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Sample records for protein tyrosine nitration

  1. Protein tyrosine nitration in the cell cycle

    SciTech Connect

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-09-23

    Highlights: {yields} Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. {yields} Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. {yields} Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

  2. Proteomic Approaches to Analyze Protein Tyrosine Nitration

    PubMed Central

    Feeney, Maria B.

    2013-01-01

    Abstract Significance: The conversion of protein-bound Tyr residues to 3-nitrotyrosine (3NY) can occur during nitrative stress and has been correlated to aging and many disease states. Proteomic analysis of this post-translational modification, using mass spectrometry-based techniques, is crucial for understanding its potential role in pathological and physiological processes. Recent Advances: To overcome some of the disadvantages inherent to well-established nitroproteomic methods using anti-3NY antibodies and gel-based separations, methods involving multidimensional chromatography, precursor ion scanning, and/or chemical derivatization have emerged for both identification and quantitation of protein nitration sites. A few of these methods have successfully detected endogenous 3NY modifications from biological samples. Critical Issues: While model systems often show promising results, identification of endogenous 3NY modifications remains largely elusive. The frequently low abundance of nitrated proteins in vivo, even under inflammatory conditions, is especially challenging, and sample loss due to derivatization and cleaning may become significant. Future Directions: Continued efforts to avoid interference from non-nitrated peptides without sacrificing recovery of nitrated peptides are needed. Quantitative methods are emerging and are crucial for identifying endogenous modifications that may have significant biological impacts. Antioxid. Redox Signal. 19, 1247–1256. PMID:23157221

  3. Enrichment and detection of tyrosine-nitrated proteins.

    PubMed

    Dekker, Frank; Abello, Nicolas; Wisastra, Rosalina; Bischoff, Rainer

    2012-08-01

    Nitrotyrosine is a post-translationally modified amino acid with distinctly different properties than tyrosine or any other of the genetically encoded amino acids. Detecting proteins containing nitrotyrosine is the first step towards a better understanding of the role of nitrotyrosine in health and disease. Moreover, quantifying the extent of nitrotyrosine and determining its location in a protein forms the basis for a better understanding of the effect of tyrosine nitration on biological function. Described in this unit is a method to detect tyrosine-nitrated proteins in tissue sections and on western blots after creating a fluorescent complex between aminotyrosine, salicylaldehyde, and Al(3+). In addition, an approach is detailed for labeling aminotyrosine with biotin to enrich peptides from complex samples. Both methods require reduction of nitrotyrosine to aminotyrosine, which can be achieved with sodium dithionite or hemin plus dithiothreitol. PMID:22851496

  4. The nature of heme/iron-induced protein tyrosine nitration

    PubMed Central

    Bian, Ka; Gao, Zhonghong; Weisbrodt, Norman; Murad, Ferid

    2003-01-01

    Recently, substantial evidence has emerged that revealed a very close association between the formation of nitrotyrosine and the presence of activated granulocytes containing peroxidases, such as myeloperoxidase. Peroxidases share heme-containing homology and can use H2O2 to oxidize substrates. Heme is a complex of iron with protoporphyrin IX, and the iron-containing structure of heme has been shown to be an oxidant in several model systems where the prooxidant effects of free iron, heme, and hemoproteins may be attributed to the formation of hypervalent states of the heme iron. In the current study, we have tested the hypothesis that free heme and iron play a crucial role in NO2-Tyr formation. The data from our study indicate that: (i) heme/iron catalyzes nitration of tyrosine residues by using hydrogen peroxide and nitrite, a reaction that revealed the mechanism underlying the protein nitration by peroxidase, H2O2, and NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document}; (ii) H2O2 plays a key role in the protein oxidation that forms the basis for the protein nitration, whereas nitrite is an essential element that facilitates nitration by the heme(Fe), H2O2, and the NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document} system; (iii) the formation of a Fe(IV) hypervalent compound may be essential for heme(Fe)-catalyzed nitration, whereas O\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage

  5. Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences.

    PubMed

    Takakusa, Hideo; Mohar, Isaac; Kavanagh, Terrance J; Kelly, Edward J; Kaspera, Rüdiger; Nelson, Sidney D

    2012-03-30

    Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an α-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-l-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondrial dysfunction. PMID:22402285

  6. Modulation of protein tyrosine nitration and inflammatory mediators by isoprenylhydroquinone glucoside.

    PubMed

    Olmos, Ana; Giner, Rosa-María; Recio, María-Carmen; Ríos, José-Luis; Máñez, Salvador

    2007-03-01

    The nitration of tyrosine caused by peroxynitrite and other reactive nitrogen species is clearly detrimental for some physiological processes; however, its signalling role is still open to controversy. Among the natural phenolics known for their ability to oppose free tyrosine nitration, isoprenylhydroquinone glucoside is investigated due to its unusual structure, which contains a simple hydroxybenzene alkylated by a hemiterpenoid moiety. This hydroquinone was shown to be an effective inhibitor of peroxynitrite-induced protein tyrosine nitration in 3T3 fibroblasts. When tested on bovine seroalbumin nitration, however, the potency was reduced by half and the effect was almost abolished in the presence of bicarbonate. In contrast, addition of this anion had no effect on the nitrite/hydrogen peroxide/hemin system. Isoprenylhydroquinone glucoside was also active in the microM range on intra- and extracellular protein-bound tyrosine nitration by phorbol 12-myristate 13-acetate-stimulated neutrophils. The effects on nitric oxide synthase expression, interleukin-1beta and tumor necrosis factor-alpha production by lipopolysaccharide-stimulated macrophages were quite moderate. Thus, isoprenylhydroquinone glucoside is an inhibitor of protein nitration in situ, but lacks effect on the generation of either nitric oxide or inflammatory cytokines. PMID:17161592

  7. Atherosclerosis: A Link Between Lipid Intake and Protein Tyrosine Nitration

    PubMed Central

    Upmacis, Rita K.

