formation of the receptor-ligand complex, is the critical regulatory event affecting the subsequent EGF-dependent cellular responses leading to DNA synthesis and cell proliferation. The molecular mechanism by which EGF-dependent activation of receptor kinase activity takes place is not clearly understood. In this study, the growth factor-dependent activation of the EGF receptor tyrosine kinase was examined in vitro using detergent-solubilized, partially purified GEF receptors from A5431 human epidermoid carcinoma cells. Evaluation of the cooperativity observed in the EGF-dependent activation of soluble receptor tyrosine kinase would suggest a mechanism requiring the binding of the EGF peptide to both ligand binding sites on a receptor dimer to induce full receptor kinase activity. Equations describing potential cooperative kinase activation pathways have been examined. The theoretical system which best simulates the allosteric regulation observed in the experimental kinase activation data is that describing multiple essential activation. In addition, studies using mutant analogs of the EGF peptide ligand appear to confirm the requirement for an essential conformational change in the receptor-ligand complex to activate the receptor kinase activity. Several mutant growth factor analogues are able to occupy the ligand binding sites on the receptor without inducing the fully active receptor conformation.

  3. Quinoxaline-Based Scaffolds Targeting Tyrosine Kinases and Their Potential Anticancer Activity.

    PubMed

    El Newahie, Aliya M S; Ismail, Nasser S M; Abou El Ella, Dalal A; Abouzid, Khaled A M

    2016-05-01

    Quinoxaline derivatives, also called benzopyrazines, are an important class of heterocyclic compounds. Quinoxalines have drawn great attention due to their wide spectrum of biological activities. They are considered as an important basis for anticancer drugs due to their potential activity as protein kinase inhibitors. In this review, we focus on the chemistry of the quinoxaline derivatives, the strategies for their synthesis, their potential activities against various tyrosine kinases, and on the structure-activity relationship studies reported to date.

  4. Activating Mutations in PIK3CA Lead to Widespread Modulation of the Tyrosine Phosphoproteome

    PubMed Central

    Blair, Brian G.; Pinto, Sneha M.; Nirujogi, Raja S.; Jelinek, Christine A.; Malhotra, Radhika; Kim, Min-Sik; Park, Ben Ho; Pandey, Akhilesh

    2015-01-01

    The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis. PMID:26267517

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

    PubMed

    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

  6. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  7. Tyrosine nitration provokes inhibition of sunflower carbonic anhydrase (β-CA) activity under high temperature stress.

    PubMed

    Chaki, Mounira; Carreras, Alfonso; López-Jaramillo, Javier; Begara-Morales, Juan C; Sánchez-Calvo, Beatriz; Valderrama, Raquel; Corpas, Francisco J; Barroso, Juan B

    2013-02-28

    Protein tyrosine nitration is a post-translational modification (PTM) mediated by reactive nitrogen species (RNS) and it is a new area of research in higher plants. Previously, it was demonstrated that the exposition of sunflower (Helianthus annuus L.) seedlings to high temperature (HT) caused both oxidative and nitrosative stress. The nitroproteome analysis under this stress condition showed the induction of 13 tyrosine-nitrated proteins being the carbonic anhydrase (CA) one of these proteins. The analysis of CA activity under high temperature showed that this stress inhibited the CA activity by a 43%. To evaluate the effect of nitration on the CA activity in sunflower it was used 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent. Thus the CA activity was inhibited by 41%. In silico analysis of the pea CA protein sequence suggests that Tyr(205) is the most likely potential target for nitration.

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

    PubMed

    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.

  9. Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry*

    PubMed Central

    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 ≥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. PMID:19887368

  10. Characterization of a Mn sup 2+ -dependent membrane serine kinase that is activated by tyrosine phosphorylation

    SciTech Connect

    Singh, T.J. )

    1991-03-11

    It is hypothesized that the insulin receptor (IR) tyrosine kinase may directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases as well as their modes of activation are unclear. The authors have described a serine kinase from rat liver membranes that copurifies with the IR on wheat germ agglutinin (WGA)-sepharose. The kinase is activated after phosphorylation of the WGA-sepharose-purified fraction by casein kinase-1, casein kinase-2, or casein kinase-3. A tyrosine kinase, possibly IR tyrosine kinase, also participates in the activation process since a phosphotyrosine phosphatase inhibitor such as vanadate, p-nitrophenyl phosphate, or phosphotyrosine is required in reaction mixtures for activation to be observed. By contrast, phosphoserine and phosphothreonine do not support activation. The activated kinase can use IR {beta}-subunit, myelin basic protein (MBP), and histones as substrates. IR {beta}-subunit phosphorylation was stimulated by MBP, histones, and polylysine, and inhibited by heparin and poly(glu, tyr). The kinase prefers Mn{sup 2+} over Mg{sup 2+} as a metal cofactor.

  11. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling.

    PubMed

    Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J; Andersson, Leif C; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

    2015-10-20

    Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy. PMID:26430242

  12. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling

    PubMed Central

    Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J.; Andersson, Leif C.; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

    2015-01-01

    Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy. PMID:26430242

  13. Mutational analysis of Agrobacterium tumefaciens virD2: tyrosine 29 is essential for endonuclease activity.

    PubMed Central

    Vogel, A M; Das, A

    1992-01-01

    Agrobacterium tumefaciens VirD2 polypeptide, in the presence of VirD1, catalyzes a site- and strand-specific nicking reaction at the T-DNA border sequences. VirD2 is found tightly attached to the 5' end of the nicked DNA. The protein-DNA complex is presumably formed via a tyrosine residue of VirD2 (F. Durrenberger, A. Crameri, B. Hohn, and Z. Koukolikova-Nicola, Proc. Natl. Acad. Sci. USA 86:9154-9158, 1989). A mutational approach was used to study whether a tyrosine residue(s) of VirD2 is required for its activity. By site-specific mutagenesis, a tyrosine (Y) residue at position 29, 68, 99, 119, 121, 160, or 195 of the octopine Ti plasmid pTiA6 VirD2 was altered to phenylalanine (F). The Y-29-F or Y-121-F mutation completely abolished nicking activity of VirD2 in vivo in Escherichia coli. Two other substitutions, Y-68-F and Y-160-F, drastically reduced VirD2 activity. A substitution at position 99, 119, or 195 had no effect on VirD2 activity. Additional mutagenesis experiments showed that at position 29, no other amino acid could substitute for tyrosine without destroying VirD2 activity. At position 121, only a tryptophan (W) residue could be substituted. This, however, yielded a mutant protein with significantly reduced VirD2 activity. The nicked DNA from strains bearing a Y-68-F, Y-99-F, Y-119-F, Y-160-F, Y-195-F, or Y-121-W mutation in VirD2 was always found to contain a tightly linked protein. Images PMID:1309520

  14. Flavonoids and tyrosine nitration: structure-activity relationship correlation with enthalpy of formation.

    PubMed

    Sadeghipour, Mitra; Terreux, Raphael; Phipps, Jenny

    2005-03-01

    The ability of 11 flavonoids, naturally occurring polyphenols, and their related structure-activity relationships (SAR's) for inhibiting peroxynitrite-induced nitration of tyrosine was investigated. The flavonoids under study could be classified into four groups having very distinct in vitro inhibition effects. We also calculated the heat of formation (DeltaH(f)) of the corresponding flavonoids radicals which supported this finding. The most effective flavonoids included: catechin, taxifolin, luteolin, quercetin, and myricetin which have a common structural feature of ortho-dihydroxyl moiety (3',4'-OH substitution). Naringenin, kaempferol, and morin were 50% less effective inhibitors than the former group of flavonoid while their activities were in the range of trolox (an alpha-tocopherol analogue). The common structural aspect of this group of flavonoids is 4'-OH substitution. Therefore, these two groups of flavonoids may have similar mechanisms for their inhibition activity. No inhibition activity was observed by galangin. Apigenin behaved as a pro-oxidant in our in vitro study. Naringin was as effective as the second group at 4 mM tyrosine concentration while did not illustrate any inhibitory effect at 1 mM concentration of tyrosine. Our study provides further evidence for the importance of the catechol B ring and to a lesser effect the importance of 4'-OH substitution. Moreover, we observed very little or no influence on activity of flavonoids by 3-OH substitution and/or a C2-C3 double bond conjugated with 4-keto group within the subgroup containing the catechol moiety. Theoretical calculation of DeltaDeltaH(f) for tyrosyl radical repair by flavonoids (TyO*+FlOH-->TyOH+FlO*) correlated well with our in vitro results (inhibition% = -10 (DeltaDeltaH(f)), R2=0.906). Furthermore, this correlation was independent of tyrosine concentration. This model can be used to accurately predict the inhibitory effect of flavonoids on nitrotyrosine formation.

  15. Inhibition by ajoene of protein tyrosine phosphatase activity in human platelets.

    PubMed

    Villar, R; Alvariño, M T; Flores, R

    1997-02-01

    The effects of ajoene (a potent antithrombotic agent obtained from garlic) on the tyrosine phosphorylation status of human platelet proteins were investigated by immunoblotting-based experiments using an anti-phosphotyrosine antibody. Incubation of platelets with ajoene enhanced the phosphorylation of at least four proteins (estimated MWs 76, 80, 84 and 120 kDa), both in resting platelets and in platelets subsequently stimulated with thrombin (0.1 U/ml). This effect was both dose- and incubation-time-dependent. High concentrations of ajoene (50 microM) or long periods of incubation (10 min) led to nonselective 'hyperphosphorylation' of numerous proteins. The effects of ajoene on protein tyrosine phosphatase (PTP) activity in platelet lysates were also investigated, PTP activity was inhibited when platelets were incubated with ajoene before lysis, but not when ajoene was added to lysates of platelets which had not been pre-exposed to ajoene.

  16. Activation of Tsk and Btk tyrosine kinases by G protein beta gamma subunits.

    PubMed Central

    Langhans-Rajasekaran, S A; Wan, Y; Huang, X Y

    1995-01-01

    Tsk/Itk and Btk are members of the pleckstrin-homology (PH) domain-containing tyrosine kinase family. The PH domain has been demonstrated to be able to interact with beta gamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) (G beta gamma) and phospholipids. Using cotransfection assays, we show here that the kinase activities of Tsk and Btk are stimulated by certain G beta gamma subunits. Furthermore, using an in vitro reconstitution assay with purified bovine brain G beta gamma subunits and the immunoprecipitated Tsk, we find that Tsk kinase activity is increased by G beta gamma subunits and another membrane factor(s). These results indicate that this family of tyrosine kinases could be an effector of heterotrimeric G proteins. Images Fig. 1 Fig. 2 Fig. 3 PMID:7567982

  17. Activation of the Syk tyrosine kinase is insufficient for downstream signal transduction in B lymphocytes

    PubMed Central

    Hsueh, Robert C; Hammill, Adrienne M; Lee, Jamie A; Uhr, Jonathan W; Scheuermann, Richard H

    2002-01-01

    Background Immature B lymphocytes and certain B cell lymphomas undergo apoptotic cell death following activation of the B cell antigen receptor (BCR) signal transduction pathway. Several biochemical changes occur in response to BCR engagement, including activation of the Syk tyrosine kinase. Although Syk activation appears to be necessary for some downstream biochemical and cellular responses, the signaling events that precede Syk activation remain ill defined. In addition, the requirements for complete activation of the Syk-dependent signaling step remain to be elucidated. Results A mutant form of Syk carrying a combination of a K395A substitution in the kinase domain and substitutions of three phenylalanines (3F) for the three C-terminal tyrosines was expressed in a murine B cell lymphoma cell line, BCL1.3B3 to interfere with normal Syk regulation as a means to examine the Syk activation step in BCR signaling. Introduction of this kinase-inactive mutant led to the constitutive activation of the endogenous wildtype Syk enzyme in the absence of receptor engagement through a 'dominant-positive' effect. Under these conditions, Syk kinase activation occurred in the absence of phosphorylation on Syk tyrosine residues. Although Syk appears to be required for BCR-induced apoptosis in several systems, no increase in spontaneous cell death was observed in these cells. Surprisingly, although the endogenous Syk kinase was enzymatically active, no enhancement in the phosphorylation of cytoplasmic proteins, including phospholipase Cγ2 (PLCγ2), a direct Syk target, was observed. Conclusion These data indicate that activation of Syk kinase enzymatic activity is insufficient for Syk-dependent signal transduction. This observation suggests that other events are required for efficient signaling. We speculate that localization of the active enzyme to a receptor complex specifically assembled for signal transduction may be the missing event. PMID:12470302

  18. Identification of therapeutic targets in ovarian cancer through active tyrosine kinase profiling

    PubMed Central

    Ocaña, Alberto; Pandiella, Atanasio

    2015-01-01

    The activation status of a set of pro-oncogenic tyrosine kinases in ovarian cancer patient samples was analyzed to define potential therapeutic targets. Frequent activation of HER family receptor tyrosine kinases, especially HER2, was observed. Studies in ovarian cancer cell lines confirmed the activation of HER2. Moreover, knockdown of HER2 caused a strong inhibition of their proliferation. Analyses of the action of agents that target HER2 indicated that the antibody drug conjugate trastuzumab-emtansine (T-DM1) caused a substantial antitumoral effect in vivo and in vitro, and potentiated the action of drugs used in the therapy of ovarian cancer. T-DM1 provoked cell cycle arrest in mitosis, and caused the appearance of aberrant mitotic spindles in cells treated with the drug. Biochemical experiments confirmed accumulation of the mitotic markers phospho-Histone H3 and phospho-BUBR1 in cells treated with the drug. Prolonged treatment of ovarian cancer cells with T-DM1 provoked the appearance of multinucleated cells which later led to cell death. Together, these data indicate that HER2 represents an important oncogene in ovarian cancer, and suggest that targeting this tyrosine kinase with T-DM1 may be therapeutically effective, especially in ovarian tumors with high content of HER2. PMID:26336133

  19. An EGFR wild type-EGFRvIII-HB-EGF feed forward loop regulates the activation of EGFRvIII

    PubMed Central

    Li, Li; Chakraborty, Sharmistha; Yang, Chin-Rang; Hatanpaa, Kimmo J.; Cipher, Daisha J.; Puliyappadamba, Vineshkumar Thidil; Rehman, Alizeh; Jiwani, Ameena J.; Mickey, Bruce; Madden, Christopher; Raisanen, Jack; Burma, Sandeep; Saha, Debabrata; Wang, Zhixiang; Pingle, Sandeep C.; Kesari, Santosh; Boothman, David A.; Habib, Amyn A.

    2014-01-01

    EGFRvIII is a key oncogene in glioblastoma (GBM). EGFRvIII results from an in frame deletion in the extracellular domain of EGFR, does not bind ligand, and is thought to be constitutively active. While EGFRvIII dimerization is known to activate EGFRvIII, the factors that drive EGFRvIII dimerization and activation are not well understood. Here we present a new model of EGFRvIII activation and propose that oncogenic activation of EGFRvIII in glioma cells is driven by co-expressed activated EGFR wild type (EGFRwt). Increasing EGFRwt leads to a striking increase in EGFRvIII tyrosine phosphorylation and activation while silencing EGFRwt inhibits EGFRvIII activation. Both the dimerization arm and the kinase activity of EGFRwt are required for EGFRvIII activation. EGFRwt activates EGFRvIII by facilitating EGFRvIII dimerization. We have previously identified HB-EGF, a ligand for EGFRwt, as a gene induced specifically by EGFRvIII. In this study we show that HB-EGF, is induced by EGFRvIII only when EGFRwt is present. Remarkably, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII activation. Thus, increasing HB-EGF leads to a striking increase in EGFRvIII tyrosine phosphorylation while silencing HB-EGF attenuates EGFRvIII phosphorylation, suggesting that an EGFRvIII-HB-EGF-EGFRwt feed forward loop regulates EGFRvIII activation. Silencing EGFRwt or HB-EGF leads to a striking inhibition of EGFRvIII induced tumorigenicity, while increasing EGFRwt or HB-EGF levels resulted in accelerated EGFRvIII mediated oncogenicity in an orthotopic mouse model. Furthermore, we demonstrate the existence of this loop in human GBM. Thus, our data demonstrate that oncogenic activation of EGFRvIII in GBM is likely maintained by a continuous EGFRwt-EGFRvIII-HBEGF loop, potentially an attractive target for therapeutic intervention. PMID:24077285

  20. Structural basis for stem cell factor–KIT signaling and activation of class III receptor tyrosine kinases

    PubMed Central

    Liu, Heli; Chen, Xiaoyan; Focia, Pamela J; He, Xiaolin

    2007-01-01

    Stem cell factor (SCF) binds to and activates the KIT receptor, a class III receptor tyrosine kinase (RTK), to stimulate diverse processes including melanogenesis, gametogenesis and hematopoeisis. Dysregulation of KIT activation is associated with many cancers. We report a 2.5 Å crystal structure of the functional core of SCF bound to the extracellular ligand-binding domains of KIT. The structure reveals a ‘wrapping' SCF-recognition mode by KIT, in which KIT adopts a bent conformation to facilitate each of its first three immunoglobulin (Ig)-like domains to interact with SCF. Three surface epitopes on SCF, an extended loop, the B and C helices, and the N-terminal segment, contact distinct KIT domains, with two of the epitopes undergoing large conformational changes upon receptor binding. The SCF/KIT complex reveals a unique RTK dimerization assembly, and a novel recognition mode between four-helix bundle cytokines and Ig-family receptors. It serves as a framework for understanding the activation mechanisms of class III RTKs. PMID:17255936

  1. LAT-1 activity of meta-substituted phenylalanine and tyrosine analogs.

    PubMed

    Augustyn, Evan; Finke, Karissa; Zur, Arik A; Hansen, Logan; Heeren, Nathan; Chien, Huan-Chieh; Lin, Lawrence; Giacomini, Kathleen M; Colas, Claire; Schlessinger, Avner; Thomas, Allen A

    2016-06-01

    The transporter protein Large-neutral Amino Acid Transporter 1 (LAT-1, SLC7A5) is responsible for transporting amino acids such as tyrosine and phenylalanine as well as thyroid hormones, and it has been exploited as a drug delivery mechanism. Recently its role in cancer has become increasingly appreciated, as it has been found to be up-regulated in many different tumor types, and its expression levels have been correlated with prognosis. Substitution at the meta position of aromatic amino acids has been reported to increase affinity for LAT-1; however, the SAR for this position has not previously been explored. Guided by newly refined computational models of the binding site, we hypothesized that groups capable of filling a hydrophobic pocket would increase binding to LAT-1, resulting in improved substrates relative to parent amino acid. Tyrosine and phenylalanine analogs substituted at the meta position with halogens, alkyl and aryl groups were synthesized and tested in cis-inhibition and trans-stimulation cell assays to determine activity. Contrary to our initial hypothesis we found that lipophilicity was correlated with diminished substrate activity and increased inhibition of the transporter. The synthesis and SAR of meta-substituted phenylalanine and tyrosine analogs is described.

  2. LAT-1 activity of meta-substituted phenylalanine and tyrosine analogs.

    PubMed

    Augustyn, Evan; Finke, Karissa; Zur, Arik A; Hansen, Logan; Heeren, Nathan; Chien, Huan-Chieh; Lin, Lawrence; Giacomini, Kathleen M; Colas, Claire; Schlessinger, Avner; Thomas, Allen A

    2016-06-01

    The transporter protein Large-neutral Amino Acid Transporter 1 (LAT-1, SLC7A5) is responsible for transporting amino acids such as tyrosine and phenylalanine as well as thyroid hormones, and it has been exploited as a drug delivery mechanism. Recently its role in cancer has become increasingly appreciated, as it has been found to be up-regulated in many different tumor types, and its expression levels have been correlated with prognosis. Substitution at the meta position of aromatic amino acids has been reported to increase affinity for LAT-1; however, the SAR for this position has not previously been explored. Guided by newly refined computational models of the binding site, we hypothesized that groups capable of filling a hydrophobic pocket would increase binding to LAT-1, resulting in improved substrates relative to parent amino acid. Tyrosine and phenylalanine analogs substituted at the meta position with halogens, alkyl and aryl groups were synthesized and tested in cis-inhibition and trans-stimulation cell assays to determine activity. Contrary to our initial hypothesis we found that lipophilicity was correlated with diminished substrate activity and increased inhibition of the transporter. The synthesis and SAR of meta-substituted phenylalanine and tyrosine analogs is described. PMID:27106710

  3. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  4. Shp2 protein tyrosine phosphatase inhibitor activity of estramustine phosphate and its triterpenoid analogs

    PubMed Central

    Scott, Latanya M.; Chen, Liwei; Daniel, Kenyon G.; Brooks, Wesley H.; Guida, Wayne C.; Lawrence, Harshani R.; Sebti, Said M.; Lawrence, Nicholas J.; Wu, Jie

    2010-01-01

    Shp2 protein tyrosine phosphate (PTP) is a novel target for anticancer drug discovery. We identified estramustine phosphate as a Shp2 PTP inhibitor from the National Cancer Institute Approved Oncology Drug set. A focused structure-activity relationship study indicated that the 17- phosphate group is required for the Shp2 PTP inhibitor activity of estramustine phosphate. A search for estramustine phosphate analogs led to identification of two triperpenoids, enoxolone and celastrol, having Shp2 PTP inhibitor activity. With the previously reported PTP1B inhibitor trodusquemine, our study reveals steroids and triterpenoids with negatively charged phosphate, carboxylate, or sulfonate groups as novel pharmacophores of selective PTP inhibitors. PMID:21193311

  5. Time-resolved luminescence detection of spleen tyrosine kinase activity through terbium sensitization.

    PubMed

    Lipchik, Andrew M; Parker, Laurie L

    2013-03-01

    Disruption of regulatory protein phosphorylation can lead to disease and is particularly prevalent in cancers. Inhibitors that target deregulated kinases are therefore a major focus of chemotherapeutic development. Achieving sensitivity and specificity in high-throughput compatible kinase assays is key to successful inhibitor development. Here, we describe the application of time-resolved luminescence detection to the direct sensing of spleen tyrosine kinase (Syk) activity and inhibition using a novel peptide substrate. Chelation and luminescence sensitization of Tb(3+) allowed the direct detection of peptide phosphorylation without any antibodies or other labeling reagents. Characterizing the Tb(3+) coordination properties of the phosphorylated vs unphosphorylated form of the peptide revealed that an inner-sphere water was displaced upon phosphorylation, which likely was responsible for both enhancing the luminescence intensity and also extending the lifetime, which enabled gating of the luminescence signal to improve the dynamic range. Furthermore, a shift in the optimal absorbance maximum for excitation was observed, from 275 nm (for the unphosphorylated tyrosine peptide) to 266 nm (for the phosphorylated tyrosine peptide). Accordingly, time-resolved measurements with excitation at 266 nm via a monochromator enabled a 16-fold improvement in base signal-to-noise for distinguishing phosphopeptide from unphosphorylated peptide. This led to a high degree of sensitivity and quantitative reproducibility, demonstrating the amenability of this method to both research laboratory and high-throughput applications.

  6. Closed-Loop and Activity-Guided Optogenetic Control

    PubMed Central

    Grosenick, Logan; Marshel, James H.; Deisseroth, Karl

    2016-01-01

    Advances in optical manipulation and observation of neural activity have set the stage for widespread implementation of closed-loop and activity-guided optical control of neural circuit dynamics. Closing the loop optogenetically (i.e., basing optogenetic stimulation on simultaneously observed dynamics in a principled way) is a powerful strategy for causal investigation of neural circuitry. In particular, observing and feeding back the effects of circuit interventions on physiologically relevant timescales is valuable for directly testing whether inferred models of dynamics, connectivity, and causation are accurate in vivo. Here we highlight technical and theoretical foundations as well as recent advances and opportunities in this area, and we review in detail the known caveats and limitations of optogenetic experimentation in the context of addressing these challenges with closed-loop optogenetic control in behaving animals. PMID:25856490

  7. Multi-Wavelength Study of Active Region Loop Dynamics

    NASA Astrophysics Data System (ADS)

    Banerjee, D.

    2006-11-01

    Observations have revealed the existence of weak transient disturbances in extended coronal loop systems. These propagating disturbances (PDs) originate from small scale brightenings at the footpoints of the loops and propagate upward along the loops. In all cases observed, the projected propagation speed is close to, but below the expected sound speed in the loops. This suggests that the PDs could be interpreted as slow mode MHD waves. Interpreting the oscillation in terms of different wave modes and/or plasma motions always depend on the line of sight as we observe in the limb or on the center of the disk. The JOP 165 campaign will address some of these questions. MDI and TRACE photospheric and UV imaging of TRACE and SPIRIT have been acquired simultaneously with high temporal and spatial coverage along with the spectroscopic data from CDS. EIT was operated in the shutter-less mode to achieve high Cadence. Some of the off- limb active region dynamics and oscillations observed during this JOP campaign will be focused in this presentation. Plasma condensations and temporal variations in active region loops will be also addressed.

  8. Zinc is a transmembrane agonist that induces platelet activation in a tyrosine phosphorylation-dependent manner.

    PubMed

    Watson, Ben R; White, Nathan A; Taylor, Kirk A; Howes, Joanna-Marie; Malcor, Jean-Daniel M; Bihan, Dominique; Sage, Stewart O; Farndale, Richard W; Pugh, Nicholas

    2016-01-01

    Following platelet adhesion and primary activation at sites of vascular injury, secondary platelet activation is induced by soluble platelet agonists, such as ADP, ATP, thrombin and thromboxane. Zinc ions are also released from platelets and damaged cells and have been shown to act as a platelet agonist. However, the mechanism of zinc-induced platelet activation is not well understood. Here we show that exogenous zinc gains access to the platelet cytosol and induces full platelet aggregation that is dependent on platelet protein tyrosine phosphorylation, PKC and integrin αIIbβ3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2β1. Low concentrations of ZnSO4 potentiated platelet aggregation by collagen-related peptide (CRP-XL), thrombin and adrenaline. Chelation of intracellular zinc reduced platelet aggregation induced by a number of different agonists, inhibited zinc-induced tyrosine phosphorylation and inhibited platelet activation in whole blood under physiologically relevant flow conditions. Our data are consistent with a transmembrane signalling role for zinc in platelet activation during thrombus formation.

  9. Astragaloside II triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity

    PubMed Central

    Wan, Chun-ping; Gao, Li-xin; Hou, Li-fei; Yang, Xiao-qian; He, Pei-lan; Yang, Yi-fu; Tang, Wei; Yue, Jian-min; Li, Jia; Zuo, Jian-ping

    2013-01-01

    Aim: To investigate the immunomodulating activity of astragalosides, the active compounds from a traditional tonic herb Astragalus membranaceus Bge, and to explore the molecular mechanisms underlying the actions, focusing on CD45 protein tyrosine phosphatase (CD45 PTPase), which plays a critical role in T lymphocyte activation. Methods: Primary splenocytes and T cells were prepared from mice. CD45 PTPase activity was assessed using a colorimetric assay. Cell proliferation was measured using a [3H]-thymidine incorporation assay. Cytokine proteins and mRNAs were examined with ELISA and RT-PCR, respectively. Activation markers, including CD25 and CD69, were analyzed using flow cytometry. Activation of LCK (Tyr505) was detected using Western blot analysis. Mice were injected with the immunosuppressant cyclophosphamide (CTX, 80 mg/kg), and administered astragaloside II (50 mg/kg). Results: Astragaloside I, II, III, and IV concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC50 values ranged from 3.33 to 10.42 μg/mL. Astragaloside II (10 and 30 nmol/L) significantly enhanced the proliferation of primary splenocytes induced by ConA, alloantigen or anti-CD3. Astragaloside II (30 nmol/L) significantly increased IL-2 and IFN-γ secretion, upregulated the mRNA levels of IFN-γ and T-bet in primary splenocytes, and promoted CD25 and CD69 expression on primary CD4+ T cells upon TCR stimulation. Furthermore, astragaloside II (100 nmol/L) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells, which could be blocked by a specific CD45 PTPase inhibitor. In CTX-induced immunosuppressed mice, oral administration of astragaloside II restored the proliferation of splenic T cells and the production of IFN-γ and IL-2. However, astragaloside II had no apparent effects on B cell proliferation. Conclusion: Astragaloside II enhances T cell activation by regulating the activity of CD45 PTPase, which may explain why Astragalus

  10. Molecular docking studies of banana flower flavonoids as insulin receptor tyrosine kinase activators as a cure for diabetes mellitus

    PubMed Central

    Ganugapati, Jayasree; Baldwa, Aashish; Lalani, Sarfaraz

    2012-01-01

    Diabetes mellitus is a metabolic disorder caused due to insulin deficiency. Banana flower is a rich source of flavonoids that exhibit anti diabetic activity. Insulin receptor is a tetramer that belongs to a family of receptor tyrosine kinases. It contains two alpha subunits that form the extracellular domain and two beta subunits that constitute the intracellular tyrosine kinase domain. Insulin binds to the extracellular region of the receptor and causes conformational changes that lead to the activation of the tyrosine kinase. This leads to autophosphorylation, a step that is crucial in insulin signaling pathway. Hence, compounds that augment insulin receptor tyrosine kinase activity would be useful in the treatment of diabetes mellitus. The 3D structure of IR tyrosine kinase was obtained from PDB database. The list of flavonoids found in banana flower was obtained from USDA database. The structures of the flavonoids were obtained from NCBI Pubchem. Docking analysis of the flavonoids was performed using Autodock 4.0 and Autodock Vina. The results indicate that few of the flavonoids may be potential activators of IR tyrosine kinase. PMID:22493522

  11. Molecular docking studies of banana flower flavonoids as insulin receptor tyrosine kinase activators as a cure for diabetes mellitus.

    PubMed

    Ganugapati, Jayasree; Baldwa, Aashish; Lalani, Sarfaraz

    2012-01-01

    Diabetes mellitus is a metabolic disorder caused due to insulin deficiency. Banana flower is a rich source of flavonoids that exhibit anti diabetic activity. Insulin receptor is a tetramer that belongs to a family of receptor tyrosine kinases. It contains two alpha subunits that form the extracellular domain and two beta subunits that constitute the intracellular tyrosine kinase domain. Insulin binds to the extracellular region of the receptor and causes conformational changes that lead to the activation of the tyrosine kinase. This leads to autophosphorylation, a step that is crucial in insulin signaling pathway. Hence, compounds that augment insulin receptor tyrosine kinase activity would be useful in the treatment of diabetes mellitus. The 3D structure of IR tyrosine kinase was obtained from PDB database. The list of flavonoids found in banana flower was obtained from USDA database. The structures of the flavonoids were obtained from NCBI Pubchem. Docking analysis of the flavonoids was performed using Autodock 4.0 and Autodock Vina. The results indicate that few of the flavonoids may be potential activators of IR tyrosine kinase.

  12. The distribution of maximum temperatures of coronal active region loops

    NASA Technical Reports Server (NTRS)

    Teske, R. G.; Mayfield, E. B.

    1981-01-01

    Starting with the integrated emission measure distributions of solar active regions, the distribution of the maximum temperature parameter which characterizes individual plasma loops is determined. The observed emission measure distributions were determined by combining EUV and X-ray data from two separate experiments on ATM/Skylab. The present work sets some limits on such an approach. It is found that the distribution of maximum temperature has approximately the same shape as the integrated emission measure distributions, a result which is expected since most of the loop emission measure is near their maximum temperatures.

  13. Slow Magnetosonic Waves and Fast Flows in Active Region Loops

    NASA Technical Reports Server (NTRS)

    Ofman, L.; Wang, T. J.; Davila, J. M.

    2012-01-01

    Recent extreme ultraviolet spectroscopic observations indicate that slow magnetosonic waves are present in active region (AR) loops. Some of the spectral data were also interpreted as evidence of fast (approx 100-300 km/s) quasiperiodic flows. We have performed three-dimensional magnetohydrodynamic (3D MHD) modeling of a bipolar AR that contains impulsively generated waves and flows in coronal loops. The model AR is initiated with a dipole magnetic field and gravitationally stratified density, with an upflow-driven steadily or periodically in localized regions at the footpoints of magnetic loops. The resulting flows along the magnetic field lines of the AR produce higher density loops compared to the surrounding plasma by injection of material into the flux tubes and the establishment of siphon flow.We find that the impulsive onset of flows with subsonic speeds result in the excitation of damped slow magnetosonic waves that propagate along the loops and coupled nonlinearly driven fast-mode waves. The phase speed of the slow magnetosonic waves is close to the coronal sound speed. When the amplitude of the driving pulses is increased we find that slow shock-like wave trains are produced. When the upflows are driven periodically, undamped oscillations are produced with periods determined by the periodicity of the upflows. Based on the results of the 3D MHD model we suggest that the observed slow magnetosonic waves and persistent upflows may be produced by the same impulsive events at the bases of ARs.

  14. SLOW MAGNETOSONIC WAVES AND FAST FLOWS IN ACTIVE REGION LOOPS

    SciTech Connect

    Ofman, L.; Wang, T. J.; Davila, J. M.

    2012-08-01

    Recent extreme ultraviolet spectroscopic observations indicate that slow magnetosonic waves are present in active region (AR) loops. Some of the spectral data were also interpreted as evidence of fast ({approx}100-300 km s{sup -1}) quasi-periodic flows. We have performed three-dimensional magnetohydrodynamic (3D MHD) modeling of a bipolar AR that contains impulsively generated waves and flows in coronal loops. The model AR is initiated with a dipole magnetic field and gravitationally stratified density, with an upflow-driven steadily or periodically in localized regions at the footpoints of magnetic loops. The resulting flows along the magnetic field lines of the AR produce higher density loops compared to the surrounding plasma by injection of material into the flux tubes and the establishment of siphon flow. We find that the impulsive onset of flows with subsonic speeds result in the excitation of damped slow magnetosonic waves that propagate along the loops and coupled nonlinearly driven fast-mode waves. The phase speed of the slow magnetosonic waves is close to the coronal sound speed. When the amplitude of the driving pulses is increased we find that slow shock-like wave trains are produced. When the upflows are driven periodically, undamped oscillations are produced with periods determined by the periodicity of the upflows. Based on the results of the 3D MHD model we suggest that the observed slow magnetosonic waves and persistent upflows may be produced by the same impulsive events at the bases of ARs.

  15. A protein tyrosine phosphatase-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activities.

    PubMed

    Takagi, T; Taylor, G S; Kusakabe, T; Charbonneau, H; Buratowski, S

    1998-08-18

    The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5'-phosphatase. BVP sequentially removes gamma and beta phosphates from the 5' end of triphosphate-terminated RNA, leaving a 5'-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA. PMID:9707557

  16. Structure and activation of MuSK, a receptor tyrosine kinase central to neuromuscular junction formation.

    PubMed

    Hubbard, Stevan R; Gnanasambandan, Kavitha

    2013-10-01

    MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in the formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases. PMID:23467009

  17. Epidermal growth factor receptor activation by diesel particles is mediated by tyrosine phosphatase inhibition

    SciTech Connect

    Tal, Tamara L.; Bromberg, Philip A.; Kim, Yumee; Samet, James M.

    2008-12-15

    Exposure to particulate matter (PM) is associated with increased cardiopulmonary morbidity and mortality. Diesel exhaust particles (DEP) are a major component of ambient PM and may contribute to PM-induced pulmonary inflammation. Proinflammatory signaling is mediated by phosphorylation-dependent signaling pathways whose activation is opposed by the activity of protein tyrosine phosphatases (PTPases) which thereby function to maintain signaling quiescence. PTPases contain an invariant catalytic cysteine that is susceptible to electrophilic attack. DEP contain electrophilic oxy-organic compounds that may contribute to the oxidant effects of PM. Therefore, we hypothesized that exposure to DEP impairs PTPase activity allowing for unopposed basal kinase activity. Here we report that exposure to 30 {mu}g/cm{sup 2} DEP for 4 h induces differential activation of signaling in primary cultures of human airway epithelial cells (HAEC), a primary target cell in PM inhalation. In-gel kinase activity assay of HAEC exposed to DEPs of low (L-DEP), intermediate (I-DEP) or high (H-DEP) organic content showed differential activation of intracellular kinases. Exposure to these DEP also induced varying levels of phosphorylation of the receptor tyrosine kinase EGFR in a manner that requires EGFR kinase activity but does not involve receptor dimerization. We demonstrate that treatment with DEP results in an impairment of total and EGFR-directed PTPase activity in HAEC with a potency that is independent of the organic content of these particles. These data show that DEP-induced EGFR phosphorylation in HAEC is the result of a loss of PTPase activities which normally function to dephosphorylate EGFR in opposition to baseline EGFR kinase activity.

  18. C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein.

    PubMed

    Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

    2011-02-15

    The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus, and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the virus-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in the extent of cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F protein fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in the extent of fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of addition of the HA tag varied with other fusion proteins, as parainfluenza virus 5 F-HA showed a decreased level of surface expression and no stimulation of fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope-tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in the modulation of the membrane fusion reaction promoted by these viral glycoproteins.

  19. The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.

    PubMed Central

    Poole, A; Gibbins, J M; Turner, M; van Vugt, M J; van de Winkel, J G; Saito, T; Tybulewicz, V L; Watson, S P

    1997-01-01

    Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor gamma-chain, the tyrosine kinase Syk and phospholipase Cgamma2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor gamma-chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cgamma2 by collagen stimulation is absent in mice lacking the Fc receptor gamma-chain. Tyrosine phosphorylation of phospholipase Cgamma2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor gamma-chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor gamma-chain or Syk. The absence of Fc receptor gamma-chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus. PMID:9171347

  20. Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase

    PubMed Central

    Klein, Tobias; Vajpai, Navratna; Phillips, Jonathan J.; Davies, Gareth; Holdgate, Geoffrey A.; Phillips, Chris; Tucker, Julie A.; Norman, Richard A.; Scott, Andrew D.; Higazi, Daniel R.; Lowe, David; Thompson, Gary S.; Breeze, Alexander L.

    2015-01-01

    Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp–Phe–Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called ‘DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a ‘DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-β4 loop and ‘molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1. PMID:26203596

  1. Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase

    NASA Astrophysics Data System (ADS)

    Klein, Tobias; Vajpai, Navratna; Phillips, Jonathan J.; Davies, Gareth; Holdgate, Geoffrey A.; Phillips, Chris; Tucker, Julie A.; Norman, Richard A.; Scott, Andrew D.; Higazi, Daniel R.; Lowe, David; Thompson, Gary S.; Breeze, Alexander L.

    2015-07-01

    Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp-Phe-Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called `DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a `DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-β4 loop and `molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.

  2. Prognostic value of cytosolic tyrosine kinase activity in 249 node-positive breast cancer patients.

    PubMed Central

    Romain, S.; Chinot, O.; Klijn, J. G.; van Putten, W. L.; Guirou, O.; Look, M.; Martin, P. M.; Foekens, J. A.

    1994-01-01

    Tyrosine-specific protein kinase (TPK) has been associated with the cytoplasmic domain of growth factor receptors as well as oncoproteins. Enzymatic activation appears to be a major initial event in these signal transduction pathways. In this study, TPK was determined in the cytosols of 249 node-positive primary breast tumours. Enzyme activity was measured using [32P]ATP and poly(glutamic acid-tyrosine) (4:1) as an artificial substrate. Levels of TPK varied from 0 to 35.9 pmol ATP min-1 mg-1 protein (median 11.4). No correlation was found with tumour size or number of positive lymph nodes. In contrast, levels of TPK were negatively associated with age (P = 0.01) and menopausal status (P < 0.05) of the patients. Higher concentrations of TPK were in addition found in tumours negative for oestradiol (P < 0.01) and progesterone (P < 0.05) receptors. Finally, a positive correlation was found between TPK and urokinase plasminogen activator (UPA) (P < 0.05). Patients whose tumours contained high levels of TPK had reduced disease-free (P = 0.01) and overall survival (P < 0.05). In Cox multivariate analysis, including patient's age, menopausal status, tumour size, number of positive lymph nodes, steroid receptors and UPA, TPK retained its independent prognostic importance. PMID:8054279

  3. Estrogen stimulates protein tyrosine phosphorylation and Src kinase activity in avian osteoclasts.

    PubMed

    Brubaker, K D; Gay, C V

    1999-12-01

    The estrogen, 17beta-estradiol, stimulated a profound increase in phosphotyrosine immunostaining of proteins that localized along the site of attachment in avian osteoclasts within 1 min of treatment. By 10 min, this rapidly occurring event had returned to basal levels. Pretreatment with 1 microM herbimycin A, a tyrosine kinase inhibitor, prevented the response. Immunoblotting revealed that Src kinase was one of the phosphorylated intermediates. Src kinase also appeared to translocate to the periphery of the cells during the 1 min 17beta-estradiol treatment and became dispersed by 10 min. Src kinase activity measurements indicated an increase in phosphotransferase activity after the 1 min estradiol treatment; this effect diminished with longer exposures to estrogen. Pretreatment of osteoclasts with 1 microg/ml cytochalasin B, an inhibitor of actin polymerization, delayed the appearance of increased phosphotyrosine immunostaining at attachment sites, possibly through inhibition of Src kinase translocation. These findings demonstrate that estrogen stimulates rapid tyrosine phosphorylation in osteoclasts, a process that involves activation and translocation of Src kinase to the plasma membrane.

  4. Structure-Functional Study of Tyrosine and Methionine Dipeptides: An Approach to Antioxidant Activity Prediction

    PubMed Central

    Torkova, Anna; Koroleva, Olga; Khrameeva, Ekaterina; Fedorova, Tatyana; Tsentalovich, Mikhail

    2015-01-01

    Quantum chemical methods allow screening and prediction of peptide antioxidant activity on the basis of known experimental data. It can be used to design the selective proteolysis of protein sources in order to obtain products with antioxidant activity. Molecular geometry and electronic descriptors of redox-active amino acids, as well as tyrosine and methionine-containing dipeptides, were studied by Density Functional Theory method. The calculated data was used to reveal several descriptors responsible for the antioxidant capacities of the model compounds based on their experimentally obtained antioxidant capacities against ABTS (2,2′-Azino-bis-(3-ethyl-benzothiazoline-6-sulfonate)) and peroxyl radical. A formula to predict antioxidant activity of peptides was proposed. PMID:26512651

  5. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    PubMed

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer. PMID:27481946

  6. Activation of tyrosine kinase c-Abl contributes to α-synuclein–induced neurodegeneration

    PubMed Central

    Lee, Su Hyun; Kim, Donghoon; Karuppagounder, Senthilkumar S.; Kumar, Manoj; Mao, Xiaobo; Shin, Joo Ho; Lee, Yunjong; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.; Ko, Han Seok

    2016-01-01

    Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein–induced neuropathology. In mice expressing a human α-synucleinopathy–associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein–induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies. PMID:27348587

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

    PubMed

    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.

  8. A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2

    PubMed Central

    1996-01-01

    Defects in the gene encoding Bruton's tyrosine kinase (Btk) result in a disease called X-linked agammaglobulinemia, in which there is a profound decrease of mature B cells due to a block in B cell development. Recent studies have shown that Btk is tyrosine phosphorylated and activated upon B cell antigen receptor (BCR) stimulation. To elucidate the functions of this kinase, we examined BCR signaling of DT40 B cells deficient in Btk. Tyrosine phosphorylation of phospholipase C (PLC)-gamma 2 upon receptor stimulation was significantly reduced in the mutant cells, leading to the loss of both BCR-coupled phosphatidylinositol hydrolysis and calcium mobilization. Pleckstrin homology and Src-homology 2 domains of Btk were required for PLC-gamma 2 activation. Since Syk is also required for the BCR-induced PLC-gamma 2 activation, our findings indicate that PLC-gamma 2 activation is regulated by Btk and Syk through their concerted actions. PMID:8691147

  9. Structure and Activation of MuSK, a Receptor Tyrosine Kinase Central to Neuromuscular Junction Formation

    PubMed Central

    Hubbard, Stevan R.; Gnanasambandan, Kavitha

    2014-01-01

    MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation. PMID:23467009

  10. The tyrosine kinase inhibitor nilotinib selectively inhibits CYP2C8 activities in human liver microsomes.

    PubMed

    Kim, Min-Jung; Lee, Jae-Won; Oh, Kyung-Suk; Choi, Chang-Soo; Kim, Kwang Hee; Han, Won Seok; Yoon, Chang-No; Chung, Eun Sook; Kim, Dong-Hyun; Shin, Jae-Gook

    2013-01-01

    The tyrosine kinase inhibitor nilotinib was examined for its inhibition of cytochrome P450s (CYPs) in human liver microsomes and in human CYPs expressed in a baculovirus-insect cell system. Nilotinib demonstrated preferential inhibition of CYP2C8-mediated paclitaxel 6α-hydroxylation, rosiglitazone hydroxylation and amodiaquine N-deethylation in human liver microsomes, with IC₅₀ values of 0.4, 7.5 and 0.7 µM, respectively. The IC₅₀ value of nilotinib for paclitaxel 6α-hydroxylation was 20-fold lower than that of the other five tyrosine-kinase inhibitors tested. Nilotinib appears to display competitive inhibition against paclitaxel 6α-hydroxylation and amodiaquine N-deethylation, with estimated mean Ki values of 0.90 and 0.15 µM in human liver microsomes and 0.10 and 0.61 µM in recombinant human CYP2C8, respectively. These results are consistent with those of molecular docking simulations, where paclitaxel could not access the CYP2C8 catalytic site in the presence of nilotinib, but the binding of midazolam, a substrate of CYP3A4, to the catalytic site of CYP3A4 was not affected by nilotinib. The demonstrated inhibitory activity of nilotinib against CYP2C8 at concentrations less than those observed in patients who received nilotinib therapy is of potential clinical relevance and further in vivo exploration is warranted.

  11. New peptide architectures through C–H activation stapling between tryptophan–phenylalanine/tyrosine residues

    PubMed Central

    Mendive-Tapia, Lorena; Preciado, Sara; García, Jesús; Ramón, Rosario; Kielland, Nicola; Albericio, Fernando; Lavilla, Rodolfo

    2015-01-01

    Natural peptides show high degrees of specificity in their biological action. However, their therapeutical profile is severely limited by their conformational freedom and metabolic instability. Stapled peptides constitute a solution to these problems and access to these structures lies on a limited number of reactions involving the use of non-natural amino acids. Here, we describe a synthetic strategy for the preparation of unique constrained peptides featuring a covalent bond between tryptophan and phenylalanine or tyrosine residues. The preparation of such peptides is achieved in solution and on solid phase directly from the corresponding sequences having an iodo-aryl amino acid through an intramolecular palladium-catalysed C–H activation process. Moreover, complex topologies arise from the internal stapling of cyclopeptides and double intramolecular arylations within a linear peptide. Finally, as a proof of principle, we report the application to this new stapling method to relevant biologically active compounds. PMID:25994485

  12. Activation of protein tyrosine kinase p72syk by Fc epsilon RI aggregation in rat basophilic leukemia cells. p72syk is a minor component but the major protein tyrosine kinase of pp72.

    PubMed

    Minoguchi, K; Benhamou, M; Swaim, W D; Kawakami, Y; Kawakami, T; Siraganian, R P

    1994-06-17

    Aggregation of the high affinity IgE receptors (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in protein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of approximately 72-kDa proteins (pp72) and that the tyrosine kinase p72syk is one component of pp72. Here we studied further the relationship of p72syk to pp72. The aggregation of Fc epsilon RI induced the activation of p72syk which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72syk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72syk was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phosphorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was p72syk. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated protens including 71-80-kDa proteins that associate with p53/56lyn. A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75HS1 (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72syk is activated after Fc epsilon RI aggregation. PMID:7515887

  13. A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.

    PubMed

    Demoulin, J B; Uyttenhove, C; Van Roost, E; DeLestré, B; Donckers, D; Van Snick, J; Renauld, J C

    1996-09-01

    Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

  14. Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity.

    PubMed

    Turner, Abigail H; Lebhar, Michael S; Proctor, Angela; Wang, Qunzhao; Lawrence, David S; Allbritton, Nancy L

    2016-02-19

    Although peptide-based reporters of protein tyrosine kinase (PTK) activity have been used to study PTK enzymology in vitro, the application of these reporters to intracellular conditions is compromised by their dephosphorylation, preventing PTK activity measurements. Nonproteinogenic amino acids may be utilized to rationally design selective peptidic ligands by accessing greater chemical and structural diversity than is available using the native amino acids. We describe a peptidic reporter that, upon phosphorylation by the epidermal growth factor receptor (EGFR), is resistant to dephosphorylation both in vitro and in cellulo. The reporter contains a conformationally constrained phosphorylatable moiety (7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in the place of L-tyrosine and is efficiently phosphorylated in A431 epidermoid carcinoma cells. Dephosphorylation of the reporter occurs 3 orders of magnitude more slowly compared with that of the conventional tyrosine-containing reporter.

  15. Bruton’s tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity

    PubMed Central

    Kawakami, Yuko; Miura, Toru; Bissonnette, Reid; Hata, Daisuke; Khan, Wasif N.; Kitamura, Toshio; Maeda-Yamamoto, Mari; Hartman, Stephen E.; Yao, Libo; Alt, Frederick W.; Kawakami, Toshiaki

    1997-01-01

    Mast cells derived from Bruton’s tyrosine kinase (Btk)-defective xid or btk null mice showed greater expansion in culture containing interleukin-3 (IL-3) than those from wild-type (wt) mice. Although the proliferative response to IL-3 was not significantly different between the wt and xid mast cells, xid and btk null mast cells died by apoptosis more slowly than their wt counterparts upon IL-3 deprivation. Consistent with these findings, the apoptosis-linked c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity was compromised in these btk-mutated cells upon FcɛRI crosslinking or upon stimulation with IL-3 or with stem cell factor. p38 activity was less severely, but significantly, affected by btk mutation, whereas extracellular signal-regulated kinases were not affected by the same mutation. Btk-mediated regulation of apoptosis and JNK activity was confirmed by reconstitution of btk null mutant mast cells with the wt btk cDNA. Furthermore, growth factor withdrawal induced the activation and sustained activity of JNK in wt mast cells, while JNK activity was consistently lower in btk-mutated mast cells. These results support the notion that Btk regulates apoptosis through the JNK activation. PMID:9108083

  16. Demonstration of protein tyrosine phosphatase activity in the second of two homologous domains of CD45.

    PubMed

    Tan, X; Stover, D R; Walsh, K A

    1993-04-01

    It has been reported that alteration of deletion of critical residues within one of the two homologous protein tyrosine phosphatase (PTPase)-like domains of CD45 completely abolishes all activity, suggesting that only the more N-terminal domain is catalytically active. However, we now demonstrate, by two independent techniques, that the second (C-terminal) domain is also a viable phosphatase. Limited proteolysis by endoproteinase Lys-C or trypsin increased the phosphatase activity toward reduced, carboxymethylated, and maleylated lysozyme approximately 8-fold. A 50-kDa fragment, isolated by ion exchange chromatography, was found to be responsible for this activity. N-terminal sequencing revealed that this fragment includes less than half of the first phosphatase domain and most, if not all, of the second. In a second experiment, 109 residues, including the presumed catalytic region, were removed from domain I by site-directed mutagenesis. Expression of this construct in a mammalian cell line resulted in increased PTPase activity over nontransfected control cells. Isolation of the recombinant CD45 by immunoprecipitation and immunoaffinity chromatography revealed that it had phosphatase activity. Both of these experimental approaches demonstrate that the second conserved PTPase domain of CD45 is a functioning PTPase, but that external regulation may be required to express its activity in the context of the native molecule. PMID:8463207

  17. Phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) influence mushroom tyrosinase activity.

    PubMed

    Lejczak, B; Kafarski, P; Makowiecka, E

    1987-02-15

    A series of phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) were synthesized in order to study their interaction with mushroom tyrosinase. 1-Amino-2-(3,4-dihydroxyphenyl)ethylphosphonic acid and 1-amino-2-(3,4-dimethoxyphenyl)ethylphosphonic acid turned out to be substrates for mushroom tyrosinase with Km values of 3.3 mM and 9.3 mM respectively. Shortening of the alkyl chain by one methylene group gave amino-(3,4-dihydroxyphenyl)methylphosphonic acid, one of the most powerful known inhibitors of this enzyme. This compound, racemic as well as in its optically active forms, exerts a mixed type of inhibition with an affinity for the enzyme one order of magnitude greater than that of the natural substrate. PMID:3109385

  18. Phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) influence mushroom tyrosinase activity.

    PubMed Central

    Lejczak, B; Kafarski, P; Makowiecka, E

    1987-01-01

    A series of phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) were synthesized in order to study their interaction with mushroom tyrosinase. 1-Amino-2-(3,4-dihydroxyphenyl)ethylphosphonic acid and 1-amino-2-(3,4-dimethoxyphenyl)ethylphosphonic acid turned out to be substrates for mushroom tyrosinase with Km values of 3.3 mM and 9.3 mM respectively. Shortening of the alkyl chain by one methylene group gave amino-(3,4-dihydroxyphenyl)methylphosphonic acid, one of the most powerful known inhibitors of this enzyme. This compound, racemic as well as in its optically active forms, exerts a mixed type of inhibition with an affinity for the enzyme one order of magnitude greater than that of the natural substrate. PMID:3109385

  19. Discovery of novel glitazones incorporated with phenylalanine and tyrosine: synthesis, antidiabetic activity and structure-activity relationships.

    PubMed

    Prashantha Kumar, B R; Baig, Nasir R; Sudhir, Sai; Kar, Koyal; Kiranmai, M; Pankaj, M; Joghee, Nanjan M

    2012-12-01

    We report a series of new glitazones incorporated with phenylalanine and tyrosine. All the compounds were tested for their in vitro glucose uptake activity using rat-hemidiaphragm, both in presence and absence of insulin. Six of the most active compounds from the in vitro screening were taken forward for their in vivo triglyceride and glucose lowering activity against dexamethazone induced hyperlipidemia and insulin resistance in Wistar rats. The liver samples of rats that received the most active compounds, 23 and 24, in the in vivo studies, were subjected to histopathological examination to assess their short term hepatotoxicity. The investigations on the in vitro glucose uptake, in vivo triglyceride and glucose lowering activity are described here along with the quantitative structure-activity relationships.

  20. Mutation at a Strictly Conserved, Active Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity

    SciTech Connect

    Chen, Zhi-wei; Datta, Saumen; DuBois, Jennifer L.; Klinman, Judith P.; Mathews, F. Scott

    2010-09-07

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

  1. The distribution of maximum temperatures of coronal active region loops

    NASA Technical Reports Server (NTRS)

    Mayfield, E. B.; Teske, R. G.

    1980-01-01

    The emission measure distribution across the range 4.5 log T 6.5 was derived for several coronal active regions by combining EUV line fluxes with broadband X-ray fluxes. The distributions of the maximum temperature was then derived using a numerical model. It is shown that the emission measure distribution can be represented over the full range 5.6 log Tm 6.5 by the superposition of simple loop models, if the models incorporate a substantial rise in their individual emission measure distributions near the maximum temperature. The unresolved loops may have substantial area ratios, since it is this ratio that fixes the extent of the rise in the emission measure distribution. Since the bulk of the emission measure is then contributed from the loop tops, the distribution of maximum temperatures has approximately the same shape as does the integrated emission measure distributions. The EUV and X-ray data used were obtained by from two separate experiments on ATM/Skylab.

  2. Structure of Escherichia coli tyrosine Kinase Etk Reveals a Novel Activation Mechanism

    SciTech Connect

    Lee,D.; Zheng, J.; She, Y.; Jia, Z.

    2008-01-01

    While protein tyrosine (Tyr) kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. The inner-membrane Wzc/Etk protein belongs to the bacterial PTK family, which has an important function in regulating the polymerization and transport of virulence-determining capsular polysaccharide (CPS). The kinase uses a unique two-step activation process involving intra-phosphorylation of a Tyr residue, although the molecular mechanism remains unknown. Herein, we report the first crystal structure of a bacterial PTK, the C-terminal kinase domain of Escherichia coli Tyr kinase (Etk) at 2.5-Angstroms resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting mass spectrometric evidence of Etk, a unique activation mechanism is proposed that involves the phosphorylated Tyr residue, Y574, at the active site and its specific interaction with a previously unidentified key Arg residue, R614, to unblock the active site. Both in vitro kinase activity and in vivo antibiotics resistance studies using structure-guided mutants further support the novel activation mechanism.

  3. Rotenone enhances N-methyl-D-aspartate currents by activating a tyrosine kinase in rat dopamine neurons.

    PubMed

    Wu, Yan-Na; Martella, Giuseppina; Johnson, Steven W

    2007-11-19

    Our previous work showed that the pesticide rotenone increases the amplitude of inward currents evoked by N-methyl-D-aspartate (NMDA) in substantia nigra dopamine neurons. Using patch pipettes to record whole-cell currents in rat brain slices, we report that the rotenone-induced potentiation of NMDA current is blocked by the tyrosine kinase inhibitors genistein and PP1. This action of rotenone is mimicked by H2O2, which is also blocked by genistein. Our results suggest that the rotenone-dependent increase in NMDA current is mediated by release of reactive oxygen species that activates a protein tyrosine kinase.

  4. Kinetic Characterization of O-Phospho-L-Tyrosine Phosphohydrolase Activity of Two Fungal Phytases.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal phytases belonging to 'Histidine Acid Phosphatase' or HAP class of phosphomonoesterase that catalyzes the hydrolysis of phytic acid could also hydrolyze O-phospho-tyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phospho-tyrosine hydrola...

  5. c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution.

    PubMed

    Badger, John; Grover, Prerna; Shi, Haibin; Panjarian, Shoghag B; Engen, John R; Smithgall, Thomas E; Makowski, Lee

    2016-06-14

    Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity. PMID:27166638

  6. c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution

    PubMed Central

    2016-01-01

    Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity. PMID:27166638

  7. Identification of canonical tyrosine-dependent and non-canonical tyrosine-independent STAT3 activation sites in the intracellular domain of the interleukin 23 receptor.

    PubMed

    Floss, Doreen M; Mrotzek, Simone; Klöcker, Tobias; Schröder, Jutta; Grötzinger, Joachim; Rose-John, Stefan; Scheller, Jürgen

    2013-07-01

    Signaling of interleukin 23 (IL-23) via the IL-23 receptor (IL-23R) and the shared IL-12 receptor β1 (IL-12Rβ1) controls innate and adaptive immune responses and is involved in the differentiation and expansion of IL-17-producing CD4(+) T helper (TH17) cells. Activation of signal transducer and activator of transcription 3 (STAT3) appears to be the major signaling pathway of IL-23, and STAT binding sites were predicted in the IL-23R but not in the IL-12Rβ1 chain. Using site-directed mutagenesis and deletion variants of the murine and human IL-23R, we showed that the predicted STAT binding sites (pYXXQ; including Tyr-504 and Tyr-626 in murine IL-23R and Tyr-484 and Tyr-611 in human IL-23R) mediated STAT3 activation. Furthermore, we identified two uncommon STAT3 binding/activation sites within the murine IL-23R. First, the murine IL-23R carried the Y(542)PNFQ sequence, which acts as an unusual Src homology 2 (SH2) domain-binding protein activation site of STAT3. Second, we identified a non-canonical, phosphotyrosine-independent STAT3 activation motif within the IL-23R. A third predicted site, Tyr-416 in murine and Tyr-397 in human IL-23R, is involved in the activation of PI3K/Akt and the MAPK pathway leading to STAT3-independent proliferation of Ba/F3 cells upon stimulation with IL-23. In contrast to IL-6-induced short term STAT3 phosphorylation, cellular activation by IL-23 resulted in a slower but long term STAT3 phosphorylation, indicating that the IL-23R might not be a major target of negative feedback inhibition by suppressor of cytokine signaling (SOCS) proteins. In summary, we characterized IL-23-dependent signal transduction with a focus on STAT3 phosphorylation and identified canonical tyrosine-dependent and non-canonical tyrosine-independent STAT3 activation sites in the IL-23R.

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

    EPA Science Inventory

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

  9. Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

    PubMed Central

    Cavalheiro, Renan P.; Machado, Daisy; Cruz, Bread L. G.; Paredes-Gamero, Edgar J.; Gomes-Marcondes, Maria C. C.; Zambuzzi, Willian F.; Vasques, Luciana; Nader, Helena B.; Souza, Ana Carolina S.; Justo, Giselle Z.

    2015-01-01

    Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death. PMID:25781955

  10. Activation of the low molecular weight protein tyrosine phosphatase in keratinocytes exposed to hyperosmotic stress.

    PubMed

    Silva, Rodrigo A; Palladino, Marcelly V; Cavalheiro, Renan P; Machado, Daisy; Cruz, Bread L G; Paredes-Gamero, Edgar J; Gomes-Marcondes, Maria C C; Zambuzzi, Willian F; Vasques, Luciana; Nader, Helena B; Souza, Ana Carolina S; Justo, Giselle Z

    2015-01-01

    Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death. PMID:25781955

  11. IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation

    PubMed Central

    Kawaai, Katsuhiro; Mizutani, Akihiro; Shoji, Hirotaka; Ogawa, Naoko; Ebisui, Etsuko; Kuroda, Yukiko; Wakana, Shigeharu; Hisatsune, Chihiro; Mikoshiba, Katsuhiko

    2015-01-01

    Inositol 1,4,5-trisphosphate receptor (IP3R) binding protein released with IP3 (IRBIT) contributes to various physiological events (electrolyte transport and fluid secretion, mRNA polyadenylation, and the maintenance of genomic integrity) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain. Here we identified calcium calmodulin-dependent kinase II alpha (CaMKIIα) as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses CaMKIIα kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In addition, we show that mice lacking IRBIT present with elevated catecholamine levels, increased locomotor activity, and social abnormalities. The level of tyrosine hydroxylase (TH) phosphorylation by CaMKIIα, which affects TH activity, was significantly increased in the ventral tegmental area of IRBIT-deficient mice. We concluded that IRBIT suppresses CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation. PMID:25922519

  12. Hot topic, warm loops, cooling plasma? Multithermal analysis of active region loops

    SciTech Connect

    Schmelz, J. T.; Pathak, S.; Christian, G. M.; Dhaliwal, R. S.; Brooks, D. H.

    2014-11-10

    We have found indications of a relationship between the differential emission measure (DEM) weighted temperature and the cross-field DEM width for coronal loops. The data come from the Hinode X-ray Telescope, the Hinode EUV Imaging Spectrometer, and the Solar Dynamics Observatory Atmospheric Imaging Assembly. These data show that cooler loops tend to have narrower DEM widths. If most loops observed by these instruments are composed of bundles of unresolved magnetic strands and are only observed in their cooling phase, as some studies have suggested, then this relationship implies that the DEM of a coronal loop narrows as it cools. This could imply that fewer strands are seen emitting in the later cooling phase, potentially resolving the long standing controversy of whether the cross-field temperatures of coronal loops are multithermal or isothermal.

  13. Hot Topic, Warm Loops, Cooling Plasma? Multithermal Analysis of Active Region Loops

    NASA Astrophysics Data System (ADS)

    Schmelz, J. T.; Pathak, S.; Brooks, D. H.; Christian, G. M.; Dhaliwal, R. S.

    2014-11-01

    We have found indications of a relationship between the differential emission measure (DEM) weighted temperature and the cross-field DEM width for coronal loops. The data come from the Hinode X-ray Telescope, the Hinode EUV Imaging Spectrometer, and the Solar Dynamics Observatory Atmospheric Imaging Assembly. These data show that cooler loops tend to have narrower DEM widths. If most loops observed by these instruments are composed of bundles of unresolved magnetic strands and are only observed in their cooling phase, as some studies have suggested, then this relationship implies that the DEM of a coronal loop narrows as it cools. This could imply that fewer strands are seen emitting in the later cooling phase, potentially resolving the long standing controversy of whether the cross-field temperatures of coronal loops are multithermal or isothermal.

  14. Short-term effects of endothelins on tyrosine hydroxylase activity and expression in the olfactory bulb of normotensive rats.

    PubMed

    Nabhen, Sabrina L; Perfume, Guadalupe; Battistone, María A; Rossi, Andrés; Abramoff, Tamara; Bianciotti, Liliana G; Vatta, Marcelo S

    2009-05-01

    The olfactory system in rats is part of the limbic region with extensive afferent connections with brain areas involved in the regulation of behaviour and autonomic responses. The existence of the endothelin system and catecholaminergic neurons in the olfactory bulb suggests that endothelins may modulate noradrenergic transmission and diverse olfactory mediated processes. In the present work we studied the effect of endothelin-1 and -3 on neuronal norepinephrine release and the short-term regulation of tyrosine hydroxylase in the olfactory bulb. Results showed that both endothelins increased tyrosine hydroxylase activity through the activation of a non-conventional endothelin G-protein coupled receptor, coupled to the stimulation of protein kinase A and C, as well as Ca(2+)/calmodulin-dependent protein kinase II. On the other hand, neither endothelin-1 nor endothelin-3 modified tyrosine hydroxylase total protein levels, but both peptides increased the phosphorylation of serine residues of the enzyme at sites 19 and 40. Furthermore, endothelins enhanced norepinephrine release in olfactory neurons suggesting that this event may contribute to increased tyrosine hydroxylase activity by reducing the feedback inhibition. Taken together present findings show a clear interaction between the endothelin system, and the catecholaminergic transmission in the olfactory bulb. Additional studies are required to evaluate the physiological functions regulated by endothelins at this brain level.

  15. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice

    PubMed Central

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  16. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice.

    PubMed

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  17. Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition.

    PubMed

    Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D; Nencioni, Alessio

    2015-05-20

    Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use.

  18. Novel Small Molecule Activators of the Trk Family of Receptor Tyrosine Kinases

    PubMed Central

    Obianyo, Obiamaka; Ye, Keqiang

    2012-01-01

    The Tropomyosin-related kinase (Trk) receptors are a subset of the receptor tyrosine kinase family with an important functionality in the regulation of neurotrophic signaling in the peripheral and central nervous system. As the receptors are able to mediate neuronal survival by associating with their respective neurotrophin ligands, many studies have focused on the therapeutic potential of generating small-molecule mimetic compounds that elicit agonistic effects similar to those of the natural protein ligands. To this end, various structure-based studies have led to the generation of bivalent peptide-based agonists and antibodies that selectively initiate Trk receptor signaling; however, these compounds do not possess the ideal characteristics of a potential drug. Additionally, the reliance of structure-based data to generate the compound libraries, limits the potential identification of novel chemical structures with desirable activity. Therefore, subsequent investigations utilized a cell-based apoptotic screen to facilitate the analysis of large, diverse chemical libraries of small molecules and quickly identify compounds with Trk-dependent antiapoptotic activity. Herein, we describe the Trk agonists that have been identified by this screening methodology and summarize their in vitro and in vivo neurotrophic activity as well as their efficacy in various neurological disease models, implicating their future utility as therapeutic compounds. PMID:22982231

  19. Effects of metformin on insulin receptor tyrosine kinase activity in rat adipocytes.

    PubMed

    Jacobs, D B; Hayes, G R; Truglia, J A; Lockwood, D H

    1986-11-01

    The cellular mechanism(s) by which the biguanide, metformin, exerts its antihyperglycaemic effect was investigated. Rat adipocytes were either treated acutely (2 h) or maintained in a biochemically defined medium (20 h) in the presence or absence of metformin (1 X 10(-4) mol/l). Exposure to the drug resulted in a significant enhancement (p less than 0.01) of hexose transport in both the absence (basal) and presence of insulin. Stimulation of transport was not explained by the increase in the basal state alone, since the incremental response to maximally effective concentrations of insulin was significantly enhanced p less than 0.025. Insulin-receptor tyrosine kinase activity was examined under the same experimental conditions. Activity of the kinase was unaltered as evaluated by phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. Furthermore, in this investigation neither insulin receptor number nor affinity was changed in adipose tissue treated with metformin. These studies indicate that metformin potentiates the effect of insulin on glucose transport at a site(s) beyond insulin receptor binding and phosphorylation.

  20. Loss of the tyrosine phosphatase PTPRD leads to aberrant STAT3 activation and promotes gliomagenesis.

    PubMed

    Ortiz, Berenice; Fabius, Armida W M; Wu, Wei H; Pedraza, Alicia; Brennan, Cameron W; Schultz, Nikolaus; Pitter, Kenneth L; Bromberg, Jacqueline F; Huse, Jason T; Holland, Eric C; Chan, Timothy A

    2014-06-01

    PTPRD, which encodes the protein tyrosine phosphatase receptor-δ, is one of the most frequently inactivated genes across human cancers, including glioblastoma multiforme (GBM). PTPRD undergoes both deletion and mutation in cancers, with copy number loss comprising the primary mode of inactivation in GBM. However, it is unknown whether loss of PTPRD promotes tumorigenesis in vivo, and the mechanistic basis of PTPRD function in tumors is unclear. Here, using genomic analysis and a glioma mouse model, we demonstrate that loss of Ptprd accelerates tumor formation and define the oncogenic context in which Ptprd loss acts. Specifically, we show that in human GBMs, heterozygous loss of PTPRD is the predominant type of lesion and that loss of PTPRD and the CDKN2A/p16(INK4A) tumor suppressor frequently co-occur. Accordingly, heterozygous loss of Ptprd cooperates with p16 deletion to drive gliomagenesis in mice. Moreover, loss of the Ptprd phosphatase resulted in phospho-Stat3 accumulation and constitutive activation of Stat3-driven genetic programs. Surprisingly, the consequences of Ptprd loss are maximal in the heterozygous state, demonstrating a tight dependence on gene dosage. Ptprd loss did not increase cell proliferation but rather altered pathways governing the macrophage response. In total, we reveal that PTPRD is a bona fide tumor suppressor, pinpoint PTPRD loss as a cause of aberrant STAT3 activation in gliomas, and establish PTPRD loss, in the setting of CDKN2A/p16(INK4A) deletion, as a driver of glioma progression.

  1. Malignant transformation of early lymphoid progenitors in mice expressing an activated Blk tyrosine kinase

    PubMed Central

    Malek, Sami N.; Dordai, Dominic I.; Reim, Johannes; Dintzis, Howard; Desiderio, Stephen

    1998-01-01

    The intracellular signals governing cellular proliferation and developmental progression during lymphocyte development are incompletely understood. The tyrosine kinase Blk is expressed preferentially in the B lineage, but its function in B cell development has been largely unexplored. We have generated transgenic mice expressing constitutively active Blk [Blk(Y495F)] in the B and T lymphoid compartments. Expression of Blk(Y495F) in the B lineage at levels similar to that of endogenous Blk induced B lymphoid tumors of limited clonality, whose phenotypes are characteristic of B cell progenitors at the proB/preB-I to preB-II transition. Expression of constitutively active Blk in the T lineage resulted in the appearance of clonal, thymic lymphomas composed of intermediate single positive cells. Taken together, these results indicate that specific B and T cell progenitor subsets are preferentially susceptible to transformation by Blk(Y495F) and suggest a role for Blk in the control of proliferation during B cell development. PMID:9636152

  2. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons.

    PubMed

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson's disease.

  3. Transcription repressor activity of spleen tyrosine kinase mediates breast tumor suppression

    PubMed Central

    Wang, Lei; Devarajan, Eswaran; He, Jin; Reddy, Sekhar P.; Le Dai, Jia

    2005-01-01

    Spleen tyrosine kinase (SYK) is a candidate tumor suppressor gene in breast. Loss of SYK expression in breast tumors as a result of DNA hypermethylation promotes tumor cell proliferation and invasion and predicts shorter survival of breast cancer patients. We previously reported that, in addition to its well-known cytoplasmic localization, the full length Syk is also present in the nucleus, and that Syk nuclear translocation is a rate-limiting step to determine Syk tumor suppressor function. Here we show that the full-length form of Syk acts as a transcription repressor in the cell nucleus. Ectopic expression of Syk down-regulates the transcription of FRA1 and cyclin D1 oncogenes. This transcription repressing activity of Syk is associated with its binding to members of the histone deacetylase family. Syk interacts with transcription factor Sp1 at the Sp1 DNA-binding site in the FRA1 promoter to repress Sp1-activated FRA1 transcription. Thus, breast tumorigenesis and progression resulting from the loss of SYK are underscored by the de-repression of Sp1-mediated oncogene transcription. PMID:16288017

  4. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons

    PubMed Central

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson’s disease. PMID:26886559

  5. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    SciTech Connect

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F.

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

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

    NASA Technical Reports Server (NTRS)

    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.

  7. Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades

    PubMed Central

    Jiang, Cheng-shi; Liang, Lin-fu; Guo, Yue-wei

    2012-01-01

    This article provides an overview of approximately 300 secondary metabolites with inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), which were isolated from various natural sources or derived from synthetic process in the last decades. The structure-activity relationship and the selectivity of some compounds against other protein phosphatases were also discussed. Potential pharmaceutical applications of several PTP1B inhibitors were presented. PMID:22941286

  8. Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity.

    PubMed Central

    Bollag, G E; Roth, R A; Beaudoin, J; Mochly-Rosen, D; Koshland, D E

    1986-01-01

    The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC 2.7.1.37). This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112). These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor. Images PMID:3526339

  9. Leptin receptor activation increases Sam68 tyrosine phosphorylation and expression in human trophoblastic cells.

    PubMed

    Sánchez-Jiménez, Flora; Pérez-Pérez, Antonio; González-Yanes, Carmen; Najib, Souad; Varone, Cecilia L; Sánchez-Margalet, Víctor

    2011-01-30

    Leptin is produced in placenta where it has been found to be an important autocrine signal for trophoblastic growth during pregnancy, promoting antiapoptotic and trophic effects. Leptin receptor is present in trophoblastic cells and leptin may fully activate signaling. We have previously implicated the RNA-binding protein Sam68 in leptin signal transduction in immune cells. In the present work, we have studied the possible role of Sam68 in leptin receptor signaling in trophoblastic cells (JEG-3 cells). Leptin dose-dependently stimulated Sam68 phosphorylation in JEG-3 cells, as assessed by immunoprecipitation and immunoblot with anti-phosphotyrosine antibodies. As previously observed in other systems, tyrosine phosphorylation of Sam68 in response to leptin inhibits its RNA binding capacity. Besides, leptin stimulation dose-dependently increases Sam68 expression in JEG-3 cells, as assessed by quantitative PCR. Consistently, the amount of Sam68 protein is increased after 24h of leptin stimulation of trophoblastic cells. In order to study the possible role of Sam68 on leptin receptor synthesis, we employed antisense strategy to knockdown the expression of Sam68. We have found that a decrease in Sam68 expression leads to a decrease in leptin receptor amount in JEG-3 cells, as assessed both by quantitative PCR and immunoblot. These results strongly suggest the participation of Sam68 in leptin receptor signaling in human trophoblastic cells, and therefore, Sam68 may mediate some of the leptin effects in placenta. PMID:21035519

  10. Insulin binding and receptor tyrosine kinase activity in skeletal muscle of carnivorous and omnivorous fish.

    PubMed

    Párrizas, M; Planas, J; Plisetskaya, E M; Gutiérrez, J

    1994-06-01

    We characterized the insulin receptors in skeletal muscle from several fish species with different nutritional preferences: brown trout (Salmo trutta fario), gilthead sea bream (Sparus aurata), tilapia (Tilapia mossambica), and carp (Cyprinus carpio), semipurified by affinity chromatography (wheat germ agglutinin-agarose). Total specific binding and number of receptors per unit weight of piscine white skeletal muscle were lower than those values found in mammalian skeletal muscle. The same parameters in carp muscle receptor preparations were severalfold higher than in trout muscle (binding capacity 440 +/- 47 fmol/mg glycoprotein in carp and 82 +/- 23 fmol/mg glycoprotein in trout). Piscine insulin receptors phosphorylated exogenous substrate poly(Glu,Tyr) but less so than mammalian receptors. Tyrosine kinase activity of receptors, calculated as percent of 32P incorporated into substrate in the presence of insulin compared with basal incorporation, was also highest in carp (210 +/- 4%) and lowest in trout (150 +/- 2%). In both trout and carp deprived of food for 15 days, specific binding of insulin decreased. Nevertheless, differences between the two species were retained. Our results demonstrate that particular properties of insulin receptors in fish skeletal muscle may be related to nutritional preferences. This finding coincides with the phenomenon of differential glucose tolerance in fish: carnivorous fish, such as trout, are less tolerant, whereas omnivorous fish, such as carp, readily utilize a carbohydrate-rich diet. PMID:8024051

  11. Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity

    PubMed Central

    1994-01-01

    The molecular mechanisms whereby hyaluronan (HA) stimulates cell motility was investigated in a C-H-ras transformed 10T 1/2 fibroblast cell line (C3). A significant (p < 0.001) stimulation of C3 cell motility with HA (10 ng/ml) was accompanied by an increase in protein tyrosine phosphorylation as detected by anti-phosphotyrosine antibodies using immunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and transient with phosphorylation occurring within 1 min of HA addition and dissipating below control levels 10-15 min later. These responses were also elicited by an antibody generated against a peptide sequence within the HA receptor RHAMM. Treatment of cells with tyrosine kinase inhibitors (genistein, 10 micrograms/ml or herbimycin A, 0.5 micrograms/ml) or microinjection of anti-phosphotyrosine antibodies inhibited the transient protein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimulated tyrosine phosphorylation and the resulting cell locomotion, cytoskeletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapid assembly and disassembly of focal adhesions as revealed by immunofluorescent localization of vinculin. The time course with which HA and antibody induced focal adhesion turnover exactly paralleled the induction of transient protein tyrosine phosphorylation. In addition, phosphotyrosine staining colocalized with vinculin within structures in the lamellapodia of these cells. Notably, the focal adhesion kinase, pp125FAK, was rapidly phosphorylated and dephosphorylated after HA stimulation. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAMM. They also provide a possible molecular basis for focal adhesion turnover, a process that is

  12. Reactive Nitrogen Species and Hydrogen Sulfide as Regulators of Protein Tyrosine Phosphatase Activity

    PubMed Central

    2014-01-01

    Abstract Significance: Redox modifications of thiols serve as a molecular code enabling precise and complex regulation of protein tyrosine phosphatases (PTPs) and other proteins. Particular gasotransmitters and even the redox modifications themselves affect each other, of which a typical example is S-nitrosylation-mediated protection against the further oxidation of protein thiols. Recent Advances: For a long time, PTPs were considered constitutively active housekeeping enzymes. This view has changed substantially over the last two decades, and the PTP family is now recognized as a group of tightly and flexibly regulated fundamental enzymes. In addition to the conventional ways in which they are regulated, including noncovalent interactions, phosphorylation, and oxidation, the evidence that has accumulated during the past two decades suggests that many of these enzymes are also modulated by gasotransmitters, namely by nitric oxide (NO) and hydrogen sulfide (H2S). Critical Issues: The specificity and selectivity of the methods used to detect nitrosylation and sulfhydration remains to be corroborated, because several researchers raised the issue of false-positive results, particularly when using the most widespread biotin switch method. Further development of robust and straightforward proteomic methods is needed to further improve our knowledge of the full extent of the gasotransmitters-mediated changes in PTP activity, selectivity, and specificity. Further Directions: Results of the hitherto performed studies on gasotransmitter-mediated PTP signaling await translation into clinical medicine and pharmacotherapeutics. In addition to directly affecting the activity of particular PTPs, the use of reversible S-nitrosylation as a protective mechanism against oxidative stress should be of high interest. Antioxid. Redox Signal. 20, 2191–2209. PMID:24328688

  13. Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 2. Cooperative binding of ATP is limited to the initial turnover of the enzyme.

    PubMed

    Sheoran, Anita; First, Eric A

    2008-05-01

    The activation of D-tyrosine by tyrosyl-tRNA synthetase has been investigated using single and multiple turnover kinetic methods. In the presence of saturating concentrations of D-tyrosine, the activation reaction displays sigmoidal kinetics with respect to ATP concentration under single turnover conditions. In contrast, when the kinetics for the activation reaction are monitored using a steady-state (multiple turnover) pyrophosphate exchange assay, Michaelis-Menten kinetics are observed. Previous investigations indicated that activation of l-tyrosine by the K233A variant of Bacillus stearothermophilus tyrosyl-tRNA synthetase displays sigmoidal kinetics similar to those observed for activation of d-tyrosine by the wild-type enzyme. Kinetic analyses indicate that the sigmoidal behavior of the d-tyrosine activation reaction is not enhanced when Lys-233 is replaced by alanine. This supports the hypothesis that the mechanistic basis for the sigmoidal behavior is the same for both d-tyrosine activation by wild-type tyrosyl-tRNA synthetase and activation of l-tyrosine by the K233A variant. The observed sigmoidal behavior presents a paradox, as tyrosyl-tRNA synthetase displays an extreme form of negative cooperativity, known as "half-of-the-sites reactivity," with respect to tyrosine binding and tyrosyl-adenylate formation. We propose that the binding of D-tyrosine weakens the affinity with which ATP binds to the functional subunit in tyrosyl-tRNA synthetase. This allows ATP to bind initially to the nonfunctional subunit, inducing a conformational change in the enzyme that enhances the affinity of the functional subunit for ATP. The observation that sigmoidal kinetics are observed only under single turnover conditions suggests that this conformational change is stable over multiple rounds of catalysis. PMID:18319246

  14. Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase.

    PubMed Central

    Hayashi, H; Miyake, N; Kanai, F; Shibasaki, F; Takenawa, T; Ebina, Y

    1991-01-01

    Insulin causes a dramatic and rapid increase in phosphatidylinositol 3-kinase activity in the anti-phosphotyrosine immunoprecipitates of cells overexpressing the human insulin receptor. This enzyme may therefore be a mediator of insulin signal transduction [Endemann, Yonezawa & Roth (1990) J. Biol. Chem. 265, 396-400; Ruderman, Kapeller, White & Cantley (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415]. At least two questions remain to be elucidated. Firstly, does the insulin receptor tyrosine kinase phosphorylate phosphatidylinositol 3-kinase directly, or does it phosphorylate a protein associated with the 3-kinase? Second, if the enzyme is a direct substrate for the insulin receptor tyrosine kinase, does tyrosine phosphorylation of phosphatidylinositol 3-kinase by the kinase alter the specific enzyme activity, or does the amount of the tyrosine-phosphorylated form of the phosphatidylinositol 3-kinase increase, with no change in the specific activity? We report here evidence that the 85 kDa subunit of highly purified phosphatidylinositol 3-kinase is phosphorylated on the tyrosine residue by the activated normal insulin receptor in vitro, but not by a mutant insulin receptor which lacks tyrosine kinase activity. We found that an increase in enzyme activity was detected in response to insulin not only in the anti-phosphotyrosine immunoprecipitates of the cytosol, but also in the cytosolic fraction before immunoprecipitation. In addition, we partially separated the tyrosine-phosphorylated form from the unphosphorylated form of the enzyme, by using a f.p.l.c. Mono Q column. The insulin-stimulated phosphatidylinositol 3-kinase activity was mainly detected in the fraction containing almost all of the tyrosine-phosphorylated form. This result suggests that tyrosine phosphorylation of phosphatidylinositol 3-kinase by the insulin receptor kinase may increase the specific activity of the former enzyme in vivo. Images Fig. 1. Fig. 2. Fig. 4. PMID:1722393

  15. The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner.

    PubMed

    Xia, K; Mukhopadhyay, N K; Inhorn, R C; Barber, D L; Rose, P E; Lee, R S; Narsimhan, R P; D'Andrea, A D; Griffin, J D; Roberts, T M

    1996-10-15

    JAK2, a member of the Janus kinase superfamily was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-gamma and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of p21ras. Under these conditions, a ternary complex of p21ras, JAK2, and Raf-1 was observed. In contrast, in the absence of p21ras, coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp60v-src. In mammalian cells treated with either erythropoietin or interferon-gamma, a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and p21ras cooperate to activate Raf-1.

  16. CEP-701 and CEP-751 inhibit constitutively activated RET tyrosine kinase activity and block medullary thyroid carcinoma cell growth.

    PubMed

    Strock, Christopher J; Park, Jong-In; Rosen, Mark; Dionne, Craig; Ruggeri, Bruce; Jones-Bolin, Susan; Denmeade, Samuel R; Ball, Douglas W; Nelkin, Barry D

    2003-09-01

    All of the cases of medullary thyroid carcinoma (MTC) express the RET receptor tyrosine kinase. In essentially all of the hereditary cases and approximately 40% of the sporadic cases of MTC, the RET kinase is constitutively activated by mutation. This suggests that RET may be an effective therapeutic target for treatment of MTC. We show that the indolocarbazole derivatives, CEP-701 and CEP-751, inhibit RET in MTC cells. These compounds effectively inhibit RET phosphorylation in a dose-dependent manner at concentrations <100 nM in 0.5% serum and at somewhat higher concentrations in the presence of 16% serum. They also blocked the growth of these MTC cells in culture. CEP-751 and its prodrug, CEP-2563, also inhibited tumor growth in MTC cell xenografts. These results show that inhibiting RET can block the growth of MTC cells and may have a therapeutic benefit in MTC.

  17. Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A.

    PubMed

    Wan, Xiaofeng; Ru, Yanfei; Chu, Chen; Ni, Zimei; Zhou, Yuchuan; Wang, Shoulin; Zhou, Zuomin; Zhang, Yonglian

    2016-06-01

    Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility. PMID:27174873

  18. Tyrosine Hydroxylase Phosphorylation in Catecholaminergic Brain Regions: A Marker of Activation following Acute Hypotension and Glucoprivation

    PubMed Central

    Damanhuri, Hanafi A.; Burke, Peter G. R.; Ong, Lin K.; Bobrovskaya, Larisa; Dickson, Phillip W.; Dunkley, Peter R.; Goodchild, Ann K.

    2012-01-01

    The expression of c-Fos defines brain regions activated by the stressors hypotension and glucoprivation however, whether this identifies all brain sites involved is unknown. Furthermore, the neurochemicals that delineate these regions, or are utilized in them when responding to these stressors remain undefined. Conscious rats were subjected to hypotension, glucoprivation or vehicle for 30, 60 or 120 min and changes in the phosphorylation of serine residues 19, 31 and 40 in the biosynthetic enzyme, tyrosine hydroxylase (TH), the activity of TH and/or, the expression of c-Fos were determined, in up to ten brain regions simultaneously that contain catecholaminergic cell bodies and/or terminals: A1, A2, caudal C1, rostral C1, A6, A8/9, A10, nucleus accumbens, dorsal striatum and medial prefrontal cortex. Glucoprivation evoked phosphorylation changes in A1, caudal C1, rostral C1 and nucleus accumbens whereas hypotension evoked changes A1, caudal C1, rostral C1, A6, A8/9, A10 and medial prefrontal cortex 30 min post stimulus whereas few changes were evident at 60 min. Although increases in pSer19, indicative of depolarization, were seen in sites where c-Fos was evoked, phosphorylation changes were a sensitive measure of activation in A8/9 and A10 regions that did not express c-Fos and in the prefrontal cortex that contains only catecholaminergic terminals. Specific patterns of serine residue phosphorylation were detected, dependent upon the stimulus and brain region, suggesting activation of distinct signaling cascades. Hypotension evoked a reduction in phosphorylation in A1 suggestive of reduced kinase activity. TH activity was increased, indicating synthesis of TH, in regions where pSer31 alone was increased (prefrontal cortex) or in conjunction with pSer40 (caudal C1). Thus, changes in phosphorylation of serine residues in TH provide a highly sensitive measure of activity, cellular signaling and catecholamine utilization in catecholaminergic brain regions, in the

  19. Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin.

    PubMed

    Moog-Lutz, Christel; Degoutin, Joffrey; Gouzi, Jean Y; Frobert, Yvelyne; Brunet-de Carvalho, Nicole; Bureau, Jocelyne; Créminon, Christophe; Vigny, Marc

    2005-07-15

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. The nature of the cognate ligands of ALK in vertebrate is still a matter of debate. We produced a panel of monoclonal antibodies (mAbs) directed against the extracellular domain of the human receptor. Two major species of ALK (220 and 140 kDa) were identified in transfected cells, and the use of our mAbs established that the 140-kDa species results from a cleavage of the 220-kDa form. Two mAbs, in the nm range, induced the differentiation of PC12 cells transiently transfected with ALK. In human embryonic kidney 293 cells stably expressing ALK, these two mAbs strongly activated the receptor and subsequently the mitogen-activated protein kinase pathway. We further showed for the first time that activation of ALK also resulted in a specific activation of STAT3. In contrast, other mAbs presented the characteristics of blocking antibodies. Finally, in these cell systems, a mitogenic form of pleiotrophin, a proposed ligand of ALK, failed to activate this receptor. Thus, in the absence of clearly established ligand(s) in vertebrates, the availability of mAbs allowing the activation or the inhibition of the receptor will be essential for a better understanding of the biological roles of ALK.

  20. Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation.

    PubMed

    Hoyt, Laura R; Ather, Jennifer L; Randall, Matthew J; DePuccio, Daniel P; Landry, Christopher C; Wewers, Mark D; Gavrilin, Mikhail A; Poynter, Matthew E

    2016-08-15

    Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multiprotein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the proinflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol, and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow-derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of γ-aminobutyric acid A receptor activation or N-methyl-d-asparate receptor inhibition but were associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, whereas administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other

  1. Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation.

    PubMed

    Hoyt, Laura R; Ather, Jennifer L; Randall, Matthew J; DePuccio, Daniel P; Landry, Christopher C; Wewers, Mark D; Gavrilin, Mikhail A; Poynter, Matthew E

    2016-08-15

    Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multiprotein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the proinflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol, and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow-derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of γ-aminobutyric acid A receptor activation or N-methyl-d-asparate receptor inhibition but were associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, whereas administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other

  2. Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells

    PubMed Central

    Sela, Meirav; Bogin, Yaron; Beach, Dvora; Oellerich, Thomas; Lehne, Johanna; Smith-Garvin, Jennifer E; Okumura, Mariko; Starosvetsky, Elina; Kosoff, Rachelle; Libman, Evgeny; Koretzky, Gary; Kambayashi, Taku; Urlaub, Henning; Wienands, Jürgen; Chernoff, Jonathan; Yablonski, Deborah

    2011-01-01

    Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1. PMID:21725281

  3. Activation of the protein-tyrosine kinase associated with the bombesin receptor complex in small cell lung carcinomas.

    PubMed Central

    Gaudino, G; Cirillo, D; Naldini, L; Rossino, P; Comoglio, P M

    1988-01-01

    It has been hypothesized that bombesin-like peptides produced by small cell lung carcinomas may sustain deregulated proliferation through an autocrine mechanism. We have shown that the neuropeptide bombesin leads to the activation of a protein-tyrosine kinase that phosphorylates a 115-kDa protein (p115) associated with the bombesin receptor complex in mouse Swiss 3T3 fibroblasts. We now report that phosphotyrosine antibodies recognize a 115-kDa protein, phosphorylated on tyrosine, in four human small cell lung carcinoma cell lines producing bombesin but not in a nonproducer "variant" line. p115 from detergent-treated small cell lung carcinoma cells binds to bombesin-Sepharose and can be phosphorylated on tyrosine in the presence of radiolabeled ATP and Mn2+. As for the p115 immunoprecipitated from mouse fibroblast, the small cell lung carcinoma p115 can be phosphorylated in an immunocomplex kinase assay. However, the latter does not require the presence of exogenous bombesin for activity. Binding data, obtained by using radiolabeled ligand, suggest receptor occupancy in the cell lines producing bombesin. These observations are consistent with the hypothesis that proliferation in some human small cell lung carcinoma lines is under autocrine control, regulated through activation of bombesin receptors. Images PMID:2451242

  4. A critical tyrosine residue determines the uncoupling protein-like activity of the yeast mitochondrial oxaloacetate carrier.

    PubMed

    Luévano-Martínez, Luis A; Barba-Ostria, Carlos; Araiza-Olivera, Daniela; Chiquete-Félix, Natalia; Guerrero-Castillo, Sergio; Rial, Eduardo; Georgellis, Dimitris; Uribe-Carvajal, Salvador

    2012-04-01

    The mitochondrial Oac (oxaloacetate carrier) found in some fungi and plants catalyses the uptake of oxaloacetate, malonate and sulfate. Despite their sequence similarity, transport specificity varies considerably between Oacs. Indeed, whereas ScOac (Saccharomyces cerevisiae Oac) is a specific anion-proton symporter, the YlOac (Yarrowia lipolytica Oac) has the added ability to transport protons, behaving as a UCP (uncoupling protein). Significantly, we identified two amino acid changes at the matrix gate of YlOac and ScOac, tyrosine to phenylalanine and methionine to leucine. We studied the role of these amino acids by expressing both wild-type and specifically mutated Oacs in an Oac-null S. cerevisiae strain. No phenotype could be associated with the methionine to leucine substitution, whereas UCP-like activity was dependent on the presence of the tyrosine residue normally expressed in the YlOac, i.e. Tyr-ScOac mediated proton transport, whereas Phe-YlOac lost its protonophoric activity. These findings indicate that the UCP-like activity of YlOac is determined by the tyrosine residue at position 146.

  5. Mechanism of dual specificity kinase activity of DYRK1A.

    PubMed

    Walte, Agnes; Rüben, Katharina; Birner-Gruenberger, Ruth; Preisinger, Christian; Bamberg-Lemper, Simone; Hilz, Nikolaus; Bracher, Franz; Becker, Walter

    2013-09-01

    The function of many protein kinases is controlled by the phosphorylation of a critical tyrosine residue in the activation loop. Dual specificity tyrosine-phosphorylation-regulated kinases (DYRKs) autophosphorylate on this tyrosine residue but phosphorylate substrates on aliphatic amino acids. This study addresses the mechanism of dual specificity kinase activity in DYRK1A and related kinases. Tyrosine autophosphorylation of DYRK1A occurred rapidly during in vitro translation and did not depend on the non-catalytic domains or other proteins. Expression in bacteria as well as in mammalian cells revealed that tyrosine kinase activity of DYRK1A is not restricted to the co-translational autophosphorylation in the activation loop. Moreover, mature DYRK1A was still capable of tyrosine autophosphorylation. Point mutants of DYRK1A and DYRK2 lacking the activation loop tyrosine showed enhanced tyrosine kinase activity. A series of structurally diverse DYRK1A inhibitors was used to pharmacologically distinguish different conformational states of the catalytic domain that are hypothesized to account for the dual specificity kinase activity. All tested compounds inhibited substrate phosphorylation with higher potency than autophosphorylation but none of the tested inhibitors differentially inhibited threonine and tyrosine kinase activity. Finally, the related cyclin-dependent kinase-like kinases (CLKs), which lack the activation loop tyrosine, autophosphorylated on tyrosine both in vitro and in living cells. We propose a model of DYRK autoactivation in which tyrosine autophosphorylation in the activation loop stabilizes a conformation of the catalytic domain with enhanced serine/threonine kinase activity without disabling tyrosine phosphorylation. The mechanism of dual specificity kinase activity probably applies to related serine/threonine kinases that depend on tyrosine autophosphorylation for maturation. PMID:23809146

  6. Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

    PubMed

    Maisonneuve, Pierre; Caillet-Saguy, Célia; Vaney, Marie-Christine; Bibi-Zainab, Edoo; Sawyer, Kristi; Raynal, Bertrand; Haouz, Ahmed; Delepierre, Muriel; Lafon, Monique; Cordier, Florence; Wolff, Nicolas

    2016-08-01

    The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction.

  7. Involvement of the protein tyrosine phosphatase SHP-1 in Ras-mediated activation of the mitogen-activated protein kinase pathway.