    2009-01-01

    Atherosclerosis, a disease characterized by plaque formation in the arterial wall that can lead to heart attack and stroke, is a principal cause of death in the world. Since the 1990’s, protein nitrotyrosine formation has been known to occur in the atherosclerotic plaque. This potentially damaging reaction occurs as a result of tyrosine modification by reactive nitrogen species, such as nitrogen dioxide radical, which forms upon peroxynitrite decomposition or nitrite oxidation by hydrogen peroxide-activated peroxidase enzymes. The presence of protein-bound nitrotyrosine can be considered an indicator of a loss in the natural balance of oxidants and antioxidants, and as such, there is an emerging view that protein-bound nitrotyrosine may be a risk factor for cardiovascular disease. This review brings together evidence that the accumulation of protein nitrotyrosine during atherogenesis is more widespread than initially thought (as its presence can be detected not only in the lesion but also in the blood stream and other organs) and is closely linked to lipid intake. PMID:20157638

  8. Protein Tyrosine Nitration and Thiol Oxidation by Peroxynitrite—Strategies to Prevent These Oxidative Modifications

    PubMed Central

    Daiber, Andreas; Daub, Steffen; Bachschmid, Markus; Schildknecht, Stefan; Oelze, Matthias; Steven, Sebastian; Schmidt, Patrick; Megner, Alexandra; Wada, Masayuki; Tanabe, Tadashi; Münzel, Thomas; Bottari, Serge; Ullrich, Volker

    2013-01-01

    The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 μM) with an EC50 value of 5 μM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 μM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 μM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite. PMID:23567270

  9. Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration

    PubMed Central

    Chaki, Mounira; Álvarez de Morales, Paz; Ruiz, Carmelo; Begara-Morales, Juan C.; Barroso, Juan B.; Corpas, Francisco J.; Palma, José M.

    2015-01-01

    Background and Aims Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper. Methods The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis. Key Results Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit. Conclusions The RNS profile reported here indicates that ripening of

  10. Mass spectrometric analysis of protein tyrosine nitration in aging and neurodegenerative diseases.

    PubMed

    Yeo, Woon-Seok; Kim, Young Jun; Kabir, Mohammad Humayun; Kang, Jeong Won; Ahsan-Ul-Bari, Md; Kim, Kwang Pyo

    2015-01-01

    This review highlights the significance of protein tyrosine nitration (PTN) in signal transduction pathways, the progress achieved in analytical methods, and the implication of nitration in the cellular pathophysiology of aging and age-related neurodegenerative diseases. Although mass spectrometry of nitrated peptides has become a powerful tool for the characterization of nitrated peptides, the low stoichiometry of this modification clearly necessitates the use of affinity chromatography to enrich modified peptides. Analysis of nitropeptides involves identification of endogenous, intact modification as well as chemical conversion of the nitro group to a chemically reactive amine group and further modifications that enable affinity capture and enhance detectability by altering molecular properties. In this review, we focus on the recent progress in chemical derivatization of nitropeptides for enrichment and mass analysis, and for detection and quantification using various analytical tools. PTN participates in physiological processes, such as aging and neurodegenerative diseases. Accumulation of 3-nitrotyrosine has been found to occur during the aging process; this was identified through mass spectrometry. Further, there are several studies implicating the presence of nitrated tyrosine in age-related diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. PMID:24889964

  11. Nano titanium dioxide photocatalytic protein tyrosine nitration: A potential hazard of TiO{sub 2} on skin

    SciTech Connect

    Lu, Naihao; Zhu Zhening; Zhao Xuqi; Tao Ran; Yang Xiangliang Gao Zhonghong

    2008-06-13

    Protein tyrosine nitration is a prevalent post-translational modification which occurs as a result of oxidative and nitrative stress, it may be directly involved in the onset and/or progression of diseases. Considering the existence of nano titanium dioxide (TiO{sub 2}) in environment and sunscreen products along with the high content of nitrite in sweat, the UV-exposed skin may be a significant target for the photosensitized damage. In this paper, tyrosine nitration of bovine serum albumin (BSA) was initiated in the UV-irradiated reaction mixture containing 0.2-3.0 mg/ml of three commercially nano TiO{sub 2} products and 0.25-1.0 mM NO{sub 2}{sup -}. It was found that anatase TiO{sub 2} and Degussa P25 TiO{sub 2} showed prominent photocatalytic activity on promoting the formation of protein tyrosine nitration, and the optimum condition for the reaction was around physiological pH. Meanwhile, the photocatalytic effect of rutile on protein tyrosine nitration was subtle. The potential physiological significance of nano TiO{sub 2}-photocatalytic protein nitration was also demonstrated in mouse skin homogenate. Although the relationship between photocatalytic protein tyrosine nitration and chronic cutaneous diseases needs further study, the toxicity of nano TiO{sub 2} to the skin disease should be paid more attention in the production and utilization process.

  12. When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

    NASA Astrophysics Data System (ADS)

    Petre, Brînduşa-Alina; Ulrich, Martina; Stumbaum, Mihaela; Bernevic, Bogdan; Moise, Adrian; Döring, Gerd; Przybylski, Michael

    2012-11-01

    Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar KD values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

  13. Detection of in vivo protein tyrosine nitration in petite mutant of Saccharomyces cerevisiae: consequence of its formation and significance.

    PubMed

    Panja, Chiranjit; Ghosh, Sanjay

    2014-09-01

    Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with physiological and pathophysiological conditions. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group. In our previous study we first time showed that PTN occurs in vivo in Saccharomyces cerevisiae. In the present study we observed occurrence of PTN in petite mutant of S. cerevisiae which indicated that PTN is not absolutely dependent on functional mitochondria. Nitration of proteins in S. cerevisiae was also first time confirmed in immunohistochemical study using spheroplasts. Using proteosomal mutants Rpn10Δ, Pre9Δ, we first time showed that the fate of protein nitration in S. cerevisiae was not dependent on proteosomal clearing and probably played vital role in modulating signaling cascades. From our study it is evident that protein tyrosine nitration is a normal physiological event of S. cerevisiae. PMID:25111815

  14. Top-Down Mass Analysis of Protein Tyrosine Nitration: Comparison of Electron Capture Dissociation with “Slow-Heating” Tandem Mass Spectrometry Methods

    PubMed Central

    2010-01-01

    Tyrosine nitration in proteins is an important post-translational modification (PTM) linked to various pathological conditions. When multiple potential sites of nitration exist, tandem mass spectrometry (MS/MS) methods provide unique tools to locate the nitro-tyrosine(s) precisely. Electron capture dissociation (ECD) is a powerful MS/MS method, different in its mechanisms to the “slow-heating” threshold fragmentation methods, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). Generally, ECD provides more homogeneous cleavage of the protein backbone and preserves labile PTMs. However recent studies in our laboratory demonstrated that ECD of doubly charged nitrated peptides is inhibited by the large electron affinity of the nitro group, while CID efficiency remains unaffected by nitration. Here, we have investigated the efficiency of ECD versus CID and IRMPD for top-down MS/MS analysis of multiply charged intact nitrated protein ions of myoglobin, lysozyme, and cytochrome c in a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the “slow-heating” methods. PMID:20677807

  15. Methotrexate administration induces differential and selective protein tyrosine nitration and cysteine nitrosylation in the subcellular organelles of the small intestinal mucosa of rats.