    PubMed

    Krautwald, S; Büscher, D; Kummer, V; Buder, S; Baccarini, M

    1996-11-01

    Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kinase receptors or their substrates and relay positive mitogenic signals via the activation of the Ras-mitogen-activated protein kinase (MAPK) pathway. Conversely, the structurally related phosphatase SHP-1 is predominantly expressed in hemopoietic cells and becomes tyrosine phosphorylated upon colony-stimulating factor 1 treatment of macrophages without associating with the colony-stimulating factor 1 receptor tyrosine kinase. Mice lacking functional SHP-1 (me/me and me(v)/me(v)) develop systemic autoimmune disease with accumulation of macrophages, suggesting that SHP-1 may be a negative regulator of hemopoietic cell growth. By using macrophages expressing dominant negative Ras and the me(v)/me(v) mouse mutant, we show that SHP-1 is activated in the course of mitogenic signal transduction in a Ras-dependent manner and that its activity is necessary for the Ras-dependent activation of the MAPK pathway but not of the Raf-1 kinase. Consistent with a role for SHP-1 as an intermediate between Ras and the MEK-MAPK pathway, Ras-independent activation of the latter kinases by bacterial lipopolysaccharide occurred normally in me(v)/me(v) cells. Our results sharply accentuate the diversity of signal transduction in mammalian cells, in which the same signaling intermediates can be rearranged to form different pathways. PMID:8887625

  8. Involvement of the protein tyrosine phosphatase SHP-1 in Ras-mediated activation of the mitogen-activated protein kinase pathway.

    PubMed Central

    Krautwald, S; Büscher, D; Kummer, V; Buder, S; Baccarini, M

    1996-01-01

    Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kinase receptors or their substrates and relay positive mitogenic signals via the activation of the Ras-mitogen-activated protein kinase (MAPK) pathway. Conversely, the structurally related phosphatase SHP-1 is predominantly expressed in hemopoietic cells and becomes tyrosine phosphorylated upon colony-stimulating factor 1 treatment of macrophages without associating with the colony-stimulating factor 1 receptor tyrosine kinase. Mice lacking functional SHP-1 (me/me and me(v)/me(v)) develop systemic autoimmune disease with accumulation of macrophages, suggesting that SHP-1 may be a negative regulator of hemopoietic cell growth. By using macrophages expressing dominant negative Ras and the me(v)/me(v) mouse mutant, we show that SHP-1 is activated in the course of mitogenic signal transduction in a Ras-dependent manner and that its activity is necessary for the Ras-dependent activation of the MAPK pathway but not of the Raf-1 kinase. Consistent with a role for SHP-1 as an intermediate between Ras and the MEK-MAPK pathway, Ras-independent activation of the latter kinases by bacterial lipopolysaccharide occurred normally in me(v)/me(v) cells. Our results sharply accentuate the diversity of signal transduction in mammalian cells, in which the same signaling intermediates can be rearranged to form different pathways. PMID:8887625

  9. Sensitivity and kinase activity of epidermal growth factor receptor (EGFR) exon 19 and others to EGFR-tyrosine kinase inhibitors.

    PubMed

    Furuyama, Kazuto; Harada, Taishi; Iwama, Eiji; Shiraishi, Yoshimasa; Okamura, Kyoko; Ijichi, Kayo; Fujii, Akiko; Ota, Keiichi; Wang, Shuo; Li, Heyan; Takayama, Koichi; Giaccone, Giuseppe; Nakanishi, Yoichi

    2013-05-01

    The presence of epidermal growth factor receptor (EGFR) somatic mutations in non-small-cell lung cancer patients is associated with response to treatment with EGFR-tyrosine kinase inhibitors, such as gefitinib and erlotinib. More than 100 mutations in the kinase domain of EGFR have been identified. In particular there are many variations of deletion mutations in exon 19. In this study, using yellow fluorescent protein-tagged fragments of the EGFR intracellular domain, we examined the differences in sensitivity to gefitinib, erlotinib and afatinib between several exon 19 mutants and other common EGFR mutations. We also used serum of patients undergoing treatment with EGFR-tyrosine kinase inhibitors in this system. In addition, we examined the relative kinase activity of these mutants by measuring relative fluorescent intensity after immunofluorescence staining. We found that both sensitivity to EGFR-tyrosine kinase inhibitors and relative kinase activity differed among several EGFR mutations found in the same region of the kinase domain. This study underscores the importance of reporting the clinical outcome of treatment in relation to different EGFR mutations.

  10. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  11. The Kaposi's Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor Contains an Immunoreceptor Tyrosine-Based Inhibitory Motif That Activates Shp2 ▿

    PubMed Central

    Philpott, Nicola; Bakken, Thomas; Pennell, Christopher; Chen, Liwei; Wu, Jie; Cannon, Mark

    2011-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) is a constitutively active, highly angiogenic homologue of the interleukin-8 (IL-8) receptors that signals in part via the cytoplasmic protein tyrosine phosphatase Shp2. We show that vGPCR contains a bona fide immunoreceptor tyrosine-based inhibitory motif (ITIM) that binds and constitutively activates Shp2. PMID:21047965

  12. Insulin-induced tyrosine dephosphorylation of paxillin and focal adhesion kinase requires active phosphotyrosine phosphatase 1D.

    PubMed Central

    Ouwens, D M; Mikkers, H M; van der Zon, G C; Stein-Gerlach, M; Ullrich, A; Maassen, J A

    1996-01-01

    Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor beta-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(L-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D. PMID:8809054

  13. Gambogic Acid Inhibits STAT3 Phosphorylation Through Activation of Protein Tyrosine Phosphatase SHP-1: Potential Role in Proliferation and Apoptosis

    PubMed Central

    Prasad, Sahdeo; Pandey, Manoj K.; Yadav, Vivek R.; Aggarwal, Bharat B.

    2011-01-01

    The transcription factor, signal transducer and activator of transcription 3 (STAT3), is associated with proliferation, survival, and metastasis of cancer cells. We investigated whether gambogic acid (GA), a xanthone derived from the resin of traditional Chinese medicine, Gamboge hanburyi (mangosteen), can regulate the STAT3 pathway, leading to suppression of growth and sensitization of cancer cells. We found that GA induced apoptosis in human multiple myeloma cells that correlated with the inhibition of both constitutive and inducible STAT3 activation. STAT3 phosphorylation at both tyrosine residue 705 and serine residue 727 was inhibited by GA. STAT3 suppression was mediated through the inhibition of activation of the protein tyrosine kinases Janus-activated kinase (JAK) 1, and JAK2. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate reversed the GA-induced down-regulation of STAT3, suggesting the involvement of a PTP. We also found that GA induced the expression of the PTP SHP-1. Deletion of the SHP-1 gene by small interfering RNA suppressed the ability of GA to inhibit STAT3 activation and to induce apoptosis, suggesting the critical role of SHP-1 in its action. Moreover, GA down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) proteins, and this correlated with suppression of proliferation and induction of apoptosis. Overall, these results suggest that GA blocks STAT3 activation, leading to suppression of tumor cell proliferation and induction of apoptosis. PMID:21490133

  14. Regulatory role of tyrosine phosphorylation in the swelling-activated chloride current in isolated rabbit articular chondrocytes.

    PubMed

    Okumura, Noriaki; Imai, Shinji; Toyoda, Futoshi; Isoya, Eiji; Kumagai, Kousuke; Matsuura, Hiroshi; Matsusue, Yoshitaka

    2009-08-01

    Articular chondrocytes are exposed in vivo to the continually changing osmotic environment and thus require volume regulatory mechanisms. The present study was designed to investigate (i) the functional role of the swelling-activated Cl(-) current (I(Cl,swell)) in the regulatory volume decrease (RVD) and (ii) the regulatory role of tyrosine phosphorylation in I(Cl,swell), in isolated rabbit articular chondrocytes. Whole-cell membrane currents were recorded from chondrocytes in isosmotic, hyposmotic and hyperosmotic external solutions under conditions where Na(+), K(+) and Ca(2+) currents were minimized. The cell surface area was also measured using microscope images from a separate set of chondrocytes and was used as an index of cell volume. The isolated chondrocytes exhibited a RVD during sustained exposure to hyposmotic solution, which was mostly inhibited by the I(Cl,swell) blocker 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl)oxobutyric acid (DCPIB) at 20 microM. Exposure to a hyposmotic solution activated I(Cl,swell), which was also largely inhibited by 20 microM DCPIB. I(Cl,swell) in rabbit articular chondrocytes had a relative taurine permeability (P(tau)/P(Cl)) of 0.21. Activation of I(Cl,swell) was significantly reduced by the protein tyrosine kinase (PTK) inhibitor genistein (30 microM) but was only weakly affected by its inactive analogue daidzein (30 microM). Intracellular application of protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate (250 and 500 microM) resulted in a gradual activation of a Cl(-) current even in isosmotic solutions. This Cl(-) current was almost completely inhibited by 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS, 500 microM) and was also largely suppressed by exposure to hyperosmotic solution, thus indicating a close similarity to I(Cl,swell). Pretreatment of chondrocytes with genistein significantly prevented the activation of the Cl(-) current by sodium orthovanadate, suggesting that the basal

  15. HYSCORE Analysis of the Effects of Substrates on Coordination of Water to the Active Site Iron in Tyrosine Hydroxylase.

    PubMed

    McCracken, John; Eser, Bekir E; Mannikko, Donald; Krzyaniak, Matthew D; Fitzpatrick, Paul F

    2015-06-23

    Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O₂ at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O₂ to poise the active site iron in an S = ³/₂ {FeNO}⁷ form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H₂O molecule characterized by proton isotropic hyperfine couplings (A(iso) = 0.0 ± 0.3 MHz) and dipolar couplings (T = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an A(iso) of 2.0 MHz and a T of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H₂O bound to {FeNO}⁷ model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH₄, the HYSCORE cross peaks attributed to H₂O and OH⁻ for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an A(iso) of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin.

  16. Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes.

    PubMed Central

    Ahmad, F; Azevedo, J L; Cortright, R; Dohm, G L; Goldstein, B J

    1997-01-01

    Obese human subjects have increased protein-tyrosine phosphatase (PTPase) activity in adipose tissue that can dephosphorylate and inactivate the insulin receptor kinase. To extend these findings to skeletal muscle, we measured PTPase activity in the skeletal muscle particulate fraction and cytosol from a series of lean controls, insulin-resistant obese (body mass index > 30) nondiabetic subjects, and obese individuals with non-insulin-dependent diabetes. PTPase activities in subcellular fractions from the nondiabetic obese subjects were increased to 140-170% of the level in lean controls (P < 0.05). In contrast, PTPase activity in both fractions from the obese subjects with non-insulin-dependent diabetes was significantly decreased to 39% of the level in controls (P < 0.05). By immunoblot analysis, leukocyte antigen related (LAR) and protein-tyrosine phosphatase 1B had the greatest increase (threefold) in the particulate fraction from obese, nondiabetic subjects, and immunodepletion of this fraction using an affinity-purified antibody directed at the cytoplasmic domain of leukocyte antigen related normalized the PTPase activity when compared to the activity from control subjects. These findings provide further support for negative regulation of insulin action by specific PTPases in the pathogenesis of insulin resistance in human obesity, while other regulatory mechanisms may be operative in the diabetic state. PMID:9218523

  17. Tyrosine administration enhances dopamine synthesis and release in light-activated rat retina

    NASA Technical Reports Server (NTRS)

    Gibson, C. J.; Watkins, C. J.; Wurtman, R. J.

    1983-01-01

    Exposure of dark-adapted albino rats to light (350 lux) significantly elevated retinal levels of the dopamine metabolite dihydroxyphenyl acetic acid during the next hour; their return to a dark environment caused dihydroxyphenyl acetic acid levels to fall. Retinal dopamine levels were increased slightly by light exposure, suggesting that the increase in dihydroxyphenyl acetic acid reflected accelerated dopamine synthesis. Administration of tyrosine (100 mg/kg, i.p.) further elevated retinal dihydroxyphenyl acetic acid among light-exposed animals, but failed to affect dopamine release among animals in the dark. These observations show that a physiological stimulus - light exposure - can cause catecholaminergic neurons to become tyrosine-dependent; they also suggest that food consumption may affect neurotransmitter release within the retina.

  18. Bacteriorhodopsin mutants containing single tyrosine to phenylalanine substitutions are all active in proton translocation.

    PubMed Central

    Mogi, T; Stern, L J; Hackett, N R; Khorana, H G

    1987-01-01

    To study the possible role of the tyrosine residues in proton translocation by bacteriorhodopsin, we have replaced these residues individually by phenylalanine. The required codon changes were introduced in the bacterioopsin gene by replacement of appropriate restriction fragments by synthetic counterparts containing the desired nucleotide changes. The denatured opsin polypeptides obtained by expression of the mutant genes in Escherichia coli were purified and treated with a mixture of detergents, phospholipids, and retinal in a previously established renaturation procedure. All of the mutant proteins folded to regenerate bacteriorhodopsin-like chromophores. Three mutants with tyrosine to phenylalanine substitutions at positions 57, 83, and 185 regenerated the chromophore more slowly than the wild-type protein, and two of these mutants, Phe-57 and -83, showed slightly blue-shifted chromophores. When reconstituted into liposomes all of the mutant proteins with single Tyr----Phe substitutions pumped protons at rates and levels comparable to those of the wild-type bacteriorhodopsin. We conclude that single substitutions of tyrosine by phenylalanine do not affect folding, retinal binding, or light-dependent proton pumping in bacteriorhodopsin. PMID:3039495

  19. Microbial Protein-tyrosine Kinases*

    PubMed Central

    Chao, Joseph D.; Wong, Dennis; Av-Gay, Yossef

    2014-01-01

    Microbial ester kinases identified in the past 3 decades came as a surprise, as protein phosphorylation on Ser, Thr, and Tyr amino acids was thought to be unique to eukaryotes. Current analysis of available microbial genomes reveals that “eukaryote-like” protein kinases are prevalent in prokaryotes and can converge in the same signaling pathway with the classical microbial “two-component” systems. Most microbial tyrosine kinases lack the “eukaryotic” Hanks domain signature and are designated tyrosine kinases based upon their biochemical activity. These include the tyrosine kinases termed bacterial tyrosine kinases (BY-kinases), which are responsible for the majority of known bacterial tyrosine phosphorylation events. Although termed generally as bacterial tyrosine kinases, BY-kinases can be considered as one family belonging to the superfamily of prokaryotic protein-tyrosine kinases in bacteria. Other members of this superfamily include atypical “odd” tyrosine kinases with diverse mechanisms of protein phosphorylation and the “eukaryote-like” Hanks-type tyrosine kinases. Here, we discuss the distribution, phylogeny, and function of the various prokaryotic protein-tyrosine kinases, focusing on the recently discovered Mycobacterium tuberculosis PtkA and its relationship with other members of this diverse family of proteins. PMID:24554699

  20. Rapid evaluation of tyrosine kinase activity of membrane-integrated human epidermal growth factor receptor using the yeast Gγ recruitment system.

    PubMed

    Fukuda, Nobuo; Honda, Shinya

    2015-04-17

    Epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family and plays key roles in the regulation of fundamental cellular processes, including cell proliferation, migration, differentiation, and survival. Deregulation of EGFR tyrosine kinase activity is involved in the development and progression of human cancers. In the present study, we attempted to develop a method to evaluate the tyrosine kinase activity of human EGFR using the yeast Gγ recruitment system. Autophosphorylation of tyrosine residues on the cytoplasmic tail of EGFR induces recruitment of Grb2-fused Gγ subunits to the inner leaflet of the plasma membrane in yeast cells, which leads to G-protein signal transduction and activation of downstream signaling events, including mating and diploid cell growth. We demonstrate that our system is applicable for the evaluation of tyrosine kinase inhibitors, which are regarded as promising drug candidates to prevent the growth of tumor cells. This approach provides a rapid and easy-to-use tool to select EGFR-targeting tyrosine kinase inhibitors that are able to permeate eukaryotic membranes and function in intracellular environments.

  1. Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton’s Tyrosine Kinase

    PubMed Central

    Novina, Carl D.; Kumar, Sanjay; Bajpai, Urmila; Cheriyath, Venugopalan; Zhang, Keming; Pillai, Shiv; Wortis, Henry H.; Roy, Ananda L.

    1999-01-01

    Bruton’s tyrosine kinase (Btk) is required for normal B-cell development, as defects in Btk lead to X-linked immunodeficiency (xid) in mice and X-linked agammaglobulinemia (XLA) in humans. Here we demonstrate a functional interaction between the multifunctional transcription factor TFII-I and Btk. Ectopic expression of wild-type Btk enhances TFII-I-mediated transcriptional activation and its tyrosine phosphorylation in transient-transfection assays. Mutation of Btk in either the PH domain (R28C, as in the murine xid mutation) or the kinase domain (K430E) compromises its ability to enhance both the tyrosine phosphorylation and the transcriptional activity of TFII-I. TFII-I associates constitutively in vivo with wild-type Btk and kinase-inactive Btk but not xid Btk. However, membrane immunoglobulin M cross-linking in B cells leads to dissociation of TFII-I from Btk. We further show that while TFII-I is found in both the nucleus and cytoplasm of wild-type and xid primary resting B cells, nuclear TFII-I is greater in xid B cells. Most strikingly, receptor cross-linking of wild-type (but not xid) B cells results in increased nuclear import of TFII-I. Taken together, these data suggest that although the PH domain of Btk is primarily responsible for its physical interaction with TFII-I, an intact kinase domain of Btk is required to enhance transcriptional activity of TFII-I in the nucleus. Thus, mutations impairing the physical and/or functional association between TFII-I and Btk may result in diminished TFII-I-dependent transcription and contribute to defective B-cell development and/or function. PMID:10373551

  2. Tyrosine hydroxylase activity of immobilized tyrosinase on enzacryl-AA and CPG-AA supports: Stabilization and properties.

    PubMed

    Vilanova, E; Manjon, A; Iborra, J L

    1984-11-01

    Frog epidermis tyrosinase has been immobilized on Enzacryl-AA (a polyacrylamide-based support) and CPG(zirclad)-Arylamine (a controlled pore glass support) in order to stabilize the tyrosine hydroxylase activity of the enzyme; in this way, the immobilized enzyme could be used to synthesize L-dopa from L-tyrosine. The activity immobilization yield Y(IME) (act) (higher than 86%), coupling efficiency (up to 90%), storage stability (no loss in 120 days), and reaction stability (t(1/2) was higher than 20 h in column reactors) were measured for tyrosinase after its immobilization. The results showed a noticeable improvement (in immobilization yield, coupling efficiency, and storage and operational stabilities) over previous reports in which tyrosinase was immobilized for L-dopa production. The activity and stability of immobilized enzyme preparations working in three different reactor types have been compared when used in equivalent conditions with respect to a new proposed parameter of the reactor (R(p)), which allows different reactor configurations to be related to the productivity of the reactor during its useful life time. The characteristic reaction inactivation which soluble tyrosinase shows after a short reaction time has been avoided by immobilization, and the stabilization was enhanced by the presence of ascorbate. However, another inactivation process appeared after a prolonged use of the immobilized enzyme. The effects of reactor type and operating conditions on immobilized enzyme activity and stability are discussed.

  3. Positive And Negative Feedback Loops Coupled By Common Transcription Activator And Repressor

    NASA Astrophysics Data System (ADS)

    Sielewiesiuk, Jan; Łopaciuk, Agata

    2015-03-01

    Dynamical systems consisting of two interlocked loops with negative and positive feedback have been studied using the linear analysis of stability and numerical solutions. Conditions for saddle-node bifurcation were formulated in a general form. Conditions for Hopf bifurcations were found in a few symmetrical cases. Auto-oscillations, when they exist, are generated by the negative feedback repressive loop. This loop determines the frequency and amplitude of oscillations. The positive feedback loop of activation slightly modifies the oscillations. Oscillations are possible when the difference between Hilll's coefficients of the repression and activation is sufficiently high. The highly cooperative activation loop with a fast turnover slows down or even makes the oscillations impossible. The system under consideration can constitute a component of epigenetic or enzymatic regulation network.

  4. New hippolide derivatives with protein tyrosine phosphatase 1B inhibitory activity from the marine sponge Hippospongia lachne.

    PubMed

    Piao, Shu-Juan; Jiao, Wei-Hua; Yang, Fan; Yi, Yang-Hua; Di, Ying-Tong; Han, Bing-Nan; Lin, Hou-Wen

    2014-07-01

    Five new sesterterpenoids, compounds 1-5, have been isolated from the sponge Hippospongia lachne off Yongxing Island in the South China Sea. The structures of compounds 1-5 were elucidated through extensive spectroscopic analysis, including HRMS, 1D, and 2D NMR experiments. The stereochemistry, including absolute configurations of these compounds, was determined by spectroscopic, chemical, and computational methods. Compounds 1 and 5 showed moderate protein tyrosine phosphatase 1B (PTP1B) inhibitory activities with IC50 values of 5.2 μM and 8.7 μM, respectively, more potent than previously reported hippolides.

  5. Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth.

    PubMed

    Giannoni, Elisa; Buricchi, Francesca; Raugei, Giovanni; Ramponi, Giampietro; Chiarugi, Paola

    2005-08-01

    Src tyrosine kinases are central components of adhesive responses and are required for cell spreading onto the extracellular matrix. Among other intracellular messengers elicited by integrin ligation are reactive oxygen species, which act as synergistic mediators of cytoskeleton rearrangement and cell spreading. We report that after integrin ligation, the tyrosine kinase Src is oxidized and activated. Src displays an early activation phase, concurrent with focal adhesion formation and driven mainly by Tyr527 dephosphorylation, and a late phase, concomitant with reactive oxygen species production, cell spreading, and integrin-elicited kinase oxidation. In addition, our results suggest that reactive oxygen species are key mediators of in vitro and in vivo v-Src tumorigenic properties, as both antioxidant treatments and the oxidant-insensitive C245A and C487A Src mutants greatly decrease invasivity, serum-independent and anchorage-independent growth, and tumor onset. Therefore we propose that, in addition to the known phosphorylation/dephosphorylation circuitry, redox regulation of Src activity is required during both cell attachment to the extracellular matrix and tumorigenesis.

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

    PubMed

    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

  7. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

    PubMed Central

    Migliaccio, A; Di Domenico, M; Castoria, G; de Falco, A; Bontempo, P; Nola, E; Auricchio, F

    1996-01-01

    The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor. Images PMID:8635462

  8. Activation of the neu tyrosine kinase induces the fos/jun transcription factor complex, the glucose transporter and ornithine decarboxylase

    PubMed Central

    1989-01-01

    We have studied the ability of the neu tyrosine kinase to induce a signal for the activation of cell growth-regulated genes. Serum-starved NIH 3T3 cells expressing an epidermal growth factor receptor (EGF- R)/neu construct encoding a hybrid receptor protein were stimulated with EGF and the activation of the neu tyrosine kinase and stimulation of growth factor inducible genes were followed at the mRNA, protein, and activity levels, and compared to the corresponding responses in the neu proto-oncogene and oncogene expressing cells. Induction of the expression of jun mRNAs was an immediate early effect of EGF stimulation, followed by a marked increase in the biosynthesis of the fos/jun transcription factor complex and an increased transcription factor activity as measured by a recombinant transcription unit using chloramphenicol acetyltransferase assays. In distinction, elevated AP- 1/PEA-1 activity in the absence of a significant increase in jun and fos expression was characteristic of the neu oncogene-expressing cells. The glucose transporter mRNA increased at 2 h of EGF stimulation and was associated with enhanced glucose transport of the EGF-treated cells. An increase of ornithine decarboxylase (ODC) mRNA and activity followed these changes. In contrast, serum-starved, EGF-treated neu proto-oncogene- and oncogene-expressing cells showed constitutively low and high glucose transporter and ODC activities, respectively. These findings demonstrate that the chimeric EGF-R/neu receptor is capable of activating the expression of both immediate early genes and biochemical activities associated with cell growth stimulation. PMID:2572601

  9. THE ROLE OF ACTIVE REGION TOPOLOGY IN EXCITATION, TRAPPING, AND DAMPING OF CORONAL LOOP OSCILLATIONS

    SciTech Connect

    Selwa, M.; Ofman, L. E-mail: Leon.Ofman@nasa.go

    2010-05-01

    We investigate the role of magnetic field topology in dense coronal loop oscillation by the means of three-dimensional magnetohydrodynamic numerical simulations of two models of idealized active regions (ARs). The first AR model is initialized as a straight cylinder surrounded by the field lines of the same length and orientation. The second model consists of a potential dipole magnetic configuration and contains a loop with a higher density than its surroundings. Dipole field lines have position-dependent length and orientation in contrary to straight ones. We study different ways of excitation of transverse loop oscillations by an external pulse and a nearly eigenmode excitation implemented inside the loop. We find that perturbation acting directly on a single loop excites oscillations both in cylindrical and dipole loops. However, the leakage of the wave energy is larger in a curved loop compared to a straight loop. External excitation of the whole AR is efficient in the excitation of oscillation in the straight field configuration, but results in less efficient excitation in the case of dipole field. We show that excitation of collective motion of straight field lines having the same wave periods and planes of the oscillations requires much less energy than excitation of dipole field lines having position-dependent orientation and wave periods and being excited individually, not having a collective mode of oscillation. We conclude that coherent motion of straight field lines is one of the factors that decrease the energy leakage from an oscillating loop, while individual motions of dipole field lines require more energy from the source to produce the loop oscillations, and also lead to higher damping rate compared to the straight field case. We discuss Transition Region and Coronal Explorer (TRACE) observations of coronal loop oscillations in view of our theoretical findings. We show several examples of time signatures of transversal loop oscillations observed

  10. Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent

    PubMed Central

    Bonenfant, Débora; Rubert, Joëlle; Vangrevelinghe, Eric; Scheufler, Clemens; Marque, Fanny; Régnier, Catherine H.; De Pover, Alain; Ryckelynck, Hugues; Bhagwat, Neha; Koppikar, Priya; Goel, Aviva; Wyder, Lorenza; Tavares, Gisele; Baffert, Fabienne; Pissot-Soldermann, Carole; Manley, Paul W.; Gaul, Christoph; Voshol, Hans; Levine, Ross L.; Sellers, William R.; Hofmann, Francesco; Radimerski, Thomas

    2016-01-01

    JAK inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type-I binding mode leads to an increase in JAK activation-loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type-II inhibition acts in the opposite manner, leading to a loss of activation-loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation-loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation-loop may or may not be elicited. PMID:22684457

  11. The heterotrimeric G q protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes.

    PubMed Central

    Sadoshima, J; Izumo, S

    1996-01-01

    p21 ras plays as important role in cell proliferation, transformation and differentiation. Recently, the requirement of p21 ras has been suggested for cellular responses induced by stimulation of heterotrimeric G protein-coupled receptors. However, it remains to be determined how agonists for G protein-coupled receptors activate p21 ras in metazoans. We show here that stimulation of the G q protein-coupled angiotensin II (Ang II) receptor causes activation of p21 ras in cardiac myocytes. The p21 ras activation by Ang II is mediated by an increase in the guanine nucleotide exchange activity, but not by an inhibition of the GTPase-activating protein. Ang II causes rapid tyrosine phosphorylation of Shc and its association with Grb2 and mSos-1, a guanine nucleotide exchange factor of p21 ras. This leads to translocation of mSos-1 to the membrane fraction. Shc associates with the SH3 domain of Fyn whose tyrosine kinase activity is activated by Ang II with a similar time course as that of tyrosine phosphorylation of Shc. Ang II-induced increase in the guanine nucleotide exchange activity was inhibited by a peptide ligand specific to the SH3 domain of the Src family tyrosine kinases. These results suggest that an agonist for a pertussis toxin-insensitive G protein-coupled receptor may initiate the cross-talk with non-receptor-type tyrosine kinases, thereby activating p21 ras using a similar mechanism as receptor tyrosine kinase-induced p21 ras activation. Images PMID:8631299

  12. Incomplete Folding upon Binding Mediates Cdk4/Cyclin D Complex Activation by Tyrosine Phosphorylation of Inhibitor p27 Protein*

    PubMed Central

    Ou, Li; Ferreira, Antonio M.; Otieno, Steve; Xiao, Limin; Bashford, Donald; Kriwacki, Richard W.

    2011-01-01

    p27Kip1 (p27), an intrinsically disordered protein, regulates the various Cdk/cyclin complexes that control cell cycle progression. The kinase inhibitory domain of p27 contains a cyclin-binding subdomain (D1), a Cdk-binding subdomain (D2), and a linker helix subdomain that connects D1 and D2. Here, we report that, despite extensive sequence conservation between Cdk4/cyclin D1 (hereafter Cdk4/cyclin D) and Cdk2/cyclin A, the thermodynamic details describing how the individual p27 subdomains contribute to equally high affinity binding to these two Cdk/cyclin complexes are strikingly different. Differences in enthalpy/entropy compensation revealed that the D2 subdomain of p27 folds incompletely when binding Cdk4/cyclin D versus Cdk2/cyclin A. Incomplete binding-induced folding exposes tyrosine 88 of p27 for phosphorylation by the nonreceptor tyrosine kinase Abl. Importantly, tyrosine phosphorylation (of p27) relieves Cdk inhibition by p27, enabling cell cycle entry. Furthermore, the interaction between a conserved hydrophobic patch on cyclin D and subdomain D1 is much weaker than that with cyclin A; consequently, a construct containing subdomains D1 and LH (p27-D1LH) does not inhibit substrate binding to Cdk4/cyclin D as it does to Cdk2/cyclin A. Our results provide a mechanism by which Cdk4 (within the p27/Cdk4/cyclin D complex) is poised to be activated by extrinsic mitogenic signals that impinge upon p27 at the earliest stage of cell division. More broadly, our results further illustrate the regulatory versatility of intrinsically disordered proteins. PMID:21715330

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

    PubMed

    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

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

    PubMed

    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.

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

    PubMed Central

    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

  16. Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases.

    PubMed

    Mu, D; Janes, S M; Smith, A J; Brown, D E; Dooley, D M; Klinman, J P

    1992-04-25

    The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.

  17. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor.

    PubMed

    Lotz-Jenne, Carina; Lüthi, Urs; Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-05-01

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered "off-target" effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo. PMID:27036020

  18. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor

    PubMed Central

    Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-01-01

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered “off-target” effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo. PMID:27036020

  19. Compaction and segregation of sister chromatids via active loop extrusion

    PubMed Central

    Goloborodko, Anton; Imakaev, Maxim V; Marko, John F; Mirny, Leonid

    2016-01-01

    The mechanism by which chromatids and chromosomes are segregated during mitosis and meiosis is a major puzzle of biology and biophysics. Using polymer simulations of chromosome dynamics, we show that a single mechanism of loop extrusion by condensins can robustly compact, segregate and disentangle chromosomes, arriving at individualized chromatids with morphology observed in vivo. Our model resolves the paradox of topological simplification concomitant with chromosome 'condensation', and explains how enzymes a few nanometers in size are able to control chromosome geometry and topology at micron length scales. We suggest that loop extrusion is a universal mechanism of genome folding that mediates functional interactions during interphase and compacts chromosomes during mitosis. DOI: http://dx.doi.org/10.7554/eLife.14864.001 PMID:27192037

  20. Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src

    PubMed Central

    Anguita, Estefanía; Benaim, Gustavo; Villalobo, Antonio

    2015-01-01

    Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred. PMID:26058065

  1. Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.

    PubMed Central

    Uckun, F M; Schieven, G L; Tuel-Ahlgren, L M; Dibirdik, I; Myers, D E; Ledbetter, J A; Song, C W

    1993-01-01

    Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation. Images PMID:8419931

  2. The catecholamines up (Catsup) protein of Drosophila melanogaster functions as a negative regulator of tyrosine hydroxylase activity.

    PubMed Central

    Stathakis, D G; Burton, D Y; McIvor, W E; Krishnakumar, S; Wright, T R; O'Donnell, J M

    1999-01-01

    We report the genetic, phenotypic, and biochemical analyses of Catecholamines up (Catsup), a gene that encodes a negative regulator of tyrosine hydroxylase (TH) activity. Mutations within this locus are semidominant lethals of variable penetrance that result in three broad, overlapping effective lethal phases (ELPs), indicating that the Catsup gene product is essential throughout development. Mutants from each ELP exhibit either cuticle defects or catecholamine-related abnormalities, such as melanotic salivary glands or pseudotumors. Additionally, Catsup mutants have significantly elevated TH activity that may arise from a post-translational modification of the enzyme. The hyperactivation of TH in Catsup mutants results in abnormally high levels of catecholamines, which can account for the lethality, visible phenotypes, and female sterility observed in these mutants. We propose that Catsup is a component of a novel system that downregulates TH activity, making Catsup the fourth locus found within the Dopa decarboxylase (Ddc) gene cluster that functions in catecholamine metabolism. PMID:10471719

  3. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    PubMed

    Saouaf, S J; Mahajan, S; Rowley, R B; Kut, S A; Fargnoli, J; Burkhardt, A L; Tsukada, S; Witte, O N; Bolen, J B

    1994-09-27

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction. PMID:7524079

  4. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    PubMed Central

    Saouaf, S J; Mahajan, S; Rowley, R B; Kut, S A; Fargnoli, J; Burkhardt, A L; Tsukada, S; Witte, O N; Bolen, J B

    1994-01-01

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction. Images PMID:7524079

  5. Probing the active site loop motif of murine ferrochelatase by random mutagenesis.

    PubMed

    Shi, Zhen; Ferreira, Gloria C

    2004-05-01

    Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. A conserved loop motif was shown to form part of the active site and contact the bound porphyrin by molecular dynamics calculations and structural analysis. We applied a random mutagenesis approach and steady-state kinetic analysis to assess the role of the loop motif in murine ferrochelatase function, particularly with respect to porphyrin interaction. Functional substitutions in the 10 consecutive loop positions Gln(248)-Leu(257) were identified by genetic complementation in Escherichia coli strain Deltavis. Lys(250), Val(251), Pro(253), Val(254), and Pro(255) tolerated a variety of replacements including single substitutions and contained low informational content. Gln(248), Ser(249), Gly(252), Trp(256), and Leu(257) possessed high informational content, since permissible replacements were limited and only observed in multiply substituted mutants. Selected active loop variants exhibited k(cat) values comparable with or higher than that of wild-type murine ferrochelatase. The K(m) values for porphyrin increased, except for the single mutant V251L. Other than a moderate increase observed in the triple mutant S249A/K250Q/V251C, the K(m) values for Fe(2+) were lowered. The k(cat)/K(m) for porphyrin remained largely unchanged, with the exception of a 10-fold reduction in the triple mutant K250M/V251L/W256Y. The k(cat)/K(m) for Fe(2+) was improved. Molecular modeling of these active loop variants indicated that loop mutations resulted in alterations of the active site architecture. However, despite the plasticity of the loop primary structure, the relative spatial positioning of the loop in the active site appeared to be maintained in functional variants, supporting a role for the loop in ferrochelatase function. PMID:14981080

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

    PubMed

    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.

  7. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    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. PMID:25706379

  8. Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4.

    PubMed Central

    Lee, J; Gray, A; Yuan, J; Luoh, S M; Avraham, H; Wood, W I

    1996-01-01

    The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney. Images Fig. 1 Fig. 2 Fig. 3 Fig. 6 PMID:8700872

  9. Activation of tyrosine aminotransferase expression in fetal liver by 5-azacytidine

    SciTech Connect

    Rothrock, R.; Perry, S.T.; Isham, K.R.; Lee, K.L.; Kenney, F.T.

    1983-06-15

    Rat fetuses of 20 days gestational age were treated in utero with the inhibitor of DNA methylation, 5-azacytidine. The liver enzyme tyrosine aminotransferase, normally expressed at very low levels until several hours after birth, was increased by the drug in the fetal livers after a lag period of about 9 hours, reaching a level 70-fold above control levels 18 hours after treatment. The high levels attained after 5-azacytidine treatment are comparable to those of glucocorticoid-treated adult livers, and were not further increased by administration of hydrocortisone to dams carrying treated fetuses. Cytidine and two other analogs, cytosine arabinoside and 6-azacytidine, were essentially without effect. 15 references, 2 figures, 1 table.

  10. GM1 ganglioside activates ERK1/2 and Akt downstream of Trk tyrosine kinase and protects PC12 cells against hydrogen peroxide toxicity.

    PubMed

    Zakharova, Irina O; Sokolova, Tatyana V; Vlasova, Yulia A; Furaev, Victor V; Rychkova, Maria P; Avrova, Natalia F

    2014-11-01

    Ganglioside GM1 at micro- and nanomolar concentrations was shown to increase the viability of pheochromocytoma PC12 cells exposed to hydrogen peroxide and diminish the accumulation of reactive oxygen species and oxidative inactivation of Na(+),K(+)-ATPase, the effects of micromolar GM1 being more pronounced than those of nanomolar GM1. These effects of GM1 were abolished by Trk receptor tyrosine kinase inhibitor and diminished by MEK1/2, phosphoinositide 3-kinase and protein kinase C inhibitors. Hydrogen peroxide activates Trk tyrosine kinase; Akt and ERK1/2 are activated downstream of this protein kinase. GM1 was found to activate Trk receptor tyrosine kinase in PC12 cells. GM1 (100 nM and 10 µM) increased the basal activity of Akt, but did not change Akt activity in cells exposed to hydrogen peroxide. Basal ERK1/2 activity in PC12 cells was increased by GM1 at a concentration of 10 µM, but not at nanomolar concentrations. Activation of ERK1/2 by hydrogen peroxide was enhanced by GM1 at a concentration of 10 µM and to a lesser extent at a concentration of 100 nM. Thus, the protective and metabolic effects of GM1 ganglioside on PC12 cells exposed to hydrogen peroxide appear to depend on the activation of Trk receptor tyrosine kinase and downstream activation of Akt and ERK1/2.

  11. Sesquiterpene Hydroquinones with Protein Tyrosine Phosphatase 1B Inhibitory Activities from a Dysidea sp. Marine Sponge Collected in Okinawa.

    PubMed

    Abdjul, Delfly B; Yamazaki, Hiroyuki; Takahashi, Ohgi; Kirikoshi, Ryota; Ukai, Kazuyo; Namikoshi, Michio

    2016-07-22

    Three new sesquiterpene hydroquinones, avapyran (1), 17-O-acetylavarol (2), and 17-O-acetylneoavarol (3), were isolated from a Dysidea sp. marine sponge collected in Okinawa together with five known congeners: avarol (4), neoavarol (5), 20-O-acetylavarol (6), 20-O-acetylneoavarol (7), and 3'-aminoavarone (8). The structures of 1-3 were assigned on the basis of their spectroscopic data. Compounds 1-3 inhibited the activity of protein tyrosine phosphatase 1B with IC50 values of 11, 9.5, and 6.5 μM, respectively, while known compounds 4-8 gave IC50 values of 12, >32, 10, 8.6, and 18 μM, respectively. In a preliminary investigation on structure-activity relationships, six ester and methoxy derivatives (9-14) were prepared from 4 and 5.

  12. Sesquiterpene Hydroquinones with Protein Tyrosine Phosphatase 1B Inhibitory Activities from a Dysidea sp. Marine Sponge Collected in Okinawa.

    PubMed

    Abdjul, Delfly B; Yamazaki, Hiroyuki; Takahashi, Ohgi; Kirikoshi, Ryota; Ukai, Kazuyo; Namikoshi, Michio

    2016-07-22

    Three new sesquiterpene hydroquinones, avapyran (1), 17-O-acetylavarol (2), and 17-O-acetylneoavarol (3), were isolated from a Dysidea sp. marine sponge collected in Okinawa together with five known congeners: avarol (4), neoavarol (5), 20-O-acetylavarol (6), 20-O-acetylneoavarol (7), and 3'-aminoavarone (8). The structures of 1-3 were assigned on the basis of their spectroscopic data. Compounds 1-3 inhibited the activity of protein tyrosine phosphatase 1B with IC50 values of 11, 9.5, and 6.5 μM, respectively, while known compounds 4-8 gave IC50 values of 12, >32, 10, 8.6, and 18 μM, respectively. In a preliminary investigation on structure-activity relationships, six ester and methoxy derivatives (9-14) were prepared from 4 and 5. PMID:27336796

  13. Antifibrotic and anti-inflammatory activity of the tyrosine kinase inhibitor nintedanib in experimental models of lung fibrosis.

    PubMed

    Wollin, Lutz; Maillet, Isabelle; Quesniaux, Valérie; Holweg, Alexander; Ryffel, Bernhard

    2014-05-01

    The tyrosine kinase inhibitor nintedanib (BIBF 1120) is in clinical development for the treatment of idiopathic pulmonary fibrosis. To explore its mode of action, nintedanib was tested in human lung fibroblasts and mouse models of lung fibrosis. Human lung fibroblasts expressing platelet-derived growth factor (PDGF) receptor-α and -β were stimulated with platelet-derived growth factor BB (homodimer) (PDGF-BB). Receptor activation was assessed by autophosphorylation and cell proliferation by bromodeoxyuridine incorporation. Transforming growth factor β (TGFβ)-induced fibroblast to myofibroblast transformation was determined by α-smooth muscle actin (αSMA) mRNA analysis. Lung fibrosis was induced in mice by intratracheal bleomycin or silica particle administration. Nintedanib was administered every day by gavage at 30, 60, or 100 mg/kg. Preventive nintedanib treatment regimen started on the day that bleomycin was administered. Therapeutic treatment regimen started at various times after the induction of lung fibrosis. Bleomycin caused increased macrophages and lymphocytes in the bronchoalveolar lavage (BAL) and elevated interleukin-1β (IL-1β), tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen in lung tissue. Histology revealed chronic inflammation and fibrosis. Silica-induced lung pathology additionally showed elevated BAL neutrophils, keratinocyte chemoattractant (KC) levels, and granuloma formation. Nintedanib inhibited PDGF receptor activation, fibroblast proliferation, and fibroblast to myofibroblast transformation. Nintedanib significantly reduced BAL lymphocytes and neutrophils but not macrophages. Furthermore, interleukin-1β, KC, TIMP-1, and lung collagen were significantly reduced. Histologic analysis showed significantly diminished lung inflammation, granuloma formation, and fibrosis. The therapeutic effect was dependent on treatment start and duration. Nintedanib inhibited receptor tyrosine kinase activation and the proliferation and

  14. Creatine and pyruvate prevent the alterations caused by tyrosine on parameters of oxidative stress and enzyme activities of phosphoryltransfer network in cerebral cortex of Wistar rats.

    PubMed

    de Andrade, Rodrigo Binkowski; Gemelli, Tanise; Rojas, Denise Bertin; Bonorino, Narielle Ferner; Costa, Bruna May Lopes; Funchal, Cláudia; Dutra-Filho, Carlos Severo; Wannmacher, Clovis Milton Duval

    2015-01-01

    Tyrosine accumulates in inborn errors of tyrosine catabolism, especially in tyrosinemia type II. In this disease caused by tyrosine aminotransferase deficiency, eyes, skin, and central nervous system disturbances are found. In the present study, we investigated the chronic effect of tyrosine methyl ester (TME) and/or creatine plus pyruvate on some parameters of oxidative stress and enzyme activities of phosphoryltransfer network in cerebral cortex homogenates of 21-day-old Wistar. Chronic administration of TME induced oxidative stress and altered the activities of adenylate kinase and mitochondrial and cytosolic creatine kinase. Total sulfhydryls content, GSH content, and GPx activity were significantly diminished, while DCFH oxidation, TBARS content, and SOD activity were significantly enhanced by TME. On the other hand, TME administration decreased the activity of CK from cytosolic and mitochondrial fractions but enhanced AK activity. In contrast, TME did not affect the carbonyl content and PK activity in cerebral cortex of rats. Co-administration of creatine plus pyruvate was effective in the prevention of alterations provoked by TME administration on the oxidative stress and the enzymes of phosphoryltransfer network, except in mitochondrial CK, AK, and SOD activities. These results indicate that chronic administration of TME may stimulate oxidative stress and alter the enzymes of phosphoryltransfer network in cerebral cortex of rats. In case this also occurs in the patients affected by these disorders, it may contribute, along with other mechanisms, to the neurological dysfunction of hypertyrosinemias, and creatine and pyruvate supplementation could be beneficial to the patients.

  15. Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase.

    PubMed

    Morris, P; Rios-Solis, L; García-Arrazola, R; Lye, G J; Dalby, P A

    2016-07-01

    Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185-192 and 382-392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60°C and 65°C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55°C or 60°C for 1h. Interestingly, H192P was also 2-times more active than wild-type TK at 25°C. Optimal activity was achieved at 60°C for H192P compared to 55°C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.

  16. CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C-gamma(1) upon B cell activation

    PubMed Central

    1996-01-01

    Cross-linking B cell antigen receptor (BCR) elicits early signal transduction events, including activation of protein tyrosine kinases, phosphorylation of receptor components, activation of phospholipase C- gamma (PLC-gamma), and increases in intracellular free Ca2+. In this article, we report that cross-linking the BCR led to a rapid translocation of cytosolic protein tyrosine phosphatase (PTP) 1C to the particulate fraction, where it became associated with a 140-150-kD tyrosyl-phosphorylated protein. Western blotting analysis identified this 140-150-kD protein to be CD22. The association of PTP-1C with CD22 was mediated by the NH2-terminal Src homology 2 (SH2) domain of PTP-1C. Complexes of either CD22/PTP-1C/Syk/PLC-gamma(1) could be isolated from B cells stimulated by BCR engagement or a mixture of hydrogen peroxidase and sodium orthovanadate, respectively. The binding of PLC- gamma(1) and Syk to tyrosyl-phosphorylated CD22 was mediated by the NH2- terminal SH2 domain of PLC-gamma(1) and the COOH-terminal SH2 domain of Syk, respectively. These observations suggest that tyrosyl- phosphorylated CD22 may downmodulate the activity of this complex by dephosphorylation of CD22, Syk, and/or PLC-gamma(1). Transient expression of CD22 and a null mutant of PTP-1C (PTP-1CM) in COS cells resulted in an increase in tyrosyl phosphorylation of CD22 and its interaction with PTP-1CM. By contrast, CD22 was not tyrosyl phosphorylated or associated with PTP-1CM in the presence of wild-type PTP-1C. These results suggest that tyrosyl-phosphorylated CD22 may be a substrate for PTP-1C regulates tyrosyl phosphorylation of CD22. PMID:8627166

  17. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    PubMed

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface. PMID:23580642

  18. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    PubMed

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. Specific dephosphorylation of Janus Kinase 2 by protein tyrosine phosphatases.

    PubMed

    Li, Jianzhuo; Liu, Xidong; Chu, Huiying; Fu, Xueqi; Li, Tianbao; Hu, Lianghai; Xing, Shu; Li, Guohui; Gu, Jingkai; Zhao, Zhizhuang Joe

    2015-01-01

    Many protein kinases are activated through phosphorylation of an activation loop thereby turning on downstream signaling pathways. Activation of JAK2, a nonreceptor tyrosine kinase with an important role in growth factor and cytokine signaling, requires phosphorylation of the 1007 and 1008 tyrosyl residues. Dephosphorylation of these two sites by phosphatases presumably inactivates the enzyme, but the underlying mechanism is not known. In this study, we employed MALDI-TOF/TOF and triple quadrupole mass spectrometers to analyze qualitatively and quantitatively the dephosphorylation process by using synthetic peptides derived from the tandem autophosphorylation sites (Y1007 and Y1008) of human JAK2. We found that tyrosine phosphatases catalyzed the dephosphorylation reaction sequentially, but different enzymes exhibited different selectivity. Protein tyrosine phosphatase 1B caused rapid dephosphorylation of Y1008 followed by Y1007, while SHP1 and SHP2 selectively dephosphorylated Y1008 only, and yet HePTP randomly removed a single phosphate from either Y1007 or Y1008, leaving behind mono-phosphorylated peptides. The specificity of dephosphorylation was further confirmed by molecular modeling. The data reveal multiple modes of JAK2 regulation by tyrosine phosphatases, reflecting a complex, and intricate interplay between protein phosphorylation and dephosphorylation.

  1. Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

    SciTech Connect

    Zhang Danhui; Kanthasamy, Arthi; Anantharam, Vellareddy; Kanthasamy, Anumantha

    2011-07-15

    Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKC{delta}) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKC{delta} negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here, we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKC{delta}-PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3-10 {mu}M) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKC{delta} specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1-1 {mu}M Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKC{delta} kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKC{delta} inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKC{delta} and PP2A activity.

  2. THE ROLE OF ACTIVE REGION LOOP GEOMETRY. I. HOW CAN IT AFFECT CORONAL SEISMOLOGY?

    SciTech Connect

    Selwa, M.; Ofman, L.; Solanki, S. K. E-mail: leon.ofman@nasa.gov

    2011-01-01

    We present numerical results of coronal loop oscillation excitation using a three-dimensional (3D) MHD model of an idealized active region (AR) field. The AR is initialized as a potential dipole magnetic configuration with gravitationally stratified density and contains a loop with a higher density than its surroundings. We study different ways of excitation of vertical kink oscillations of this loop by velocity: as an initial condition, and as an impulsive excitation with a pulse of a given position, duration, and amplitude. We vary the geometry of the loop in the 3D MHD model and find that it affects both the period of oscillations and the synthetic observations (difference images) that we get from oscillations. Due to the overestimated effective length of the loop in the case of loops which have maximum separation between their legs above the footpoints (>50% of observed loops), the magnetic field obtained from coronal seismology can also be overestimated. The 3D MHD model shows how the accuracy of magnetic field strength determined from coronal seismology can be improved. We study the damping mechanism of the oscillations and find that vertical kink waves in 3D stratified geometry are damped mainly due to wave leakage in the horizontal direction.

  3. THE ROLE OF MAGNETIC TOPOLOGY IN THE HEATING OF ACTIVE REGION CORONAL LOOPS

    SciTech Connect

    Lee, J.-Y.; Reeves, Katharine K.; Korreck, K. E.; Golub, L.; DeLuca, E. E.; Barnes, Graham; Leka, K. D.

    2010-11-10

    We investigate the evolution of coronal loop emission in the context of the coronal magnetic field topology. New modeling techniques allow us to investigate the magnetic field structure and energy release in active regions (ARs). Using these models and high-resolution multi-wavelength coronal observations from the Transition Region and Coronal Explorer and the X-ray Telescope on Hinode, we are able to establish a relationship between the light curves of coronal loops and their associated magnetic topologies for NOAA AR 10963. We examine loops that show both transient and steady emission, and we find that loops that show many transient brightenings are located in domains associated with a high number of separators. This topology provides an environment for continual impulsive heating events through magnetic reconnection at the separators. A loop with relatively constant X-ray and EUV emission, on the other hand, is located in domains that are not associated with separators. This result implies that larger-scale magnetic field reconnections are not involved in heating plasma in these regions, and the heating in these loops must come from another mechanism, such as small-scale reconnections (i.e., nanoflares) or wave heating. Additionally, we find that loops that undergo repeated transient brightenings are associated with separators that have enhanced free energy. In contrast, we find one case of an isolated transient brightening that seems to be associated with separators with a smaller free energy.

  4. Oncogenic activation of the Met receptor tyrosine kinase fusion protein, Tpr-Met, involves exclusion from the endocytic degradative pathway.

    PubMed

    Mak, H H L; Peschard, P; Lin, T; Naujokas, M A; Zuo, D; Park, M

    2007-11-01

    Multiple mechanisms of dysregulation of receptor tyrosine kinases (RTKs) are observed in human cancers. In addition to gain-of-function, loss of negative regulation also contributes to oncogenic activation of RTKs. Negative regulation of many RTKs involves their internalization and degradation in the lysosome, a process regulated through ubiquitination. RTK oncoproteins activated following chromosomal translocation, are no longer transmembrane proteins, and are predicted to escape lysosomal degradation. To test this, we used the Tpr-Met oncogene, generated following chromosomal translocation of the hepatocyte growth factor receptor (Met). Unlike Met, Tpr-Met is localized in the cytoplasm and also lacks the binding site for Cbl ubiquitin ligases. We determined whether subcellular localization of Tpr-Met, and/or loss of its Cbl-binding site, is important for oncogenic activity. Presence of a Cbl-binding site and ubiquitination of cytosolic Tpr-Met oncoproteins does not alter their transforming activity. In contrast, plasma membrane targeting allows Tpr-Met to enter the endocytic pathway, and Tpr-Met transforming activity as well as protein stability are decreased in a Cbl-dependent manner. We show that transformation by Tpr-Met is in part dependent on its ability to escape normal downregulatory mechanisms. This provides a paradigm for many RTK oncoproteins activated following chromosomal translocation.

  5. Receptor Tyrosine Kinases, TYRO3, AXL, and MER, Demonstrate Distinct Patterns and Complex Regulation of Ligand-induced Activation*

    PubMed Central

    Tsou, Wen-I; Nguyen, Khanh-Quynh N.; Calarese, Daniel A.; Garforth, Scott J.; Antes, Anita L.; Smirnov, Sergey V.; Almo, Steve C.; Birge, Raymond B.; Kotenko, Sergei V.

    2014-01-01

    TYRO3, AXL, and MER receptors (TAMs) are three homologous type I receptor-tyrosine kinases that are activated by endogenous ligands, protein S (PROS1) and growth arrest-specific gene 6 (GAS6). These ligands can either activate TAMs as soluble factors, or, in turn, opsonize phosphatidylserine (PS) on apoptotic cells (ACs) and serve as bridging molecules between ACs and TAMs. Abnormal expression and activation of TAMs have been implicated in promoting proliferation and survival of cancer cells, as well as in suppressing anti-tumor immunity. Despite the fact that TAM receptors share significant similarity, little is known about the specificity of interaction between TAM receptors and their ligands, particularly in the context of ACs, and about the functional diversity of TAM receptors. To study ligand-mediated activation of TAMs, we generated a series of reporter cell lines expressing chimeric TAM receptors. Using this system, we found that each TAM receptor has a unique pattern of interaction with and activation by GAS6 and PROS1, which is also differentially affected by the presence of ACs, PS-containing lipid vesicles and enveloped virus. We also demonstrated that γ-carboxylation of ligands is essential for the full activation of TAMs and that soluble immunoglobulin-like TAM domains act as specific ligand antagonists. These studies demonstrate that, despite their similarity, TYRO3, AXL, and MER are likely to perform distinct functions in both immunoregulation and the recognition and removal of ACs. PMID:25074926

  6. Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

    PubMed

    Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

    2015-05-30

    Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

  7. Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER)

    PubMed Central

    Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L.; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

    2015-01-01

    Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics. PMID:26079946

  8. Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

    PubMed Central

    Peraldi, P; Zhao, Z; Filloux, C; Fischer, E H; Van Obberghen, E

    1994-01-01

    Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP. Images PMID:8197172

  9. Tyrosine kinase inhibitors. 13. Structure-activity relationships for soluble 7-substituted 4-[(3-bromophenyl)amino]pyrido[4,3-d]pyrimidines designed as inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor.

    PubMed

    Thompson, A M; Murray, D K; Elliott, W L; Fry, D W; Nelson, J A; Showalter, H D; Roberts, B J; Vincent, P W; Denny, W A

    1997-11-21

    The general class of 4-(phenylamino)quinazolines are potent (some members with IC50 values < 1 nM) and selective inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), via competitive binding at the ATP site of the enzyme, but many of the early analogues had poor aqueous solubility (< 1 mM). A series of 7-substituted 4-[(3-bromophenyl)-amino]pyrido[4,3-d]pyrimidines, together with selected (3-methylphenyl)amino analogues, were prepared by reaction of the analogous 7-fluoro derivatives with appropriate amine nucleophiles in 2-BuOH or aqueous 1-PrOH. All of the compounds were evaluated for their ability to inhibit the tyrosine-phosphorylating action of EGF-stimulated full-length EGFR enzyme. Selected analogues were also evaluated for their inhibition of autophosphorylation of the EGF receptor in A431 human epidermoid carcinoma cells in culture and against A431 tumor xenografts in mice. Analogues bearing a wide variety of polyol, cationic, and anionic solubilizing substituents retained activity, but the most effective in terms of both increased aqueous solubility (> 40 mM) and retention of overall inhibitory activity (IC50's of 0.5-10 nM against isolated enzyme and 8-40 nM for inhibition of EGFR autophosphorylation in A431 cells) were weakly basic amine derivatives. These results are broadly consistent with a proposed model for the binding of these compounds to EGFR, in which the 6- and 7-positions of the pyridopyrimidine ring are in a largely hydrophobic binding region of considerable steric freedom, at the entrance of the adenine binding cleft. The most active cationic analogues have a weakly basic side chain where the amine moiety is three or more carbon atoms away from the nucleus. Two of the compounds (bearing weakly basic morpholinopropyl and strongly basic (dimethylamino)butyl solubilizing groups) produced in vivo tumor growth delays of 13-21 days against advanced stage A431 epidermoid xenografts in nude mice, when

  10. CD45 cross-linking regulates phospholipase C activation and tyrosine phosphorylation of specific substrates in CD3/Ti-stimulated T cells.

    PubMed

    Ledbetter, J A; Schieven, G L; Uckun, F M; Imboden, J B

    1991-03-01

    In lymphocytes, CD45 regulates the increase in cytoplasmic calcium concentration that occurs after receptor cross-linking. Here we show that T cell receptor complex (CD3/Ti)-mediated inositol phosphate production was inhibited by CD45 ligation in Jurkat cells. CD3/Ti signaling in normal T cells was also inhibited by CD45 ligation, but coupling of CD4 with CD3/Ti gave augmented calcium signals that were entirely resistant to the inhibitory effect of CD45. In contrast, CD3-induced T cell proliferation was suppressed by immobilized CD45 mAb even in the presence of CD4 mAb. The effect of CD45 and CD4 ligation on tyrosine phosphorylation during T cell activation was directly examined by immunoblotting with anti-phosphotyrosine. Using immobilized mAb, CD45 ligation suppressed the tyrosine phosphorylation of specific substrates induced by CD3/Ti stimulation, including almost complete suppression of 150-, 36-, and 35-kDa proteins and partial suppression of 76- and 80-kDa proteins. Other tyrosine-phosphorylated proteins induced by CD3/Ti stimulation, including 135- and 21-kDa proteins, were not suppressed by simultaneous ligation of CD3/Ti and CD45. Simultaneous ligation of CD3 and CD4 enhanced tyrosine phosphorylation of all substrates, but did not overcome the CD45-mediated suppression of tyrosine phosphorylation of the 35- and 36-kDa proteins. The CD45-mediated suppression of phospholipase C activation is therefore modulated by association with CD4 without altering the specific inhibition of tyrosine phosphorylation and T cell proliferation after co-ligation of CD45 and CD3/Ti.

  11. SRC-DEPENDENT PHOSPHORYLATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR ON TYROSINE 845 IS REQUIRED FOR ZINC-INDUCED RAS ACTIVATION

    EPA Science Inventory

    Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor on Tyrosine 845 Is Required for Zinc-induced Ras Activation
    Weidong Wu 1 , Lee M. Graves 2 , Gordon N. Gill 3 , Sarah J. Parsons 4 , and James M. Samet 5
    1 Center for Environmental Medicine and Lung Biolo...

  12. PTP-PEST targets a novel tyrosine site in p120 catenin to control epithelial cell motility and Rho GTPase activity

    PubMed Central

    Espejo, Rosario; Jeng, Yowjiun; Paulucci-Holthauzen, Adriana; Rengifo-Cam, William; Honkus, Krysta; Anastasiadis, Panos Z.; Sastry, Sarita K.

    2014-01-01

    ABSTRACT Tyrosine phosphorylation is implicated in regulating the adherens junction protein, p120 catenin (p120), however, the mechanisms are not well defined. Here, we show, using substrate trapping, that p120 is a direct target of the protein tyrosine phosphatase, PTP-PEST, in epithelial cells. Stable shRNA knockdown of PTP-PEST in colon carcinoma cells results in an increased cytosolic pool of p120 concomitant with its enhanced tyrosine phosphorylation and decreased association with E-cadherin. Consistent with this, PTP-PEST knockdown cells exhibit increased motility, enhanced Rac1 and decreased RhoA activity on a collagen substrate. Furthermore, p120 localization is enhanced at actin-rich protrusions and lamellipodia and has an increased association with the guanine nucleotide exchange factor, VAV2, and cortactin. Exchange factor activity of VAV2 is enhanced by PTP-PEST knockdown whereas overexpression of a VAV2 C-terminal domain or DH domain mutant blocks cell motility. Analysis of point mutations identified tyrosine 335 in the N-terminal domain of p120 as the site of PTP-PEST dephosphorylation. A Y335F mutant of p120 failed to induce the ‘p120 phenotype’, interact with VAV2, stimulate cell motility or activate Rac1. Together, these data suggest that PTP-PEST affects epithelial cell motility by controlling the distribution and phosphorylation of p120 and its availability to control Rho GTPase activity. PMID:24284071

  13. Bacterial lipopolysaccharide increases tyrosine phosphorylation of zonula adherens proteins and opens the paracellular pathway in lung microvascular endothelia through TLR4, TRAF6, and src family kinase activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: LPS is a key mediator in vascular leak syndromes associated with Gram-negative bacterial infections and opens the pulmonary vascular endothelial paracellular pathway through protein tyrosine kinase (PTK) activation. We asked which PTKs and signaling molecules mediate LPS-induced endothel...

  14. Polymorphism in the Tyrosine Hydroxylase (TH) Gene Is Associated with Activity-Impulsivity in German Shepherd Dogs

    PubMed Central

    Kubinyi, Enikő; Vas, Judit; Hejjas, Krisztina; Ronai, Zsolt; Brúder, Ildikó; Turcsán, Borbála; Sasvari-Szekely, Maria; Miklósi, Ádám

    2012-01-01

    We investigated the association between repeat polymorphism in intron 4 of the tyrosine hydroxylase (TH) gene and two personality traits, activity-impulsivity and inattention, in German Shepherd Dogs. The behaviour of 104 dogs was characterized by two instruments: (1) the previously validated Dog-Attention Deficit Hyperactivity Disorder Rating Scale (Dog-ADHD RS) filled in by the dog owners and (2) the newly developed Activity-impulsivity Behavioural Scale (AIBS) containing four subtests, scored by the experimenters. Internal consistency, inter-observer reliability, test-retest reliability and convergent validity were demonstrated for AIBS. Dogs possessing at least one short allele were proved to be more active-impulsive by both instruments, compared to dogs carrying two copies of the long allele (activity-impulsivity scale of Dog-ADHD RS: p = 0.007; AIBS: p = 0.023). The results have some potential to support human studies; however, further research should reveal the molecular function of the TH gene variants, and look for the effect in more breeds. PMID:22272320

  15. rlk/TXK Encodes Two Forms of a Novel Cysteine String Tyrosine Kinase Activated by Src Family Kinases

    PubMed Central

    Debnath, Jayantha; Chamorro, Mario; Czar, Michael J.; Schaeffer, Edward M.; Lenardo, Michael J.; Varmus, Harold E.; Schwartzberg, Pamela L.

    1999-01-01

    Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR. PMID:9891083

  16. Intrinsic resistance to EGFR tyrosine kinase inhibitors in advanced non-small-cell lung cancer with activating EGFR mutations

    PubMed Central

    Wang, Jun; Wang, Baocheng; Chu, Huili; Yao, Yunfeng

    2016-01-01

    Identifying activating EGFR mutations is a useful predictive strategy that helps select a population of advanced non-small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors (TKIs). Patients with sensitizing EGFR mutations (predominantly an in-frame deletion in exon 19 and an L858R substitution) are highly responsive to first-generation EGFR TKIs, such as gefitinib and erlotinib, and show improved progression-free survival without serious side effects. However, all patients with activating EGFR mutations who are initially responsive to EGFR TKIs eventually develop acquired resistance after a median progression-free survival of 10–16 months, followed by disease progression. Moreover, ~20%–30% of NSCLC patients have no objective tumor regression on initial EGFR TKI treatment, although they harbor an activating EGFR mutation. These patients represent an NSCLC subgroup that is defined as having intrinsic or primary resistance to EGFR TKIs. Different mechanisms of acquired EGFR TKI resistance have been identified, and several novel compounds have been developed to reverse acquired resistance, but little is known about EGFR TKI intrinsic resistance. In this review, we summarize the latest findings involving mechanisms of intrinsic resistance to EGFR TKIs in advanced NSCLC with activating EGFR mutations and present possible therapeutic strategies to overcome this resistance. PMID:27382309

  17. Heating Mechanisms for Intermittent Loops in Active Region Cores from AIA/SDO EUV Observations

    NASA Astrophysics Data System (ADS)

    Cadavid, A. C.; Lawrence, J. K.; Christian, D. J.; Jess, D. B.; Nigro, G.

    2014-11-01

    We investigate intensity variations and energy deposition in five coronal loops in active region cores. These were selected for their strong variability in the AIA/SDO 94 Å intensity channel. We isolate the hot Fe XVIII and Fe XXI components of the 94 Å and 131 Å by modeling and subtracting the "warm" contributions to the emission. HMI/SDO data allow us to focus on "inter-moss" regions in the loops. The detailed evolution of the inter-moss intensity time series reveals loops that are impulsively heated in a mode compatible with a nanoflare storm, with a spike in the hot 131 Å signals leading and the other five EUV emission channels following in progressive cooling order. A sharp increase in electron temperature tends to follow closely after the hot 131 Å signal confirming the impulsive nature of the process. A cooler process of growing emission measure follows more slowly. The Fourier power spectra of the hot 131 Å signals, when averaged over the five loops, present three scaling regimes with break frequencies near 0.1 min-1 and 0.7 min-1. The low frequency regime corresponds to 1/f noise; the intermediate indicates a persistent scaling process and the high frequencies show white noise. Very similar results are found for the energy dissipation in a 2D "hybrid" shell model of loop magneto-turbulence, based on reduced magnetohydrodynamics, that is compatible with nanoflare statistics. We suggest that such turbulent dissipation is the energy source for our loops.

  18. Scaling laws of coronal loops compared to a 3D MHD model of an active region

    NASA Astrophysics Data System (ADS)

    Bourdin, Ph.-A.; Bingert, S.; Peter, H.

    2016-05-01

    Context. The structure and heating of coronal loops have been investigated for decades. Established scaling laws relate fundamental quantities like the loop apex temperature, pressure, length, and coronal heating. Aims: We test these scaling laws against a large-scale 3D magneto-hydrodynamics (MHD) model of the solar corona, which became feasible with current high-performance computing. Methods: We drove an active region simulation with photospheric observations and find strong similarities to the observed coronal loops in X-rays and extreme-ultraviolet (EUV) wavelength. A 3D reconstruction of stereoscopic observations shows that our model loops have a realistic spatial structure. We compared scaling laws to our model data extracted along an ensemble of field lines. Finally, we fit a new scaling law that represents hot loops and also cooler structures, which was not possible before based only on observations. Results: Our model data gives some support for scaling laws that were established for hot and EUV-emissive coronal loops. For the Rosner-Tucker-Vaiana (RTV) scaling law we find an offset to our model data, which can be explained by 1D considerations of a static loop with a constant heat input and conduction. With a fit to our model data we set up a new scaling law for the coronal heat input along magnetic field lines. Conclusions: RTV-like scaling laws were fitted to hot loops and therefore do not predict well the coronal heat input for cooler structures that are barely observable. The basic differences between 1D and self-consistent 3D modeling account for deviations between earlier scaling laws and ours. We also conclude that a heating mechanism by MHD-turbulent dissipation within a braided flux tube would heat the corona stronger than is consistent with our model corona.

  19. The anti-esophageal cancer cell activity by a novel tyrosine/phosphoinositide kinase inhibitor PP121

    SciTech Connect

    Peng, Yi; Zhou, Yajuan; Cheng, Long; Hu, Desheng; Zhou, Xiaoyi; Wang, Zhaohua; Xie, Conghua; Zhou, Fuxiang

    2015-09-11

    Here we explored the potential effect of PP121, a novel dual inhibitor of tyrosine and phosphoinositide kinases, against human esophageal cancer cells. We showed that PP121 exerted potent cytotoxic effect in primary (patient-derived) and established (Eca-109, TE-1 and TE-3 lines) esophageal cancer cells, possibly through activating caspase-3-dependnent apoptosis. PP121 was, however, non-cytotoxic to the normal human esophageal epithelial cells (EECs). At the molecular level, we showed that PP121 blocked Akt-mTOR (mammalian target of rapamycin) activation in esophageal cancer cells, which was restored by introducing a constitutively-active Akt (CA-Akt). Yet, CA-Akt only partly inhibited cytotoxicity by PP121 in Eca-109 cells. Importantly, we showed that PP121 inhibited nuclear factor kappa B (NFκB) signaling activation in esophageal cancer cells, which appeared independent of Akt-mTOR blockage. In vivo, oral administration of PP121 remarkably inhibited Eca-109 xenograft growth in nude mice, and significantly improved mice survival. Further, the immunohistochemistry (IHC) and Western blot assays analyzing xenografted tumors showed that PP121 inhibited Akt-mTOR and NFκB activations in vivo. Together, we demonstrate that PP121 potently inhibits esophageal cancer cells in vitro and in vivo, possibly through concurrently inhibiting Akt-mTOR and NFκB signalings. - Highlights: • PP121 is cytotoxic against primary and established esophageal cancer cells. • PP121 induces caspase-3-dependnent apoptosis in esophageal cancer cells. • PP121 blocks Akt-mTOR activation in esophageal cancer cells. • PP121 inhibits NFκB activation, independent of Akt-mTOR blockage. • PP121 inhibits Eca-109 xenograft growth and Akt-mTOR/NFκB activation in vivo.

  20. Elevated protein tyrosine phosphatase activity provokes Eph/ephrin-facilitated adhesion of pre-B leukemia cells.

    PubMed

    Wimmer-Kleikamp, Sabine H; Nievergall, Eva; Gegenbauer, Kristina; Adikari, Samantha; Mansour, Mariam; Yeadon, Trina; Boyd, Andrew W; Patani, Neill R; Lackmann, Martin

    2008-08-01

    Signaling by Eph receptors and cell-surface ephrin ligands modulates adhesive cell properties and thereby coordinates cell movement and positioning in normal and oncogenic development. While cell contact-dependent Eph activation frequently leads to cell-cell repulsion, also the diametrically opposite response, cell-cell adhesion, is a probable outcome. However, the molecular principles regulating such disparate functions have remained controversial. We have examined cell-biologic mechanisms underlying this switch by analyzing ephrin-A5-induced cell-morphologic changes of EphA3-positive LK63 pre-B acute lymphoblastic leukemia cells. Their exposure to ephrin-A5 surfaces leads to a rapid conversion from a suspended/nonpolarized to an adherent/polarized cell type, a transition that relies on EphA3 functions operating in the absence of Eph-kinase signaling. Cell morphology change and adhesion of LK63 cells are effectively attenuated by endogenous protein tyrosine phosphatase (PTP) activity, whereby PTP inhibition and productive EphA3-phosphotyrosine signaling reverse the phenotype to nonadherent cells with a condensed cytoskeleton. Our findings suggest that Eph-associated PTP activities not only control receptor phosphorylation levels, but as a result switch the response to ephrin contact from repulsion to adhesion, which may play a role in the pathology of hematopoietic tumors. PMID:18385452

  1. Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

    NASA Astrophysics Data System (ADS)

    Cui, Yongchao; Shi, Dayong; Hu, Zhiqiang

    2011-11-01

    3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol ( 1) is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B (PTP1B). Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from 1H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-(2'-bromo-6'-(3″,4″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 2), 2,3-dibromo-1-(2'-bromo-6'-(2″-bromo-4″,5″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 3), 3,4-dibromo-5-(2'-bromo-6'-(2″-bromo-4″,5″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 4) and 3,4-dibromo-5-(2'-bromo-6'-(3″,4″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 5). PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay, and compounds 3 and 4 demonstrated interesting activity against PTP1B.

  2. TOPK promotes lung cancer resistance to EGFR tyrosine kinase inhibitors by phosphorylating and activating c-Jun

    PubMed Central

    Wang, Tao; Wang, Ting; Niu, Mengjie; Zhang, Shengli; Jia, Lintao; Li, Shengqing

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have shown promising clinical efficacy in non-squamous non-small cell lung cancer (NSCLC); however, resistance is frequently observed in malignant cells, operating through a mechanism that remains largely unknown. The present study shows that T-lymphokine-activated killer cell-originated protein kinase (TOPK) is upregulated in NSCLC and excessively activated in TKI-refractory cells. TOPK dictates the responsiveness of lung cancers to the EGFR-targeted TKI gefitinib through the transcription factor AP-1 component c-Jun. TOPK binds directly to and phosphorylates c-Jun, which consequently activates the transcription of AP-1 target genes, including CCND1 and CDC2. TOPK silencing sensitizes EGFR-TKI-resistant lung cancer cells to gefitinib and increases gefitinib efficacy in preclinical lung adenocarcinoma xenograft models. These findings represent a novel mechanism of lung cancer resistance to TKIs and suggest that TOPK may have value both as a predictive biomarker and as a therapeutic target: TOPK-targeted therapy may synergize with EGFR-targeted therapy in lung cancers. PMID:26745678

  3. Specific oncogenic activity of the Src-family tyrosine kinase c-Yes in colon carcinoma cells.

    PubMed

    Sancier, Florence; Dumont, Aurélie; Sirvent, Audrey; Paquay de Plater, Ludmilla; Edmonds, Thomas; David, Géraldine; Jan, Michel; de Montrion, Catherine; Cogé, Francis; Léonce, Stéphane; Burbridge, Michael; Bruno, Alain; Boutin, Jean A; Lockhart, Brian; Roche, Serge; Cruzalegui, Francisco

    2011-02-24

    c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

  4. Tyrosine phosphorylation and bacterial virulence

    PubMed Central

    Whitmore, Sarah E; Lamont, Richard J

    2012-01-01

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

  5. CD16-mediated p21ras activation is associated with Shc and p36 tyrosine phosphorylation and their binding with Grb2 in human natural killer cells

    PubMed Central

    1996-01-01

    The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the oncoprotein Shc have been implicated in a highly conserved mechanism that regulates p21ras activation. We investigated the involvement of these adaptor proteins in the signaling pathway induced by CD16 or interleukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphorylated upon CD16 or IL-2 stimulation with different kinetics. Shc immunoprecipitates from lysates of CD16- or IL-2-stimulated NK cells contained Grb2 and an unidentified 145-kD tyrosine phosphoprotein. Grb2 immunoprecipitates from anti-CD16-stimulated NK cells contained not only Shc, but also a 36-kD tyrosine phosphoprotein (p36). The interaction between Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated by in vitro binding assays using a bacteriologically synthesized glutathione S-transferase-Grb2SH2 fusion protein. We also present evidence that p21ras is activated by CD16 and IL-2R cross-linking. Accumulation of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphorylation and Grb2 association of Shc and p36 adaptor proteins. PMID:8551221

  6. Loop substitution as a tool to identify active sites of interleukin-1 beta.

    PubMed

    Palla, E; Bensi, G; Solito, E; Buonamassa, D T; Fassina, G; Raugei, G; Spano, F; Galeotti, C; Mora, M; Domenighini, M

    1993-06-25

    By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor. PMID:7685764

  7. PYK2: A Calcium-sensitive Protein Tyrosine Kinase Activated in Response to Fertilization of the Zebrafish Oocyte

    PubMed Central

    Sharma, Dipika; Kinsey, William H.

    2012-01-01

    Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone. PMID:23084926

  8. PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

    PubMed

    Sharma, Dipika; Kinsey, William H

    2013-01-01

    Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone.

  9. Ovary ecdysteroidogenic hormone requires a receptor tyrosine kinase to activate egg formation in the mosquito Aedes aegypti

    PubMed Central

    Vogel, Kevin J.; Brown, Mark R.; Strand, Michael R.

    2015-01-01

    Mosquitoes are major disease vectors because most species must feed on blood from a vertebrate host to produce eggs. Blood feeding by the vector mosquito Aedes aegypti triggers the release of two neurohormones, ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs), which activate multiple processes required for egg formation. ILPs function by binding to the insulin receptor, which activates downstream components in the canonical insulin signaling pathway. OEH in contrast belongs to a neuropeptide family called neuroparsins, whose receptor is unknown. Here we demonstrate that a previously orphanized receptor tyrosine kinase (RTK) from A. aegypti encoded by the gene AAEL001915 is an OEH receptor. Phylogenetic studies indicated that the protein encoded by this gene, designated AAEL001915, belongs to a clade of RTKs related to the insulin receptor, which are distinguished by an extracellular Venus flytrap module. Knockdown of AAEL001915 by RNAi disabled OEH-mediated egg formation in A. aegypti. AAEL001915 was primarily detected in the mosquito ovary in association with follicular epithelial cells. Both monomeric and dimeric AAEL001915 were detected in mosquito ovaries and transfected Drosophila S2 cells. Functional assays further indicated that OEH bound to dimeric AAEL001915, which resulted in downstream phosphorylation of Ak strain transforming factor (Akt). We hypothesize that orthologs of AAEL001915 in other insects are neuroparsin receptors. PMID:25848040

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

    PubMed

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

    2011-11-01

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

  11. Phosphorylation of serine palmitoyltransferase long chain-1 (SPTLC1) on tyrosine 164 inhibits its activity and promotes cell survival.

    PubMed

    Taouji, Saïd; Higa, Arisa; Delom, Frédéric; Palcy, Sandrine; Mahon, François-Xavier; Pasquet, Jean-Max; Bossé, Roger; Ségui, Bruno; Chevet, Eric

    2013-06-14

    In BCR-ABL-expressing cells, sphingolipid metabolism is altered. Because the first step of sphingolipid biosynthesis occurs in the endoplasmic reticulum (ER), our objective was to identify ABL targets in the ER. A phosphoproteomic analysis of canine pancreatic ER microsomes identified 49 high scoring phosphotyrosine-containing peptides. These were then categorized in silico and validated in vitro. We demonstrated that the ER-resident human protein serine palmitoyltransferase long chain-1 (SPTLC1), which is the first enzyme of sphingolipid biosynthesis, is phosphorylated at Tyr(164) by the tyrosine kinase ABL. Inhibition of BCR-ABL using either imatinib or shRNA-mediated silencing led to the activation of SPTLC1 and to increased apoptosis in both K562 and LAMA-84 cells. Finally, we demonstrated that mutation of Tyr(164) to Phe in SPTLC1 increased serine palmitoyltransferase activity. The Y164F mutation also promoted the remodeling of cellular sphingolipid content, thereby sensitizing K562 cells to apoptosis. Our observations provide a mechanistic explanation for imatinib-mediated cell death and a novel avenue for therapeutic strategies. PMID:23629659

  12. Antidepressant Activity of Enicostemma littorale Blume in Shp2 (Protein Tyrosine Phosphatase)-inhibited Animal Model of Depression

    PubMed Central

    Doss, VA; Kuberapandian, Dharaniyambigai

    2016-01-01

    Background: The objective of this study is to develop a new animal model based on signaling pathways to understand the pathophysiology, therapy of depression, and to investigate the antidepressant activity of Enicostemma littorale which is not yet established. Methods: Animal models of depression were raised by physical methods and administration of methyl isobutyl ketone (100 mg/kg b.w., i.p.,) and a protein tyrosine phosphatase inhibitor, sodium orthovanadate (30 mg/kg b.w., i.p.,) to young Wistar rats. E. littorale aqueous extract (100 mg/kg b.w., oral) was administered. Forced swimming test (FST), biochemical, and histopathological parameters were performed with reference to fluoxetine (20 mg/kg b.w., oral) treatment. Results: High-performance thin-layer chromatography confirmed the presence of swertiamarin, a unique glycoside present in the Gentianaceae family. FST indicated high rates of immobility in depressed groups and low rates in plant extract-administered group with reference to fluoxetine. Biochemical assays indicated significantly (P < 0.05) increased levels of total protein, superoxide dismutase, triglycerides, and total serum cholesterol, whereas significant reduction (P < 0.05) of glutathione peroxidase, catalase, and lipid peroxidation in plant extract-administered groups in comparison to the depressed groups. Histopathological analysis indicated disorganized neuronal architecture during depression whereas rejuvenation of neuronal patterns was observed during treatment with plant extract and fluoxetine. Conclusions: This study shows that sodium orthovanadate induces depression in animals and also establishes the antidepressant activity of E. littorale. PMID:27761214

  13. 1,2-Naphthoquinone activates vanilloid receptor 1 through increased protein tyrosine phosphorylation, leading to contraction of guinea pig trachea

    SciTech Connect

    Kikuno, Shota; Taguchi, Keiko; Iwamoto, Noriko; Yamano, Shigeru; Cho, Arthur K.; Froines, John R.; Kumagai, Yoshito . E-mail: yk-em-tu@md.tsukuba.ac.jp

    2006-01-15

    1,2-Naphthoquinone (1,2-NQ) has recently been identified as an environmental quinone in diesel exhaust particles (DEP) and atmospheric PM{sub 2.5}. We have found that this quinone is capable of causing a concentration-dependent contraction of tracheal smooth muscle in guinea pigs with EC{sub 5} value of 18.7 {mu}M. The contraction required extracellular calcium and was suppressed by L-type calcium channel blockers nifedipine and diltiazem. It was found that 1,2-NQ activated phospholipase A2 (PLA2)/lipoxygenase (LO)/vanilloid receptor (VR1) signaling. Additionally, 1,2-NQ was capable of transactivating protein tyrosine kinases (PTKs) such as epidermal growth factor receptor (EGFR) in guinea pig trachea, suggesting that phosphorylation of PTKs contributes to 1,2-NQ-induced tracheal contraction. Consistent with this notion, this action was blocked by the PTKs inhibitor genistein and the EGFR antagonist PD153035, indicating that contraction was, at least in part, attributable to PTKs phosphorylation that activates VR1, resulting in increased intracellular calcium content in the smooth muscle cells.

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

    PubMed

    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

  15. Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway.

    PubMed

    Marzec, M; Kasprzycka, M; Liu, X; El-Salem, M; Halasa, K; Raghunath, P N; Bucki, R; Wlodarski, P; Wasik, M A

    2007-08-16

    The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.

  16. Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf.

    PubMed

    Marzec, M; Kasprzycka, M; Liu, X; Raghunath, P N; Wlodarski, P; Wasik, M A

    2007-02-01

    The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma (ALK+ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126-mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK+ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cycle-promoting CDK4 and phospho-RB. Finally, siRNA-mediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies.

  17. Molecular dissection of egg fertilization signaling with the aid of tyrosine kinase-specific inhibitor and activator strategies.

    PubMed

    Sato, Ken-ichi; Iwasaki, Tetsushi; Hirahara, Shino; Nishihira, Yusuke; Fukami, Yasuo

    2004-03-11

    Fertilization is triggered by sperm-egg interaction and fusion that initiate a transient rise(s) in the free intracellular calcium ([Ca(2+)](i)) that is responsible for a series of biochemical and cell biological events, so-called "egg activation". Calcium-dependent egg activation leads to the initiation of developmental program that culminates in the birth of individuals. A growing body of knowledge has uncovered the molecular mechanisms underlying sperm-induced transient [Ca(2+)](i) increase(s) to some extent; namely, in most animals so far studied, a second messenger inositol 1,4,5-trisphosphate (IP(3)) seems to play a pivotal role in inducing [Ca(2+)](i) transient(s) at fertilization. However, signaling mechanisms used by sperm to initiate IP(3)-[Ca(2+)](i) transient pathway have not been elucidated. To approach this problem, we have employed African clawed frog, Xenopus laevis, as a model animal and conducted experiments designed specifically to determine the role of the Src family protein-tyrosine kinases (SFKs or Src family PTKs) in the sperm-induced egg activation. This review compiles information about the use of PTK-specific inhibitors and activators for analyzing signal transduction events in egg fertilization. Specifically, we focus on molecular identification of Xenopus Src and the signaling mechanism of the Src-dependent egg activation that has been established recently. We also summarize recent advances in understanding the role of the Src family kinases in egg fertilization of other model organisms, and discuss future directions of the field.

  18. Complexity of Receptor Tyrosine Kinase Signal Processing

    PubMed Central

    Volinsky, Natalia; Kholodenko, Boris N.

    2013-01-01

    Our knowledge of molecular mechanisms of receptor tyrosine kinase (RTK) signaling advances with ever-increasing pace. Yet our understanding of how the spatiotemporal dynamics of RTK signaling control specific cellular outcomes has lagged behind. Systems-centered experimental and computational approaches can help reveal how overlapping networks of signal transducers downstream of RTKs orchestrate specific cell-fate decisions. We discuss how RTK network regulatory structures, which involve the immediate posttranslational and delayed transcriptional controls by multiple feed forward and feedback loops together with pathway cross talk, adapt cells to the combinatorial variety of external cues and conditions. This intricate network circuitry endows cells with emerging capabilities for RTK signal processing and decoding. We illustrate how mathematical modeling facilitates our understanding of RTK network behaviors by unraveling specific systems properties, including bistability, oscillations, excitable responses, and generation of intricate landscapes of signaling activities. PMID:23906711

  19. Oxidative inhibition of receptor-type protein-tyrosine phosphatase kappa by ultraviolet irradiation activates epidermal growth factor receptor in human keratinocytes.

    PubMed

    Xu, Yiru; Shao, Yuan; Voorhees, John J; Fisher, Gary J

    2006-09-15

    Ultraviolet (UV) irradiation rapidly increases tyrosine phosphorylation (i.e. activates) of epidermal growth factor receptors (EGFR) in human skin. EGFR-dependent signaling pathways drive increased expression of matrix metalloproteinases, whose actions fragment collagen and elastin fibers, the primary structural protein components in skin connective tissue. Connective tissue fragmentation, which results from chronic exposure to solar UV irradiation, is a major determinant of premature skin aging (photoaging). UV irradiation generates reactive oxygen species, which readily react with conserved cysteine residues in the active site of protein-tyrosine phosphatases (PTP). We report here that EGFR activation by UV irradiation results from oxidative inhibition of receptor type PTP-kappa (RPTP-kappa). RPTP-kappa directly counters intrinsic EGFR tyrosine kinase activity, thereby maintaining EGFR in an inactive state. Reversible, oxidative inactivation of RPTP-kappa activity by UV irradiation shifts the kinase-phosphatase balance in favor of EGFR activation. These data delineate a novel mechanism of EGFR regulation and identify RPTP-kappa as a key molecular target for antioxidant protection against skin aging.

  20. Protein Tyrosine Phosphatase Activity in Insulin-Resistant Rodent Psammomys Obesus

    PubMed Central

    Meyerovitch, Joseph; Balta, Yigal; Ziv, Ehud; Sack, Joseph

    2002-01-01

    Phosphotyrosine phosphatase (PTPase) activity and its regulation by overnight food deprivation were studied in Psammomys obesus (sand rat), a gerbil model of insulin resistance and nutritionally induced diabetes mellitus. PTPase activity was measured using a phosphopeptide substrate containing a sequence identical to that of the major site of insulin receptor (IR) β-subunit autophosphorylation. The PTPase activity in membrane fractions was 3.5-, 8.3-, and 5.9-fold lower in liver, fat, and skeletal muscle, respectively, compared with corresponding tissues of albino rat.Western blotting of tissue membrane fractions in Psammomys showed lower PTPase and IR than in albino rats. The density of PTPase transmembrane protein band was 5.5-fold lower in liver and 12-fold lower in adipose tissue. Leukocyte antigen receptor (LAR) and IR were determined by specific immunoblotting and protein bands densitometry and were also found to be 6.3-fold lower in the liver and 22-fold lower in the adipose tissue in the hepatic membrane fractions. Liver cytosolic PTPase activity after an overnight food deprivation in the nondiabetic Psammomys rose 3.7-fold compared with postprandial PTPase activity, but it did not change significantly in diabetic fasted animals. Similar fasting-related changes were detected in the activity of PTPase derived from membrane fraction. In conclusion, the above data demonstrate that despite the insulin resistance, Psammomys is characterized by low level of PTPase activities in membrane and cytosolic fractions in all 3 major insulin responsive tissues, as well as in liver. PTPase activity does not rise in activity as a result of insulin resistance and nutritionally induced diabetes. PMID:12458662

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

    PubMed Central

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

    2001-01-01

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

  2. Comparative Analysis of Mutant Tyrosine Kinase Chemical Rescue†

    PubMed Central

    Muratore, Kathryn E.; Seeliger, Markus A.; Wang, Zhihong; Fomina, Dina; Neiswinger, Johnathan; Havranek, James J.; Baker, David; Kuriyan, John; Cole, Philip A.

    2009-01-01

    Protein tyrosine kinases are critical cell signaling enzymes. These enzymes have a highly conserved Arg residue in their catalytic loop which is present two residues or four residues downstream from an absolutely conserved Asp catalytic base. Prior studies on protein tyrosine kinases Csk and Src revealed the potential for chemical rescue of catalytically-deficient mutant kinases (Arg to Ala mutations) by small diamino compounds, particularly imidazole, however the potency and efficiency of rescue was greater for Src. This current study further examines the structural and kinetic basis of rescue for mutant Src as compared to mutant Abl tyrosine kinase. An X-ray crystal structure of R388A Src revealed the surprising finding that a histidine residue of the N-terminus of a symmetry-related kinase inserts into the active site of the adjacent Src and mimics the hydrogen bonding pattern seen in wild-type protein tyrosine kinases. Abl R367A shows potent and efficient rescue more comparable to Src, even though its catalytic loop is more like that of Csk. Various enzyme redesigns of the active sites indicate that the degree and specificity of rescue is somewhat flexible, but the overall properties of the enzymes and rescue agents play an overarching role. The newly discovered rescue agent 2-aminoimidazole is about as efficient as imidazole in rescuing R/A Src and Abl. Rate vs. pH studies with these imidazole analogs suggest that the protonated imidazolium is the preferred form for chemical rescue, consistent with structural models. The efficient rescue seen with mutant Abl points to the potential of this approach to be used effectively to analyze Abl phosphorylation pathways in cells. PMID:19260709

  3. LRRK2 Inhibits FAK Activity by Promoting FERM-mediated Autoinhibition of FAK and Recruiting the Tyrosine Phosphatase, SHP-2

    PubMed Central

    Choi, Insup; Byun, Ji-won; Park, Sang Myun; Jou, Ilo

    2016-01-01

    Mutation of leucine-rich repeat kinase 2 (LRRK2) causes an autosomal dominant and late-onset familial Parkinson's disease (PD). Recently, we reported that LRRK2 directly binds to and phosphorylates the threonine 474 (T474)-containing Thr-X-Arg(Lys) (TXR) motif of focal adhesion kinase (FAK), thereby inhibiting the phosphorylation of FAK at tyrosine (Y) 397 residue (pY397-FAK), which is a marker of its activation. Mechanistically, however, it remained unclear how T474-FAK phosphorylation suppressed FAK activation. Here, we report that T474-FAK phosphorylation could inhibit FAK activation via at least two different mechanisms. First, T474 phosphorylation appears to induce a conformational change of FAK, enabling its N-terminal FERM domain to autoinhibit Y397 phosphorylation. This is supported by the observation that the levels of pY397-FAK were increased by deletion of the FERM domain and/or mutation of the FERM domain to prevent its interaction with the kinase domain of FAK. Second, pT474-FAK appears to recruit SHP-2, which is a phosphatase responsible for dephosphorylating pY397-FAK. We found that mutation of T474 into glutamate (T474E-FAK) to mimic phosphorylation induced more strong interaction with SHP-2 than WT-FAK, and that pharmacological inhibition of SHP-2 with NSC-87877 rescued the level of pY397 in HEK293T cells. These results collectively show that LRRK2 suppresses FAK activation through diverse mechanisms that include the promotion of autoinhibition and/or the recruitment of phosphatases, such as SHP-2. PMID:27790061

  4. Spatial coupling of JNK activation to the B cell antigen receptor by tyrosine-phosphorylated ezrin1

    PubMed Central

    Parameswaran, Neetha; Enyindah-Asonye, Gospel; Bagheri, Nayer; Shah, Neilay B.; Gupta, Neetu

    2013-01-01

    The Ezrin-Radixin-Moesin (ERM) proteins regulate B lymphocyte activation via their effect on BCR diffusion and microclustering. This relies on their ability to dynamically tether the plasma membrane with actin filaments that is in turn facilitated by phosphorylation of the conserved threonine residue in the actin-binding domain. Here, we describe a novel function of ezrin in regulating JNK activation that is mediated by phosphorylation of a tyrosine (Y353) residue that is unconserved with moesin and radixin. BCR, but not CD40, TLR4 or CXCR5 stimulation, induced phosphorylation of ezrin at Y353 in mouse splenic B cells. Ezrin existed in a preformed complex with Syk in unstimulated B cells and underwent Syk-dependent phosphorylation upon anti-IgM stimulation. Y353-phosphorylated ezrin co-localized with the BCR within minutes of stimulation and co-trafficked with the endocytosed BCRs through the early and late endosomes. The T567 residue of ezrin was rephosphorylated in late endosomes and at the plasma membrane at later times of BCR stimulation. Expression of a non-phosphorylatable Y353F mutant of ezrin specifically impaired JNK activation. BCR crosslinking induced the association of Y353-phosphorylated ezrin with JNK and its kinase MKK7, and spatial co-localization with phosphorylated JNK in the endosomes. The YFP-tagged Y353F mutant displayed reduced co-localization with the endocytosed BCR as compared to wild type Ezrin-YFP. Taken together, our data identify a novel role for ezrin as a spatial adaptor that couples JNK signaling components to the BCR signalosome, thus facilitating JNK activation. PMID:23338238

  5. Mycobacterial Protein Tyrosine Phosphatases A and B Inhibitors Augment the Bactericidal Activity of the Standard Anti-tuberculosis Regimen

    PubMed Central

    Dutta, Noton K.; He, Rongjun; Pinn, Michael L.; He, Yantao; Burrows, Francis; Zhang, Zhong-Yin; Karakousis, Petros C.

    2016-01-01

    Novel drugs are required to shorten the duration of treatment for tuberculosis (TB) and to combat the emergence of drug resistance. One approach has been to identify and target Mycobacterium tuberculosis (Mtb) virulence factors, which promote the establishment of TB infection and pathogenesis. Mtb produces a number of virulence factors, including two protein tyrosine phosphatases (PTPs), mPTPA and mPTPB, to evade the antimicrobial functions of host macrophages. To assess the therapeutic potential of targeting the virulent Mtb PTPs, we developed highly potent and selective inhibitors of mPTPA (L335-M34) and mPTPB (L01-Z08) with drug-like properties. We tested the bactericidal activity of L335-M34 and L01-Z08 alone or together in combination with the standard antitubercular regimen of isoniazid-rifampicin-pyrazinamide (HRZ) in the guinea pig model of chronic TB infection, which faithfully recapitulates some of the key histological features of human TB lesions. Following a single dose of L335-M34 50mg/kg and L01-Z08 20 mg/kg, plasma levels were maintained at levels 10-fold greater than the biochemical IC50 for 12–24 hours. Although neither PTP inhibitor alone significantly enhanced the antibacterial activity of HRZ, dual inhibition of mPTPA and mPTPB in combination with HRZ showed modest synergy, even after 2 weeks of treatment. After 6 weeks of treatment, the degree of lung inflammation correlated with the bactericidal activity of each drug regimen. This study highlights the potential utility of targeting Mtb virulence factors, and specifically the Mtb PTPs, as a strategy for enhancing the activity of standard anti-TB treatment. PMID:27478867

  6. Cisplatin stimulates protein tyrosine phosphorylation in macrophages.

    PubMed

    Kumar, R; Shrivastava, A; Sodhi, A

    1995-03-01

    Cisplatin [cis-dichlorodiamine platinum (II)], a potent anti-tumor compound, stimulates immune responses by activating monocyte-macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event that mediates cellular responses, we examined whether cisplatin alters tyrosine phosphorylation in macrophages. We found that cisplatin increased tyrosine phosphorylation of several proteins in peritoneal macrophages and in P388D1 and IC-21 macrophage cell lines. Treatment of macrophages with tyrosine kinase inhibitors, genestein and lavendustin A, inhibited cisplatin-stimulated protein tyrosine phosphorylation in macrophages. Macrophages treated with cisplatin also exhibit increased fluorescence with anti-phosphotyrosine-FITC antibody. These data indicate that protein tyrosine phosphorylation plays a role in cisplatin-induced activation of macrophages. PMID:7539662

  7. Cdk5/p35 phosphorylates lemur tyrosine kinase-2 to regulate protein phosphatase-1C phosphorylation and activity.

    PubMed

    Manser, Catherine; Vagnoni, Alessio; Guillot, Florence; Davies, Jennifer; Miller, Christopher C J

    2012-05-01

    Cyclin-dependent kinase-5 (cdk5)/p35 and protein phosphatase-1 (PP1) are two major enzymes that control a variety of physiological processes within the nervous system including neuronal differentiation, synaptic plasticity and axonal transport. Defective cdk5/p35 and PP1 function are also implicated in several major human neurodegenerative diseases. Cdk5/p35 and the catalytic subunit of PP1 (PP1C) both bind to the brain-enriched, serine-threonine kinase lemur tyrosine kinase-2 (LMTK2). Moreover, LMTK2 phosphorylates PP1C on threonine-320 (PP1Cthr³²⁰) to inhibit its activity. Here, we demonstrate that LMTK2 is phosphorylated on serine-1418 (LMTK2ser¹⁴¹⁸) by cdk5/p35 and present evidence that this regulates its ability to phosphorylate PP1Cthr³²⁰. We thus describe a new signalling pathway within the nervous system that links cdk5/p35 with PP1C and which has implications for a number of neuronal functions and neuronal dysfunction.