    PubMed

    Natarajan, Kasthuri; Abraham, Premila

    2016-05-01

    Gastrointestinal toxicity is one of the most frequent dose limiting side effects of methotrexate (MTX), a commonly used chemotherapeutic drug. Peroxynitrite (PON) overproduction is reported to contribute to MTX induced gastrointestinal mucositis. However, the consequence of PON overproduction i.e. protein tyrosine nitration and protein cysteine nitrosylation, the subcellular distribution of these modified proteins and their molecular weights have not been investigated yet. Mucositis was induced in Wistar rats by the administration of 3 consecutive i.p. injections of MTX. Tyrosine nitrated proteins and cysteine nitrosylated proteins were determined in the subcellular organelles fractions of mucosa using immunoprecipitation and western blot. The proteins in the subcellular fractions were separated by 1D electrophoresis, and probed with anti -nitrotyrosine antibody and anti-nitrosocysteine antibody. After MTX treatment, a general increase in protein tyrosine nitration as well as a change in the spectrum of proteins that underwent nitration was observed. The relative densities of the 3 nitrotyrosine protein adducts were as follows: Mitochondria > cytosol > microsomes > nucleus. In the mitochondrial fraction increased nitration of 12 kDa, 25 kDa 29Kda, 47 kDa, and 62Kda proteins, in the cytosol increased nitration of 12 kDa, 19 kDa, 45 kDa, and 60 kDa proteins and in the nuclear fraction increased nitration of 17 kDa, 35 kDa, and 58 kDa proteins was observed. On the other hand, MTX treatment resulted to a general decrease in protein cysteine nitrosylation in all the subcellular fractions. These results suggest that MTX induced, PON mediated small intestinal injury is mediated by differential nitration and nitrosylation of proteins in the subcellular organelles with increased protein tyrosine nitration and decreased cysteine nitrosylation. In addition MTX treatment results in selective nitration and nitrosylation of proteins in the intestinal mucosa. This

  16. Tubulin tyrosine nitration regulates microtubule organization in plant cells

    PubMed Central

    Blume, Yaroslav B.; Krasylenko, Yuliya A.; Demchuk, Oleh M.; Yemets, Alla I.

    2013-01-01

    During last years, selective tyrosine nitration of plant proteins gains importance as well-recognized pathway of direct nitric oxide (NO) signal transduction. Plant microtubules are one of the intracellular signaling targets for NO, however, the molecular mechanisms of NO signal transduction with the involvement of cytoskeletal proteins remain to be elucidated. Since biochemical evidence of plant α-tubulin tyrosine nitration has been obtained recently, potential role of this posttranslational modification in regulation of microtubules organization in plant cell is estimated in current paper. It was shown that 3-nitrotyrosine (3-NO2-Tyr) induced partially reversible Arabidopsis primary root growth inhibition, alterations of root hairs morphology and organization of microtubules in root cells. It was also revealed that 3-NO2-Tyr intensively decorates such highly dynamic microtubular arrays as preprophase bands, mitotic spindles and phragmoplasts of Nicotiana tabacum Bright Yellow-2 (BY-2) cells under physiological conditions. Moreover, 3D models of the mitotic kinesin-8 complexes with the tail of detyrosinated, tyrosinated and tyrosine nitrated α-tubulin (on C-terminal Tyr 450 residue) from Arabidopsis were reconstructed in silico to investigate the potential influence of tubulin nitrotyrosination on the molecular dynamics of α-tubulin and kinesin-8 interaction. Generally, presented data suggest that plant α-tubulin tyrosine nitration can be considered as its common posttranslational modification, the direct mechanism of NO signal transduction with the participation of microtubules under physiological conditions and one of the hallmarks of the increased microtubule dynamics. PMID:24421781

  17. Protein Tyrosine Nitration of the Flavin Subunit Is Associated with Oxidative Modification of Mitochondrial Complex II in the Post-ischemic Myocardium*S⃞

    PubMed Central

    Chen, Chwen-Lih; Chen, Jingfeng; Rawale, Sharad; Varadharaj, Saradhadevi; Kaumaya, Pravin P. T.; Zweier, Jay L.; Chen, Yeong-Renn

    2008-01-01

    Increased \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2}^{\\overline{.}}\\end{equation*}\\end{document} and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. A crucial segment of the mitochondrial electron transport chain is succinate ubiquinone reductase (SQR or Complex II). In SQR, oxidative impairment and deglutathionylation of the 70-kDa flavin protein occurs in the post-ischemic heart (Chen, Y. R., Chen, C. L., Pfeiffer, D. R., and Zweier, J. L. (2007) J. Biol. Chem. 282,32640 -3265417848555). To gain insights into the oxidative modification of the 70-kDa protein in the post-ischemic myocardium, we used the identified S-glutathionylated peptide (77AAFGLSEAGFNTACVTK93) of the 70-kDa protein as a chimeric epitope incorporating a “promiscuous” T cell epitope to generate a high titer polyclonal antibody, AbGSC90. Purified AbGSC90 showed a high binding affinity to isolated SQR. Antibodies of AbGSC90 moderately inhibited the electron transfer and superoxide generation activities of SQR. To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90 and probed with antibodies against 3-nitrotyrosine. Enhancement of protein tyrosine nitration was detected in the post-ischemic myocardium. Isolated SQR was subjected to in vitro protein nitration with peroxynitrite, leading to site-specific nitration at the 70-kDa polypeptide and impairment of SQR electron transfer activity. Protein nitration of SQR further impaired its protein-protein interaction with Complex III. Liquid chromatography/tandem mass spectrometry analysis indicated that Tyr-56 and Tyr

  18. MUSCLE PROTEIN TYROSINE NITRATION PATTERNS DURING CHRONIC SUBCLINICAL INTRAMUSCULAR PARASITISM: CO-LOCALIZATION TO FIBER TYPE AND UBIQUITIN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study was conducted to determine whether the inflammatory oxidative response to chronic intramuscular parasitism, as modeled with the protozoan parasite Sarcocystis cruzi, results in protein nitration damage and whether a pattern to it localization can be characterized. Holstein steer c...

  19. Conversion of 3-nitrotyrosine to 3-aminotyrosine residues facilitates mapping of tyrosine nitration in proteins by electrospray ionization-tandem mass spectrometry using electron capture dissociation.