  8. A new activity of doublecortin in recognition of the phospho-FIGQY tyrosine in the cytoplasmic domain of neurofascin.

    PubMed

    Kizhatil, Krishnakumar; Wu, Yi-Xin; Sen, Anindita; Bennett, Vann

    2002-09-15

    Doublecortin is a cytoplasmic protein mutated in the neuronal migration disorder X-linked lissencephaly. This study describes a novel activity of doublecortin in recognition of the FIGQY-phosphotyrosine motif present in the cytoplasmic domain of the L1 cell adhesion molecule neurofascin. Phospho-FIGQY-neurofascin (186 kDa) coimmunoprecipitated with doublecortin from detergent extracts of embryonic brain membranes, and this doublecortin-phospho-FIGQY neurofascin complex was disassociated by a synthetic phospho-FIGQY neurofascin peptide but not by a dephospho-FIGQY peptide. Doublecortin specifically recognized the phospho-FIGQY tyrosine in the context of a synthetic phospho-FIGQY neurofascin peptide and in phospho-FIGQY neurofascin isolated from cells treated with pervanadate. Mutations of doublecortin causing lissencephaly (R59H, D62N, and G253D) abolished binding to the phospho-FIGQY peptide and to phospho-FIGQY neurofascin. Finally, phospho-FIGQY neurofascin and doublecortin colocalize in developing axon tracts and in zones enriched in migrating neurons in the embryonic cerebral cortex. In the adult rostral migratory stream, doublecortin colocalizes in migrating neurons with a phospho-FIGQY bearing L1 CAM different from neurofascin. The finding that doublecortin associates with FIGQY-phosphorylated neurofascin provides the first connection of doublecortin with the plasma membrane and could be important for a function of doublecortin in directing neuronal migration.

  9. Effect of 2',6'-dimethyl-L-tyrosine (Dmt) on pharmacological activity of cyclic endomorphin-2 and morphiceptin analogs.

    PubMed

    Fichna, Jakub; Perlikowska, Renata; Wyrębska, Anna; Gach, Katarzyna; Piekielna, Justyna; do-Rego, Jean Claude; Toth, Geza; Kluczyk, Alicja; Janecki, Tomasz; Janecka, Anna

    2011-12-01

    This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-Yaa)NH(2) or Tyr-c(Xaa-Phe-D-Pro-Yaa)NH(2), respectively, where Xaa and Yaa were L/D Asp or L/D Lys. Further modification of these analogs was achieved by introduction of 2',6'-dimethyl-L-tyrosine (Dmt) instead of Tyr in position 1. Peptides were synthesized by solid phase method and cleaved from the resin by a microwave-assisted procedure. Dmt(1)-substituted analogs displayed high affinity at the μ-opioid receptors, remained intact after incubation with the rat brain homogenate and showed remarkable, long-lasting μ-opioid receptor-mediated antinociceptive activity after central, but not peripheral administration. Our results demonstrate that cyclization is a promising strategy in the development of new opioid analgesics, but further modifications are necessary to enhance the blood-brain barrier permeability.

  10. Src Tyrosine Kinase Activation by 4-Hydroxynonenal Upregulates p38, ERK/AP-1 Signaling and COX-2 Expression in YPEN-1 Cells

    PubMed Central

    Jang, Eun Ji; Jeong, Hyoung Oh; Park, Daeui; Kim, Dae Hyun; Choi, Yeon Ja; Chung, Ki Wung; Park, Min Hi; Yu, Byung Pal; Chung, Hae Young

    2015-01-01

    4-Hydroxynonenal (4-HNE), a major end product of lipid peroxidation, is highly reactive and involved in various cellular processes, such as inflammatory signaling. However, to date, the mechanistic roles of 4-HNE in inflammatory signaling related to protein tyrosine kinases have not been elucidated. In the present study, we investigated the interaction between 4-HNE and Src (a non-receptor tyrosine kinase) for its involvement in the molecular modulation of the inflammatory signaling pathway utilizing the YPEN-1 cell system. Immunoprecipitation experiments showed that 4-HNE phosphorylates (activates) Src at Tyr416 via adduct formation. In addition, LC-MS/MS and a docking simulation model revealed an addiction site at the Cys248 residue of Src, resulting in the stimulation of downstream p38, ERK/AP-1 and cyclooxygenase-2 (COX-2) signaling in YPEN-1 cells. The role of 4-HNE-activated Src in downstream inflammatory signaling was further investigated using dasatinib (a Src inhibitor) and by siRNA knockdown of Src. p38 and ERK were directly regulated by Src, as revealed by immunoblotting of the phosphorylated forms of mitogen-activated protein kinases (MAPKs), which are key elements in the signaling transduction pathway initiated by Src. The study also shows that Src modulates the HNE-enhanced activation of AP-1 and the expression of COX-2 (a target gene of AP-1). Together, the results of this study show that 4-HNE stimulates Src tyrosine kinase in activation of the inflammation process. PMID:26466383

  11. The tyrosine phosphatase PTPRO sensitizes colon cancer cells to anti-EGFR therapy through activation of SRC-mediated EGFR signaling.

    PubMed

    Asbagh, Layka Abbasi; Vazquez, Iria; Vecchione, Loredana; Budinska, Eva; De Vriendt, Veerle; Baietti, Maria Francesca; Steklov, Mikhail; Jacobs, Bart; Hoe, Nicholas; Singh, Sharat; Imjeti, Naga-Sailaja; Zimmermann, Pascale; Sablina, Anna; Tejpar, Sabine

    2014-10-30

    Inappropriate activation of epidermal growth factor receptor (EGFR) plays a causal role in many cancers including colon cancer. The activation of EGFR by phosphorylation is balanced by receptor kinase and protein tyrosine phosphatase activities. However, the mechanisms of negative EGFR regulation by tyrosine phosphatases remain largely unexplored. Our previous results indicate that protein tyrosine phosphatase receptor type O (PTPRO) is down-regulated in a subset of colorectal cancer (CRC) patients with a poor prognosis. Here we identified PTPRO as a phosphatase that negatively regulates SRC by directly dephosphorylating Y416 phosphorylation site. SRC activation triggered by PTPRO down-regulation induces phosphorylation of both EGFR at Y845 and the c-CBL ubiquitin ligase at Y731. Increased EGFR phosphorylation at Y845 promotes its receptor activity, whereas enhanced phosphorylation of c-CBL triggers its degradation promoting EGFR stability. Importantly, hyperactivation of SRC/EGFR signaling triggered by loss of PTPRO leads to high resistance of colon cancer to EGFR inhibitors. Our results not only highlight the PTPRO contribution in negative regulation of SRC/EGFR signaling but also suggest that tumors with low PTPRO expression may be therapeutically targetable by anti-SRC therapies. PMID:25301722

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

    PubMed

    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 CjWRKY(Y115E) 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

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

    PubMed Central

    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

  14. Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the Fc gamma-IIA receptor.

    PubMed Central

    Yanaga, F; Poole, A; Asselin, J; Blake, R; Schieven, G L; Clark, E A; Law, C L; Watson, S P

    1995-01-01

    Activation of human platelets by cross-linking of the platelet low-affinity IgG receptor, the Fc gamma receptor IIA (Fc gamma-RIIA), or by collagen is associated with rapid phosphorylation on tyrosine of the non-receptor tyrosine kinase syk. Phosphorylation is still observed, albeit sometimes reduced, in the presence of a combination of a protein kinase C inhibitor, Ro 31-8220, and the intracellular calcium chelator, BAPTA-AM, demonstrating independence from phosphoinositide-specific phospholipase C (PLC) activity. In contrast, the combination of Ro 31-8220 and BAPTA-AM completely inhibits phosphorylation of syk in thrombin-stimulated platelets. Phosphorylation of syk increases its autophosphorylation activity measured in a kinase assay performed on syk immunoprecipitates. Fc gamma-RIIA also undergoes phosphorylation in syk immunoprecipitates from platelets activated by cross-linking of Fc gamma-RIIA but not by collagen, suggesting that it associates with the kinase. Consistent with this, tyrosine-phosphorylated Fc gamma-RIIA is precipitated by a glutathione S-transferase (GST) fusion protein containing the tandem src homology (SH2) domains of syk from Fc gamma-RIIA- but not collagen-activated cells. Two uncharacterized tyrosine-phosphorylated proteins of 40 and 65 kDa are uniquely precipitated by a GST fusion protein containing the tandem syk-SH2 domains in collagen-stimulated platelets. A peptide based on the antigen recognition activation motif (ARAM) of Fc gamma-RIIA, and phosphorylated on the two tyrosine residues found within this region, selectively binds syk from lysates of resting platelets; this interaction is not seen with a non-phosphorylated peptide. Kinase assays on Fc gamma-RIIA immunoprecipitates reveal the constitutive association of an unidentified kinase activity in resting cells which phosphorylates a 67 kDa protein. Syk is not detected in Fc gamma-RIIA immunoprecipitates from resting cells but associates with the receptor following activation

  15. Src-Mediated Phosphorylation of the Tyrosine Phosphatase PRL-3 Is Required for PRL-3 Promotion of Rho Activation, Motility and Invasion

    PubMed Central

    Fiordalisi, James J.; Dewar, Brian J.; Graves, Lee M.; Madigan, James P.; Cox, Adrienne D.

    2013-01-01

    The metastasis-associated tyrosine phosphatase PRL-3/PTP4A is upregulated in numerous cancers, but the mechanisms modulating PRL-3 activity other than its expression levels have not been investigated. Here we report evidence for both Src-dependent tyrosine phosphorylation of PRL-3 and Src-mediated regulation of PRL-3 biological activities. We used structural mutants, pharmacological inhibitors and siRNA to demonstrate Src-dependent phosphorylation of endogenous PRL-3 in SW480 colon cancer cells. We also demonstrated that PRL-3 was not tyrosine phosphorylated in SYF mouse embryo fibroblasts deficient in Src, Yes and Fyn unless Src was re-expressed. Further, we show that platelet-derived growth factor (PDGF) can stimulate PRL-3 phosphorylation in a Src-dependent manner. Finally, we show that PRL-3-induced cell motility, Matrigel invasion and activation of the cytoskeleton-regulating small GTPase RhoC were abrogated in the presence of the phosphodeficient PRL-3 mutant Y53F, or by use of a Src inhibitor. Thus, PRL-3 requires the activity of a Src kinase, likely Src itself, to promote these cancer-associated phenotypes. Our data establish a model for the regulation of PRL-3 by Src that supports the possibility of their coordinate roles in signaling pathways promoting invasion and metastasis, and supports simultaneous use of novel molecularly targeted therapeutics directed at these proteins. PMID:23691193

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

    PubMed Central

    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

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

    PubMed

    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.

  18. Activation of platelet-activating factor receptor and pleiotropic effects on tyrosine phospho-EGFR/Src/FAK/paxillin in ovarian cancer.

    PubMed

    Aponte, Margarita; Jiang, Wei; Lakkis, Montaha; Li, Ming-Jiang; Edwards, Dale; Albitar, Lina; Vitonis, Allison; Mok, Samuel C; Cramer, Daniel W; Ye, Bin

    2008-07-15

    Among the proinflammatory mediators, platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a major primary and secondary messenger involved in intracellular and extracellular communication. Evidence suggests that PAF plays a significant role in oncogenic transformation, tumor growth, angiogenesis, and metastasis. However, PAF, with its receptor (PAFR) and their downstream signaling targets, has not been thoroughly studied in cancer. Here, we characterized the PAFR expression pattern in 4 normal human ovarian surface epithelial (HOSE) cell lines, 13 ovarian cancer cell lines, paraffin blocks (n = 84), and tissue microarrays (n = 230) from patients with ovarian cancer. Overexpression of PAFR was found in most nonmucinous types of ovarian cancer but not in HOSE and mucinous cancer cells. Correspondingly, PAF significantly induced cell proliferation and invasion only in PAFR-positive cells (i.e., OVCA429 and OVCA432), but not in PAFR-negative ovarian cells (HOSE and mucinous RMUG-L). The dependency of cell proliferation and invasion on PAFR was further confirmed using PAFR-specific small interfering RNA gene silencing probes, antibodies against PAFR and PAFR antagonist, ginkgolide B. Using quantitative multiplex phospho-antibody array technology, we found that tyrosine phosphorylation of EGFR/Src/FAK/paxillin was coordinately activated by PAF treatment, which was correlated with the activation of phosphatidylinositol 3-kinase and cyclin D1 as markers for cell proliferation, as well as matrix metalloproteinase 2 and 9 for invasion. Specific tyrosine Src inhibitor (PP2) reversibly blocked PAF-activated cancer cell proliferation and invasion. We suggest that PAFR is an essential upstream target of Src and other signal pathways to control the PAF-mediated cancer progression.

  19. Motor activity affects adult skeletal muscle re-innervation acting via tyrosine kinase receptors.

    PubMed

    Sartini, Stefano; Bartolini, Fanny; Ambrogini, Patrizia; Betti, Michele; Ciuffoli, Stefano; Lattanzi, Davide; Di Palma, Michael; Cuppini, Riccardo

    2013-05-01

    Recently, muscle expression of brain-derived neurotrophic factor (BDNF) mRNA and protein under activity control has been reported. BDNF is a neurotrophin known to be involved in axon sprouting in the CNS. Hence, we set out to study the effect of chronic treadmill mid-intensity running on adult rat muscle re-innervation, and to explore the involvement of BDNF and tropomyosin-related kinase (Trk) receptors. After nerve crush, muscle re-innervation was evaluated using intracellular recordings, tension recordings, immunostaining and Western blot analyses. An enhanced muscle multiple innervation was found in running rats that was fully reversed to control values blocking Trk receptors or interrupting the running activity. An increase in muscle multiple innervation was also found in sedentary rats treated with a selective TrkB receptor agonist. The expression of TrkB receptors by intramuscular axons was demonstrated, and increased muscle expression of BDNF was found in running animals. The increase in muscle multiple innervation was consistent with the faster muscle re-innervation that we found in running animals. We conclude that, when regenerating axons contact muscle cells, muscle activity progressively increases modulating BDNF and possibly other growth factors, which in turn, acting via Trk receptors, induce axon sprouting to re-innervate skeletal muscle.

  20. Synaptic generation of an intracellular retrograde signal requires activation of the tyrosine kinase and mitogen-activated protein kinase signaling cascades in Aplysia.

    PubMed

    Stough, Shara; Kopec, Ashley M; Carew, Thomas J

    2015-11-01

    Cellular changes underlying memory formation can be generated in an activity-dependent manner at specific synapses. Thus an important question concerns the mechanisms by which synaptic signals communicate with the cell body to mediate these cellular changes. A monosynaptic circuit that is enhanced by sensitization in Aplysia is well-suited to study this question because three different subcellular compartments: (i) the sensorimotor SN-MN synapses, (ii) the SN projections to MNs via axonal connections, (iii) the SN cell bodies, can all be manipulated and studied independently. Here, we report that activity-dependent (AD) training in either the entire SN-MN circuit or in only the synaptic compartment, activates MAPK in a temporally and spatially specific pattern. Specifically, we find (i) MAPK activation is first transiently generated at SN-MN synapses during training, (ii) immediately after training MAPK is transiently activated in SN-MN axonal connections and persistently activated in SN cell bodies, and finally, (iii) MAPK is activated in SN cell bodies and SN-MN synapses 1h after training. These data suggest that there is an intracellularly transported retrograde signal generated at the synapse which is later responsible for delayed MAPK activation at SN somata. Finally, we find that this retrograde signal requires activation of tyrosine kinase (TK) and MEK signaling cascades at the synapses.

  1. The pepsin residue glycine-76 contributes to active-site loop flexibility and participates in catalysis.

    PubMed Central

    Okoniewska, M; Tanaka, T; Yada, R Y

    2000-01-01

    Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71--80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes. PMID:10861225

  2. Heating mechanisms for intermittent loops in active region cores from AIA/SDO EUV observations

    SciTech Connect

    Cadavid, A. C.; Lawrence, J. K.; Christian, D. J.; Jess, D. B.; Nigro, G.

    2014-11-01

    We investigate intensity variations and energy deposition in five coronal loops in active region cores. These were selected for their strong variability in the AIA/SDO 94 Å intensity channel. We isolate the hot Fe XVIII and Fe XXI components of the 94 Å and 131 Å by modeling and subtracting the 'warm' contributions to the emission. HMI/SDO data allow us to focus on 'inter-moss' regions in the loops. The detailed evolution of the inter-moss intensity time series reveals loops that are impulsively heated in a mode compatible with a nanoflare storm, with a spike in the hot 131 Å signals leading and the other five EUV emission channels following in progressive cooling order. A sharp increase in electron temperature tends to follow closely after the hot 131 Å signal confirming the impulsive nature of the process. A cooler process of growing emission measure follows more slowly. The Fourier power spectra of the hot 131 Å signals, when averaged over the five loops, present three scaling regimes with break frequencies near 0.1 min{sup –1} and 0.7 min{sup –1}. The low frequency regime corresponds to 1/f noise; the intermediate indicates a persistent scaling process and the high frequencies show white noise. Very similar results are found for the energy dissipation in a 2D 'hybrid' shell model of loop magneto-turbulence, based on reduced magnetohydrodynamics, that is compatible with nanoflare statistics. We suggest that such turbulent dissipation is the energy source for our loops.

  3. NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site

    PubMed Central

    Bonneau, Eric; Girard, Nicolas; Boisbouvier, Jérôme; Legault, Pascale

    2011-01-01

    The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem–loop VI fragment containing the A730 internal loop, which forms part of the active site. In the presence of magnesium ions, the A730 loop adopts a structure that is consistent with existing biochemical data and most likely reflects its conformation in the VS ribozyme prior to docking with the cleavage site internal loop. Interestingly, the A730 loop adopts an S-turn motif that is also present in loop B within the hairpin ribozyme active site. The S-turn appears necessary to expose the Watson–Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis. The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme. These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme. PMID:21266483

  4. Selective antiviral activity of synthetic soluble L-tyrosine and L-dopa melanins against human immunodeficiency virus in vitro.

    PubMed

    Montefiori, D C; Zhou, J Y

    1991-01-01

    Melanins are pigments found in hair, skin, irides of the eye, and brain. Their functions in mammals include protection from exposure to sunlight, camouflage from predators, sexual recognition within species, and possible electron transfer reactants. Most natural melanins exist in an insoluble form, which is one reason there is little information on the biological properties of soluble melanins. Here, synthetic soluble melanins were obtained by chemical oxidation of L-tyrosine or spontaneous oxidation of L-beta-3,4-dihydroxyphenylalanine (L-dopa). Replication of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) was inhibited by soluble melanin in two human lymphoblastoid cell lines (MT-2 and H9) and in phytohemagglutinin-stimulated human T cells. Effective concentrations of 0.15-10 micrograms/ml had no cell toxicity. Melanin blocked infection by cell-free virus and interfered with HIV-induced syncytium formation and cytopathic effects when fusion-susceptible, uninfected cells, were mixed with chronically infected cells. Melanin also impeded the HIV-1 envelope surface glycoprotein, and T cell specific monoclonal antibody leu-3a (CD4), but not leu-5b (CD2), from binding to the surface of MT-2 cells. No effect on HIV-1 reverse transcriptase activity in viral lysates was observed. These results identify a unique biological property of melanin, and suggest that soluble melanins may represent a new class of pharmacologically active substances which should be further investigated for potential therapeutic utility in the treatment of Acquired Immune Deficiency Syndrome (AIDS).

  5. Protease activated receptor 1 (PAR1) enhances Src-mediated tyrosine phosphorylation of NMDA receptor in intracerebral hemorrhage (ICH)

    PubMed Central

    Duan, Zhen-Zhen; Zhang, Feng; Li, Feng-Ying; Luan, Yi-Fei; Guo, Peng; Li, Yi-Hang; Liu, Yong; Qi, Su-Hua

    2016-01-01

    It has been demonstrated that Src could modulate NMDA receptor, and PAR1 could also affect NMDAR signaling. However, whether PAR1 could regulate NMDAR through Src under ICH has not yet been investigated. In this study, we demonstrated the role of Src-PSD95-GluN2A signaling cascades in rat ICH model and in vitro thrombin challenged model. Using the PAR1 agonist SFLLR, antagonist RLLFS and Src inhibitor PP2, electrophysiological analysis showed that PAR1 regulated NMDA-induced whole-cell currents (INMDA) though Src in primary cultured neurons. Both in vivo and in vitro results showed the elevated phosphorylation of tyrosine in Src and GluN2A and enhanced interaction of the Src-PSD95-GluN2A under model conditions. Treatment with the PAR1 antagonist RLLFS, AS-PSD95 (Antisense oligonucleotide against PSD95) and Src inhibitor PP2 inhibited the interaction among Src-PSD95-GluN2A, and p-Src, p-GluN2A. Co-application of SFLLR and AS-PSD95, PP2, or MK801 (NMDAR inhibitor) abolished the effect of SF. In conclusion, our results demonstrated that activated thrombin receptor PAR1 induced Src activation, enhanced the interaction among Src-PSD95-GluN2A signaling modules, and up-regulated GluN2A phosphorylation after ICH injury. Elucidation of such signaling cascades would possibly provide novel targets for ICH treatment. PMID:27385592

  6. Validation of in vivo pharmacodynamic activity of a novel PDGF receptor tyrosine kinase inhibitor using immunohistochemistry and quantitative image analysis.

    PubMed

    D'Andrea, Michael R; Mei, Jay M; Tuman, Robert W; Galemmo, Robert A; Johnson, Dana L

    2005-08-01

    With the advent of agents directed against specific molecular targets in drug discovery, it has become imperative to show a compound's cellular impact on the intended biomolecule in vivo. The objective of the present study was to determine if we could develop an assay to validate the in vivo effects of a compound. Hence, we investigated the in vivo pharmacodynamic activity of JNJ-10198409, a relatively selective inhibitor of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK), in tumor tissues after administering the compound orally in a nude mouse xenograft model of human LoVo colon cancer. We developed a novel assay to quantify the in vivo anti-PDGF-RTK activity of the inhibitor in tumor tissue by determining the phosphorylation status of phospholipase Cgamma1 (PLCgamma1), a key downstream cellular molecule in the PDGF-RTK signaling cascade. We used two antibodies, one specific for the total (phosphorylated and unphosphorylated forms) PLCgamma1 (pan-PLCgamma1) and the other, specific for phosphorylated form of PLCgamma1 (ph-PLCgamma1) to immunohistochemically detect their expression in tumor tissues. Computer-assisted image analysis was then used to directly compare the ratio of ph-PLCgamma1 to pan-PLCgamma1 immunolabeling intensities in serial sections (5 mum) of tumors obtained from vehicle- and JNJ-10198409-treated tumor-bearing mice. Our data showed statistically significant, dose-dependent differences in the ph-PLC/pan-PLC ratio among the four treatment groups (vehicle, 25, 50, and 100 mg/kg b.i.d.). These results confirmed this compound's ability to suppress PDGF-RTK downstream signaling in tumor tissues in vivo. In addition to this specific application of this in vivo validation approach to those targets that use PLCgamma as a downstream signaling partner, these methods may also benefit other drug discovery targets. PMID:16093435

  7. Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

    SciTech Connect

    Ikeda, Kikuko; Nakayama, Yuji; Togashi, Yuuki; Obata, Yuuki; Kuga, Takahisa; Kasahara, Kousuke; Fukumoto, Yasunori; Yamaguchi, Naoto

    2008-11-01

    Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification.

  8. Tyrosine kinase activity of CD4-associated p56lck may not be required for CD4-dependent T-cell activation.

    PubMed Central

    Collins, T L; Burakoff, S J

    1993-01-01

    The lymphoid-specific tyrosine kinase p56lck (Lck) is critical for the development and activation of T lymphocytes, and Lck kinase activity has been implicated in both T-cell antigen receptor/CD3- and CD4-mediated signaling. CD4-dependent T-cell activation has been demonstrated to be dependent upon the association of CD4 with Lck. To examine the role of the kinase activity of Lck in CD4-dependent T-cell activation, we have generated several kinase-deficient mutants of Lck. When transfected into CD4+ murine T-cell hybridoma cells, these mutants cause approximately 90% diminution in CD4-associated Lck kinase activity. Specifically, upon CD4 crosslinking there is decreased Lck autophosphorylation and decreased phosphorylation of an exogenous substrate. When CD4 is crosslinked to the T-cell antigen receptor-CD3 complex, decreased phosphorylation of associated substrates is also observed. In spite of this striking inhibition of Lck kinase function, cells expressing the kinase-deficient mutants demonstrate normal or enhanced CD4-dependent antigen responsiveness. These data demonstrate that the level of Lck kinase activity does not correlate with its CD4-associated function and suggest that the kinase activity of Lck may not be required for CD4-mediated signaling. Images Fig. 1 Fig. 2 Fig. 3 PMID:7505449

  9. Regulation of alternative macrophage activation in the liver following acetaminophen intoxication by stem cell-derived tyrosine kinase

    SciTech Connect

    Gardner, Carol R.; Hankey, Pamela; Mishin, Vladimir; Francis, Mary; Yu, Shan; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-07-15

    Stem cell-derived tyrosine kinase (STK) is a transmembrane receptor reported to play a role in macrophage switching from a classically activated/proinflammatory phenotype to an alternatively activated/wound repair phenotype. In the present studies, STK{sup −/−} mice were used to assess the role of STK in acetaminophen-induced hepatotoxicity as evidence suggests that the pathogenic process involves both of these macrophage subpopulations. In wild type mice, centrilobular hepatic necrosis and increases in serum transaminase levels were observed within 6 h of acetaminophen administration (300 mg/kg, i.p.). Loss of STK resulted in a significant increase in sensitivity of mice to the hepatotoxic effects of acetaminophen and increased mortality, effects independent of its metabolism. This was associated with reduced levels of hepatic glutathione, rapid upregulation of inducible nitric oxide synthase, and prolonged induction of heme oxygenase-1, suggesting excessive oxidative stress in STK{sup −/−} mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK{sup −/−} mice. Whereas F4/80{sup +} macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK{sup −/−} mice. In wild type mice hepatic expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12, products of classically activated macrophages, increased after acetaminophen administration. Monocyte chemotactic protein-1 (MCP-1) and its receptor, CCR2, as well as IL-10, mediators involved in recruiting and activating anti-inflammatory/wound repair macrophages, also increased in wild type mice after acetaminophen. Loss of STK blunted the effects of acetaminophen on expression of TNFα, IL-1β, IL-12, MCP-1 and CCR2, while expression of IL-10 increased. Hepatic expression of CX3CL1, and its receptor, CX3CR1 also increased in STK{sup −/−} mice

  10. Chemical constituents of Blumea balsamifera of Indonesia and their protein tyrosine phosphatase 1B inhibitory activity.

    PubMed

    Saifudin, Azis; Tanaka, Ken; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2012-07-01

    A methanol extract of the leaves of Blumea balsamifera (L.) DC. (Asteraceae) afforded a new guaian-type sesquiterpene, epiblumeaene K (1), together with four known guaian-type sesquiterpenes (2-5), three known sesquiterpenes (6-8), and nine known flavonoids (9-17) by a combination of chromatography and preparative TLC techniques. Their structures were elucidated by extensive spectroscopic methods and comparison with the literature data. Among the isolated compounds, a known sesquiterpene, beta-caryophyllene 8R,9R-oxide (6), exhibited a significant PTP1B inhibitory activity in a dose-dependent manner, with an IC50 value of 25.8 microM (5.62 microg/mL). PMID:22908553

  11. The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases

    PubMed Central

    Otieno, Steve; Lelli, Moreno; Kriwacki, Richard W.

    2014-01-01

    The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21Cip1, p27Kip1 and p57Kip2. Their kinase inhibitory activities are mediated by a homologous N-terminal kinase-inhibitory domain (KID). The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the KID and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, and its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation. PMID:25463440

  12. The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.

    PubMed

    Anufrieva, Natalya V; Faleev, Nicolai G; Morozova, Elena A; Bazhulina, Natalia P; Revtovich, Svetlana V; Timofeev, Vladimir P; Tkachev, Yaroslav V; Nikulin, Alexei D; Demidkina, Tatyana V

    2015-09-01

    In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and β-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-β- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and β-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  13. Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcgammaRIIA and alphaIIbbeta3.

    PubMed

    Pampolina, Caroline; McNicol, Archibald

    2005-05-01

    The low-affinity IgG receptor, FcgammaRIIA, has been implicated in Streptococcus sanguis-induced platelet aggregation. Therefore, it is likely that signal transduction is at least partly mediated by FcgammaRIIA activation and a tyrosine kinase-dependent pathway. In this study the signal transduction mechanisms associated with platelet activation in response to the oral bacterium, S. sanguis were characterised. In the presence of IgG, S. sanguis strain 2017-78 caused the tyrosine phosphorylation of FcgammaRIIA 30s following stimulation, which led to the phosphorylation of Syk, LAT, and PLCgamma2. These early events were dependent on Src family kinases but independent of either TxA(2) or the engagement of the alpha(IIb)beta(3) integrin. During the lag phase prior to platelet aggregation, FcgammaRIIA, Syk, LAT, and PLCgamma2 were each dephosphorylated, but were re-phosphorylated as aggregation occurred. Platelet stimulation by 2017-78 also induced the tyrosine phosphorylation of PECAM-1, an ITIM-containing receptor that recruits protein tyrosine phosphatases. PECAM-1 co-precipitated with the protein tyrosine phosphatase SHP-1 in the lag phase. SHP-1 was also maximally tyrosine phosphorylated during this phase, suggesting a possible role for SHP-1 in the observed dephosphorylation events. As aggregation occurred, SHP-1 was dephosphorylated, while FcgammaRIIA, Syk, LAT, and PLCgamma2 were rephosphorylated in an RGDS-sensitive, and therefore alpha(IIb)beta(3)-dependent, manner. Additionally, TxA(2) release, 5-hydroxytryptamine secretion and phosphatidic acid formation were all blocked by RGDS. Aspirin also abolished these events, but only partially inhibited alpha(IIb)beta(3) -mediated re-phosphorylation. Therefore, S. sanguis -bound IgG cross links FcgammaRIIA and initiates a signaling pathway that is down-regulated by PECAM-1-bound SHP-1. Subsequent engagement of alpha(IIb)beta(3) leads to SHP-1 dephosphorylation permiting a second wave of signaling leading to TxA(2

  14. Anomalous pH-dependence of the activity of human matrilysin (matrix metalloproteinase-7) as revealed by nitration and amination of its tyrosine residues

    PubMed Central

    2004-01-01

    Matrilysin activity exhibits a broad bell-shaped pH-dependence profile, with pKa values of 4.0 and 9.8. A maximum of five out of eight tyrosine residues in matrilysin were nitrated with tetranitromethane. On nitration of between one and five tyrosines, pKa at the alkaline side (pKe2) was shifted from 9.8 to 10.3–10.6, while that at the acidic side (pKe1) was not altered. The pKe2 that was shifted by nitration to 10.3–10.6 was restored to 9.4–9.7 by subsequent amination, suggesting that the shift in pKe2 is induced by a negative charge introduced on the most reactive tyrosine, Tyr-150. The Michaelis constant (Km) observed at pH 10 was decreased by nitration as a result of the increase in pKe2, suggesting that the residue with pKe2 may play a role in the recognition of substrate. When four or five tyrosines were nitrated, the activity at pH <7 decreased significantly, while that at pH 7–10 was unchanged, and thus the pH-dependence was not bell-shaped, but anomalous, with a third pKa (pKe3) of 6.2–6.4 in addition to pKe1 and pKe2. This suggests the possibility that a newly introduced nitrotyrosine residue has a strong influence on the activity as an ionizable group. PMID:15487974

  15. A conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate.

    PubMed

    Ostrander, Elizabeth L; Larson, John D; Schuermann, Jonathan P; Tanner, John J

    2009-02-10

    Proline dehydrogenase (PRODH) catalyzes the oxidation of l-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-l-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analogue l-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 A, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding the substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.

  16. A Conserved Active Site Tyrosine Residue of Proline Dehydrogenase Helps Enforce the Preference for Proline over Hydroxyproline as the Substrate

    SciTech Connect

    Ostrander, E.L.; Larson, J.D.; Schuermann, J.P.; Tanner, J.J.

    2009-03-02

    Proline dehydrogenase (PRODH) catalyzes the oxidation of L-proline to {Delta}-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-L-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analogue L-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 {angstrom}, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding the substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.

  17. EFA6 controls Arf1 and Arf6 activation through a negative feedback loop.

    PubMed

    Padovani, Dominique; Folly-Klan, Marcia; Labarde, Audrey; Boulakirba, Sonia; Campanacci, Valérie; Franco, Michel; Zeghouf, Mahel; Cherfils, Jacqueline

    2014-08-26

    Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.

  18. The second-sphere residue T263 is important for the function and catalytic activity of PTP1B via interaction with the WPD-loop.

    PubMed

    Xiao, Peng; Wang, Xiao; Wang, Hong-Mei; Fu, Xiao-Lei; Cui, Fu-ai; Yu, Xiao; Wen, Shi-shuai; Bi, Wen-Xiang; Sun, Jin-Peng

    2014-12-01

    Protein tyrosine phosphatases have diverse substrate specificities and intrinsic activities that lay the foundations for the fine-tuning of a phosphorylation network to precisely regulate cellular signal transduction. All classical PTPs share common catalytic mechanisms, and the important catalytic residues in the first sphere of their active sites have been well characterized. However, little attention has been paid to the second-sphere residues that are potentially important in defining the intrinsic activity and substrate specificity of PTPs. Here, we find that a conserved second-sphere residue, Thr263, located in the surface Q-loop is important for both the function and activity of PTPs. Using PTP1B as a study model, we found that mutations of Thr263 impaired the negative regulation role of PTP1B in insulin signaling. A detailed mechanistic study utilizing steady-state kinetics, Brønsted analysis and pH dependence in the presence of pNPP or phosphopeptide substrates revealed that Thr263 is required for the stabilization of the leaving group during catalysis. Further crystallographic studies and structural comparison revealed that Thr263 regulates the general acid function through modulation of the WPD-loop by the T263:F182/Y/H interaction pair, which is conserved in 26 out of 32 classical PTPs. In addition, the hydrophobic interaction between Thr263 and Arg1159 of the insulin receptor contributes to the substrate specificity of PTP1B. Taken together, our findings demonstrate the general role of the second-sphere residue Thr263 in PTP catalysis. Our findings suggest that the second sphere residues of PTP active site may play important roles in PTP-mediated function in both normal and diseased states. PMID:25450460

  19. Protein tyrosine phosphatase 1B inhibitory activity of Indonesian herbal medicines and constituents of Cinnamomum burmannii and Zingiber aromaticum.

    PubMed

    Saifudin, Azis; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2013-04-01

    We screened water and methanol extracts of 28 Indonesian medicinal plants for their protein tyrosine phosphatase 1B (PTP1B) inhibitory activities. Nine water extracts, i.e., Alstonia scholaris leaf, Blumea balsamifera, Cinnamomum burmannii, Cymbopogon nardus, Melaleuca leucadendra, Phyllanthus niruri, Piper nigrum, Syzygium aromaticum, and Sy. polyanthum, exhibited ≥70 % inhibition at 25 μg/mL, whereas 11 methanol extracts, i.e., Als. scholaris, Andrographis paniculata, B. balsamifera, Ci. burmannii, Curcuma heyneana, Glycyrrhiza glabra, M. leucadendra, Punica granatum, Rheum palmatum, Sy. polyanthum, and Z. aromaticum, exhibited ≥70 % inhibition at 25 μg/mL. Water extracts of B. balsamifera (IC50, 2.26 μg/mL) and M. leucadendra (IC50, 2.05 μg/mL), and methanol extracts of Ci. burmannii (IC50, 2.47 μg/mL), Pu. granatum (IC50, 2.40 μg/mL), and Sy. polyanthum (IC50, 1.03 μg/mL) exhibited strong inhibitory activity, which was comparable with that of the positive control, RK-682 (IC50, 2.05 μg/mL). The PTP1B inhibitory activity of the constituents of Ci. burmannii and Z. aromaticum was then evaluated. 5'-Hydroxy-5-hydroxymethyl-4″,5″-methylenedioxy-1,2,3,4-dibenzo-1,3,5-cycloheptatriene (2; IC50, 29.7 μM) and trans-cinnamaldehyde (5; IC50, 57.6 μM) were the active constituents of Ci. burmannii, while humulatrien-5-ol-8-one (21; IC50, 27.7 μM), kaempferol-3,4'-di-O-methyl ether (32; IC50, 17.5 μM), and (S)-6-gingerol (33; IC50, 28.1 μM) were those of Z. aromaticum. These results suggest that these medicinal plants may contribute to the treatment and/or prevention of type II diabetes and/or obesity through PTP1B inhibition. PMID:22645080

  20. Ginsenoside-Rp1 inhibits platelet activation and thrombus formation via impaired glycoprotein VI signalling pathway, tyrosine phosphorylation and MAPK activation

    PubMed Central

    Endale, M; Lee, WM; Kamruzzaman, SM; Kim, SD; Park, JY; Park, MH; Park, TY; Park, HJ; Cho, JY; Rhee, MH

    2012-01-01

    BACKGROUND AND PURPOSE Ginsenosides are the main constituents for the pharmacological effects of Panax ginseng. Such effects of ginsenosides including cardioprotective and anti-platelet activities have shown stability and bioavailability limitations. However, information on the anti-platelet activity of ginsenoside-Rp1 (G-Rp1), a stable derivative of ginsenoside-Rg3, is scarce. We examined the ability of G-Rp1 to modulate agonist-induced platelet activation. EXPERIMENTAL APPROACH G-Rp1 in vitro and ex vivo effects on agonist-induced platelet-aggregation, granule-secretion, [Ca2+]i mobilization, integrin-αIIbβ3 activation were examined. Vasodilator-stimulated phosphoprotein (VASP) and MAPK expressions and levels of tyrosine phosphorylation of the glycoprotein VI (GPVI) signalling pathway components were also studied. G-Rp1 effects on arteriovenous shunt thrombus formation in rats or tail bleeding time and ex vivo coagulation time in mice were determined. KEY RESULT G-Rp1 markedly inhibited platelet aggregation induced by collagen, thrombin or ADP. While G-Rp1 elevated cAMP levels, it dose-dependently suppressed collagen-induced ATP-release, thromboxane secretion, p-selectin expression, [Ca2+]i mobilization and αIIbβ3 activation and attenuated p38MAPK and ERK2 activation. Furthermore, G-Rp1 inhibited tyrosine phosphorylation of multiple components (Fyn, Lyn, Syk, LAT, PI3K and PLCγ2) of the GPVI signalling pathway. G-Rp1 inhibited in vivo thrombus formation and ex vivo platelet aggregation and ATP secretion without affecting tail bleeding time and coagulation time, respectively. CONCLUSION AND IMPLICATIONS G-Rp1 inhibits collagen-induced platelet activation and thrombus formation through modulation of early GPVI signalling events, and this effect involves VASP stimulation, and ERK2 and p38-MAPK inhibition. These data suggest that G-Rp1 may have therapeutic potential for the treatment of cardiovascular diseases involving aberrant platelet activation. PMID

  1. Heat stress activates the yeast high-osmolarity glycerol mitogen-activated protein kinase pathway, and protein tyrosine phosphatases are essential under heat stress.

    PubMed

    Winkler, Astrid; Arkind, Christopher; Mattison, Christopher P; Burkholder, Anne; Knoche, Kathryn; Ota, Irene

    2002-04-01

    The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1 delta strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways. PMID:12455951

  2. Heat Stress Activates the Yeast High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway, and Protein Tyrosine Phosphatases Are Essential under Heat Stress

    PubMed Central

    Winkler, Astrid; Arkind, Christopher; Mattison, Christopher P.; Burkholder, Anne; Knoche, Kathryn; Ota, Irene

    2002-01-01

    The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1Δ strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways. PMID:12455951

  3. Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

    PubMed Central

    Stahlhut, Steen Gustav; Li, Mingji; Gaspar, Paula; Siedler, Solvej; Förster, Jochen; Maury, Jérôme; Borodina, Irina

    2015-01-01

    Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 μM p-coumaric acid OD600 unit−1 in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases. PMID:25911487

  4. Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct from one another, and thus while most animal receptor kinases are tyrosin...

  5. Rosiglitazone ameliorates abnormal expression and activity of protein tyrosine phosphatase 1B in the skeletal muscle of fat-fed, streptozotocin-treated diabetic rats

    PubMed Central

    Wu, Yong; Ouyang, Jing Ping; Wu, Ke; Wang, Shi Shun; Wen, Chong Yuan; Xia, Zheng Yuan

    2005-01-01

    Protein tyrosine phosphatase 1B (PTP1B) acts as a physiological negative regulator of insulin signaling by dephosphorylating the activated insulin receptor (IR). Here we examine the role of PTP1B in the insulin-sensitizing action of rosiglitazone (RSG) in skeletal muscle and liver. Fat-fed, streptozotocin-treated rats (10-week-old), an animal model of type II diabetes, and age-matched, nondiabetic controls were treated with RSG (10 μmol kg−1 day−1) for 2 weeks. After RSG treatment, the diabetic rats showed a significant decrease in blood glucose and improved insulin sensitivity. Diabetic rats showed significantly increased levels and activities of PTP1B in the skeletal muscle (1.6- and 2-fold, respectively) and liver (1.7- and 1.8-fold, respectively), thus diminishing insulin signaling in the target tissues. We found that the decreases in insulin-stimulated glucose uptake (55%), tyrosine phosphorylation of IRβ-subunits (48%), and IR substrate-1 (IRS-1) (39%) in muscles of diabetic rats were normalized after RSG treatment. These effects were associated with 34 and 30% decreases in increased PTP1B levels and activities, respectively, in skeletal muscles of diabetic rats. In contrast, RSG did not affect the increased PTP1B levels and activities or the already reduced insulin-stimulated glycogen synthesis and tyrosine phosphorylation of IRβ-subunits and IRS-2 in livers of diabetic rats. RSG treatment in normal rats did not significantly change PTP1B activities and levels or protein levels of IRβ, IRS-1, and -2 in diabetic rats. These data suggest that RSG enhances insulin activity in skeletal muscle of diabetic rats possibly by ameliorating abnormal levels and activities of PTP1B. PMID:15997237

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

    PubMed

    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 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 ventilation. The model

  7. A New Family of Receptor Tyrosine Kinases with a Venus Flytrap Binding Domain in Insects and Other Invertebrates Activated by Aminoacids

    PubMed Central

    Ahier, Arnaud; Rondard, Philippe; Gouignard, Nadège; Khayath, Naji; Huang, Siluo; Trolet, Jacques; Donoghue, Daniel J.; Gauthier, Monique; Pin, Jean-Philippe; Dissous, Colette

    2009-01-01

    Background Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. Methods and Findings Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABAB receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. Conclusion The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases. PMID:19461966

  8. Immunoreceptor tyrosine-based activation motif phosphorylation during engulfment of Neisseria gonorrhoeae by the neutrophil-restricted CEACAM3 (CD66d) receptor.

    PubMed

    McCaw, Shannon E; Schneider, Jutta; Liao, Edward H; Zimmermann, Wolfgang; Gray-Owen, Scott D

    2003-08-01

    Gonorrhea is characterized by a purulent urethral or cervical discharge consisting primarily of neutrophils associated with Neisseria gonorrhoeae. These interactions are facilitated by gonococcal colony opacity-associated (Opa) protein binding to host cellular CEACAM receptors. Of these, CEACAM3 is restricted to neutrophils and contains an immunoreceptor tyrosine-based activation motif (ITAM) reminiscent of that found within certain phagocytic Fc receptors. CEACAM3 was tyrosine phosphorylated by a Src family kinase-dependent process upon infection by gonococci expressing CEACAM-specific Opa proteins. This phosphorylation was necessary for efficient bacterial uptake; however, a less efficient uptake process became evident when kinase inhibitors or mutagenesis of the ITAM were used to prevent phosphorylation. Ligated CEACAM3 was recruited to a cytoskeleton-containing fraction, intense foci of polymerized actin were evident where bacteria attached to HeLa-CEACAM3, and disruption of polymerized actin by cytochalasin D blocked all bacterial uptake by these cells. These data support a model whereby CEACAM3 can mediate the Opa-dependent uptake of N. gonorrhoeae via either an efficient, ITAM phosphorylation-dependent process that resembles phagocytosis or a less efficient, tyrosine phosphorylation-independent mechanism. PMID:12864848

  9. Calcium alginate bead immobilization of cells containing tyrosine ammonia lyase activity for use in the production of p-hydroxycinnamic acid.

    PubMed

    Trotman, Robert J; Camp, Carl E; Ben-Bassat, Arie; DiCosimo, Robert; Huang, Lixuan; Crum, Grace A; Sariaslani, F Sima; Haynie, Sharon L

    2007-01-01

    An Escherichia coli catalyst with tyrosine ammonia lyase activity (TAL) has been stabilized for repeated use in batch conversions of high tyrosine solids to p-hydroxycinnamic acid (pHCA). The TAL biocatalyst was stabilized by controlling the reaction pH to 9.8 +/- 0.1 and immobilizing the cells within a calcium alginate matrix that was cross-linked with glutaraldehyde and polyethyleneimine (GA/PEI). We found a GA range where the bead-encapsulated TAL was not inactivated, and the resulting cross-linking provided the beads with the mechanical stability necessary for repeated use in consecutive batch reactions with catalyst recycle. The GA/PEI calcium alginate TAL catalyst was used in 41 1-L batch reactions where 50 g L(-1) tyrosine was converted to 39 +/- 4 g L(-1) pHCA in each batch. The practical usefulness and ease of this process was demonstrated by scaling up the TAL bead immobilization and using the immobilized TAL catalyst in four 125-L bioconversion reactions to produce over 12 kg of purified pHCA.

  10. Breast tumor kinase (protein tyrosine kinase 6) regulates heregulin-induced activation of ERK5 and p38 MAP kinases in breast cancer cells.

    PubMed

    Ostrander, Julie Hanson; Daniel, Andrea R; Lofgren, Kristopher; Kleer, Celina G; Lange, Carol A

    2007-05-01

    Total tyrosine kinase activity is often elevated in both cytosolic and membrane fractions of malignant breast tissue and correlates with a decrease in disease-free survival. Breast tumor kinase (Brk; protein tyrosine kinase 6) is a soluble tyrosine kinase that was cloned from a metastatic breast tumor and found to be overexpressed in a majority of breast tumors. Herein, we show that Brk is overexpressed in 86% of invasive ductal breast tumors and coexpressed with ErbB family members in breast cancer cell lines. Additionally, the ErbB ligand, heregulin, activates Brk kinase activity. Knockdown of Brk by stable expression of short hairpin RNA (shRNA) in T47D breast cancer cells decreases proliferation and blocks epidermal growth factor (EGF)- and heregulin-induced activation of Rac GTPase, extracellular signal-regulated kinase (ERK) 5, and p38 mitogen-activated protein kinase (MAPK) but not Akt, ERK1/2, or c-Jun NH(2)-terminal kinase. Furthermore, EGF- and heregulin-induced cyclin D1 expression is dependent on p38 signaling and inhibited by Brk shRNA knockdown. The myocyte enhancer factor 2 transcription factor target of p38 MAPK and ERK5 signaling is also sensitive to altered Brk expression. Finally, heregulin-induced migration of T47D cells requires p38 MAPK activity and is blocked by Brk knockdown. These results place Brk in a novel signaling pathway downstream of ErbB receptors and upstream of Rac, p38 MAPK, and ERK5 and establish the ErbB-Brk-Rac-p38 MAPK pathway as a critical mediator of breast cancer cell migration.