    PubMed

    Guo, Jia; Prokai, Laszlo

    2012-12-01

    Protein tyrosine nitration is associated with oxidative stress and various human diseases. Tandem mass spectrometry has been the method of choice for the identification and localization of this posttranslational modification to understand the underlying mechanisms and functional consequences. Due to the electron predator effect of the nitro group limiting fragmentation of the peptide backbone, electron-based dissociation has not been applicable, however, to nitrotyrosine-containing peptides. A straightforward conversion of the nitrotyrosine to the aminotyrosine residues is introduced to address this limitation. When tested with nitrated ubiquitin and human serum albumin as model proteins in top-down and bottom-up approaches, respectively, this chemical derivatization enhanced backbone fragmentation of the corresponding nitroproteins and nitropeptides by electron capture dissociation (ECD). Increased sequence coverage has been obtained by combining in the bottom-up strategy the conversion of nitrotyrosine to aminotyrosine and introducing, in addition to trypsin, a further digesting enzyme of complementary specificity, when protein nitration was mapped by liquid chromatography-electrospray ionization tandem mass spectrometry using both collision-induced dissociation (CID) and ECD. PMID:23280749

  20. Fully automated multidimensional reversed-phase liquid chromatography with tandem anion/cation exchange columns for simultaneous global endogenous tyrosine nitration detection, integral membrane protein characterization, and quantitative proteomics mapping in cerebral infarcts.

    PubMed

    Quan, Quan; Szeto, Samuel S W; Law, Henry C H; Zhang, Zaijun; Wang, Yuqiang; Chu, Ivan K

    2015-10-01

    Protein tyrosine nitration (PTN) is a signature hallmark of radical-induced nitrative stress in a wide range of pathophysiological conditions, with naturally occurring abundances at substoichiometric levels. In this present study, a fully automated four-dimensional platform, consisting of high-/low-pH reversed-phase dimensions with two additional complementary, strong anion (SAX) and cation exchange (SCX), chromatographic separation stages inserted in tandem, was implemented for the simultaneous mapping of endogenous nitrated tyrosine-containing peptides within the global proteomic context of a Macaca fascicularis cerebral ischemic stroke model. This integrated RP-SA(C)X-RP platform was initially benchmarked through proteomic analyses of Saccharomyces cerevisiae, revealing extended proteome and protein coverage. A total of 27 144 unique peptides from 3684 nonredundant proteins [1% global false discovery rate (FDR)] were identified from M. fascicularis cerebral cortex tissue. The inclusion of the S(A/C)X columns contributed to the increased detection of acidic, hydrophilic, and hydrophobic peptide populations; these separation features enabled the concomitant identification of 127 endogenous nitrated peptides and 137 transmembrane domain-containing peptides corresponding to integral membrane proteins, without the need for specific targeted enrichment strategies. The enhanced diversity of the peptide inventory obtained from the RP-SA(C)X-RP platform also improved analytical confidence in isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses. PMID:26335518

  1. Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity.

    PubMed Central

    Lanone, Sophie; Manivet, Philippe; Callebert, Jacques; Launay, Jean-Marie; Payen, Didier; Aubier, Michel; Boczkowski, Jorge; Mebazaa, Alexandre

    2002-01-01

    Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity. PMID:12097137

  2. Peroxynitrite Mediates Active Site Tyrosine Nitration in Manganese Superoxide Dismutase. Evidence of a Role for the Carbonate Radical Anion

    PubMed Central

    Surmeli, N. Basak; Litterman, Nadia K.; Miller, Anne Frances; Groves, John T.

    2010-01-01

    Protein tyrosine nitration has been observed in a variety of human diseases associated with oxidative stress, such as inflammatory, neurodegenerative and cardiovascular conditions. However, the pathways leading to nitration of tyrosine residues are still unclear. Recent studies have shown that peroxynitrite (PN), produced by the reaction of superoxide and nitric oxide, can lead to protein nitration and inactivation. Tyrosine nitration may also be mediated by nitrogen dioxide produced by the oxidation of nitrite by peroxidases. Manganese superoxide dismutase (MnSOD), which plays a critical role in cellular defense against oxidative stress by decomposing superoxide within mitochondria, is nitrated and inactivated under pathological conditions. In this study, MnSOD is shown to catalyze PN-mediated self-nitration. Direct, spectroscopic observation of the kinetics of PN decay and nitrotyrosine formation (kcat = 9.3 × 102 M-1s-1) indicates that the mechanism involves redox cycling between Mn2+ and Mn3+, similar to that observed with superoxide. Distinctive patterns of tyrosine nitration within MnSOD by various reagents were revealed and quantified by MS/MS analysis of MnSOD trypsin digest peptides. These analyses showed that three of the seven tyrosine residues of MnSOD (Tyr34, Tyr9, and Tyr11) were most susceptible to nitration and that the relative amounts of nitration of these residues varied widely depending upon the nature of the nitrating agent. Notably, nitration mediated by PN, both in the presence and absence of CO2, resulted in nitration of the active site tyrosine, Tyr34, while nitration by freely diffusing nitrogen dioxide led to surface nitration at Tyr9 and Tyr11. Flux analysis of the nitration of Tyr34 by PN-CO2 showed that the nitration rate coincided with the kinetics of the reaction of PN with CO2. These kinetics and the 20-fold increase in the efficiency of tyrosine nitration in the presence of CO2 suggest a specific role for the carbonate radical

  3. Endogenously Nitrated Proteins in Mouse Brain: Links To Neurodegenerative Disease

    SciTech Connect

    Sacksteder, Colette A.; Qian, Weijun; Knyushko, Tanya V.; Wang, Haixing H.; Chin, Mark H.; Lacan, Goran; Melega, William P.; Camp, David G.; Smith, Richard D.; Smith, Desmond J.; Squier, Thomas C.; Bigelow, Diana J.

    2006-07-04

    Increased nitrotyrosine modification of proteins has been documented in multiple pathologies in a variety of tissue types; emerging evidence suggests its additional role in redox regulation of normal metabolism. In order to identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic dataset identifying 7,792 proteins from whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in identification of 31 unique nitrotyrosine sites within 29 different proteins. Over half of the nitrated proteins identified have been reported to be involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces increased nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, characteristics consistent with peroxynitrite-induced tyrosine modification. More striking is the five-fold greater nitration of tyrosines having nearby basic sidechains, suggesting electrostatic attraction of basic groups with the negative charge of peroxynitrite. Together, these results suggest that elevated peroxynitrite generation plays a role in neurodegenerative changes in the brain and provides a predictive tool of functionally important sites of nitration.

  4. Tyrosine Nitration within the Proline-Rich Region of Tau in Alzheimer's Disease

    PubMed Central

    Reyes, Juan F.; Fu, Yifan; Vana, Laurel; Kanaan, Nicholas M.; Binder, Lester I.