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

    SciTech Connect

    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.

  12. Temperature Analysis of an Active Region Core Loop Using AIA and XRT Data

    NASA Astrophysics Data System (ADS)

    Garst, Jennifer W.; Schmelz, J.; Kimble, J.

    2012-05-01

    Data obtained on December 10, 2010 by both the Atmospheric Imaging Assembly (AIA) and the X-Ray Telescope (XRT) are co-aligned and appropriately scaled in order to do a differential emission measure analysis of the combined data. This project uses Hybrid abundances from Fludra & Schmelz and atomic data from the CHIANTI atomic physics database to analyze an active region core loop and report on the multithermal analysis of the combined data set. The loop being analyzed is visible in the 94, 131, 171, 193, 211, 335 Å passbands on AIA; and the Al-thick, Ti-poly, Al-mesh, Al-poly/Ti-poly, C-Poly/Ti-poly, C-poly, Be-thin, Be-med, Al-med, and Al-poly filters on XRT. Solar physics research at the University of Memphis is supported by NSF ATM-0402729 as well as a Hinode subcontract from NASA/SAO.

  13. Tracking performance of unbalanced QPSK demodulators. II - Biphase Costas loop with active arm filters

    NASA Technical Reports Server (NTRS)

    Simon, M. K.

    1978-01-01

    In a Costas loop study for biphase modulation conducted by Simon and Lindsey (1977), it was demonstrated that considerable improvement in tracking performance could be obtained by employing active arm filters of the integrate-and-dump type as opposed to passive arm filters. An investigation is conducted concerning the possibility to obtain a similar performance improvement for an unbalanced quadriphase-shift-keying (QPSK) modulation. It is found that the biphase Costas loop can be used as an efficient demodulator of QPSK in cases in which the ratio of data rates is of the same order of magnitude as the inverse of the power ratio. These cases involve approximately equal signal energies in the two channels.

  14. Sensory and decision-related activity propagate in a cortical feedback loop during touch perception.

    PubMed

    Kwon, Sung Eun; Yang, Hongdian; Minamisawa, Genki; O'Connor, Daniel H

    2016-09-01

    The brain transforms physical sensory stimuli into meaningful perceptions. In animals making choices about sensory stimuli, neuronal activity in successive cortical stages reflects a progression from sensation to decision. Feedforward and feedback pathways connecting cortical areas are critical for this transformation. However, the computational functions of these pathways are poorly understood because pathway-specific activity has rarely been monitored during a perceptual task. Using cellular-resolution, pathway-specific imaging, we measured neuronal activity across primary (S1) and secondary (S2) somatosensory cortices of mice performing a tactile detection task. S1 encoded the stimulus better than S2, while S2 activity more strongly reflected perceptual choice. S1 neurons projecting to S2 fed forward activity that predicted choice. Activity encoding touch and choice propagated in an S1-S2 loop along feedforward and feedback axons. Our results suggest that sensory inputs converge into a perceptual outcome as feedforward computations are reinforced in a feedback loop. PMID:27437910

  15. Possible C1q bypass loop activation in the haemolytic uraemic syndrome.

    PubMed Central

    Nolin, L; O'Regan, S; Pelletier, M; Rivard, G E; Mongeau, J G; Robitaille, P

    1979-01-01

    Ultrastructural and immunofluorescent microscopic studies were performed on renal tissue obtained from nine patients during the acute and convalescent phase of the haemolytic uraemic syndrome (HUS). All had glomerular deposits of IgM in the absence of circulating immune complexes. This was associated with deposition of C1q during the acute phase, and properdin and C3 during the convalescent phase. C4 was consistently absent. Since such a pattern of complement deposition does not fulfil criteria either for alternate or classical pathway activation, the possibility of C1q bypass loop activation by IgM is suggested. PMID:371879

  16. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  17. Tyrosine-assisted preparation of Ag/ZnO nanocomposites with enhanced photocatalytic performance and synergistic antibacterial activities

    NASA Astrophysics Data System (ADS)

    Lu, Weiwei; Liu, Guosheng; Gao, Shuyan; Xing, Shantao; Wang, Jianji

    2008-11-01

    In this paper, Ag/ZnO metal-semiconductor nanocomposites were prepared through a facile one-pot hydrothermal method with the assistance of tyrosine. The synthesized samples were structurally characterized by x-ray diffraction, scanning electron microscope, transmission electron microscope and x-ray photoelectron spectroscopy. It was shown that the added tyrosine served both as a shape conductor for the formation of ZnO faceted nanorods and as a reducing agent of Ag+ ions. In the reaction process, the complexation of Ag+ with NH3 and OH- decreased the redox potential of Ag+/Ag, which prevented the formation of isolated Ag nanoparticles in solution. The prepared Ag/ZnO nanocomposites showed potential applications in photodegradation of organic dye pollutants and destruction of bacteria.

  18. The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma

    PubMed Central

    Boregowda, Rajeev K.; Medina, Daniel J.; Markert, Elke; Bryan, Michael A.; Chen, Wenjin; Chen, Suzie; Rabkin, Anna; Vido, Michael J.; Gunderson, Samuel I.; Chekmareva, Marina; Foran, David J.; Lasfar, Ahmed; Goydos, James S.; Cohen-Solal, Karine A.

    2016-01-01

    Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma. PMID:27102439

  19. The active site loop of S-adenosylmethionine synthetase modulates catalytic efficiency.

    PubMed

    Taylor, John C; Takusagawa, Fusao; Markham, George D

    2002-07-30

    Crystallographic studies of Escherichia coli S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase, MAT) have defined a flexible polypeptide loop that can gate access to the active site without contacting the substrates. The influence of the length and sequence of this active site loop on catalytic efficiency has been characterized in a mutant in which the E. coli MAT sequence (DRADPLEQ) has been replaced with the distinct sequence of the corresponding region of the otherwise highly homologous rat liver enzyme (HDLRNEEDV). Four additional mutants in which the entire DRADPLEQ sequence was replaced by five, six, seven, or eight glycines have been studied to unveil the effects of loop length and the influence of side chains. In all of the mutants, the maximal rate of S-adenosylmethionine formation (k(cat)) is diminished by more than 200-fold whereas the rate of hydrolysis of the tripolyphosphate intermediate is decreased by less than 3-fold. Thus, the function of the loop is localized to the first step in the overall reaction. The K(m) for methionine increases in all of the oligoglycine mutants, whereas the K(m) values for ATP are not substantially different. The k(cat) for the wild-type enzyme is decreased by increases in solution microviscosity with 55% of the maximal dependence. Thus, a diffusional event is coupled to the chemical step of AdoMet formation, which is known to be rate-limiting. The results indicate that a conformational change, possibly loop closure, is associated with AdoMet synthesis. The data integrate a previously discovered conformational change associated with PPP(i) binding to the E x AdoMet complex into the reaction sequence, reflecting a difference in protein conformation in the E x AdoMet x PPP(i) complex whether it is formed from the E x ATP x methionine complex or from binding of exogenous PPP(i). The temperature dependence of the k(cat) for S-adenosylmethionine formation shows that the removal of the side chains in the

  20. p-Hydroxyphenylacetaldehyde is the major product of L-tyrosine oxidation by activated human phagocytes. A chloride-dependent mechanism for the conversion of free amino acids into reactive aldehydes by myeloperoxidase.

    PubMed

    Hazen, S L; Hsu, F F; Heinecke, J W

    1996-01-26

    Reactive aldehydes generated during lipid peroxidation have been implicated in the pathogenesis of atherosclerosis as well as other inflammatory diseases. A potential catalyst for such reactions is myeloperoxidase, a hemeprotein secreted by activated phagocytes. We now report that activated neutrophils utilize the myeloperoxidase-H2O2-chloride system to convert L-tyrosine to p-hydroxyphenylacetaldehyde. Production of p-hydroxyphenylacetaldehyde was nearly quantitative at physiological concentrations of L-tyrosine and chloride. Aldehyde generation required myeloperoxidase, H2O2, L-tyrosine, and chloride ion; it was inhibited by the H2O2 scavenger catalase and by the heme poisons azide and cyanide. Phorbol ester- and calcium ionophore-stimulated human neutrophils likewise generated p-hydroxyphenylacetaldehyde from L-tyrosine by a pathway inhibited by azide, cyanide, and catalase. Aldehyde production accounted for 75% of H2O2 generated by optimally stimulated neutrophils at plasma concentrations of L-tyrosine and chloride. Collectively, these results indicate that activated phagocytes, under physiological conditions, utilize myeloperoxidase to execute the chloride-dependent conversion of L-tyrosine to the lipid-soluble aldehyde, p-hydroxyphenylacetaldehyde, in near quantitative yield. Moreover, like aldehydes derived from lipid peroxidation, amino acid-derived aldehydes may exert potent biological effects in vascular lesions and other sites of inflammation.

  1. Enhancer-like long-range transcriptional activation by λ CI-mediated DNA looping

    PubMed Central

    Cui, Lun; Murchland, Iain; Shearwin, Keith E.; Dodd, Ian B.

    2013-01-01

    How distant enhancer elements regulate the assembly of a transcription complex at a promoter remains poorly understood. Here, we use long-range gene regulation by the bacteriophage λ CI protein as a powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed by CI bridging its binding sites at OR and OL, is known already to enhance repression at the lysogenic promoter PRM, located at OR. Here, we show that CI looping also activates PRM by allowing the C-terminal domain of the α subunit of the RNA polymerase bound at PRM to contact a DNA site adjacent to the distal CI sites at OL. Our results establish OL as a multifaceted enhancer element, able to activate transcription from long distances independently of orientation and position. We develop a physicochemical model of our in vivo data and use it to show that the observed activation is consistent with a simple recruitment mechanism, where the α–C-terminal domain to DNA contact need only provide ∼2.7 kcal/mol of additional binding energy for RNA polymerase. Structural modeling of this complete enhancer–promoter complex reveals how the contact is achieved and regulated, and suggests that distal enhancer elements, once appropriately positioned at the promoter, can function in essentially the same way as proximal promoter elements. PMID:23382214

  2. Enhancer-like long-range transcriptional activation by λ CI-mediated DNA looping.

    PubMed

    Cui, Lun; Murchland, Iain; Shearwin, Keith E; Dodd, Ian B

    2013-02-19

    How distant enhancer elements regulate the assembly of a transcription complex at a promoter remains poorly understood. Here, we use long-range gene regulation by the bacteriophage λ CI protein as a powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed by CI bridging its binding sites at OR and OL, is known already to enhance repression at the lysogenic promoter PRM, located at OR. Here, we show that CI looping also activates PRM by allowing the C-terminal domain of the α subunit of the RNA polymerase bound at PRM to contact a DNA site adjacent to the distal CI sites at OL. Our results establish OL as a multifaceted enhancer element, able to activate transcription from long distances independently of orientation and position. We develop a physicochemical model of our in vivo data and use it to show that the observed activation is consistent with a simple recruitment mechanism, where the α-C-terminal domain to DNA contact need only provide ∼2.7 kcal/mol of additional binding energy for RNA polymerase. Structural modeling of this complete enhancer-promoter complex reveals how the contact is achieved and regulated, and suggests that distal enhancer elements, once appropriately positioned at the promoter, can function in essentially the same way as proximal promoter elements. PMID:23382214

  3. The CD3 gamma epsilon/delta epsilon signaling module provides normal T cell functions in the absence of the TCR zeta immunoreceptor tyrosine-based activation motifs.

    PubMed

    Pitcher, Lisa A; Mathis, Meredith A; Young, Jennifer A; DeFord, Laura M; Purtic, Bozidar; Wulfing, Christoph; van Oers, Nicolai S C

    2005-12-01

    T cell receptor (TCR) signal transduction is mediated by the immunoreceptor tyrosine-based activation motifs (ITAM). The ten ITAM in the TCR complex are distributed in two distinct signaling modules termed TCR zetazeta and CD3 gammaepsilon/deltaepsilon. To delineate the specific role of the zeta ITAM in T cell development and TCR signal transmission, we compared the properties of T cells from different TCR zeta-transgenic lines wherein tyrosine-to-phenylalanine substitutions had been introduced in the zeta subunit. These lines lack selected phosphorylated forms of TCR zeta including just p23, both p21 and p23, or all phospho-zeta derivatives. We report herein that the efficiency of positive selection in HY TCR-transgenic female mice was directly related to the number of zeta ITAM in the TCR. In contrast, TCR-mediated signal transmission and T cell proliferative responses following agonist peptide stimulation were similar and independent of the zeta ITAM. Only the duration of MAPK activation was affected by multiple zeta ITAM substitutions. These results strongly suggest that the ITAM in the CD3 gammaepsilon/deltaepsilon module can provide normal TCR signal transmission, with zeta ITAM providing a secondary function facilitating MAPK activation and positive selection.

  4. TTT-3002 is a novel FLT3 tyrosine kinase inhibitor with activity against FLT3-associated leukemias in vitro and in vivo.

    PubMed

    Ma, Hayley; Nguyen, Bao; Li, Li; Greenblatt, Sarah; Williams, Allen; Zhao, Ming; Levis, Mark; Rudek, Michelle; Duffield, Amy; Small, Donald

    2014-03-01

    More than 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FMS-like tyrosine kinase-3 (FLT3). The most common type, internal tandem duplication (ITD), confers poor prognosis. We report for the first time on TTT-3002, a tyrosine kinase inhibitor (TKI) that is one of the most potent FLT3 inhibitors discovered to date. Studies using human FLT3/ITD mutant leukemia cell lines revealed the half maximal inhibitory concentration (IC50) for inhibiting FLT3 autophosphorylation is from 100 to 250 pM. The proliferation IC50 for TTT-3002 in these same cells was from 490 to 920 pM. TTT-3002 also showed potent activity when tested against the most frequently occurring FLT3-activating point mutation, FLT3/D835Y, against which many current TKIs are ineffective. These findings were validated in vivo by using mouse models of FLT3-associated AML. Survival and tumor burden of mice in several FLT3/ITD transplantation models is significantly improved by administration of TTT-3002 via oral dosing. Finally, we demonstrated that TTT-3002 is cytotoxic to leukemic blasts isolated from FLT3/ITD-expressing AML patients, while displaying minimal toxicity to normal hematopoietic stem/progenitor cells from healthy blood and bone marrow donors. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI in the treatment of FLT3-mutant AML.

  5. Bruton's Tyrosine Kinase Regulates the Activation of Gene Rearrangements at the λ Light Chain Locus in Precursor B Cells in the Mouse

    PubMed Central

    Dingjan, Gemma M.; Middendorp, Sabine; Dahlenborg, Katarina; Maas, Alex; Grosveld, Frank; Hendriks, Rudolf W.

    2001-01-01

    Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an ∼50% reduction in the frequency of immunoglobulin (Ig) λ light chain expression, already at the immature B cell stage in the bone marrow. Conversely, transgenic mice expressing the activated mutant BtkE41K showed increased λ usage. As the κ/λ ratio is dependent on (a) the level and kinetics of κ and λ locus activation, (b) the life span of pre-B cells, and (c) the extent of receptor editing, we analyzed the role of Btk in these processes. Enforced expression of the Bcl-2 apoptosis inhibitor did not alter the Btk dependence of λ usage. Crossing 3-83μδ autoantibody transgenic mice into Btk-deficient mice showed that Btk is not essential for receptor editing. Also, Btk-deficient surface Ig+ B cells that were generated in vitro in interleukin 7-driven bone marrow cultures manifested reduced λ usage. An intrinsic defect in λ locus recombination was further supported by the finding in Btk-deficient mice of reduced λ usage in the fraction of pre-B cells that express light chains in their cytoplasm. These results implicate Btk in the regulation of the activation of the λ locus for V(D)J recombination in pre-B cells. PMID:11369788

  6. The role of the K-channel and the active-site tyrosine in the catalytic mechanism of cytochrome c oxidase.

    PubMed

    Sharma, Vivek; Wikström, Mårten

    2016-08-01

    The active site of cytochrome c oxidase (CcO) comprises an oxygen-binding heme, a nearby copper ion (CuB), and a tyrosine residue that is covalently linked to one of the histidine ligands of CuB. Two proton-conducting pathways are observed in CcO, namely the D- and the K-channels, which are used to transfer protons either to the active site of oxygen reduction (substrate protons) or for pumping. Proton transfer through the D-channel is very fast, and its role in efficient transfer of both substrate and pumped protons is well established. However, it has not been fully clear why a separate K-channel is required, apparently for the supply of substrate protons only. In this work, we have analysed the available experimental and computational data, based on which we provide new perspectives on the role of the K-channel. Our analysis suggests that proton transfer in the K-channel may be gated by the protonation state of the active-site tyrosine (Tyr244) and that the neutral radical form of this residue has a more general role in the CcO mechanism than thought previously. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26898520

  7. Multiple receptor tyrosine kinase activation attenuates therapeutic efficacy of the fibroblast growth factor receptor 2 inhibitor AZD4547 in FGFR2 amplified gastric cancer

    PubMed Central

    Zhang, Zhe; Liu, Xinyang; Wu, Zheng; Geng, Ruixuan; Ge, Xiaoxiao; Dai, Congqi; Liu, Rujiao; Zhang, Qunling; Li, Wenhua; Li, Jin

    2015-01-01

    Fibroblast growth factor receptor 2 (FGFR2)-targeted therapy has attracted considerable attention as novel anticancer agents in gastric cancer (GC). However, intrinsic or acquired drug resistance has emerged as a major challenge to their clinical use. In this study, we demonstrated that several receptor tyrosine kinase (RTK), including EGFR, HER3 and MET, activations contributed to AZD4547 (a selective FGFR2 inhibitor) hyposensitivity in FGFR2 amplified GC cells. The rescue effect was abrogated by inhibiting these RTKs with their targeted tyrosine kinase inhibitors (TKIs). In addition, synergy in growth inhibition was observed when the GC cells were treated with a combination of AZD4547 and cetuximab (an EGFR monoclonal antibody) both in vitro and in vivo. More importantly, tissue microarray analysis revealed that these resistance-conferring RTKs were highly expressed in FGFR2 positive GC patients. Taken together, these observations demonstrated RTKs including EGFR, HER3 and MET activations as novel mechanisms of hyposensitivity to AZD4547. It will be clinically valuable to investigate the involvement of RTK-mediated signaling in intrinsicor acquired resistance to FGFR2 TKIs in GC. A combination targeted therapeutic strategy may be recommended for treating FGFR2 amplified GC patients with these RTK activations. PMID:25576915

  8. Effect of reactive site loop elongation on the inhibitory activity of C1-inhibitor.

    PubMed

    Bos, Ineke G A; Lubbers, Yvonne T P; Eldering, Eric; Abrahams, Jan Pieter; Hack, C Erik

    2004-06-01

    The serine protease inhibitor C1-Inhibitor (C1-Inh) inhibits several complement- and contact-system proteases, which play an important role in inflammation. C1-Inh has a short reactive site loop (RSL) compared to other serpins. RSL length determines the inhibitory activity of serpins. We investigated the effect of RSL elongation on inhibitory activity of C1-Inh by insertion of one or two alanine residues in the RSL. One of five mutants had an increased association rate with kallikrein, but was nevertheless a poor inhibitor because of a simultaneous high stoichiometry of inhibition (>10). The association rate of the other variants was lower than that of wild-type C1-Inh. These data suggest that the relatively weak inhibitory activity of C1-Inh is not the result of its short RSL. The short RSL of C1-Inh has, surprisingly, the optimal length for inhibition.

  9. A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

    SciTech Connect

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Peterson, Scott N.; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon

    2012-04-18

    Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

  10. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    SciTech Connect

    Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  11. Two Closely Spaced Tyrosines Regulate NFAT Signaling in B Cells via Syk Association with Vav▿

    PubMed Central

    Chen, Chih-Hong; Martin, Victoria A.; Gorenstein, Nina M.; Geahlen, Robert L.; Post, Carol Beth

    2011-01-01

    Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2. PMID:21606197

  12. Design, Synthesis, Biological Activity and Molecular Dynamics Studies of Specific Protein Tyrosine Phosphatase 1B Inhibitors over SHP-2

    PubMed Central

    Sun, Su-Xia; Li, Xiao-Bo; Liu, Wen-Bo; Ma, Ying; Wang, Run-Ling; Cheng, Xian-Chao; Wang, Shu-Qing; Liu, Wei

    2013-01-01

    Over expressing in PTPN1 (encoding Protein tyrosine phosphatase 1B, PTP1B), a protein tyrosine phosphatase (PTP) that plays an overall positive role in insulin signaling, is linked to the pathogenesis of diabetes and obesity. The relationship between PTP1B and human diseases exhibits PTP1B as the target to treat these diseases. In this article, small weight molecules of the imidazolidine series were screened from databases and optimized on silicon as the inhibitors of PTP1B based on the steric conformation and electronic configuration of thiazolidinedione (TZD) compounds. The top three candidates were tested using an in vitro biological assay after synthesis. Finally, we report a novel inhibitor, Compound 13, that specifically inhibits PTP1B over the closely related phosphatase Src homology 2 (SH2) domain-containing phosphatase 2 (SHP-2) at 80 μM. Its IC50 values are reported in this paper as well. This compound was further verified by computer analysis for its ability to combine the catalytic domains of PTP1B and SHP-2 by molecular dynamics (MD) simulations. PMID:23774838

  13. Multiple loop activations and continuous energy release in the solar flare of June 15, 1973

    NASA Technical Reports Server (NTRS)

    Widing, K. G.; Dere, K. P.

    1977-01-01

    The spatial and temporal evolution of the high-temperature plasma in the solar flare of June 15, 1973, is studied using XUV spectroheliograms and X-ray filtergrams obtained from Skylab. The analysis focuses on the changing forms and brightness of Fe XXIII 263-A and Fe XXIV 255-A images. Temperatures and emission measures computed for different times during the flare are compared with those derived from Solrad-9 flux data, the electron temperature in the bright compact core of the Fe XXIV image is determined, and a coronal origin is suggested for this bright core. The observational evidence shows that the overall flare event involved a number of different preexisting loops and arches which were activated in succession. The activation and heating are found to have persisted well past the end of the burst phase, implying that the energy release did not end when the impulsive phase was over. The overall development of the flare is summarized on the basis of the observed order of appearance of the loops.

  14. Parsing the phonological loop: activation timing in the dorsal speech stream determines accuracy in speech reproduction.

    PubMed

    Herman, Alexander B; Houde, John F; Vinogradov, Sophia; Nagarajan, Srikantan S

    2013-03-27

    Despite significant research and important clinical correlates, direct neural evidence for a phonological loop linking speech perception, short-term memory and production remains elusive. To investigate these processes, we acquired whole-head magnetoencephalographic (MEG) recordings from human subjects performing a variable-length syllable sequence reproduction task. The MEG sensor data were source localized using a time-frequency optimized spatially adaptive filter, and we examined the time courses of cortical oscillatory power and the correlations of oscillatory power with behavior between onset of the audio stimulus and the overt speech response. We found dissociations between time courses of behaviorally relevant activations in a network of regions falling primarily within the dorsal speech stream. In particular, verbal working memory load modulated high gamma power in both Sylvian-parietal-temporal and Broca's areas. The time courses of the correlations between high gamma power and subject performance clearly alternated between these two regions throughout the task. Our results provide the first evidence of a reverberating input-output buffer system in the dorsal stream underlying speech sensorimotor integration, consistent with recent phonological loop, competitive queuing, and speech-motor control models. These findings also shed new light on potential sources of speech dysfunction in aphasia and neuropsychiatric disorders, identifying anatomically and behaviorally dissociable activation time windows critical for successful speech reproduction. PMID:23536060

  15. Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

    PubMed Central

    Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

    2015-01-01

    The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

  16. Asymmetry of the active site loop conformation between subunits of glutamate-1-semialdehyde aminomutase in solution.

    PubMed

    Campanini, Barbara; Bettati, Stefano; di Salvo, Martino Luigi; Mozzarelli, Andrea; Contestabile, Roberto

    2013-01-01

    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.

  17. Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response.

    PubMed

    Chan, Tung O; Zhang, Jin; Tiegs, Brian C; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M; Armen, Roger S; Rodeck, Ulrich; Penn, Raymond B

    2015-10-01

    The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr(308) in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr(308) dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser(473)) increased phosphatase resistance of the phosphorylated activation loop (pThr(308)) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr(308) phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin.

  18. Enhanced insulin-receptor tyrosine kinase activity associated with chromosomal translocation (1;19) in a pre-B-cell leukemia line.

    PubMed

    Newman, J D; Harrison, L C; Eckardt, G S; Jack, I

    1992-02-01

    The gene for the insulin receptor has been assigned to chromosome 19 near the breakpoint of the translocation t(1;19) which occurs in 25% of pre-B-cell leukemias. Insulin receptors in a pre-B-cell leukemia cell line (ACV) with t(1;19) were found to have 2-fold higher affinity for insulin, 5-fold higher basal and insulin-stimulated beta sub-unit autophosphorylation, and 2-fold higher basal and 4-fold higher insulin-stimulated beta sub-unit kinase activity on the synthetic peptide poly(Glu,Tyr), compared to receptors in a B-cell line (ADD) with normal karyotype from the same patient. ACV cells had a novel 13-kb receptor mRNA species and expressed a DNA polymorphism localized to the tyrosine kinase domain of the receptor gene. These findings suggest that t(1;19) in the ACV cell may result in rearrangement of the insulin receptor gene and translation of a receptor with enhanced tyrosine kinase activity. PMID:1310491

  19. Phosphorylation of the protein kinase C-theta activation loop and hydrophobic motif regulates its kinase activity, but only activation loop phosphorylation is critical to in vivo nuclear-factor-kappaB induction.

    PubMed Central

    Liu, Yin; Graham, Caroline; Li, Aiqun; Fisher, Robert J; Shaw, Stephen

    2002-01-01

    Protein kinase C (PKC)-theta, a member of the 'novel' subfamily of PKC isoforms, is of singular importance in transducing signals in T-lymphocytes. Since understanding of regulatory phosphorylation of novel PKCs is fragmentary and inconsistent with findings for 'classical' PKC isoforms, we investigated three potential phosphorylation sites on PKC-theta; in the activation loop (Thr(538)), turn motif (Ser(676)) and hydrophobic motif (Ser(695)). Combined evidence from phospho-specific antisera and MS demonstrates phosphorylation at all three sites. Unlike its closest paralogue, PKC-delta, lack of negative charge in the activation loop of PKC-theta results in a profound catalytic defect (>100-fold reduction in the T538A mutant); the high sequence similarity between PKC-theta and -delta assists in the formulation of structural hypotheses to account for this major difference. In contrast with mechanisms proposed for other PKC isoforms, phosphorylation at the other two sites does not reconstitute catalytic activity. Activation loop phosphorylation is critical in vivo, since the T538A mutant completely lost its capacity to mediate T-cell receptor-stimulation of nuclear factor kappaB (NF-kappaB) activation in Jurkat T-cells. Hydrophobic motif phosphorylation also substantially influences PKC-theta catalytic activity (5-fold reduction in the S695A mutant), but does not impair NF-kappaB activation in Jurkat T-cells. Its mechanism is independent of secondary effects on activation loop phosphorylation and cannot be explained by thermal instability. Turn motif phosphorylation has a limited effect on kinase activity, but negatively regulates other aspects of PKC-theta function, since the S676A mutant is more efficient than wild-type in inducing NF-kappaB activation in Jurkat T-cells. These findings expand our understanding of the roles of phosphorylation in novel PKCs, and indicate that PKC-theta is a constitutively competent kinase as a consequence of constitutive

  20. pSer40 tyrosine hydroxylase immunohistochemistry identifies the anatomical location of C1 neurons in rat RVLM that are activated by hypotension.

    PubMed

    Nedoboy, P E; Mohammed, S; Kapoor, K; Bhandare, A M; Farnham, M M J; Pilowsky, P M

    2016-03-11

    Identification of neurons, and their phenotype, that are activated in response to specific stimuli is a critical step in understanding how neural networks integrate inputs to produce specific outputs. Here, we developed novel mouse monoclonal antibodies of different IgG isotypes that are specific to tyrosine hydroxylase (TH), and to tyrosine hydroxylase activated at its serine 40 position (pSer40TH), in order to assess changes in the activity of phenotypically identified cardiovascular neurons using fluorescence immunohistochemistry. We find that the proportion of C1 pSer40TH-positive neurons in the central and medial region of the rat rostral ventrolateral medulla (RVLM) increases dramatically following hydralazine treatment, whereas phenylephrine treatment does not significantly change the pSer40TH/TH ratio in these regions compared to control. This finding suggests that there is a mediolateral topology associated with the activation of C1 neurons following baroreceptor loading or unloading. Overall, we conclude first, that our newly characterized monoclonal antibodies are specific, and selective, against TH and pSer40TH. Secondly, that they can be used to label TH and pSer40TH immunoreactive neurons simultaneously, and thirdly that that they can be used to identify the activation state of catecholamine synthetizing neurons after physiological stimuli. Finally, we find that there is basal level of activation of TH neurons in the lateral, central and medial regions (∼ 70%, 30% and 45%, respectively) of the C1 area, but that following unloading of the baroreceptors there is a marked increase in activation of central (∼ 80%) and medial (∼ 90%) C1 neurons in the RVLM.

  1. A Natural Hepatocyte Growth Factor/Scatter Factor Autocrine Loop in Myoblast Cells and the Effect of the Constitutive Met Kinase Activation on Myogenic Differentiation

    PubMed Central

    Anastasi, Sergio; Giordano, Silvia; Sthandier, Olga; Gambarotta, Giovanna; Maione, Rossella; Comoglio, Paolo; Amati, Paolo

    1997-01-01

    As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way. To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65tpr-met) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65tpr-met highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand–receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65tpr-met kinase. These data indicate that a met kinase signal released from differentiation

  2. Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

    PubMed Central

    Camurdanoglu, B. Z.; Hrovat, C.; Dürnberger, G.; Madalinski, M.; Mechtler, K.; Herbst, R.

    2016-01-01

    The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance. PMID:27666825

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

    PubMed Central

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

    2015-01-01

    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. PMID:25588787

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

    PubMed

    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.

  5. Active opsin loci adopt intrachromosomal loops that depend on the photoreceptor transcription factor network.

    PubMed

    Peng, Guang-Hua; Chen, Shiming

    2011-10-25

    Rod and cone opsin genes are expressed in a mutually exclusive manner in their respective photoreceptor subtypes in the mammalian retina. Previous transgenic mouse studies showed that functional interactions between the distal enhancer and proximal promoter of rhodopsin and long/medium-wavelength (L/M) opsin genes are essential for regulating their cell-type-specific transcription. We have used chromosomal conformation capture assays in mouse retinas to investigate the molecular mechanism responsible for this interaction. Here we show that each opsin gene forms intrachromosomal loops in the appropriate photoreceptor subtype, while maintaining a linear configuration in other cell types where it is silent. The enhancer forms physical contacts not only with the promoter but also with the coding regions of each opsin locus. ChIP assays showed that cell-type-specific target binding by three key photoreceptor transcription factors-cone--rod homeobox (CRX), neural retina leucine zipper (NRL), and nuclear receptor subfamily 2, group E, member 3 (NR2E3)--is required for the appropriate local chromosomal organization and transcription of rod and cone opsins. Similar correlations between chromosomal loops and active transcription of opsin genes were also observed in human photoreceptors. Furthermore, quantitative chromosomal conformation capture on human retinas from two male donors showed that the L/M enhancer locus control region (LCR) loops with either the L or M promoter in a near 3:1 ratio, supporting distance-dependent competition between L and M for LCR. Altogether, our results suggest that the photoreceptor transcription factor network cooperatively regulates the chromosomal organization of target genes to precisely control photoreceptor subtype-specific gene expression.

  6. The solar extreme ultra-violet corona: Resolved loops and the unresolved active region corona

    NASA Astrophysics Data System (ADS)

    Cirtain, Jonathan Wesley

    In this work, physical characteristics of the solar corona as observed in the Extreme Ultra-Violet (EUV) regime are investigated. The focus will be the regions of intense EUV radiation generally found near the locations of sunspots. These regions are commonly called active regions. Multiple space- based observing platforms have been deployed in the last decade; it is possible to use several of these observatories in combination to develop a more complete picture of the solar corona. Joint Observing Program 146 was created to collect spectroscopic intensities using the Coronal Diagnostic Spectrometer on Solar and Heliospheric Observatory and EUV images using NASA's Transition Region and Coronal Explorer. The emission line intensities are analyzed to develop an understanding of the temperature and density of the active region coronal plasma. However, the performance of the CDS instrument in the spatial and temporal domains is limited and to compensate for these limitations, data collected by the TRACE instrument provide a high spatial and temporal resolution set of observations. One of the most exciting unsolved problems in solar astrophysics is to understand why the corona maintains a temperature roughly two orders of magnitude higher than the underlying material. A detailed investigation of the coronal emission has provided constraints on models of the heating mechanism, since the temperature, density and evolution of emission rates for multiple ionic species are indicative of the mechanism(s) working to heat the corona. The corona appears to consist of multiple unresolved structures as well as resolved active region structures, called coronal loops. The purpose of the present work is to determine the characteristics of the unresolved background corona. Using the characterizations of the coronal unresolved background, results for loops after background subtraction are also presented. This work demonstrates the magnitude of the unresolved coronal emission with

  7. Plasiatine, an Unprecedented Indole–Phenylpropanoid Hybrid from Plantago asiatica as a Potent Activator of the Nonreceptor Protein Tyrosine Phosphatase Shp2

    PubMed Central

    Gao, Zhong-Hua; Shi, Yi-Ming; Qiang, Zhe; Wang, Xia; Shang, Shan-Zhai; Yang, Yan; Du, Bao-Wen; Peng, Hui-Pan; Ji, Xu; Li, Honglin; Wang, Fei; Xiao, Wei-Lie

    2016-01-01

    Plasiatine (1), isolated from the seeds of Plantago asiatica, is an unprecedented indole analogue linked to a phenylpropanoid moiety via a carbon bond that builds up a novel heteromeric construction with a C19N2 scaffold. Its structure was determined by spectroscopic data and computational evidence. Notably, experimental assay demonstrated that 1 significantly enhanced the activity of the nonreceptor protein tyrosine phosphatase Shp2 in vitro in a concentration-dependent manner with an EC50 value of 0.97 μM, and activated phosphorylation of ERK, a known target of Shp2. Moreover, plasiatine (1) promoted hepatocellular HepG2 cells migration. Molecular docking suggested that plasiatine (1) binds to the catalytic cleft of Shp2. These results identified plasiatine (1) as the first small molecule Shp2 activator, and it warrants further investigation as a novel pharmaceutical tool to study the function of Shp2 in tumorigenesis. PMID:27101899

  8. Plasiatine, an Unprecedented Indole-Phenylpropanoid Hybrid from Plantago asiatica as a Potent Activator of the Nonreceptor Protein Tyrosine Phosphatase Shp2.

    PubMed

    Gao, Zhong-Hua; Shi, Yi-Ming; Qiang, Zhe; Wang, Xia; Shang, Shan-Zhai; Yang, Yan; Du, Bao-Wen; Peng, Hui-Pan; Ji, Xu; Li, Honglin; Wang, Fei; Xiao, Wei-Lie

    2016-01-01

    Plasiatine (1), isolated from the seeds of Plantago asiatica, is an unprecedented indole analogue linked to a phenylpropanoid moiety via a carbon bond that builds up a novel heteromeric construction with a C19N2 scaffold. Its structure was determined by spectroscopic data and computational evidence. Notably, experimental assay demonstrated that 1 significantly enhanced the activity of the nonreceptor protein tyrosine phosphatase Shp2 in vitro in a concentration-dependent manner with an EC50 value of 0.97 μM, and activated phosphorylation of ERK, a known target of Shp2. Moreover, plasiatine (1) promoted hepatocellular HepG2 cells migration. Molecular docking suggested that plasiatine (1) binds to the catalytic cleft of Shp2. These results identified plasiatine (1) as the first small molecule Shp2 activator, and it warrants further investigation as a novel pharmaceutical tool to study the function of Shp2 in tumorigenesis. PMID:27101899

  9. Plasiatine, an Unprecedented Indole–Phenylpropanoid Hybrid from Plantago asiatica as a Potent Activator of the Nonreceptor Protein Tyrosine Phosphatase Shp2

    NASA Astrophysics Data System (ADS)

    Gao, Zhong-Hua; Shi, Yi-Ming; Qiang, Zhe; Wang, Xia; Shang, Shan-Zhai; Yang, Yan; Du, Bao-Wen; Peng, Hui-Pan; Ji, Xu; Li, Honglin; Wang, Fei; Xiao, Wei-Lie

    2016-04-01

    Plasiatine (1), isolated from the seeds of Plantago asiatica, is an unprecedented indole analogue linked to a phenylpropanoid moiety via a carbon bond that builds up a novel heteromeric construction with a C19N2 scaffold. Its structure was determined by spectroscopic data and computational evidence. Notably, experimental assay demonstrated that 1 significantly enhanced the activity of the nonreceptor protein tyrosine phosphatase Shp2 in vitro in a concentration-dependent manner with an EC50 value of 0.97 μM, and activated phosphorylation of ERK, a known target of Shp2. Moreover, plasiatine (1) promoted hepatocellular HepG2 cells migration. Molecular docking suggested that plasiatine (1) binds to the catalytic cleft of Shp2. These results identified plasiatine (1) as the first small molecule Shp2 activator, and it warrants further investigation as a novel pharmaceutical tool to study the function of Shp2 in tumorigenesis.

  10. The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

    PubMed Central

    Gu, Changkyu; Park, Soochul

    2001-01-01

    Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via α5β1- or β3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110γ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110γ. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110γ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110γ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion. PMID:11416136

  11. Variants of the yeast MAPK Mpk1 are fully functional independently of activation loop phosphorylation.

    PubMed

    Goshen-Lago, Tal; Goldberg-Carp, Anat; Melamed, Dganit; Darlyuk-Saadon, Ilona; Bai, Chen; Ahn, Natalie G; Admon, Arie; Engelberg, David

    2016-09-01

    MAP kinases of the ERK family are conserved from yeast to humans. Their catalytic activity is dependent on dual phosphorylation of their activation loop's TEY motif, catalyzed by MAPK kinases (MEKs). Here we studied variants of Mpk1, a yeast orthologue of Erk, which is essential for cell wall integrity. Cells lacking MPK1, or the genes encoding the relevant MEKs, MKK1 and MKK2, do not proliferate under cell wall stress, imposed, for example, by caffeine. Mutants of Mpk1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1∆mkk2∆ and mkk1∆mkk2∆pbs2∆ste7∆ cells, suggesting that they autophosphorylate. However, strikingly, when Y268C/A mutations were combined with the kinase-dead mutation, K54R, or mutations at the TEY motif, T190A+Y192F, the resulting proteins still allowed mkk1∆mkk2∆ cells to proliferate under caffeine stress. Mutating the equivalent residue, Tyr-280/Tyr-261, in Erk1/Erk2 significantly impaired Erk1/2's catalytic activity. This study describes the first case in which a MAPK, Erk/Mpk1, imposes a phenotype via a mechanism that is independent of TEY phosphorylation and an unusual case in which an equivalent mutation in a highly conserved domain of yeast and mammalian Erks causes an opposite effect. PMID:27413009

  12. Endocytosis of Receptor Tyrosine Kinases

    PubMed Central

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    1995-10-01

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

  15. Thermal and Kinetic Properties of Motions in a Prominence Activation and Nearby Loop

    NASA Technical Reports Server (NTRS)

    Kucera, Therese; Landi, E.

    2005-01-01

    We perform a quantitative analysis of the thermal properties of a prominence activation and motions in a nearby loop. In order to make measurements of the quickly moving features seen in prominences in the UV we use the SOHO/SUMER spectrograph to take a time series of exposures from a single pointing position, providing a measurement of spectral line properties as a function of time and position along the slit. The lines observed cover a broad range of temperatures from 80,000 - 1.6 million K. These measurements are combined with TRACE movies in transition region and coronal temperature bands to obtain more complete information concerning prominence structure and motions. The resulting observations allow us to analyze the thermal and kinetic energy of the moving sources as functions of time. The loop and prominence are most apparent in lines formed at temperatures below 250,000 K. We find that in most cases the temperature distribution of plasma in a moving feature changes relatively little over time periods of about 20 minutes.

  16. Modeling active region transient brightenings observed with X-ray telescope as multi-stranded loops

    SciTech Connect

    Kobelski, Adam R.; McKenzie, David E.; Donachie, Martin

    2014-05-10

    Strong evidence exists that coronal loops as observed in extreme ultraviolet and soft X-rays may not be monolithic isotropic structures, but can often be more accurately modeled as bundles of independent strands. Modeling the observed active region transient brightenings (ARTBs) within this framework allows for the exploration of the energetic ramifications and characteristics of these stratified structures. Here we present a simple method of detecting and modeling ARTBs observed with the Hinode X-Ray Telescope (XRT) as groups of zero-dimensional strands, which allows us to probe parameter space to better understand the spatial and temporal dependence of strand heating in impulsively heated loops. This partially automated method can be used to analyze a large number of observations to gain a statistical insight into the parameters of coronal structures, including the number of heating events required in a given model to fit the observations. In this article, we present the methodology and demonstrate its use in detecting and modeling ARTBs in a sample data set from Hinode/XRT. These initial results show that, in general, multiple heating events are necessary to reproduce observed ARTBs, but the spatial dependence of these heating events cannot yet be established.

  17. Adaptable Single Active Loop Thermal Control System (TCS) for Future Space Missions

    NASA Technical Reports Server (NTRS)

    Mudawar, Issam; Lee, Seunghyun; Hasan, Mohammad

    2015-01-01

    This presentation will examine the development of a thermal control system (TCS) for future space missions utilizing a single active cooling loop. The system architecture enables the TCS to be reconfigured during the various mission phases to respond, not only to varying heat load, but to heat rejection temperature as well. The system will consist of an accumulator, pump, cold plates (evaporators), condenser radiator, and compressor, in addition to control, bypass and throttling valves. For cold environments, the heat will be rejected by radiation, during which the compressor will be bypassed, reducing the system to a simple pumped loop that, depending on heat load, can operate in either a single-phase liquid mode or two-phase mode. For warmer environments, the pump will be bypassed, enabling the TCS to operate as a heat pump. This presentation will focus on recent findings concerning two-phase flow regimes, pressure drop, and heat transfer coefficient trends in the cabin and avionics micro-channel heat exchangers when using the heat pump mode. Also discussed will be practical implications of using micro-channel evaporators for the heat pump.

  18. Rhodotorula glutinis Phenylalanine/Tyrosine Ammonia Lyase Enzyme Catalyzed Synthesis of the Methyl Ester of para-Hydroxycinnamic Acid and its Potential Antibacterial Activity.

    PubMed

    MacDonald, Marybeth C; Arivalagan, Pugazhendhi; Barre, Douglas E; MacInnis, Judith A; D'Cunha, Godwin B

    2016-01-01

    Biotransformation of L-tyrosine methyl ester (L-TM) to the methyl ester of para- hydroxycinnamic acid (p-HCAM) using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase (PTAL; EC 4.3.1.26) enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5), temperature (37°C), speed of agitation (50 rpm), enzyme concentration (0.080 μM), and substrate concentration (0.50 mM). Under these conditions, the yield of the reaction was ∼15% in 1 h incubation period and ∼63% after an overnight (∼18 h) incubation period. The product (p-HCAM) of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR). Fourier Transform Infra-Red spectroscopy (FTIR) was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram-positive and Gram-negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications.

  19. Rhodotorula glutinis Phenylalanine/Tyrosine Ammonia Lyase Enzyme Catalyzed Synthesis of the Methyl Ester of para-Hydroxycinnamic Acid and its Potential Antibacterial Activity

    PubMed Central

    MacDonald, Marybeth C.; Arivalagan, Pugazhendhi; Barre, Douglas E.; MacInnis, Judith A.; D’Cunha, Godwin B.

    2016-01-01

    Biotransformation of L-tyrosine methyl ester (L-TM) to the methyl ester of para- hydroxycinnamic acid (p-HCAM) using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase (PTAL; EC 4.3.1.26) enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5), temperature (37°C), speed of agitation (50 rpm), enzyme concentration (0.080 μM), and substrate concentration (0.50 mM). Under these conditions, the yield of the reaction was ∼15% in 1 h incubation period and ∼63% after an overnight (∼18 h) incubation period. The product (p-HCAM) of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR). Fourier Transform Infra-Red spectroscopy (FTIR) was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram-positive and Gram-negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications. PMID:27014206

  20. A Mutational Analysis of the Active Site Loop Residues in cis-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Schroeder, Gottfried K.; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to

  1. Human-in-the-loop evaluation of RMS Active Damping Augmentation

    NASA Technical Reports Server (NTRS)

    Demeo, Martha E.; Gilbert, Michael G.; Scott, Michael A.; Lepanto, Janet A.; Bains, Elizabeth M.; Jensen, Mary C.

    1993-01-01

    Active Damping Augmentation is the insertion of Controls-Structures Integration Technology to benefit the on-orbit performance of the Space Shuttle Remote Manipulator System. The goal is to reduce the vibration decay time of the Remote Manipulator System following normal payload maneuvers and operations. Simulation of Active Damping Augmentation was conducted in the realtime human-in-the-loop Systems Engineering Simulator at the NASA Johnson Space Center. The objective of this study was to obtain a qualitative measure of operational performance improvement from astronaut operators and to obtain supporting quantitative performance data. Sensing of vibratory motions was simulated using a three-axis accelerometer mounted at the end of the lower boom of the Remote Manipulator System. The sensed motions were used in a feedback control law to generate commands to the joint servo mechanisms which reduced the unwanted oscillations. Active damping of the Remote Manipulator System with an attached 3990 lb. payload was successfully demonstrated. Six astronaut operators examined the performance of an Active Damping Augmentation control law following single-joint and coordinated six-joint translational and rotational maneuvers. Active Damping Augmentation disturbance rejection of Orbiter thruster firings was also evaluated. Significant reductions in the dynamic response of the 3990 lb. payload were observed. Astronaut operators recommended investigation of Active Damping Augmentation benefits to heavier payloads where oscillations are a bigger problem (e.g. Space Station Freedom assembly operators).

  2. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  3. Cytokine-mediated induction of cyclo-oxygenase-2 by activation of tyrosine kinase in bovine endothelial cells stimulated by bacterial lipopolysaccharide.

    PubMed Central

    Akarasereenont, P.; Bakhle, Y. S.; Thiemermann, C.; Vane, J. R.

    1995-01-01

    1. The induction of cyclo-oxygenase-2 (COX-2) afforded by bacterial lipopolysaccharide (LPS, endotoxin) in bovine aortic endothelial cells (BAEC) is mediated by tyrosine kinase. LPS also causes the generation of several cytokines including interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). This study investigates whether endogenous IL-1 beta, TNF-alpha, EGF or PDGF contribute to the induction of COX-2 elicited by LPS in BAEC and if their action is due to activation of tyrosine kinase. Furthermore, we have studied the induction of COX-2 by exogenous cytokines. 2. Accumulation of 6-oxo-prostaglandin (PG) F1 alpha in cultures of BAEC was measured by radioimmunoassay at 24 h after addition of either LPS (1 microgram ml-1) alone or LPS together with a polyclonal antibody to one of the various cytokines. In experiments designed to measure 'COX activity', 6-oxo-PGF1 alpha generated by BAEC activated with recombinant human IL-1 beta, TNF-alpha, EGF or PDGF for 12 h was measured after incubation of washed cells with exogenous arachidonic acid (30 microM for 15 min). Western blot analysis determined the expression of COX-2 protein in BAEC. 3. The accumulation of 6-oxo-PGF1 alpha caused by LPS in BAEC was attenuated by co-incubation with one of the polyclonal antibodies, anti-IL-1 beta, anti-TNF-alpha, anti-EGF, anti-PDGF or with the IL-1 receptor antagonist, in a dose-dependent manner. Exogenous IL-1 beta, TNF-alpha or EGF also caused an increase in COX activity, while PDGF was ineffective.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4 PMID:7582449

  4. Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene.

    PubMed Central

    Chiaramello, A; Neuman, K; Palm, K; Metsis, M; Neuman, T

    1995-01-01

    Sequence analysis of rat and human low-affinity nerve growth factor receptor p75LNGFR gene promoter regions revealed a single E-box cis-acting element, located upstream of the major transcription start sites. Deletion analysis of the E-box sequence demonstrated that it significantly contributes to p75LNGFR promoter activity. This E box has a dual function; it mediates either activation or repression of the p75LNGFR promoter activity, depending on the interacting transcription factors. We showed that the two isoforms of the class A basic helix-loop-helix (bHLH) transcription factor ME1 (ME1a and ME1b), the murine homolog of the human HEB transcription factor, specifically repress p75LNGFR promoter activity. This repression can be released by coexpression of the HLH Id2 transcriptional regulator. In vitro analyses demonstrated that ME1a forms a stable complex with the p75LNGFR E box and likely competes with activating E-box-binding proteins. By using ME1a-overexpressing PC12 cells, we showed that the endogenous p75LNGFR gene is a target of ME1a repression. Together, these data demonstrate that the p75LNGFR E box and the interacting bHLH transcription factors are involved in the regulation of p75LNGFR gene expression. These results also show that class A bHLH transcription factors can repress and Id-like negative regulators can stimulate gene expression. PMID:7565756

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

    NASA Technical Reports Server (NTRS)

    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.

  6. Potent quinoxaline-based inhibitors of PDGF receptor tyrosine kinase activity. Part 2: the synthesis and biological activities of RPR127963 an orally bioavailable inhibitor.

    PubMed

    He, Wei; Myers, Michael R; Hanney, Barbara; Spada, Alfred P; Bilder, Glenda; Galzcinski, Helen; Amin, Dilip; Needle, Saul; Page, Ken; Jayyosi, Zaid; Perrone, Mark H

    2003-09-15

    RPR127963 demonstrates an excellent pharmacokinetic profile in several species and was found to be efficacious in the prevention of restenosis in a Yucatan mini-pig model upon oral administration of 1-5 mg/kg. The in vitro selectivity profile and SAR of the highly optimized PDGF-R tyrosine kinase inhibitor are highlighted.

  7. Closed Loop Active Flow Separation Detection and Control in a Multistage Compressor

    NASA Technical Reports Server (NTRS)

    Bright, Michelle M.; Culley, Dennis E.; Braunscheidel, Edward P.; Welch, Gerard E.

    2005-01-01

    Active closed loop flow control was successfully demonstrated on a full annulus of stator vanes in a low speed axial compressor. Two independent methods of detecting separated flow conditions on the vane suction surface were developed. The first technique detects changes in static pressure along the vane suction surface, while the second method monitors variation in the potential field of the downstream rotor. Both methods may feasibly be used in future engines employing embedded flow control technology. In response to the detection of separated conditions, injection along the suction surface of each vane was used. Injected mass flow on the suction surface of stator vanes is known to reduce separation and the resulting limitation on static pressure rise due to lowered diffusion in the vane passage. A control algorithm was developed which provided a proportional response of the injected mass flow to the degree of separation, thereby minimizing the performance penalty on the compressor system.

  8. PD166326, a novel tyrosine kinase inhibitor, has greater antileukemic activity than imatinib mesylate in a murine model of chronic myeloid leukemia.

    PubMed

    Wolff, Nicholas C; Veach, Darren R; Tong, William P; Bornmann, William G; Clarkson, Bayard; Ilaria, Robert L

    2005-05-15

    Imatinib mesylate is highly effective in newly diagnosed chronic myeloid leukemia (CML), but BCR/ABL (breakpoint cluster region/abelson murine leukemia)-positive progenitors persist in most patients with CML treated with imatinib mesylate, indicating the need for novel therapeutic approaches. In this study, we have used the murine CML-like myeloproliferative disorder as a platform to characterize the pharmacokinetic, signal transduction, and antileukemic properties of PD166326, one of the most potent members of the pyridopyrimidine class of protein tyrosine kinase inhibitors. In mice with the CML-like disease, PD166326 rapidly inhibited Bcr/Abl kinase activity after a single oral dose and demonstrated marked antileukemic activity in vivo. Seventy percent of PD166326-treated mice achieved a white blood cell (WBC) count less than 20.0 x 10(9)/L (20,000/microL) at necropsy, compared with only 8% of imatinib mesylate-treated animals. Further, two thirds of PD166326-treated animals had complete resolution of splenomegaly, compared with none of the imatinib mesylate-treated animals. Consistent with its more potent antileukemic effect in vivo, PD166326 was also superior to imatinib mesylate in inhibiting the constitutive tyrosine phosphorylation of numerous leukemia-cell proteins, including the src family member Lyn. PD166326 also prolonged the survival of mice with imatinib mesylate-resistant CML induced by the Bcr/Abl mutants P210/H396P and P210/M351T. Altogether, these findings demonstrate the potential of more potent Bcr/Abl inhibitors to provide more effective antileukemic activity. Clinical development of PD166326 or a related analog may lead to more effective drugs for the treatment of de novo and imatinib mesylate-resistant CML.

  9. Flavonoids with epidermal growth factor-receptor tyrosine kinase inhibitory activity stimulate PEPT1-mediated cefixime uptake into human intestinal epithelial cells.

    PubMed

    Wenzel, U; Kuntz, S; Daniel, H

    2001-10-01

    We have tested 33 flavonoids, occurring ubiquitously in foods of plant origin, for their ability to alter the transport of the beta-lactam antibiotic cefixime via the H+-coupled intestinal peptide transporter PEPT1 in the human intestinal epithelial cell line Caco-2. Of the flavonoids tested, quercetin, genistein, naringin, diosmin, acacetin, and chrysin increased uptake of [14C]cefixime dose dependently by up to 60%. All other flavonoids were either without effect or decreased the absorption of cefixime. Quercetin was shown to increase the Vmax of cefixime influx without changing the apparent Km for transport. However, the expected concomitant increase in intracellular acidification due to PEPT1-mediated cefixime/H+-cotransport was less pronounced in the presence of quercetin. This suggested that pH regulatory systems such as apical Na+/H+-exchange could be activated by quercetin and maintain the proton-motive driving force for cefixime uptake. Since quercetin and genistein have been shown to inhibit epidermal growth factor (EGF)-receptor tyrosine kinases, we applied tyrphostin 25 to prove whether such an inhibition could explain the stimulatory effects seen on cefixime uptake. It was found that tyrphostin 25 simulated the effects of quercetin by increasing cefixime absorption due to maintenance of the transmembrane pH gradient. In conclusion, our studies show that flavonoids with EGF-receptor tyrosine kinase inhibitory activities enhance the intestinal absorption of the beta-lactam antibiotic cefixime in Caco-2 cells by activation of apical Na+/H+-exchange and a concomitant increase of the driving force for PEPT1.

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

    PubMed

    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

  11. Tyrosine kinase inhibitors target cancer stem cells in renal cell cancer.

    PubMed

    Czarnecka, Anna M; Solarek, Wojciech; Kornakiewicz, Anna; Szczylik, Cezary

    2016-03-01

    This study was designed to analyze the impact of multi-targeted tyrosine kinase inhibitors on the cancer stem cell subpopulation in renal cell cancer. The second objective was to evaluate the effect of tumor growth inhibition related to a tumor niche factor - oxygen deprivation - as hypoxia develops along with the anti-angiogenic activity of tyrosine kinase inhibitors in renal tumors. Cells were treated with tyrosine kinase inhibitors, sunitinib, sorafenib and axitinib, in 2D and 3D culture conditions. Cell proliferation along with drug toxicity were evaluated. It was shown that the proliferation rate of cancer stem cells was decreased by the tyrosine kinase inhibitors. The efficacy of the growth inhibition was limited by hypoxic conditions and 3D intratumoral cell-cell interactions. We conclude that understanding the complex molecular interaction feedback loops between differentiated cancer cells, cancer stem cells and the tumor microenvironment in 3D culture should aid the identification of novel treatment targets and to evalute the efficacy of renal cancer therapies. Cell-cell interaction may represent a critical microenvironmental factor regulating cancer stem cell self-renewal potential, enhancing the stem cell phenotype and limiting drug toxicity. At the same time the role of hypoxia in renal cancer stem cell biology is also significant.

  12. Perindopril increases the swallowing reflex by inhibiting substance P degradation and tyrosine hydroxylase activation in a rat model of dysphagia.

    PubMed

    Ikeda, Jun-ichi; Kojima, Natsuki; Saeki, Kohji; Ishihara, Miki; Takayama, Makoto

    2015-01-01

    Patients with hypertension have a high risk of ischemic stroke and subsequent stroke-associated pneumonia. Stroke-associated pneumonia is most likely to develop in patients with dysphagia. The present study was designed to compare the ameliorative effects of different treatments in rat model of dysphagia. Spontaneously hypertensive rats were treated with bilateral common carotid artery occlusion (BCAO) to induce chronic cerebral hypoperfusion causing disorders of the swallowing reflex. Angiotensin-converting enzyme (ACE) inhibitors (perindopril, imidapril and enalapril), an angiotensin II type 1-receptor blocker (losartan), a vasodilator (hydralazine) and an indirect dopamine agonist (amantadine) were dissolved in drinking water and administered to the rats for six weeks. The blood pressure, the swallowing reflex under anesthesia, the substance P content in the striatum and the tyrosine hydroxylase (TH) expression in the substantial nigra were measured. Compared to the vehicle control, the decrease in the swallowing reflex induced by BCAO was attenuated significantly by enalapril, imidapril and perindopril, but only slightly by losartan. Hydralazine had no effect on the swallowing reflex. Amantadine significantly attenuated the decreased swallowing reflex but increased the blood pressure. Cerebral hypoperfusion for six weeks decreased the TH expression and substance P level. Perindopril improved both the TH expressions and substance P level, but imidapril, enalapril and amantadine only improved the substance P level. The present findings indicate that perindopril could be useful for preventing dysphagia in the chronic stage of stroke by attenuating the decrease in TH expression and the decrease in the substance P level.

  13. Transforming and Tumorigenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosine-Kinase

    PubMed Central

    Ormazábal, Cristina; Santos-Roncero, Matilde; Galán-Díez, Marta; Steegmann, Juan Luis; Figuera, Ángela; Arranz, Eva; Vizmanos, José Luis; Bueren, Juan A.; Río, Paula; Fernández-Ruiz, Elena

    2012-01-01

    Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a BCR-JAK2 fusion gene in fatal chronic and acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies. PMID:22384256

  14. Tyrosine Nitration of PA700 Activates the 26S Proteasome to Induce Endothelial Dysfunction in Mice With Angiotensin II–Induced Hypertension

    PubMed Central

    Xu, Jian; Wang, Shuangxi; Wu, Yong; Song, Ping; Zou, Ming-Hui

    2010-01-01

    The ubiquitin-proteasome system has been implicated in oxidative stress–induced endothelial dysfunction in cardiovascular diseases. However, the mechanism by which oxidative stress alters the ubiquitin-proteasome system is poorly defined. The present study was conducted to determine whether oxidative modifications of PA700, a 26S proteasome regulatory subunit, contributes to angiotensin II (Ang II)–induced endothelial dysfunction. Exposure of human umbilical vein endothelial cells to low concentrations of Ang II, but not vehicle, for 6 hours significantly decreased the levels of tetrahydro-L-biopterin (BH4), an essential cofactor of endothelial NO synthase, which was accompanied by a decrease in GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis. In addition, Ang II increased both tyrosine nitration of PA700 and the 26S proteasome activity, which were paralleled by increased coimmunoprecipitation of PA700 and the 20S proteasome. Genetic inhibition of NAD(P)H oxidase or administration of uric acid (a peroxynitrite scavenger) or NG-nitro-L-arginine methyl ester (nonselective NO synthase inhibitor) significantly attenuated Ang II–induced PA700 nitration, 26S proteasome activation, and reduction of GTP cyclohydrolase I and BH4. Finally, Ang II infusion in mice decreased the levels of both BH4 and GTP cyclohydrolase I and impaired endothelial-dependent relaxation in isolated aortas, and all of these effects were prevented by the administration of MG132, a potent inhibitor for 26S proteasome. We conclude that Ang II increases tyrosine nitration of PA700 resulting in accelerated GTP cyclohydrolase I degradation, BH4 deficiency, and consequent endothelial dysfunction in hypertension. PMID:19597039

  15. Neisseria meningitidis Opc Invasin Binds to the Sulphated Tyrosines of Activated Vitronectin to Attach to and Invade Human Brain Endothelial Cells

    PubMed Central

    Virji, Mumtaz

    2010-01-01

    The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y56 and Y59. Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43–68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain 45RGD47 was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may

  16. A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity*

    PubMed Central

    Guo, Shihui; Skala, Wolfgang; Magdolen, Viktor; Briza, Peter; Biniossek, Martin L.; Schilling, Oliver; Kellermann, Josef; Brandstetter, Hans; Goettig, Peter

    2016-01-01

    Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology. PMID:26582203

  17. Enhancer RNAs participate in androgen receptor-driven looping that selectively enhances gene activation.

    PubMed

    Hsieh, Chen-Lin; Fei, Teng; Chen, Yiwen; Li, Tiantian; Gao, Yanfei; Wang, Xiaodong; Sun, Tong; Sweeney, Christopher J; Lee, Gwo-Shu Mary; Chen, Shaoyong; Balk, Steven P; Liu, Xiaole Shirley; Brown, Myles; Kantoff, Philip W

    2014-05-20

    The androgen receptor (AR) is a key factor that regulates the behavior and fate of prostate cancer cells. The AR-regulated network is activated when AR binds enhancer elements and modulates specific enhancer-promoter looping. Kallikrein-related peptidase 3 (KLK3), which codes for prostate-specific antigen (PSA), is a well-known AR-regulated gene and its upstream enhancers produce bidirectional enhancer RNAs (eRNAs), termed KLK3e. Here, we demonstrate that KLK3e facilitates the spatial interaction of the KLK3 enhancer and the KLK2 promoter and enhances long-distance KLK2 transcriptional activation. KLK3e carries the core enhancer element derived from the androgen response element III (ARE III), which is required for the interaction of AR and Mediator 1 (Med1). Furthermore, we show that KLK3e processes RNA-dependent enhancer activity depending on the integrity of core enhancer elements. The transcription of KLK3e was detectable and its expression is significantly correlated with KLK3 (R(2) = 0.6213, P < 5 × 10(-11)) and KLK2 (R(2) = 0.5893, P < 5 × 10(-10)) in human prostate tissues. Interestingly, RNAi silencing of KLK3e resulted in a modest negative effect on prostate cancer cell proliferation. Accordingly, we report that an androgen-induced eRNA scaffolds the AR-associated protein complex that modulates chromosomal architecture and selectively enhances AR-dependent gene expression. PMID:24778216

  18. Transcriptional Activation by ETS and Leucine Zipper-Containing Basic Helix-Loop-Helix Proteins

    PubMed Central

    Tian, Gang; Erman, Batu; Ishii, Haruhiko; Gangopadhyay, Samudra S.; Sen, Ranjan

    1999-01-01

    The immunoglobulin μ heavy-chain gene enhancer contains closely juxtaposed binding sites for ETS and leucine zipper-containing basic helix-loop-helix (bHLH-zip) proteins. To understand the μ enhancer function, we have investigated transcription activation by the combination of ETS and bHLH-zip proteins. The bHLH-zip protein TFE3, but not USF, cooperated with the ETS domain proteins PU.1 and Ets-1 to activate a tripartite domain of this enhancer. Deletion mutants were used to identify the domains of the proteins involved. Both TFE3 and USF enhanced Ets-1 DNA binding in vitro by relieving the influence of an autoinhibitory domain in Ets-1 by direct protein-protein associations. Several regions of Ets-1 were found to be necessary, whereas the bHLH-zip domain was sufficient for this effect. Our studies define novel interactions between ETS and bHLH-zip proteins that may regulate combinatorial transcription activation by these protein families. PMID:10082562

  19. The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers

    PubMed Central

    Kennedy, Dylan P.; McRobb, Fiona M.; Leonhardt, Susan A.; Purdy, Michael; Figler, Heidi; Marshall, Melissa A.; Chordia, Mahendra; Figler, Robert; Linden, Joel

    2014-01-01

    Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists. PMID:24217444

  20. Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps

    NASA Technical Reports Server (NTRS)

    Guerra, D.; Anderson, A. J.; Salisbury, F. B.

    1985-01-01

    Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.

  1. Regulative Loops, Step Loops and Task Loops

    ERIC Educational Resources Information Center

    VanLehn, Kurt

    2016-01-01

    This commentary suggests a generalization of the conception of the behavior of tutoring systems, which the target article characterized as having an outer loop that was executed once per task and an inner loop that was executed once per step of the task. A more general conception sees these two loops as instances of regulative loops, which…

  2. A dynamic loop at the active center of the Escherichia coli pyruvate dehydrogenase complex E1 component modulates substrate utilization and chemical communication with the E2 component.

    PubMed

    Kale, Sachin; Arjunan, Palaniappa; Furey, William; Jordan, Frank

    2007-09-21

    Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.

  3. Antidepressants increase glial cell line-derived neurotrophic factor production through monoamine-independent activation of protein tyrosine kinase and extracellular signal-regulated kinase in glial cells.

    PubMed

    Hisaoka, Kazue; Takebayashi, Minoru; Tsuchioka, Mami; Maeda, Natsuko; Nakata, Yoshihiro; Yamawaki, Shigeto

    2007-04-01

    Recent studies show that neuronal and glial plasticity are important for therapeutic action of antidepressants. We previously reported that antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production in rat C6 glioma cells (C6 cells). Here, we found that amitriptyline, a tricyclic antidepressant, increased both GDNF mRNA expression and release, which were selectively and completely inhibited by mitogen-activated protein kinase kinase inhibitors. Indeed, treatment of amitriptyline rapidly increased extracellular signal-regulated kinase (ERK) activity, as well as p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase activities. Furthermore, different classes of antidepressants also rapidly increased ERK activity. The extent of acute ERK activation and GDNF release were significantly correlated to each other in individual antidepressants, suggesting an important role of acute ERK activation in GDNF production. Furthermore, antidepressants increased the acute ERK activation and GDNF mRNA expression in normal human astrocytes as well as C6 cells. Although 5-hydroxytryptamine (serotonin) (5-HT), but not noradrenaline or dopamine, increased ERK activation and GDNF release via 5-HT2A receptors, ketanserin, a 5-HT2A receptor antagonist, did not have any effect on the amitriptyline-induced ERK activation. Thus, GDNF production by amitriptyline was independent of monoamine. Both of the amitriptyline-induced ERK activation and GDNF mRNA expression were blocked by genistein, a general protein tyrosine kinase (PTK) inhibitor. Actually, we found that amitriptyline acutely increased phosphorylation levels of several phosphotyrosine-containing proteins. Taken together, these findings indicate that ERK activation through PTK regulates antidepressant-induced GDNF production and that the GDNF production in glial cells may be a novel action of the antidepressant, which is independent of monoamine. PMID:17210798

  4. Structural basis of tubulin tyrosination by tubulin tyrosine ligase.

    PubMed

    Prota, Andrea E; Magiera, Maria M; Kuijpers, Marijn; Bargsten, Katja; Frey, Daniel; Wieser, Mara; Jaussi, Rolf; Hoogenraad, Casper C; Kammerer, Richard A; Janke, Carsten; Steinmetz, Michel O

    2013-02-01

    Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL-tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton. PMID:23358242

  5. Differential activation and tyrosine hydroxylase distribution in the hippocampal, pallial and midbrain brain regions in response to cognitive performance in Indian house crows exposed to abrupt light environment.

    PubMed

    Taufique, S K Tahajjul; Kumar, Vinod

    2016-11-01

    Disruption of the cyclic feature of the day-night environment can cause negative effects on daily activity and advanced brain functions such as learning, memory and decision-making behaviour. These functions in songbirds, including corvids, involve the hippocampus, pallium and midbrain, as revealed by ZENK (a neuronal activation marker) and tyrosine hydroxylase (TH) expressions. TH is rate-limiting marker enzyme of the biosynthesis of dopamine, widely implicated in learning and memory. Here, we measured ZENK and TH immunoreactivity in the hippocampal, pallial and midbrain regions in response to cognitive performance (learning-memory retrieval) tests in Indian house crows (Corvus splendens) exposed to constant light environment (LL) with controls on 12h light:12h darkness. Along with the decay of circadian rhythm in activity behaviour, LL caused a significant decline in the cognitive performance. There was also a decrease under LL in the activity of neurons in the hippocampus, medial and central caudal nidopallium, and hyperpallium apicale, which are widely distributed with TH-immunoreactive fibres. Further, under LL, TH- immunoreactive neurons were reduced in number in midbrain dopamine synthesis sites, the venteral tegmental area (VTA) and substantia nigra (SN), with a negative correlation of co-localized ZENK/TH- immunoreactive cells on errors during the association tasks. These results show decreased activity of learning and memory neural systems, and underscore the role of dopamine in reduced cognitive performance of diurnal corvids with disrupted circadian rhythms under an abrupt light environment. PMID:27478138

  6. Proline-rich tyrosine kinase 2 via enhancing signal transducer and activator of transcription 3-dependent cJun expression mediates retinal neovascularization

    PubMed Central

    Kumar, Raj; Singh, Nikhlesh K.; Rao, Gadiparthi N.

    2016-01-01

    Despite the involvement of proline-rich tyrosine kinase 2 (Pyk2) in endothelial cell angiogenic responses, its role in pathological retinal angiogenesis is not known. In the present study, we show that vascular endothelial growth factor A (VEGFA) induces Pyk2 activation in mediating human retinal microvascular endothelial cell (HRMVEC) migration, sprouting and tube formation. Downstream to Pyk2, VEGFA induced signal transducer and activator of transcription 3 (STAT3) activation and cJun expression in the modulation of HRMVEC migration, sprouting and tube formation. Consistent with these observations, hypoxia induced activation of Pyk2-STAT3-cJun signaling axis and siRNA-mediated downregulation of Pyk2, STAT3 or cJun levels substantially inhibited hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization. Together, these observations suggest that activation of Pyk2-mediated STAT3-cJun signaling is required for VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization in vivo. PMID:27210483

  7. Differential activation and tyrosine hydroxylase distribution in the hippocampal, pallial and midbrain brain regions in response to cognitive performance in Indian house crows exposed to abrupt light environment.

    PubMed

    Taufique, S K Tahajjul; Kumar, Vinod

    2016-11-01

    Disruption of the cyclic feature of the day-night environment can cause negative effects on daily activity and advanced brain functions such as learning, memory and decision-making behaviour. These functions in songbirds, including corvids, involve the hippocampus, pallium and midbrain, as revealed by ZENK (a neuronal activation marker) and tyrosine hydroxylase (TH) expressions. TH is rate-limiting marker enzyme of the biosynthesis of dopamine, widely implicated in learning and memory. Here, we measured ZENK and TH immunoreactivity in the hippocampal, pallial and midbrain regions in response to cognitive performance (learning-memory retrieval) tests in Indian house crows (Corvus splendens) exposed to constant light environment (LL) with controls on 12h light:12h darkness. Along with the decay of circadian rhythm in activity behaviour, LL caused a significant decline in the cognitive performance. There was also a decrease under LL in the activity of neurons in the hippocampus, medial and central caudal nidopallium, and hyperpallium apicale, which are widely distributed with TH-immunoreactive fibres. Further, under LL, TH- immunoreactive neurons were reduced in number in midbrain dopamine synthesis sites, the venteral tegmental area (VTA) and substantia nigra (SN), with a negative correlation of co-localized ZENK/TH- immunoreactive cells on errors during the association tasks. These results show decreased activity of learning and memory neural systems, and underscore the role of dopamine in reduced cognitive performance of diurnal corvids with disrupted circadian rhythms under an abrupt light environment.

  8. Estradiol-17β modulates dose-dependently hypothalamic tyrosine hydroxylase activity inhibited by α-methylparatyrosine in the catfish Heteropneustes fossilis.

    PubMed

    Chaube, Radha; Joy, Keerikkattil P

    2011-12-01

    The brain is a target for organizational and activational effects of oestrogens synthesized de novo or transported from the peripheral organs. A neuroprotective role of oestrogens has been documented in a variety of vertebrates. In the present study in the catfish Heteropneustes fossilis, we have demonstrated that estradiol-17β (E(2)), the major circulating oestrogen at low dosages (0.05 and 0.1 μg/g body weight of fish for 3 days) stimulated hypothalamic tyrosine hydroxylase (TH) activity, and countered the negative effects of ovariectomy (3-week) or α-methylparatyrosine (α-MPT: 250 μg/g body weight, a competitive inhibitor of TH). In contrast, high dosages of E(2) (1 and 2 μg/g body weight of fish for 3 days) were inhibitory and further amplified the inhibitory effects of ovariectomy and α-MPT. The inhibiting role of E(2) was higher in gonad-active (prespawning) phase than gonad-inactive (resting phase) phase. The dual roles of E(2) may ensure a tight regulation of catecholaminergic activity, activating and inhibiting the system against wide fluctuations that are characteristic of seasonally breeding animals.

  9. In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos.

    PubMed Central

    Girbau, M; Bassas, L; Alemany, J; de Pablo, F

    1989-01-01

    We previously reported specific cross-linking of 125I-labeled insulin and 125I-labeled insulin-like growth factor I (IGF-I) to the alpha subunit of their respective receptors in chicken embryos of 20 somites and older. To achieve adequate sensitivity and localize spatially the receptors in younger embryos, we adapted an autoradiographic technique using whole-mounted chicken blastoderms. Insulin receptors and IGF-I receptors were expressed and could be localized as early as gastrulation, before the first somite is formed. Relative density was analyzed by a computer-assisted image system, revealing overall slightly higher binding of IGF-I than of insulin. Structures rich in both types of receptors were predominantly of ectodermal origin: Hensen's node in gastrulating embryos and neural folds, neural tube and optic vesicles during neurulation. The signal transduction capability of the receptors in early organogenesis was assessed by their ability to phosphorylate the exogenous substrate poly(Glu80Tyr20). Ligand-dependent tyrosine phosphorylation was demonstrable with both insulin and IGF-I in glycoprotein-enriched preparations from embryos at days 2 through 6 of embryogenesis. There was a developmentally regulated change in ligand-dependent tyrosine kinase activity, with a sharp increase from day 2 to day 4, in contrast with a small increase in the ligand binding. Binding of 125I-labeled IGF-I was, with the solubilized receptors, severalfold higher than binding of 125I-labeled insulin. However, the insulin-dependent phosphorylation was as high as the IGF-I-dependent phosphorylation at each developmental stage. Images PMID:2548191

  10. Calcineurin-NFAT activation and DSCR-1 auto-inhibitory loop: how is homoeostasis regulated?

    PubMed

    Minami, Takashi

    2014-04-01

    Calcineurin-nuclear factor of activated T cells (NFAT) signalling plays a critical role not only in the immune and nervous systems, but also in cardiovascular development and pathological endothelial cell activation during angiogenesis or inflammation. Studies in NFAT-null mice demonstrated that there is high redundancy between functions of the different NFAT family members. Deletion of only one NFAT causes mild phenotypes, but compound deletions of multiple NFAT family members leads to severe abnormalities in multiple organ systems. Genome-wide transcription analysis revealed that many NFAT target genes are related to cell growth and inflammation, whereas the gene most strongly induced by NFAT in endothelial cells is an auto-inhibitory molecule, Down syndrome critical region (DSCR)-1. The NFAT-DSCR-1 signalling axis may vary depending on the cell-type or signal dosage level under the microenvironment. In the endothelium, stable expression of the DSCR-1 short isoform attenuates septic inflammatory shock, tumour growth and tumour metastasis to lung. Moreover, dysfunction of DSCR-1 and the NFAT priming kinase, DYRK1A, prevents NFAT nuclear occupancy. This change in NFAT nuclear localization is responsible for many of the features of Down syndrome. Thus, fine-tuning of the NFAT-DSCR-1 negative feedback loop may enable therapeutic manipulation in vasculopathic diseases.

  11. Electromagnetic design of magneto-rheological mount and its open-loop semi-active control

    NASA Astrophysics Data System (ADS)

    Zheng, Ling; Li, Yinong; Deng, Zhaoxue

    2009-07-01

    Magneto-Rheological mount is a new type of semi-active and intelligent vibration isolator. It can adjust damping to reduce unwanted vibration from engine by supplying required input currents. The performance of Magneto-Rheological (MR) mount is much better than the conventional rubber mount or hydraulic mount due to its controllability and flexibility. In this paper, a novel MR engine mount with the flow mode-type for a sedan is devised, manufactured and characterized. Some important parameters are optimized to meet the requirements of MR mount by using Electromagnetic design methodology.. Electromagnetic finite element analysis verifies the effectiveness of the magnetic circuit design. The dynamic performances of MR engine mount in frequency domain are investigated experimentally. The results show that the dynamic stiffness and phase lag of MR engine mount can change continuously. They are frequency-dependent. In additional, a open-loop control strategy based on engine rotational speed measurement is proposed and the control system is performed in hardware and software. The experimental and theoretical results identified the effectiveness of such a semi-active vibration isolation system.

  12. Calcineurin-NFAT activation and DSCR-1 auto-inhibitory loop: how is homoeostasis regulated?

    PubMed

    Minami, Takashi

    2014-04-01

    Calcineurin-nuclear factor of activated T cells (NFAT) signalling plays a critical role not only in the immune and nervous systems, but also in cardiovascular development and pathological endothelial cell activation during angiogenesis or inflammation. Studies in NFAT-null mice demonstrated that there is high redundancy between functions of the different NFAT family members. Deletion of only one NFAT causes mild phenotypes, but compound deletions of multiple NFAT family members leads to severe abnormalities in multiple organ systems. Genome-wide transcription analysis revealed that many NFAT target genes are related to cell growth and inflammation, whereas the gene most strongly induced by NFAT in endothelial cells is an auto-inhibitory molecule, Down syndrome critical region (DSCR)-1. The NFAT-DSCR-1 signalling axis may vary depending on the cell-type or signal dosage level under the microenvironment. In the endothelium, stable expression of the DSCR-1 short isoform attenuates septic inflammatory shock, tumour growth and tumour metastasis to lung. Moreover, dysfunction of DSCR-1 and the NFAT priming kinase, DYRK1A, prevents NFAT nuclear occupancy. This change in NFAT nuclear localization is responsible for many of the features of Down syndrome. Thus, fine-tuning of the NFAT-DSCR-1 negative feedback loop may enable therapeutic manipulation in vasculopathic diseases. PMID:24505143

  13. CpG oligodeoxynucleotides with double stem-loops show strong immunostimulatory activity.

    PubMed

    Yang, Liang; Wu, Xiuli; Wan, Min; Yu, Yue; Yu, Yongli; Wang, Liying

    2013-01-01

    Based on the current understanding of TLR9 recognition of CpG ODN, we have tried to design a series of CpG ODNs that display double stem-loops when being analyzed for their secondary structures using 'mfold web server'. Proliferation of human PBMC and bioassay for IFN production were used as technical platforms in primary screening. Interestingly, two of them, designated as DSL01 and D-SL03, belonging to B class CpG ODN and C class CpG ODN respectively, showed vigorous immunostimulatory activity and were chosen for further tests. Flow cytometry analysis showed that both of them could activate human B cells, NK cells, mononuclear cells and T cells and up-regulate expression of CD80, CD86 and HLA-DR on the surface of subsets in human PBMCs. Furthermore, we demonstrated that those two ODNs potently stimulated proliferation of PBMC/splenocytes obtained from diverse vertebrate species. Noticeably, both of them displayed anti-breast cancer effect in mice when administered by peritumoral injection. PMID:23142503

  14. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  15. Genome-Wide Identification of Mitogen-Activated Protein Kinase Gene Family across Fungal Lineage Shows Presence of Novel and Diverse Activation Loop Motifs

    PubMed Central

    Mohanta, Tapan Kumar; Mohanta, Nibedita; Parida, Pratap; Panda, Sujogya Kumar; Ponpandian, Lakshmi Narayanan; Bae, Hanhong

    2016-01-01

    The mitogen-activated protein kinase (MAPK) is characterized by the presence of the T-E-Y, T-D-Y, and T-G-Y motifs in its activation loop region and plays a significant role in regulating diverse cellular responses in eukaryotic organisms. Availability of large-scale genome data in the fungal kingdom encouraged us to identify and analyse the fungal MAPK gene family consisting of 173 fungal species. The analysis of the MAPK gene family resulted in the discovery of several novel activation loop motifs (T-T-Y, T-I-Y, T-N-Y, T-H-Y, T-S-Y, K-G-Y, T-Q-Y, S-E-Y and S-D-Y) in fungal MAPKs. The phylogenetic analysis suggests that fungal MAPKs are non-polymorphic, had evolved from their common ancestors around 1500 million years ago, and are distantly related to plant MAPKs. We are the first to report the presence of nine novel activation loop motifs in fungal MAPKs. The specificity of the activation loop motif plays a significant role in controlling different growth and stress related pathways in fungi. Hence, the presences of these nine novel activation loop motifs in fungi are of special interest. PMID:26918378

  16. An Autoinhibitory Tyrosine Motif in the Cell-Cycle-Regulated Nek7 Kinase Is Released through Binding of Nek9

    PubMed Central

    Richards, Mark W.; O'Regan, Laura; Mas-Droux, Corine; Blot, Joelle M.Y.; Cheung, Jack; Hoelder, Swen; Fry, Andrew M.; Bayliss, Richard

    2009-01-01

    Summary Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the αC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors. PMID:19941817

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

    PubMed

    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.

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

    PubMed

    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

  19. Low-order design and high-order simulation of active closed-loop control for aerospace structures under construction

    NASA Technical Reports Server (NTRS)

    Balas, Mark J.

    1989-01-01

    Partially constructed/assembled structures in space are complicated enough but their dynamics will also be operating in closed-loop with feedback controllers. The dynamics of such structures are modeled by large-scale finite element models. The model dimension L is extremely large (approximately 10,000) while the numbers of actuators (M) and sensors (P) are small. The model parameters M(sub m) mass matrix, D(sub o) damping matrix, and K(sub o) stiffness matrix, are all symmetric and sparse (banded). Thus simulation of open-loop structure models of very large dimension can be accomplished by special integration techniques for sparse matrices. The problem of simulation of closed-loop control of such structures is complicated by the addition of controllers. Simulation of closed-loop controlled structures is an essential part of the controller design and evaluation process. Current research in the following areas is presented: high-order simulation of actively controlled aerospace structures; low-order controller design and SCI compensation for unmodeled dynamics; prediction of closed-loop stability using asymptotic eigenvalue series; and flexible robot manipulator control experiment.

  20. Spectroscopic study of a dark lane and a cool loop in a solar limb active region by Hinode/EIS

    SciTech Connect

    Lee, Kyoung-Sun; Imada, S.; Moon, Y.-J.; Lee, Jin-Yi

    2014-01-10

    We investigated a cool loop and a dark lane over a limb active region on 2007 March 14 using the Hinode/EUV Imaging Spectrometer. The cool loop is clearly seen in the spectral lines formed at the transition region temperature. The dark lane is characterized by an elongated faint structure in the coronal spectral lines and is rooted on a bright point. We examined their electron densities, Doppler velocities, and nonthermal velocities as a function of distance from the limb. We derived electron densities using the density sensitive line pairs of Mg VII, Si X, Fe XII, Fe XIII, and Fe XIV spectra. We also compared the observed density scale heights with the calculated scale heights from each peak formation temperatures of the spectral lines under the hydrostatic equilibrium. We noted that the observed density scale heights of the cool loop are consistent with the calculated heights, with the exception of one observed cooler temperature; we also found that the observed scale heights of the dark lane are much lower than their calculated scale heights. The nonthermal velocity in the cool loop slightly decreases along the loop, while nonthermal velocity in the dark lane sharply falls off with height. Such a decrease in the nonthermal velocity may be explained by wave damping near the solar surface or by turbulence due to magnetic reconnection near the bright point.

  1. SDO/AIA AND HINODE/EIS OBSERVATIONS OF INTERACTION BETWEEN AN EUV WAVE AND ACTIVE REGION LOOPS

    SciTech Connect

    Yang, Liheng; Zhang, Jun; Li, Ting; Liu, Wei; Shen, Yuandeng E-mail: zjun@bao.ac.cn

    2013-09-20

    We present detailed analysis of an extreme-ultraviolet (EUV) wave and its interaction with active region (AR) loops observed by the Solar Dynamics Observatory/Atmospheric Imaging Assembly and the Hinode EUV Imaging Spectrometer (EIS). This wave was initiated from AR 11261 on 2011 August 4 and propagated at velocities of 430-910 km s{sup –1}. It was observed to traverse another AR and cross over a filament channel on its path. The EUV wave perturbed neighboring AR loops and excited a disturbance that propagated toward the footpoints of these loops. EIS observations of AR loops revealed that at the time of the wave transit, the original redshift increased by about 3 km s{sup –1}, while the original blueshift decreased slightly. After the wave transit, these changes were reversed. When the EUV wave arrived at the boundary of a polar coronal hole, two reflected waves were successively produced and part of them propagated above the solar limb. The first reflected wave above the solar limb encountered a large-scale loop system on its path, and a secondary wave rapidly emerged 144 Mm ahead of it at a higher speed. These findings can be explained in the framework of a fast-mode magnetosonic wave interpretation for EUV waves, in which observed EUV waves are generated by expanding coronal mass ejections.

  2. Effect of radicicol infusion on the Src tyrosine kinase activity of rat hippocampus before and after training in an inhibitory avoidance task.

    PubMed

    Pereira, Patrícia; Vinadé, Elsa; Rodrigues, Letícia; De David e Silva, Tiago L; Ardenghi, Patrícia; da Silva Brum, Lucimar Filot; Gonçalves, Carlos Alberto; Izquierdo, Iván

    2007-07-01

    The participation of protein serine/threonine kinases in memory formation and retrieval is well established. In contrast, relatively little is known on the role of protein tyrosine kinases (PTKs). Previous work showed that intra-hippocampal infusion of the Src-PTK inhibitor radicicol inhibits memory acquisition, consolidation, and retrieval of one-trial step-down inhibitory avoidance task. In this study, we investigated the possible interaction between levels of Src-PTK activity in hippocampus and memory acquisition, formation, and retrieval of this task. Radicicol (0.5 microg/ml) was infused into the CA1 region of the hippocampus of rats trained in a one-trial step-down inhibitory avoidance task. Radicicol infused 15 min before training decreased Src-PTK activity, as measured 0, 1.5, and 24 h after training, and impaired memory acquisition of the task. When given immediately after training, there was a decrease in Src-PTK activity 1.5 h, but not 0 or 24 h after training. This treatment depressed memory consolidation. Radicicol infused into CA1 10 min prior to retrieval testing inhibited hippocampal Src-PTK activity, as measured immediately after the test session. The results suggest that Src-PTKs participate in memory acquisition, consolidation, and retrieval processes, but the timing of the role of the enzyme is different in each case.

  3. Activation of protein tyrosine kinase: a possible requirement for fixed-bacteria and lipopolysaccharide-induced increase in human natural killer cell activity.

    PubMed

    Puente, J; Salas, M A; Canon, C; Miranda, D; Wolf, M E; Mosnaim, A D

    1996-05-01

    Preincubation of peripheral blood lymphocytes (PBL) from drug-free, healthy volunteers with either the protein tyrosine kinase inhibitor genistein (GNT, n = 10, final concentration 200 microM) or the protein kinase A activator dybutiryl-cyclic-AMP (cAMP, n = 11, final concentration 10 microM), resulted in a significant inhibition of natural killer cell activity (NKCA, expressed as percentage of specific chromium release). With the exception of 4 out of the 11 cAMP-treated samples, individual values for NKCA in the drug preincubated specimens were at least 20% below the same subject baseline activity; furthermore, NKC lytic function was non-detectable in 4 out of the 10 and in 1 out of the 11 samples pretreated with either GNT or cAMP, respectively. PBL preincubation with glutaraldehyde-fixed Gram-negative bacteria (GNB, n = 13, final GNB-to-effector cell ratio of 50 : 1) resulted in a statistically significant increase in NKCA (baseline (x +/- SD) of 21.6 +/- 16.4 and bacteria treated samples of 41.5 +/- 24.6, respectively, Student's paired t-test p < 0.05). At least a 20% increase in NKC lytic function over its own baseline value was recorded for 11 out of the 13 samples tested (Table 1). Preincubation with GNB and GNT (5 samples) not only blocked the immunostimulant effects of GNB (Student's paired t-test p < 0.05), but in most cases individual values for NKCA were similar to those recorded for GNT-only treated samples. Use of cAMP instead of GNT also blocked, but to a smaller extent, the GNB-produced increases in NKC lytic function (paired Student's t-test < 0.05). PBL preincubation with lipopolysaccharide (LPS, n = 11, final concentration 50 micrograms/ml) resulted in a statistically significant increase in NKCA (baseline (x +/- SD) of 20.7 +/- 14.1 and LPS treated samples of 39.2 +/- 18.5, respectively, Student's paired t-test < 0.05). At least a 20% increase in NKCA over its own baseline value was observed for each and everyone of the 11 samples studied

  4. Regulation of Human CYP2C9 Expression by Electrophilic Stress Involves Activator Protein 1 Activation and DNA Looping

    PubMed Central

    Makia, Ngome L.; Surapureddi, Sailesh; Monostory, Katalin; Prough, Russell A.

    2014-01-01

    Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drugs, environmental chemicals, and physiologically important endogenous compounds. Initial studies using primary human hepatocytes showed induction of both the CYP2C9 and CYP2C19 genes by tert-butylhydroquinone (tBHQ). As a pro-oxidant, tBHQ regulates the expression of cytoprotective genes by activation of redox-sensing transcription factors, such as the nuclear factor E2-related factor 2 (Nrf2) and members of the activator protein 1 (AP-1) family of proteins. The promoter region of CYP2C9 contains two putative AP-1 sites (TGAGTCA) at positions −2201 and −1930, which are also highly conserved in CYP2C19. The CYP2C9 promoter is activated by ectopic expression of cFos and JunD, whereas Nrf2 had no effect. Using specific kinase inhibitors for mitogen-activated protein kinase, we showed that extracellular signal-regulated kinase and Jun N-terminal kinase are essential for tBHQ-induced expression of CYP2C9. Electrophoretic mobility shift assays demonstrate that cFos distinctly interacts with the distal AP-1 site and JunD with the proximal site. Because cFos regulates target genes as heterodimers with Jun proteins, we hypothesized that DNA looping might be required to bring the distal and proximal AP-1 sites together to activate the CYP2C9 promoter. Chromosome conformation capture analyses confirmed the formation of a DNA loop in the CYP2C9 promoter, possibly allowing interaction between cFos at the distal site and JunD at the proximal site to activate CYP2C9 transcription in response to electrophiles. These results indicate that oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes. PMID:24830941

  5. Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn{sup 2+}

    SciTech Connect

    Tal, T.L.; Graves, L.M.; Silbajoris, R.; Bromberg, P.A.; Wu, W.; Samet, J.M. . E-mail: samet.james@epa.gov

    2006-07-01

    Epidemiological studies have implicated zinc (Zn{sup 2+}) in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn{sup 2+}-induced EGFR activation in HAEC, we treated HAEC with 500 {mu}M ZnSO{sub 4} for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn{sup 2+} results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn{sup 2+}-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn{sup 2+} treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn{sup 2+}. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn{sup 2+} or V{sup 4+} was significantly diminished. Moreover, exposure of HAEC to Zn{sup 2+} also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn{sup 2+}-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn{sup 2+} exposure.

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

    PubMed

    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.

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

    PubMed Central

    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 Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) 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, Thr890, Ser893, Thr894 and Thr899, 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

  8. Phosphorylation of threonine 290 in the activation loop of Tpl2/Cot is necessary but not sufficient for kinase activity.

    PubMed

    Luciano, Brenda S; Hsu, Sang; Channavajhala, Padma L; Lin, Lih-Ling; Cuozzo, John W

    2004-12-10

    Cot/Tpl2/MAP3K8 is a serine/threonine kinase known to activate the ERK, p38, and JNK kinase pathways. Studies of Tpl2 knock-out mice reveal a clear defect in tumor necrosis factor-alpha production, although very little detail is known about its regulation and the signaling events involved. In the present study we demonstrated that phosphorylation of Cot was required for its maximal activity as phosphatase treatment of Cot decreased its kinase activity. The Cot sequence contains a conserved threonine at position 290 in the activation loop of the kinase domain. We found that mutation of this residue to alanine eliminated its ability to activate MEK/ERK and NF-kappaB pathways, whereas a phosphomimetic mutation to aspartic acid could rescue the ability to activate MEK. Thr-290 was also required for robust autophosphorylation of Cot. Antibody generated to phospho-Thr-290-Cot recognized both wild-type and kinase-dead Cot, suggesting that phosphorylation of Thr-290 did not occur through autophosphorylation but via another kinase. We showed that Cot was constitutively phosphorylated at Thr-290 in transfected human embryonic kidney 293T cells as well as human monocytes as this residue was phosphorylated in unstimulated and lipopolysaccharide-stimulated cells to the same degree. Treatment with herbimycin A inhibited Cot activity in the MEK/ERK pathway but did not inhibit phosphorylation at Thr-290. Together these results showed that phosphorylation of Cot at Thr-290 is necessary but not sufficient for full kinase activity in the MEK/ERK pathway. PMID:15466476

  9. Suppressing epileptic activity in a neural mass model using a closed-loop proportional-integral controller

    NASA Astrophysics Data System (ADS)

    Wang, Junsong; Niebur, Ernst; Hu, Jinyu; Li, Xiaoli

    2016-06-01

    Closed-loop control is a promising deep brain stimulation (DBS) strategy that could be used to suppress high-amplitude epileptic activity. However, there are currently no analytical approaches to determine the stimulation parameters for effective and safe treatment protocols. Proportional-integral (PI) control is the most extensively used closed-loop control scheme in the field of control engineering because of its simple implementation and perfect performance. In this study, we took Jansen’s neural mass model (NMM) as a test bed to develop a PI-type closed-loop controller for suppressing epileptic activity. A graphical stability analysis method was employed to determine the stabilizing region of the PI controller in the control parameter space, which provided a theoretical guideline for the choice of the PI control parameters. Furthermore, we established the relationship between the parameters of the PI controller and the parameters of the NMM in the form of a stabilizing region, which provided insights into the mechanisms that may suppress epileptic activity in the NMM. The simulation results demonstrated the validity and effectiveness of the proposed closed-loop PI control scheme.