    2011-01-01

    A substantial body of evidence suggests that nitrative injury contributes to neurodegeneration in Alzheimer's disease (AD) and other neurodegenerative disorders. Previously, we showed in vitro that within the tau protein the N-terminal tyrosine residues (Y18 and Y29) are more susceptible to nitrative modifications than other tyrosine sites (Y197 and Y394). Using site-specific antibodies to nitrated tau at Y18 and Y29, we identified tau nitrated in both glial (Y18) and neuronal (Y29) tau pathologies. In this study, we report the characterization of two novel monoclonal antibodies, Tau-nY197 and Tau-nY394, recognizing tau nitrated at Y197 and Y394, respectively. By Western blot analysis, Tau-nY197 labeled soluble tau and insoluble paired helical filament proteins (PHF-tau) nitrated at Y197 from control and AD brain samples. Tau-nY394 failed to label soluble tau isolated from control or severe AD samples, but labeled insoluble PHF-tau to a limited extent. Immunohistochemical analysis using Tau-nY197 revealed the hallmark tau pathology associated with AD; Tau-nY394 did not detect any pathological lesions characteristic of the disorder. These data suggest that a subset of the hallmark pathological inclusions of AD contain tau nitrated at Y197. However, nitration at Y197 was also identified in soluble tau from all control samples, including those at Braak stage 0, suggesting that nitration at this site in the proline-rich region of tau may have normal biological functions in the human brain. PMID:21514440

  5. Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy.

    PubMed

    Rezende Valim, Lays; Davies, Julia A; Tveen Jensen, Karina; Guo, Rui; Willison, Keith R; Spickett, Corinne M; Pitt, Andrew R; Klug, David R

    2014-11-13

    Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy. PMID:25347525

  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. Structural characterisation of tyrosine-nitrated peptides by ultraviolet and infrared matrix-assisted laser desorption/ionisation Fourier transform ion cyclotron resonance mass spectrometry.

    PubMed

    Petre, Brínduşa-Alina; Youhnovski, Nikolay; Lukkari, Juho; Weber, Reinhold; Przybylski, Michael

    2005-01-01

    Nitration of tyrosine residues in proteins may occur in cells upon oxidative stress and inflammation processes mediated through generation of reactive nitroxyl from peroxynitrite. Tyrosine nitration from oxidative pathways may generate cytotoxic species that cause protein dysfunction and pathogenesis. A number of protein nitrations in vivo have been reported and some specific Tyrosine nitration sites have been recently identified using mass spectrometric methods. High-resolution Fourier transform ion cyclotron resonance mass spectrometry (MALDI) FT-ICR-MS) is shown here to be a highly efficient method in the determination of protein nitrations. Following the identification of nitration of the catalytic site Tyr-430 residue of bovine prostacyclin synthase, we synthesised several model peptides containing both unmodified tyrosine and 3-nitro-tyrosine residues, using solid-phase peptide synthesis (SPPS). The structures of the nitrotyrosine peptides were characterised both by ESI- and by matrix-assisted laser desorption/ionisation (MALDI)-FT-ICR-MS, using a standard ultraviolet (UV) nitrogen nitrogen laser and a 2.97 microm Nd-YAG infrared laser. Using UV-MALDI-MS, 3-nitrotyrosyl-peptides were found to undergo extensive photochemical fragmentation at the nitrophenyl group, which may hamper or prevent the unequivocal identification of Tyr-nitrations in cellular proteins. In contrast, infrared-MALDI-FT-ICR-MS did not produce fragmentation of molecular ions of Tyr-nitrated peptides. PMID:16322657

  8. Quantification of nitrotyrosine in nitrated proteins

    PubMed Central

    Zhang, Yingyi; Pöschl, Ulrich

    2010-01-01

    For kinetic studies of protein nitration reactions, we have developed a method for the quantification of nitrotyrosine residues in protein molecules by liquid chromatography coupled to a diode array detector of ultraviolet-visible absorption. Nitrated bovine serum albumin (BSA) and nitrated ovalbumin (OVA) were synthesized and used as standards for the determination of the protein nitration degree (ND), which is defined as the average number of nitrotyrosine residues divided by the total number of tyrosine residues in a protein molecule. The obtained calibration curves of the ratio of chromatographic peak areas of absorbance at 357 and at 280 nm vs. nitration degree are nearly the same for BSA and OVA (relative deviations <5%). They are near-linear at low ND (< 0.1) and can be described by a second-order polynomial fit up to \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ {\\hbox{ND}} = 0.5\\left( {{R^2} > 0.99} \\right) $$\\end{document}. A change of chromatographic column led to changes in absolute peak areas but not in the peak area ratios and related calibration functions, which confirms the robustness of the analytical method. First results of laboratory experiments confirm that the method is applicable for the investigation of the reaction kinetics of protein nitration. The main advantage over alternative methods is that nitration degrees can be efficiently determined without hydrolysis or digestion of the investigated protein molecules. PMID:20300739

  9. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite.

    PubMed

    Reiter, C D; Teng, R J; Beckman, J S

    2000-10-20

    Tyrosine nitration is a widely used marker of peroxynitrite (ONOO(-)) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkaline cis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo. PMID:10906340

  10. Effects of tyrosine-26 and tyrosine-64 nitration on the photoreactions of bacteriorhodopsin

    NASA Technical Reports Server (NTRS)

    Scherrer, P.; Stoeckenius, W.

    1985-01-01

    The photoreactions of nitrated bacteriorhodopsin (bR) are examined. Flash-induced difference spectra of bR, bR with aminotyrosine in position 26 (bR-N26R) and bR with aminotyrosine in position 64 are analyzed. It is observed that changes in the actinic wavelength (from 520 to 500 or 580 nm) have no affect on the shape of the spectra and the formation and decay kinetics of the O and M intermediates. Nitration of tyrosine-64 decreases the chromophore absorbance, shifts the absorption maximum to 535 nm, and affects photocycle kinetics independent of the pK of its phenolic group. Light-dark adaptation spectra for bR are studied. The kinetics of the M and O intermediates in bR with nitrotyrosine in position 64 (bR-N64) and bR with aminotyrosine in position 64 and bR with nitrotyrosine in position 26 and bR-N26R are described and compared to bR; the pH dependence and M and O decay rates are considered. The deprotonation of bR-N64 during the photoreaction cycle and the effects of nitration on the activity of proton pumping are investigated.

  11. Heterogeneous Nitration of Tyrosine by NO­3 and N2O5: Rates, Mechanisms and Product Yields

    NASA Astrophysics Data System (ADS)

    Talukdar, R. K.; Witkowski, B.; Burkholder, J. B.; Roberts, J. M.

    2015-12-01

    Nitration of protein-bound tyrosine has been identified as a casual connection between air pollution and human health. Tyrosine is a common amino acid, 4-hydroxyphenylalanine, HO-C6H4-CH2-CH(NH2)-C(O)OH), and is present in many atmospheric bio-aerosols. Nitration of the aromatic units of protein molecules in polluted air enhances their allergenicity. The mechanism of heterogeneous nitration process of bio-aerosols by common nitrating agents in the atmosphere, O3/NO2, NO3, N2O5 is not well understood. This chemistry is thought to proceed via reactions with O3 and NO2 on particle surfaces, through mechanisms that are still uncertain. The possible role of higher nitrogen oxides also remains uncertain, partly due to a lack of measurements of fundamental chemical and physical parameters. In this work, we undertook measurements of reactive uptake of NO3, N2O5, as a function of relative humidity and temperature in a tyrosine coated flow tube reactor with chemical ionization mass spectrometric (CIMS) detection. Uptake coefficients on tyrosine coated flow tube were small under low relative humidity but were enhanced by an order of magnitude in the presence of high relative humidity, particularly for N2O5. The measured uptake coefficients were mostly due to reaction with water adsorbed on the surface of the flow tube. Only ~10% of the reactive uptake could be attributed to reaction with tyrosine. Following uptake, the contents of the flow tube were extracted, and analyzed using electrospray ionization - mass spectrometer (ESI-MS) to identify and quantify the products of the nitration reaction. The only organic reaction product detected was 3-nitro-tyrosine (3-NT). The measured uptake coefficients, mechanism of the title reactions and the possible atmospheric implications of these findings will be discussed.

  12. Exploring the sensitivity of ZnO nanotubes to tyrosine nitration: A DFT approach

    NASA Astrophysics Data System (ADS)

    Maddahi, Pari Sadat; Shahtahmassebi, Nasser; Rezaee Roknabadi, Mahmood; Moosavi, Fatemeh

    2016-05-01

    Due to association of protein tyrosine nitration (PTN) with development of some serious human disorders and diseases, in this paper, the possible applications of ZnO-based nanobiosensors in nitrated tyrosine (nTyr) detection were explored within the density functional framework. With this motivation, the interaction of nTyr with ZnO single walled nanotubes via all possible active sites of nTyr was investigated. The results show the tendency of nTyr to interact through its nitro site (forming nitro-site configuration) with ZnO SWNTs as it has the highest binding energy; while, the charge-solvent configuration involving the interaction of nTyr's phenolic ring has the second place in terms of binding energy magnitude. Regardless of which active site contributes in interaction, the binding energies exhibit an ascending trend with decrease of SWNTs' curvature. Electronic properties analysis indicates that nTyr interaction via its nitro group results in formation of some flat bands inside the band gap region leading to significant reduction of overall band gap energy. Similar behavior is also observed in charge-solvent configuration but the band gap energy is larger. These red shifts are mainly attributed to contribution of 2p orbitals of species present in nTyr. Also, the hybridization of 3d orbital of Zn atom with 2p orbitals of nitro group atomic species is found responsible for bonding formation in bioconjugated system possessing the highest binding energy. Comparison of the electronic band structure of ZnO SWNT-Tyr with that of ZnO SWNT-nTyr indicates the sensitivity of ZnO SWNTs toward tyrosine nitration hence, a considerable change in its optical spectra is expectable. This introduces ZnO SWNTs as a promising candidate for PTN detection.

  13. The Extended Family of Protein Tyrosine Phosphatases.

    PubMed

    Alonso, Andrés; Nunes-Xavier, Caroline E; Bayón, Yolanda; Pulido, Rafael

    2016-01-01

    In higher eukaryotes, the Tyr phosphorylation status of cellular proteins results from the coordinated action of Protein Tyrosine Kinases (PTKs) and Protein Tyrosine Phosphatases (PTPs). PTPs have emerged as highly regulated enzymes with diverse substrate specificity, and proteins with Tyr-dephosphorylation or Tyr-dephosphorylation-like properties can be clustered as the PTPome. This includes proteins from the PTP superfamily, which display a Cys-based catalytic mechanism, as well as enzymes from other gene families (Asp-based phosphatases, His-based phosphatases) that have converged in protein Tyr-dephosphorylation-related functions by using non-Cys-based catalytic mechanisms. Within the Cys-based members of the PTPome, classical PTPs dephosphorylate specific phosphoTyr (pTyr) residues from protein substrates, whereas VH1-like dual-specificity PTPs dephosphorylate pTyr, pSer, and pThr residues, as well as nonproteinaceous substrates, including phosphoinositides and phosphorylated carbohydrates. In addition, several PTPs have impaired catalytic activity as a result of amino acid substitutions at their active sites, but retain regulatory functions related with pTyr signaling. As a result of their relevant biological activity, many PTPs are linked to human disease, including cancer, neurodevelopmental, and metabolic diseases, making these proteins important drug targets and molecular markers in the clinic. Here, a brief overview on the biochemistry and physiology of the different groups of proteins that belong to the mammalian PTPome is presented. PMID:27514797

  14. Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives.

    PubMed

    De Caro, J D; Behnke, W D; Bonicel, J J; Desnuelle, P A; Rovery, M

    1983-09-28

    The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5. PMID:6615844

  15. Role of Protein Tyrosine Phosphatases in Plants

    PubMed Central

    Shankar, Alka; Agrawal, Nisha; Sharma, Manisha; Pandey, Amita; Pandey, Girdhar K.

    2015-01-01

    Reversible protein phosphorylation is a crucial regulatory mechanism that controls many biological processes in eukaryotes. In plants, phosphorylation events primarily occur on serine (Ser) and threonine (Thr) residues, while in certain cases, it was also discovered on tyrosine (Tyr) residues. In contrary to plants, extensive reports on Tyr phosphorylation regulating a large numbers of biological processes exist in animals. Despite of such prodigious function in animals, Tyr phosphorylation is a least studied mechanism of protein regulation in plants. Recently, various chemical analytical procedures have strengthened the view that Tyr phosphorylation is equally prevalent in plants as in animals. However, regardless of Tyr phosphorylation events occuring in plants, no evidence could be found for the existence of gene encoding for Tyr phosphorylation i.e. the typical Tyr kinases. Various methodologies have suggested that plant responses to stress signals and developmental processes involved modifications in protein Tyr phosphorylation. Correspondingly, various reports have established the role of PTPs (Protein Tyrosine Phosphatases) in the dephosphorylation and inactivation of mitogen activated protein kinases (MAPKs) hence, in the regulation of MAPK signaling cascade. Besides this, many dual specificity protein phosphatases (DSPs) are also known to bind starch and regulate starch metabolism through reversible phosphorylation. Here, we are emphasizing the significant progress on protein Tyr phosphatases to understand the role of these enzymes in the regulation of post-translational modification in plant physiology and development. PMID:26962298

  16. Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking.