  10. Suppressing epileptic activity in a neural mass model using a closed-loop proportional-integral controller

    PubMed Central

    Wang, Junsong; Niebur, Ernst; Hu, Jinyu; Li, Xiaoli

    2016-01-01

    Closed-loop control is a promising deep brain stimulation (DBS) strategy that could be used to suppress high-amplitude epileptic activity. However, there are currently no analytical approaches to determine the stimulation parameters for effective and safe treatment protocols. Proportional-integral (PI) control is the most extensively used closed-loop control scheme in the field of control engineering because of its simple implementation and perfect performance. In this study, we took Jansen’s neural mass model (NMM) as a test bed to develop a PI-type closed-loop controller for suppressing epileptic activity. A graphical stability analysis method was employed to determine the stabilizing region of the PI controller in the control parameter space, which provided a theoretical guideline for the choice of the PI control parameters. Furthermore, we established the relationship between the parameters of the PI controller and the parameters of the NMM in the form of a stabilizing region, which provided insights into the mechanisms that may suppress epileptic activity in the NMM. The simulation results demonstrated the validity and effectiveness of the proposed closed-loop PI control scheme. PMID:27273563

  11. Tyrosine Kinase Inhibition: An Approach to Drug Development

    NASA Astrophysics Data System (ADS)

    Levitzki, Alexander; Gazit, Aviv

    1995-03-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.

  12. Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

    PubMed Central

    Sette, Claudio; Bevilacqua, Arturo; Geremia, Raffaele; Rossi, Pellegrino

    1998-01-01

    Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit– induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267–2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the γ1 isoform of PLC (PLCγ1) competitively inhibits tr-kit– induced egg activation. A GST fusion protein containing the SH3 domain of PLCγ1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit–induced egg activation, showing that the effect of the SH3 domain of PLCγ1 is specific. Tr-kit–induced egg activation is also suppressed by co-injection of antibodies raised against the PLCγ1 SH domains, but not against the PLCγ1 COOH-terminal region. In transfected COS cells, coexpression of PLCγ1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCγ1 with respect to cells expressing PLCγ1 alone. These data indicate that tr-kit activates PLCγ1, and that the SH3 domain of PLCγ1 is essential for tr-kit–induced egg activation. PMID:9722617

  13. Involvement of nitric oxide pathways in short term modulation of tyrosine hydroxylase activity by endothelins 1 and 3 in the rat anterior hypothalamus.

    PubMed

    Morgazo, Carolina; Perfume, Guadalupe; Legaz, Guillermina; di Nunzio, Andrea; Hope, Sandra I; Bianciotti, Liliana G; Vatta, Marcelo S

    2005-09-01

    The ability of endothelins 1 and 3 (ET-1 and ET-3) to reduce neuronal norepinephrine release through ETB receptor activation involving nitric oxide (NO) pathways in the rat anterior hypothalamus region (AHR) was previously reported. In the present work, we studied the effects of ET-1 and -3 on tyrosine hydroxylase (TH) activity and the possible involvement of NO pathways. Results showed that ET-1 and -3 (10 nM) diminished TH activity in AHR and this effect was blocked by a selective ETB receptor antagonist (100 nM BQ-788), but not by a ET(A) receptor antagonist (BQ-610). To confirm these results, 1 microM IRL-1620 (ET(B) agonist) reduced TH activity whereas 300 nM sarafotoxin S6b falled to modify it. N(omega)-Nitro-L-arginine methyl ester (10 microM), 7-nitroindazole (10 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-ona (10 microM), KT5823 (2 microM), inhibitors of nitric oxide synthase, neuronal nitric oxide synthase, NO-sensitive-guanylyl cyclase, and protein kinase G, respectively, did not modify the reduction of TH activity produced by ETs. In addition, both 100 microM sodium nitroprusside and 50 microM 8-bromoguanosine-3',5'-cyclic monophosphate (NO donor and guanosine-3',5'-cyclic monophosphate analog, respectively) diminished TH activity. Present results showed that ET-1 and ET-3 diminished TH activity through the activation of ET(B) receptors involving the NO/guanosine-3',5'-cyclic monophosphate/protein kinase G pathway. Taken jointly present and previous results it can be concluded that both ETs play an important role as modulators of norepinephrine neurotransmission in the rat AHR.

  14. Mitogen-activated protein kinase kinase 1 (MEK1) stabilizes MyoD through direct phosphorylation at tyrosine 156 during myogenic differentiation.

    PubMed

    Jo, Chulman; Cho, Sun-Jung; Jo, Sangmee Ahn

    2011-05-27

    Previously, we reported that mitogen-activated protein kinase kinase 1 (MEK1) activated in the mid-stage of skeletal muscle differentiation promotes myogenic differentiation. To elucidate the molecular mechanism, we investigated an activity of MEK1 for MyoD. Activated MEK1 associates with MyoD in the nucleus of differentiating myoblasts. In vitro kinase assay using active MEK1, a (32)P-labeled protein band corresponding to GST-MyoD was observed but not to mutant GST-MyoD-Y156F. Tyrosine phosphorylation of endogenous MyoD was detected with a specific anti-pMyoD-Y156 antibody; however, this response was blocked by PD184352, a MEK-specific inhibitor. These results indicate that activated MEK1 phosphorylates the MyoD-Y156 residue directly. Interestingly, the protein level of mutant MyoD-Y156F decreased compared with that of wild type but was recovered in the presence of lactacystin, a proteasome inhibitor. The protein level of MyoD-Y156E, which mimics phosphorylation at Tyr-156, was above that of wild type, indicating that the phosphorylation protects MyoD from the ubiquitin proteasome-mediated degradation. In addition, the low protein level of MyoD-Y156F was recovered over that of wild type by an additional mutation at Leu-164, a critical binding residue of MAFbx/AT-1, a Skp, Cullin, F-box (SCF) E3-ubiquitin ligase. The amount of MyoD co-precipitated with MAFbx/AT-1 also was reduced in the presence of active MEK1. Thus, these results suggested that the phosphorylation probably interrupts the binding of MAFbx/AT-1 to MyoD and thereby increases its stability. Collectively, our results suggest that MEK1 activated in differentiating myoblasts stimulates muscle differentiation by phosphorylating MyoD-Y156, which results in MyoD stabilization.

  15. Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

    SciTech Connect

    Kishore, R.; Raghothama, S.; Balaram, P.

    1988-04-05

    Two cyclic peptide disulfides have been synthesized as models for the 14-membered redox-active disulfide loop glutaredoxin. /sup 1/H NMR studies at 270 MHz in chloroform solutions establish a type I ..beta..-turn conformation for the Pro-X segment in both peptides, stabilized by a 4 ..-->.. 1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II ..beta..-turn structures with -Pro-Tyr(Phe)-as the corner resides. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S-n-sigma* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.

  16. Peptide Microarrays for Real-Time Kinetic Profiling of Tyrosine Phosphatase Activity of Recombinant Phosphatases and Phosphatases in Lysates of Cells or Tissue Samples.

    PubMed

    Hovestad-Bijl, Liesbeth; van Ameijde, Jeroen; Pijnenburg, Dirk; Hilhorst, Riet; Liskamp, Rob; Ruijtenbeek, Rob

    2016-01-01

    A high-throughput method for the determination of the kinetics of protein tyrosine phosphatase (PTP) activity in a microarray format is presented, allowing real-time monitoring of the dephosphorylation of a 3-nitro-phosphotyrosine residue. The 3-nitro-phosphotyrosine residue is incorporated in potential PTP substrates. The peptide substrates are immobilized onto a porous surface in discrete spots. After dephosphorylation by a PTP, a 3-nitrotyrosine residue is formed that can be detected by a specific, sequence-independent antibody. The rate of dephosphorylation can be measured simultaneously on 12 microarrays, each comprising three concentrations of 48 clinically relevant peptides, using 1.0-5.0 μg of protein from a cell or tissue lysate or 0.1-2.0 μg of purified phosphatase. The data obtained compare well with solution phase assays involving the corresponding unmodified phosphotyrosine substrates. This technology, characterized by high-throughput (12 assays in less than 2 h), multiplexing and low sample requirements, facilitates convenient and unbiased investigation of the enzymatic activity of the PTP enzyme family, for instance by profiling of PTP substrate specificities, evaluation of PTP inhibitors and pinpointing changes in PTP activity in biological samples related to diseases. PMID:27514800

  17. Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5

    PubMed Central

    Xu, Kai; Tzvetkova-Robev, Dorothea; Xu, Yan; Goldgur, Yehuda; Chan, Yee-Peng; Himanen, Juha P.; Nikolov, Dimitar B.

    2013-01-01

    Eph receptor tyrosine kinases and their ephrin ligands mediate cell signaling during normal and oncogenic development. Eph signaling is initiated in a multistep process leading to the assembly of higher-order Eph/ephrin clusters that set off bidirectional signaling in interacting cells. Eph and ephrins are divided in two subclasses based on their abilities to bind and activate each other and on sequence conservation. EphA4 is an exception to the general rule because it can be activated by both A- and B-class ephrin ligands. Here we present high-resolution structures of the complete EphA4 ectodomain and its complexes with ephrin-A5. The structures reveal how ligand binding promotes conformational changes in the EphA4 ligand-binding domain allowing the formation of signaling clusters at the sites of cell–cell contact. In addition, the structural data, combined with structure-based mutagenesis, reveal a previously undescribed receptor–receptor interaction between the EphA4 ligand-binding and membrane-proximal fibronectin domains, which is functionally important for efficient receptor activation. PMID:23959867

  18. Induction of a tumor-associated activating mutation in protein tyrosine phosphatase Ptpn11 (Shp2) enhances mitochondrial metabolism, leading to oxidative stress and senescence.

    PubMed

    Zheng, Hong; Li, Shanhu; Hsu, Peter; Qu, Cheng-Kui

    2013-09-01

    Activating mutations in Ptpn11 (Shp2), a protein tyrosine phosphatase involved in diverse cell signaling pathways, are associated with pediatric leukemias and solid tumors. However, the pathogenic effects of these mutations have not been fully characterized. Here, we report that induction of the Ptpn11(E76K/+) mutation, the most common and active Ptpn11 mutation found in leukemias and solid tumors, in primary mouse embryonic fibroblasts resulted in proliferative arrest and premature senescence. As a result, apoptosis was markedly increased. These cellular responses were accompanied and mediated by up-regulation of p53 and p21. Moreover, intracellular levels of reactive oxygen species (ROS), byproducts of mitochondrial oxidative phosphorylation, were elevated in Ptpn11(E76K/+) cells. Since Shp2 is also distributed to the mitochondria (in addition to the cytosol), the impact of the Ptpn11(E76K/+) mutation on mitochondrial function was analyzed. These analyses revealed that oxygen consumption of Ptpn11(E76K/+) cells and the respiratory function of Ptpn11(E76K/+) mitochondria were significantly increased. Furthermore, we found that phosphorylation of mitochondrial Stat3, one of the substrates of Shp2 phosphatase, was greatly decreased in the mutant cells with the activating mutation Ptpn11(E76K/+). This study provides novel insights into the initial effects of tumor-associated Ptpn11 mutations. PMID:23884424

  19. Signal transduction in macrophages by glycosylphosphatidylinositols of Plasmodium, Trypanosoma, and Leishmania: Activation of protein tyrosine kinases and protein kinase C by inositolglycan and diacylglycerol moieties

    PubMed Central

    Tachado, Souvenir D.; Gerold, Peter; Schwarz, Ralph; Novakovic, Suzanna; McConville, Malcolm; Schofield, Louis

    1997-01-01

    The perturbation of various glycosylphosphatidylinositol (GPI)-anchored surface proteins imparts profound regulatory signals to macrophages, lymphocytes and other cell types. The specific contribution of the GPI moieties to these events however is unclear. This study demonstrates that purified GPIs of Plasmodium falciparum, Trypanosoma brucei, and Leishmania mexicana origin are sufficient to initiate signal transduction when added alone to host cells as chemically defined agonists. GPIs (10 nM–1 μM) induce rapid activation of the protein tyrosine kinase (PTK) p59hck in macrophages. The minimal structural requirement for PTK activation is the evolutionarily conserved core glycan sequence Manα1-2Manα1-6Manα1-4GlcN1-6myo-inositol. GPI-associated diacylglycerols independently activate the calcium-independent ɛ isoform of protein kinase C. Both signals collaborate in regulating the downstream NF-κB/rel-dependent gene expression of interleukin 1α, tumor necrosis factor (TNF) α, and inducible NO synthase. The alkylacylglycerol-containing iM4 GIPL of L. mexicana, however, is unable to activate protein kinase C and inhibits TNF expression in response to other agonists, establishing signaling specificity among structurally distinct GPIs. GPI alone appears sufficient to mimic the activities of malaria parasite extracts in the signaling pathway leading to TNF expression. A mAb to GPI blocks TNF induction by parasite extracts indicating that GPI is a necessary agent in this response. As protozoal GPIs are closely related to their mammalian counterparts, the data indicate that GPIs do indeed constitute a novel outside-in signaling system, acting as both agonists and second messenger substrates, and imparting at least two separate signals through the structurally distinct glycan and fatty acid domains. These activities may underlie aspects of pathology and immune regulation in protozoal infections. PMID:9108098

  20. An Assessment of Magnetic Conditions for Strong Coronal Heating in Solar Active Regions by Comparing Observed Loops with Computed Potential Field Lines

    NASA Technical Reports Server (NTRS)

    Falconer, D. A.; Gary, G. A.; Moore, R. L.; Porter, J. G.

    1998-01-01

    We report further results on the magnetic origins of coronal heating found from combining coronal images with photospheric magnetograms. Here, for two complementary active regions, we compare the measured photospheric magnetic roots, extrapolated potential fields, and the distribution of bright coronal loops, to examine the global nonpotentiality of bright extended coronal loops and the three-dimensional structure of the magnetic field at their feet and to assess the role of these magnetic conditions in the strong coronal heating in these loops.

  1. A novel Bruton's tyrosine kinase inhibitor CC-292 in combination with the proteasome inhibitor carfilzomib impacts the bone microenvironment in a multiple myeloma model with resultant antimyeloma activity.

    PubMed

    Eda, H; Santo, L; Cirstea, D D; Yee, A J; Scullen, T A; Nemani, N; Mishima, Y; Waterman, P R; Arastu-Kapur, S; Evans, E; Singh, J; Kirk, C J; Westlin, W F; Raje, N S

    2014-09-01

    Bruton's tyrosine kinase (Btk) modulates B-cell development and activation and has an important role in antibody production. Interestingly, Btk may also affect human osteoclast (OC) function; however, the mechanism was unknown. Here we studied a potent and specific Btk inhibitor, CC-292, in multiple myeloma (MM). In this report, we demonstrate that, although CC-292 increased OC differentiation, it inhibited OC function via inhibition of c-Src, Pyk2 and cortactin, all involved in OC-sealing zone formation. As CC-292 did not show potent in vitro anti-MM activity, we next evaluated it in combination with the proteasome inhibitor, carfilzomib. We first studied the effect of carfilzomib on OC. Carfilzomib did not have an impact on OC-sealing zone formation but significantly inhibited OC differentiation. CC-292 combined with carfilzomib inhibited both sealing zone formation and OC differentiation, resulting in more profound inhibition of OC function than carfilzomib alone. Moreover, the combination treatment in an in vivo MM mouse model inhibited tumor burden compared with CC-292 alone; it also increased bone volume compared with carfilzomib alone. These results suggest that CC-292 combined with carfilzomib augments the inhibitory effects against OC within the bone microenvironment and has promising therapeutic potential for the treatment of MM and related bone disease.

  2. Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation--diversity, ductility, and destiny.

    PubMed

    Suda, Kenichi; Mizuuchi, Hiroshi; Maehara, Yoshihiko; Mitsudomi, Tetsuya

    2012-12-01

    Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder.

  3. Tyrosine hydroxylase is activated and phosphorylated at different sites in rat pheochromocytoma PC 12 cells treated with phorbol ester and forskolin

    SciTech Connect

    Tachikawa, E.; Tank, A.W.; Weiner, D.H.; Mosimann, W.F.; Yanagihara, N.; Weiner, N.

    1986-03-01

    The effects of phorbol ester (4..beta..-phorbol, 12..beta..-myristate, 13..cap alpha..-acetate; TPA), an activator of Ca/sup + +//phospholipid-dependent protein kinase (PK-C), and forskolin, which stimulates adenylate cyclase and cyclic AMP-dependent protein kinase (cAMP-PK), on the activation and phosphorylation of tyrosine hydroxylase (TH) in rat pheochromocytoma (PC 12) cells were examined. Incubation of the cells with TPA (0.01-1 ..mu..M) or forskolin (0.01-0.1 ..mu..M) produces increases in activation and phosphorylation of TH in a concentration-dependent manner. The stimulatory effects of TPA are dependent on extracellular Ca/sup + +/ and are inhibited by pretreatment of the cells with trifluoperazine (TFP). The effects of forskolin are independent of Ca/sup + +/ and are not inhibited by TFP. In cells treated with forskolin, the time course of the increase in cAMP correlates with the increases in TH activity and phosphorylation. cAMP levels do not increase in cells treated with TPA. There is an increase in the phosphorylation of only one tryptic phosphopeptide derived from TH in cells treated with either forskolin or TPA. The peptide phosphorylated in TPA-treated cells exhibits different elution characteristics on HPLC from that in forskolin-treated cells. The authors conclude that TH in PC 12 cells is phosphorylated on different sites by cAMP-PK and PK-C. Phosphorylation of either of these sites is associated with enzyme activation.

  4. Long-term modulation of tyrosine hydroxylase activity and expression by endothelin-1 and -3 in the rat anterior and posterior hypothalamus.

    PubMed

    Perfume, Guadalupe; Nabhen, Sabrina L; Barrera, Karla Riquelme; Otero, María G; Bianciotti, Liliana G; Vatta, Marcelo S

    2008-03-01

    Brain catecholamines are involved in the regulation of biological functions, including cardiovascular activity. The hypothalamus presents areas with high density of catecholaminergic neurons and the endothelin system. Two hypothalamic regions intimately related with the cardiovascular control are distinguished: the anterior (AHR) and posterior (PHR) hypothalamus, considered to be sympathoinhibitory and sympathoexcitatory regions, respectively. We previously reported that endothelins (ETs) are involved in the short-term tyrosine hydroxylase (TH) regulation in both the AHR and PHR. TH is crucial for catecholaminergic transmission and is tightly regulated by well-characterized mechanisms. In the present study, we sought to establish the effects and underlying mechanisms of ET-1 and ET-3 on TH long-term modulation. Results showed that in the AHR, ETs decreased TH activity through ET(B) receptor activation coupled to the nitric oxide, phosphoinositide, and CaMK-II pathways. They also reduced total TH level and TH phosphorylated forms (Ser 19 and 40). Conversely, in the PHR, ETs increased TH activity through a G protein-coupled receptor, likely an atypical ET receptor or the ET(C) receptor, which stimulated the phosphoinositide and adenylyl cyclase pathways, as well as CaMK-II. ETs also increased total TH level and the Ser 19, 31, and 40 phosphorylated sites of the enzyme. These findings support that ETs are involved in the long-term regulation of TH activity, leading to reduced sympathoinhibition in the AHR and increased sympathoexcitation in the PHR. Present and previous studies may partially explain the cardiovascular effects produced by ETs when applied to the brain.

  5. Intracerebroventricular administration of ouabain, a Na/K-ATPase inhibitor, activates tyrosine hydroxylase through extracellular signal-regulated kinase in rat striatum.

    PubMed

    Yu, Hyun Sook; Kim, Se Hyun; Park, Hong Geun; Kim, Yong Sik; Ahn, Yong Min

    2011-11-01

    Alteration in dopamine neurotransmission has been reported to be involved in the mania of bipolar disorder. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that is crucial for dopamine biosynthesis, and its activity is tightly regulated by phosphorylation at multiple N-terminal serine residues. Previously, we have reported that intracerebroventricular (ICV) injection of ouabain, a selective Na/K-ATPase inhibitor, induces hyperactivity in rats that mimics manic symptoms related to the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2), which plays crucial roles in the modulation of TH phosphorylation. In this study, we investigated the effects of ICV injection of ouabain on TH phosphorylation in rat striatum and the involvement of ERK1/2 in ouabain-induced TH activation. ICV ouabain induced an acute dose-dependent increase in locomotor activity and in TH phosphorylation in rat striatum. TH phosphorylation at Ser19 was significantly increased with 100, 500, and 1000μM ouabain, and phosphorylation at Ser31 and Ser40 was significantly increased with 500 and 1000μM. We also found that ICV pretreatment with U0126, a specific MEK1/2 inhibitor, attenuated the 1000μM ouabain-induced increase in TH phosphorylation at Ser19, Ser31, and Ser40, as well as the hyperactivity of rats. Moreover, the increased phosphorylation of TH (Ser19, Ser31, and Ser40) was maintained until 8h after single administration ouabain was accompanied by increased phosphorylation of ERK1/2 (Thr202/Tyr204) and p90RSK (Thr359/Ser363). These findings imply that TH activation of the ERK1/2 signal pathway could play an important role in ouabain-induced hyperactivity of rats, a mania model. PMID:21871514

  6. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

    PubMed

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

    2016-04-15

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment.

  7. Long-term modulation of tyrosine hydroxylase activity and expression by endothelin-1 and -3 in the rat anterior and posterior hypothalamus.

    PubMed

    Perfume, Guadalupe; Nabhen, Sabrina L; Barrera, Karla Riquelme; Otero, María G; Bianciotti, Liliana G; Vatta, Marcelo S

    2008-03-01

    Brain catecholamines are involved in the regulation of biological functions, including cardiovascular activity. The hypothalamus presents areas with high density of catecholaminergic neurons and the endothelin system. Two hypothalamic regions intimately related with the cardiovascular control are distinguished: the anterior (AHR) and posterior (PHR) hypothalamus, considered to be sympathoinhibitory and sympathoexcitatory regions, respectively. We previously reported that endothelins (ETs) are involved in the short-term tyrosine hydroxylase (TH) regulation in both the AHR and PHR. TH is crucial for catecholaminergic transmission and is tightly regulated by well-characterized mechanisms. In the present study, we sought to establish the effects and underlying mechanisms of ET-1 and ET-3 on TH long-term modulation. Results showed that in the AHR, ETs decreased TH activity through ET(B) receptor activation coupled to the nitric oxide, phosphoinositide, and CaMK-II pathways. They also reduced total TH level and TH phosphorylated forms (Ser 19 and 40). Conversely, in the PHR, ETs increased TH activity through a G protein-coupled receptor, likely an atypical ET receptor or the ET(C) receptor, which stimulated the phosphoinositide and adenylyl cyclase pathways, as well as CaMK-II. ETs also increased total TH level and the Ser 19, 31, and 40 phosphorylated sites of the enzyme. These findings support that ETs are involved in the long-term regulation of TH activity, leading to reduced sympathoinhibition in the AHR and increased sympathoexcitation in the PHR. Present and previous studies may partially explain the cardiovascular effects produced by ETs when applied to the brain. PMID:18094067

  8. Cell signaling by receptor-tyrosine kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph

    2010-01-01

    Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses. PMID:20602996

  9. Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site.

    PubMed

    Truongvan, Ngoc; Chung, Hye-Shin; Jang, Sei-Heon; Lee, ChangWoo

    2016-03-01

    An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures. PMID:26838013

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

    PubMed

    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

  11. SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

    SciTech Connect

    Buchanan, Sean G.; Hendle, Jorg; Lee, Patrick S.; Smith, Christopher R.; Bounaud, Pierre-Yves; Jessen, Katti A.; Tang, Crystal M.; Huser, Nanni H.; Felce, Jeremy D.; Froning, Karen J.; Peterman, Marshall C.; Aubol, Brandon E.; Gessert, Steve F.; Sauder, J. Michael; Schwinn, Kenneth D.; Russell, Marijane; Rooney, Isabelle A.; Adams, Jason; Leon, Barbara C.; Do, Tuan H.; Blaney, Jeff M.; Sprengeler, Paul A.; Thompson, Devon A.; Smyth, Lydia; Pelletier, Laura A.; Atwell, Shane; Holme, Kevin; Wasserman, Stephen R.; Emtage, Spencer; Burley, Stephen K.; Reich, Siegfried H.

    2010-01-12

    The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC{sub 50} of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.

  12. Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes: The role of oxidative stress and protein tyrosine kinase activation

    SciTech Connect

    Wan Rong; Mo Yiqun; Zhang Xing; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2008-12-01

    Recently, many studies have shown that nanoparticles can translocate from the lungs to the circulatory system. As a particulate foreign body, nanoparticles could induce host responses such as reactive oxygen species (ROS) generation, inflammatory cytokine and matrix metalloproteinase (MMP) release which play a major role in tissue destruction and remodeling. However, the direct effects of nanoparticles on leukocytes, especially monocytes, are still unclear. The objective of the present study was to compare the ability of Nano-Co and Nano-TiO{sub 2} to cause alteration of transcription and activity of MMPs and to explore possible mechanisms. We hypothesized that non-toxic doses of some transition metal nanoparticles stimulate an imbalance of MMP/TIMP that cause MMP production that may contribute to their health effects. To test this hypothesis, U937 cells were treated with Nano-Co and Nano-TiO{sub 2} and cytotoxic effects and ROS generation were measured. The alteration of MMP-2 and MMP-9 expression and activity of MMP-2 and MMP-9 after exposure to these metal nanoparticles were subsequently determined. To investigate the potential signaling pathways involved in the Nano-Co-induced MMP activation, the ROS scavengers or inhibitors, AP-1 inhibitor, and protein tyrosine kinase (PTK) inhibitors were also used to pre-treat U937 cells. Our results demonstrated that exposure of U937 cells to Nano-Co, but not to Nano-TiO{sub 2}, at a dose that does not cause cytotoxicity, resulted in ROS generation and up-regulation of MMP-2 and MMP-9 mRNA expression{sub ..} Our results also showed dose- and time-related increases in pro-MMP-2 and pro-MMP-9 gelatinolytic activities in conditioned media after exposure of U937 cells to Nano-Co, but not to Nano-TiO{sub 2}. Nano-Co-induced pro-MMP-2 and pro-MMP-9 activity increases were inhibited by pre-treatment with ROS scavengers or inhibitors. We also demonstrated dose- and time-related decreases in tissue inhibitors of metalloproteinases 2

  13. 24-Week Exposure to Oxidized Tyrosine Induces Hepatic Fibrosis Involving Activation of the MAPK/TGF-β1 Signaling Pathway in Sprague-Dawley Rats Model

    PubMed Central

    Li, Zhuqing Leslie; Shi, Yonghui; Le, Guowei; Ding, Yinyi; Zhao, Qi

    2016-01-01

    Scope. Oxidized tyrosine (O-Tyr) has been widely detected in many consumer protein products. O-Tyr products such as dityrosine (Dityr) and 3-nitrotyrosine (3-NT) are universal biomarkers of protein oxidation and have been demonstrated to be associated with metabolic disorders in biological system. Evaluation of potential intracorporal effects of dietary O-Tyr is important since the mechanism of biological impacts induced by oral oxidized protein products (OPPs) is still limited although we have proved that some dietary OPPs would induce oxidative injury to liver and kidney. Methods and Results. The present study aimed to investigate the dose-dependent hepatic injury caused by oral O-Tyr in rats. 24-week feeding of O-Tyr enhanced aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, increased total bilirubin (TBiL) content, and led to oxidative damage in rats liver. Besides, O-Tyr distinctly increased the phosphorylation of p38 and ERK2 MAPKs and enhanced fibrosis-related TGF-β1 and Smad2/3 levels. Higher extracellular matrix (ECM) indexes (ICTP, PIIINP) and histological examination (HE and Masson staining) also supported dose-dependent hepatic fibrosis caused by O-Tyr. Conclusion. These findings reveal that O-Tyr may induce oxidative damage and hepatic fibrosis via MAPK/TGF-β1 signaling pathway, in which ROS together with malondialdehyde (MDA) and OPPs act as the pivotal mediators. PMID:26788244

  14. Synthesis and characterization of new optically active poly(amide-imide)s derived from N,N'-(pyromellitoyl) bis-L-tyrosine and various diamines

    NASA Astrophysics Data System (ADS)

    Khalaf, H. I.; Wady, A. N.; Daham, H. K.

    2013-01-01

    Five new optically active poly(amide-imide)s(PAIs) 5a-e were prepared by direct polycondensation reaction of N,N'- (pyromellitoyl) bis-L-tyrosine 3 as chiral dicarboxylic acid with various aromatic diamines 4a-e. Triphenylphosphite(TPP)/pyridine(py) in the presence of calcium chloride (CaCl2) and N-methyl-2-pyrrolidone (NMP) were successfully applied to direct polycondensation reaction. The resulting new polymers were obtained in good yields with inherent viscosities ranging between 0.48 dL/g and 0.6 dL/g. They were analyzed with a C.H.N. elemental analyzer, FTIR, 1H-NMR, UV-VIS spectroscopy and polarimeter (specific rotation measurement, [α]{D/25}). Thermogravimetric analysis (TGA) indicated that the residual weight percentage of polymers at 600 °C were between 48.66 % and 64.21 %, which showed their thermal stability. These polymers are attractive to be used as packing materials in chromatography columns for separation of enantiomers.

  15. AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in Ube1l Deficient Cells.

    PubMed

    Now, Hesung; Yoo, Joo-Yeon

    2016-01-01

    A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l-/- MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity. PMID:27427993

  16. Geranylated 2-arylbenzofurans from Morus alba var. tatarica and their α-glucosidase and protein tyrosine phosphatase 1B inhibitory activities.

    PubMed

    Zhang, Ya-Long; Luo, Jian-Guang; Wan, Chuan-Xing; Zhou, Zhong-Bo; Kong, Ling-Yi

    2014-01-01

    Ten new geranylated 2-arylbenzofuran derivatives, including two monoterpenoid 2-arylbenzofurans (1 and 2), two geranylated 2-arylbenzofuran enantiomers (3a and 3b), and six geranylated 2-arylbenzofurans (4-9), along with four known 2-arylbenzofurans (10-13) were isolated from the root bark of Morus alba var. tatarica. Their structures and relative configurations were established on the basis of spectroscopic data analysis. Compounds 3-7 with one asymmetric carbon at C-7″ were supposed to be enantiomeric mixtures confirmed by chiral HPLC analysis, and the absolute configurations of each enantiomer in 3-7 were determined by Rh2(OCOCF3)4-induced CD and Snatzke's method. The enantiomers with the substituting group at C-2' exhibited better resolutions on a Chiralpak AD-H column than those with the substituting group at C-4'. Compounds 1-7, 10, 11 and 13, showed α-glucosidase inhibitory activities with IC50 values of 11.9-131.9 μM, and compounds 1 and 9-13 inhibited protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 7.9-38.1 μM. PMID:24216050

  17. Phosphorylation-Dependent Interaction of Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein (YWHA) with PADI6 Following Oocyte Maturation in Mice1

    PubMed Central

    Snow, Alan J.; Puri, Pawan; Acker-Palmer, Amparo; Bouwmeester, Tewis; Vijayaraghavan, Srinivasan; Kline, Douglas

    2008-01-01

    Proteins in the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein family (YWHA; also known as 14-3-3) are involved in the regulation of many intracellular processes. We have examined the interaction of YWHA with peptidylarginine deiminase type VI (PADI6), an abundant protein in mammalian oocytes, eggs, and early embryos. Peptidylarginine deiminases catalyze the posttranslational modification of peptidylarginine to citrulline. PADI6 is associated with oocyte cytoplasmic sheets, and PADI6-deficient mice are infertile because of disruption of development beyond the two-cell stage. We found that PADI6 undergoes a dramatic developmental change in phosphorylation during oocyte maturation. This change in phosphorylation is linked to an interaction of PADI6 with YWHA in the mature egg. Recombinant glutathione S-transferase YWHA pull-down experiments and transgenic tandem affinity purification with liquid chromatography-mass spectrometry demonstrate a binding interaction between YWHA and PADI6 in mature eggs. YWHA proteins modulate or complement intracellular events involving phosphorylation-dependent switching or protein modification. These results indicate that phosphorylation and/or YWHA binding may serve as a means of intracellular PADI6 regulation. PMID:18463355

  18. The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants☆

    PubMed Central

    Tsoumpra, Maria K.; Muniz, Joao R.; Barnett, Bobby L.; Kwaasi, Aaron A.; Pilka, Ewa S.; Kavanagh, Kathryn L.; Evdokimov, Artem; Walter, Richard L.; Von Delft, Frank; Ebetino, Frank H.; Oppermann, Udo; Russell, R. Graham G.; Dunford, James E.

    2015-01-01

    Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding. PMID:26318908

  19. The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants.

    PubMed

    Tsoumpra, Maria K; Muniz, Joao R; Barnett, Bobby L; Kwaasi, Aaron A; Pilka, Ewa S; Kavanagh, Kathryn L; Evdokimov, Artem; Walter, Richard L; Von Delft, Frank; Ebetino, Frank H; Oppermann, Udo; Russell, R Graham G; Dunford, James E

    2015-12-01

    Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding. PMID:26318908

  20. Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes.

    PubMed Central

    Kawamura, M; McVicar, D W; Johnston, J A; Blake, T B; Chen, Y Q; Lal, B K; Lloyd, A R; Kelvin, D J; Staples, J E; Ortaldo, J R

    1994-01-01

    Protein-tyrosine kinases (PTKs) are critical enzymes for receptor-mediated signaling in lymphocytes. Because natural killer (NK) cells are large granular lymphocytes with specialized effector function, we set out to identify PTKs preferentially expressed in these cells. One such PTK was identified and molecularly cloned. The predicted amino acid sequence shows that this kinase lacks SH2 or SH3 domains typical of src family kinases but has tandem nonidentical catalytic domains, indicating that it is a member of the Janus family of PTKs. Immunoprecipitation using antiserum generated against a peptide corresponding to the deduced amino acid sequence of this gene revealed a kinase with a molecular weight of approximately 125,000. The pattern of expression of this kinase contrasted sharply with that of other Janus kinases, which are ubiquitously expressed. The kinase described in the present study was found to be more limited in its expression; expression was found in NK cells and an NK-like cell line but not in resting T cells or in other tissues. In contrast, stimulated and transformed T cells expressed the gene, suggesting a role in lymphoid activation. Because of its homology and tissue expression, we have tentatively termed this PTK gene L-JAK for leukocyte Janus kinase. Images PMID:8022790

  1. AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in Ube1l Deficient Cells

    PubMed Central

    Now, Hesung; Yoo, Joo-Yeon

    2016-01-01

    A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l−/− MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity. PMID:27427993

  2. Closed-loop control of epileptiform activities in a neural population model using a proportional-derivative controller

    NASA Astrophysics Data System (ADS)

    Wang, Jun-Song; Wang, Mei-Li; Li, Xiao-Li; Ernst, Niebur

    2015-03-01

    Epilepsy is believed to be caused by a lack of balance between excitation and inhibitation in the brain. A promising strategy for the control of the disease is closed-loop brain stimulation. How to determine the stimulation control parameters for effective and safe treatment protocols remains, however, an unsolved question. To constrain the complex dynamics of the biological brain, we use a neural population model (NPM). We propose that a proportional-derivative (PD) type closed-loop control can successfully suppress epileptiform activities. First, we determine the stability of root loci, which reveals that the dynamical mechanism underlying epilepsy in the NPM is the loss of homeostatic control caused by the lack of balance between excitation and inhibition. Then, we design a PD type closed-loop controller to stabilize the unstable NPM such that the homeostatic equilibriums are maintained; we show that epileptiform activities are successfully suppressed. A graphical approach is employed to determine the stabilizing region of the PD controller in the parameter space, providing a theoretical guideline for the selection of the PD control parameters. Furthermore, we establish the relationship between the control parameters and the model parameters in the form of stabilizing regions to help understand the mechanism of suppressing epileptiform activities in the NPM. Simulations show that the PD-type closed-loop control strategy can effectively suppress epileptiform activities in the NPM. Project supported by the National Natural Science Foundation of China (Grant Nos. 61473208, 61025019, and 91132722), ONR MURI N000141010278, and NIH grant R01EY016281.

  3. Activation of the lutropin/choriogonadotropin receptor (LHR) in MA-10 cells leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) by a pathway that involves Src family kinases*

    PubMed Central

    Mizutani, Tetsuya; Shiraishi, Koji; Welsh, Toni; Ascoli, Mario

    2006-01-01

    We show that activation of the endogenous or recombinant LHR in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577 but it does not affect the phosphorylation of Tyr397, Tyr861 or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes and overexpression of either of these two tyrosine kinases enhances the LHR-mediate phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Gα subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways. PMID:16293639

  4. Ginkgetin Blocks Constitutive STAT3 Activation and Induces Apoptosis through Induction of SHP-1 and PTEN Tyrosine Phosphatases.

    PubMed

    Baek, Seung Ho; Lee, Jae Hwi; Ko, Jeong-Hyeon; Lee, Hanwool; Nam, Dongwoo; Lee, Seok Geun; Yang, Woong Mo; Um, Jae-Young; Lee, Junhee; Kim, Sung-Hoon; Shim, Bum Sang; Ahn, Kwang Seok

    2016-04-01

    Ginkgetin, a biflavone from Ginkgo biloba leaves, is known to exhibit antiinflammatory, antifungal, neuroprotective, and antitumor activities, but its precise mechanism of action has not been fully elucidated. Because the aberrant activation of STAT3 has been linked with regulation of inflammation, proliferation, invasion, and metastasis of tumors, we hypothesized that ginkgetin modulates the activation of STAT3 in tumor cells. We found that ginkgetin clearly suppressed constitutive phosphorylation of STAT3 through inhibition of the activation of upstream JAK1 and c-Src kinases and nuclear translocation of STAT3 on both A549 and FaDu cells. Treatment with sodium pervanadate reversed the ginkgetin-induced down-modulation of STAT3, thereby indicating a critical role for a PTP. We also found that ginkgetin strongly induced the expression of the SHP-1 and PTEN proteins and its mRNAs. Further, deletion of SHP-1 and PTEN genes by siRNA suppressed the induction of SHP-1 and PTEN, and reversed the inhibition of STAT3 activation. Ginkgetin induced apoptosis as characterized by an increased accumulation of cells in subG1 phase, positive Annexin V binding, loss of mitochondrial membrane potential, down-regulation of STAT3-regulated gene products, and cleavage of PARP. Overall, ginkgetin abrogates STAT3 signaling pathway through induction of SHP-1 and PTEN proteins, thus attenuating STAT3 phosphorylation and tumorigenesis. PMID:27059688

  5. An investigation of coronal active region loop structures using AS&E rocket X-ray images

    NASA Technical Reports Server (NTRS)

    Webb, D. F.

    1983-01-01

    Simultaneous high spatial resolution observations at 6 cm in soft X-rays, in photospheric magnetograms, and in optical filtergrams were used to compare the most intense sources of centimetric emission in two active regions to coronal loops, sunspots, chromospheric structures, and photospheric magnetic fields. Results show that the majority of the bright microwave components are not associated with sunspots or X-ray emission. A nonthermal mechanism appears necessary to explain the brightest microwave components, discrete regions of continuous particle acceleration may be common in active regions. Studies of the plasma parameters of selected loops imply that the radio emission is consistent with gyro-resonance absorption at the third and fourth harmonic, at least from part of each loop. Results are presented for: (1) X-ray and microwave observations of active regions; (2) comparison of coronal holes observed in soft X-rays and Hel 10830 A spectrosheliograms; and (3) the reappearance of polar coronal holes and the evolution of the solar magnetic field.

  6. An Assessment of Magnetic Conditions for Strong Coronal Heating in Solar Active Regions by Comparing Observed Loops with Computed Potential Field Lines

    NASA Technical Reports Server (NTRS)

    Gary, G. A.; Moore, R. L.; Porter, J. G.; Falconer, D. A.

    1999-01-01

    We report further results on the magnetic origins of coronal heating found from registering coronal images with photospheric vector magnetograms. For two complementary active regions, we use computed potential field lines to examine the global non-potentiality of bright extended coronal loops and the three-dimensional structure of the magnetic field at their feet, and assess the role of these magnetic conditions in the strong coronal heating in these loops. The two active regions are complementary, in that one is globally potential and the other is globally nonpotential, while each is predominantly bipolar, and each has an island of included polarity in its trailing polarity domain. We find the following: (1) The brightest main-arch loops of the globally potential active region are brighter than the brightest main- arch loops of the globally strongly nonpotential active region. (2) In each active region, only a few of the mainarch magnetic loops are strongly heated, and these are all rooted near the island. (3) The end of each main-arch bright loop apparently bifurcates above the island, so that it embraces the island and the magnetic null above the island. (4) At any one time, there are other main-arch magnetic loops that embrace the island in the same manner as do the bright loops but that are not selected for strong coronal heating. (5) There is continual microflaring in sheared core fields around the island, but the main-arch bright loops show little response to these microflares. From these observational and modeling results we draw the following conclusions: (1) The heating of the main-arch bright loops arises mainly from conditions at the island end of these loops and not from their global non-potentiality. (2) There is, at most, only a loose coupling between the coronal heating in the bright loops of the main arch and the coronal heating in the sheared core fields at their feet, although in both the heating is driven by conditions/events in and around the

  7. Proteinase-activated receptors 1 and 2 and the regulation of porcine coronary artery contractility: a role for distinct tyrosine kinase pathways

    PubMed Central

    El-Daly, Mahmoud; Saifeddine, Mahmoud; Mihara, Koichiro; Ramachandran, Rithwik; Triggle, Christopher R; Hollenberg, Morley D

    2014-01-01

    Background and Purpose Because angiotensin-II-mediated porcine coronary artery (PCA) vasoconstriction is blocked by protein tyrosine kinase (PYK) inhibitors, we hypothesized that proteinase-activated receptors (PARs), known to regulate vascular tension, like angiotensin-II, would also cause PCA contractions via PYK-dependent signalling pathways. Experimental Approach Contractions of intact and endothelium-free isolated PCA rings, stimulated by PAR1/PAR2-activating peptides, angiotensin-II, PGF2α, EGF, PDGF and KCl, were monitored with/without multiple signalling pathway inhibitors, including AG-tyrphostins AG18 (non-specific PYKs), AG1478 (EGF-receptor kinase), AG1296 (PDGF receptor kinase), PP1 (Src kinase), U0126 and PD98059 (MEK/MAPKinase kinase), indomethacin/SC-560/NS-398 (COX-1/2) and L-NAME (NOS). Key Results AG18 inhibited the contractions induced by all the agonists except KCl, whereas U0126 attenuated contractions induced by PAR1/PAR2 agonists, EGF and angiotensin-II, but not by PGF2α, the COX-produced metabolites of arachidonate and KCl. PP1 only affected the responses to PAR1/PAR2-activating peptides and angiotensin-II. The EGF-kinase inhibitor, AG1478, attenuated contractions initiated by the PARs (PAR2 >> PAR1) and EGF itself, but not by angiotensin-II, PGF2α or KCl. COX-1/2 inhibitors blocked the contractions induced by all the agonists, except KCl and PGF2α. Conclusion and Implications PAR1/2-mediated contractions of the PCA are dependent on Src and MAPKinase and, in part, involve EGF-receptor-kinase transactivation and the generation of a COX-derived contractile agonist. However, the PYK signalling pathways used by PARs are distinct from each other and from those triggered by angiotensin-II and EGF. These signalling pathways may be therapeutic targets for managing coagulation-proteinase-induced coronary vasospasm. PMID:24506284

  8. Toxicological disruption of signaling homeostasis: Tyrosine phosphatses as targets

    EPA Science Inventory

    The protein tyrosine phosphatases (PTP) comprised a diverse group of enzymes whose activity opposes that of the tyrosine kinases. As such, the PTP have critical roles in maintaining signaling quiescence in resting cells and in restoring homeostasis by effecting signal termination...

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

    SciTech Connect

    Pegan, Scott D.; Rukseree, Kamolchanok; Capodagli, Glenn C.; Baker, Erica A.; Krasnykh, Olga; Franzblau, Scott G.; Mesecar, Andrew D.

    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.

  10. Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis.

    PubMed

    Pegan, Scott D; Rukseree, Kamolchanok; Capodagli, Glenn C; Baker, Erica A; Krasnykh, Olga; Franzblau, Scott G; Mesecar, Andrew D

    2013-02-01

    Class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprise 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. Also, 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.

  11. Protein-tyrosine phosphatase activity in human adipocytes is strongly correlated with insulin-stimulated glucose uptake and is a target of insulin-induced oxidative inhibition.

    PubMed

    Wu, Xiangdong; Hardy, V Elise; Joseph, Jeffrey I; Jabbour, Serge; Mahadev, Kalyankar; Zhu, Li; Goldstein, Barry J

    2003-06-01

    Protein-tyrosine phosphatases (PTPases), in particular PTP1B, have been shown to modulate insulin signal transduction in liver and skeletal muscle in animal models; however, their role in human adipose tissue remains unclear. The uptake of (14)C-D-glucose in response to 10 or 100 nmol/L insulin was measured in isolated subcutaneous adipocytes from subjects with a mean age of 44 years (range, 26 to 58) and mean body mass index (BMI) of 35.6 (range, 29.7 to 45.5). The endogenous activity of total PTPases and specifically of PTP1B in immunoprecipitates was measured in cell lysates under an inert atmosphere with and without added reducing agents. Using nonlinear regression analysis, higher BMI was significantly correlated with lower adipocyte glucose uptake (r = 0.73, P =.01) and with increased endogenous total PTPase activity (r = 0.64, P =.04). Correlation with waist circumference gave similar results. The endogenous total PTPase activity also strongly correlated with insulin-stimulated glucose uptake (R =.89, P <.0001); however, the activity of PTP1B was unrelated to the level of glucose uptake. Consistent with the insulin-stimulated oxidative inhibition of thiol-dependent PTPases reported for 3T3-L1 adipocytes and hepatoma cells, treatment of human adipocytes with 100 nmol/L insulin for 5 minutes lowered endogenous PTPase activity to 37% of control (P <.001), which was increased 25% by subsequent treatment with dithiothreitol in vitro. Cellular treatment with diphenyleneiodonium (DPI), an NADPH oxidase inhibitor that blocks the cellular generation of H(2)O(2) and reduces the insulin-induced reduction of cellular PTPase activity, also diminished insulin-stimulated glucose uptake by 82% (P =.001). These data suggest that total cellular PTPase activity, but not the activity of PTP1B, is higher in more obese subjects and is negatively associated with insulin-stimulated glucose transport. The insulin-stimulated oxidative inhibition of PTPases may also have an important

  12. The Tyrosine Kinome Dictates Breast Cancer Heterogeneity and Therapeutic Responsiveness.

    PubMed

    Ha, Jacqueline R; Siegel, Peter M; Ursini-Siegel, Josie

    2016-09-01

    Phospho-tyrosine signaling networks control numerous biological processes including cellular differentiation, cell growth and survival, motility, and invasion. Aberrant regulation of the tyrosine kinome is a hallmark of malignancy and influences all stages of breast cancer progression, from initiation to the development of metastatic disease. The success of specific tyrosine kinase inhibitors strongly validates the clinical relevance of tyrosine phosphorylation networks in breast cancer pathology. However, a significant degree of redundancy exists within the tyrosine kinome. Numerous receptor and cytoplasmic tyrosine kinases converge on a core set of signaling regulators, including adaptor proteins and tyrosine phosphatases, to amplify pro-tumorigenic signal transduction pathways. Mutational activation, amplification, or overexpression of one or more components of the tyrosine kinome represents key contributing events responsible for the tumor heterogeneity that is observed in breast cancers. It is this molecular heterogeneity that has become the most significant barrier to durable clinical responses due to the development of therapeutic resistance. This review focuses on recent literature that supports a prominent role for specific components of the tyrosine kinome in the emergence of unique breast cancer subtypes and in shaping breast cancer plasticity, sensitivity to targeted therapies, and the eventual emergence of acquired resistance. J. Cell. Biochem. 117: 1971-1990, 2016. © 2016 Wiley Periodicals, Inc.

  13. The Tyrosine Kinome Dictates Breast Cancer Heterogeneity and Therapeutic Responsiveness.

    PubMed

    Ha, Jacqueline R; Siegel, Peter M; Ursini-Siegel, Josie

    2016-09-01

    Phospho-tyrosine signaling networks control numerous biological processes including cellular differentiation, cell growth and survival, motility, and invasion. Aberrant regulation of the tyrosine kinome is a hallmark of malignancy and influences all stages of breast cancer progression, from initiation to the development of metastatic disease. The success of specific tyrosine kinase inhibitors strongly validates the clinical relevance of