    PubMed Central

    Bristow, A F; Virden, R

    1978-01-01

    1. Nitration of tyrosine residues of staphylococal penicillinase was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. 2. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. 3. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers respectively showed major bands in corresponding positions in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicating that the increase in molecular weight was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, whereas higher oligomers retained an average of 8-15% of normal activity. 4. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72.5. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product with full catalytic activity. 6. It is concluded that tyrosine-72 plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This poperty may make it useful for attachment of a reporter group. PMID:629760

  17. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases

    SciTech Connect

    Kanner, S.B.; Reynolds, A.B.; Vines, R.R.; Parsons, J.T. )

    1990-05-01

    Cellular transformation by oncogenic retroviruses encoding protein tyrosine kinases coincides with the tyrosine-specific phosphorylation of multiple protein substrates. Previous studies have shown that tyrosine phosphorylation of a protein of 120 kDa, p120, correlated with src transformation in chicken embryo fibroblasts. Additionally, the authors previously identified two phosphotyrosine-containing cellular proteins, p130 and p110, that formed stable complexes with activated variants of pp60{sup src}, the src-encoded tyrosine kinase. To study transformation-relevant tyrosine kinase substrates, they have generated monoclonal antibodies to individual tyrosine phosphoproteins, including p130, p120, p110, and five additional phosphoproteins (p210, p125, p118, p85, and p185/p64). These antibodies detected several of the same tyrosine phosphoproteins in chicken embryo fibroblasts transformed by avian retroviruses Y73 and CT10, encoding the yes and crk oncogenes, respectively. Protein substrates in mouse, rat, hamster, and human cells overexpressing activated variants of chicken pp60{sup src} were also detected by several of the monoclonal antibodies.

  18. Detection of Sequence-Specific Tyrosine Nitration of Manganese SOD and SERCA in Cardiovascular Disease and Aging

    SciTech Connect

    Xu, Shanqin; Ying, Jia; Jiang, Bingbing; Guo, Wei; Adachi, Takeshi; Sharov, Victor; Lazar, Harold; Menzoian, James; Knyushko, Tanya V.; Bigelow, Diana J.; Schoneich, Christian; Cohen, Richard

    2006-06-01

    Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies towards site-specific nY-modified proteins and to use histochemical and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies towards peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with angiotensin II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite that chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site, but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates, but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.

  19. Aniline-induced nitrosative stress in rat spleen: Proteomic identification of nitrated proteins

    SciTech Connect

    Fan Xiuzhen; Wang Jianling; Soman, Kizhake V.; Ansari, G.A.S.; Khan, M. Firoze

    2011-08-15

    Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6 kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity. - Highlights: > Proteomic approaches are used to identify nitrated proteins in the spleen. > Twenty five nitrated proteins were found only in the spleen of aniline-treated rats. > Aniline exposure led to increased iNOS mRNA and protein expression in

  20. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    SciTech Connect

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  1. Genetic alterations of protein tyrosine phosphatases in human cancers

    PubMed Central

    Zhao, Shuliang; Sedwick, David; Wang, Zhenghe

    2014-01-01

    Protein tyrosine phosphatases (PTPs) are enzymes that remove phosphate from tyrosine residues in proteins. Recent whole-exome sequencing of human cancer genomes reveals that many PTPs are frequently mutated in a variety of cancers. Among these mutated PTPs, protein tyrosine phosphatase T (PTPRT) appears to be the most frequently mutated PTP in human cancers. Beside PTPN11 which functions as an oncogene in leukemia, genetic and functional studies indicate that most of mutant PTPs are tumor suppressor genes. Identification of the substrates and corresponding kinases of the mutant PTPs may provide novel therapeutic targets for cancers harboring these mutant PTPs. PMID:25263441

  2. Alterations in connexin 43 during diabetic cardiomyopathy: competition of tyrosine nitration versus phosphorylation

    PubMed Central

    COOK, Angela C.; SCHANBACHER, Brandon L.; BAUER, John Anthony

    2014-01-01

    Objective Cardiac conduction abnormalities are observed early in the progression of Type I diabetes, but the mechanism(s) involved are undefined. Connexin 43, a critical component of ventricular gap junctions, depends on tyrosine phosphorylation status to modulate channel conductance - alterations in connexin 43 content, distributions, and/or phosphorylation status may be involved in cardiac rhythm disturbances. We tested the hypothesis that cardiac content/distribution of connexin 43 are altered in a rat model of Type I diabetic cardiomyopathy, investigating a mechanistic role for tyrosine. Methods We conducted electrocardiographic analyses during the progression of diabetic cardiomyopathy in rats dosed with streptozotocin (65mg/kg), at 3, 7, and 35 days post-induction of diabetes. Following functional analyses, we conducted immunohistochemical and immunoprecipitation studies to assess alterations in connexin 43. Results We observed significant evidence of ventricular conduction abnormalities (QRS complex, Q-T interval) as early as 7 days post-streptozotocin, persisting throughout the study. Connexin 43 levels were increased 7d post- streptozotocin and remained elevated throughout the study. Connexin 40 content was unchanged relative to controls throughout the study. Changes in Connexin 43 distribution were also observed; connexin 43 staining was dispersed from myocyte short axis junctions. Connexin 43 tyrosine phosphorylation declined during the progression of diabetes, with concurrent increases in tyrosine nitration. Conclusions These data suggest that alterations in connexin 43 content and distribution occur during experimental diabetes and likely contribute to alterations in cardiac function, and that oxidative modification of tyrosine-mediated signaling may play a mechanistic role. PMID:24796789

  3. Differential inhibition of Arabidopsis superoxide dismutases by peroxynitrite-mediated tyrosine nitration

    PubMed Central

    Holzmeister, Christian; Gaupels, Frank; Geerlof, Arie; Sarioglu, Hakan; Sattler, Michael; Durner, Jörg; Lindermayr, Christian

    2015-01-01

    Despite the importance of superoxide dismutases (SODs) in the plant antioxidant defence system little is known about their regulation by post-translational modifications. Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana. S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities. By contrast, peroxynitrite inhibited the mitochondrial manganese SOD1 (MSD1), peroxisomal copper/zinc SOD3 (CSD3), and chloroplastic iron SOD3 (FSD3), but no other SODs. MSD1 was inhibited by up to 90% but CSD3 and FSD3 only by a maximum of 30%. Down-regulation of these SOD isoforms correlated with tyrosine (Tyr) nitration and both could be prevented by the peroxynitrite scavenger urate. Site-directed mutagenesis revealed that—amongst the 10 Tyr residues present in MSD1—Tyr63 was the main target responsible for nitration and inactivation of the enzyme. Tyr63 is located nearby the active centre at a distance of only 5.26 Å indicating that nitration could affect accessibility of the substrate binding pocket. The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs. PMID:25428993

  4. Nitration is exclusive to defense-related PR-1, PR-3 and PR-5 proteins in tobacco leaves.

    PubMed

    Takahashi, Misa; Shigeto, Jun; Izumi, Shunsuke; Yoshizato, Katsutoshi; Morikawa, Hiromichi

    2016-07-01

    Protein tyrosine nitration is an important post-translational modification. A variety of nitrated proteins are reported in Arabidopsis leaves and seedlings, sunflower hypocotyls, and pea roots. The identities of nitrated proteins are species-/organ-specific, and chloroplast proteins are most nitratable in leaves. However, precise mechanism is unclear. Here, we investigated nitroproteome in tobacco leaves following exposure to nitrogen dioxide. Proteins were extracted, electrophoresed and immunoblotted using an anti-3-nitrotyrosine antibody. Mass spectrometry and FASTA search identified for the first time an exclusive nitration of pathogenesis-related proteins, PR-1, PR-3 and PR-5, which are reportedly located in the apoplast or the vacuole. Furthermore, Tyr(36) of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed. PMID:27301959

  5. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa

    SciTech Connect

    Kelly-Wintenberg, K.; Anderson, T.; Montie, T.C. )

    1990-09-01

    Purified flagella from two strains of {sup 32}P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.

  6. Microtubule-associated protein 1B interaction with tubulin tyrosine ligase contributes to the control of microtubule tyrosination.

    PubMed

    Utreras, Elías; Jiménez-Mateos, Eva Maria; Contreras-Vallejos, Erick; Tortosa, Elena; Pérez, Mar; Rojas, Sebastián; Saragoni, Lorena; Maccioni, Ricardo B; Avila, Jesús; González-Billault, Christian

    2008-01-01

    Microtubule-associated protein 1B (MAP1B) is the first microtubule-associated protein to be expressed during nervous system development. MAP1B belongs to a large family of proteins that contribute to the stabilization and/or enhancement of microtubule polymerization. These functions are related to the control of the dynamic properties of microtubules. The C-terminal domain of the neuronal alpha-tubulin isotype is characterized by the presence of an acidic polypeptide, with the last amino acid being tyrosine. This tyrosine residue may be enzymatically removed from the protein by an unknown carboxypeptidase activity. Subsequently, the tyrosine residue is again incorporated into this tubulin by another enzyme, tubulin tyrosine ligase, to yield tyrosinated tubulin. Because neurons lacking MAP1B have a reduced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the interaction is not affected by MAP1B phosphorylation. Additionally, neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal. These results suggest that MAP1B regulates tyrosination of alpha-tubulin in neuronal microtubules. This regulation may be important for general processes involved in nervous system development such as axonal guidance and neuronal migration. PMID:18075266

  7. Functional Roles of Protein Nitration in Acute and Chronic Liver Diseases

    PubMed Central

    Abdelmegeed, Mohamed A.; Song, Byoung-Joon

    2014-01-01

    Nitric oxide, when combined with superoxide, produces peroxynitrite, which is known to be an important mediator for a number of diseases including various liver diseases. Peroxynitrite can modify tyrosine residue(s) of many proteins resulting in protein nitration, which may alter structure and function of each target protein. Various proteomics and immunological methods including mass spectrometry combined with both high pressure liquid chromatography and 2D PAGE have been employed to identify and characterize nitrated proteins from pathological tissue samples to determine their roles. However, these methods contain a few technical problems such as low efficiencies with the detection of a limited number of nitrated proteins and labor intensiveness. Therefore, a systematic approach to efficiently identify nitrated proteins and characterize their functional roles is likely to shed new insights into understanding of the mechanisms of hepatic disease pathophysiology and subsequent development of new therapeutics. The aims of this review are to briefly describe the mechanisms of hepatic diseases. In addition, we specifically describe a systematic approach to efficiently identify nitrated proteins to study their causal roles or functional consequences in promoting acute and chronic liver diseases including alcoholic and nonalcoholic fatty liver diseases. We finally discuss translational research applications by analyzing nitrated proteins in evaluating the efficacies of potentially beneficial agents to prevent or treat various diseases in the liver and other tissues. PMID:24876909

  8. Co-expression of protein tyrosine kinases EGFR-2 and PDGFRβ with protein tyrosine phosphatase 1B in Pichia pastoris.

    PubMed

    Tu, Pham Ngoc; Wang, Yamin; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing

    2014-02-28

    The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Coexpression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and PDGFRβ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and PDGFRβ fusion proteins were purified by Ni(2+) affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and PDGFRβ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics. PMID:24248091

  9. Characterization of the PEST family protein tyrosine phosphatase BDP1.

    PubMed

    Kim, Y W; Wang, H; Sures, I; Lammers, R; Martell, K J; Ullrich, A

    1996-11-21

    Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues. PMID:8950995

  10. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    SciTech Connect

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. )

    1990-04-01

    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  11. Mycobacterium tuberculosis supports protein tyrosine phosphorylation

    PubMed Central

    Kusebauch, Ulrike; Ortega, Corrie; Ollodart, Anja; Rogers, Richard S.; Sherman, David R.; Moritz, Robert L.; Grundner, Christoph

    2014-01-01

    Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ∼1.4 million lives every year. PMID:24927537

  12. NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22

    PubMed Central

    Spalinger, Marianne R.; Kasper, Stephanie; Gottier, Claudia; Lang, Silvia; Atrott, Kirstin; Vavricka, Stephan R.; Scharl, Sylvie; Gutte, Petrus M.; Grütter, Markus G.; Beer, Hans-Dietmar; Contassot, Emmanuel; Chan, Andrew C.; Dai, Xuezhi; Rawlings, David J.; Mair, Florian; Becher, Burkhard; Falk, Werner; Fried, Michael; Rogler, Gerhard

    2016-01-01

    Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1β, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1β secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1β release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1β. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1β levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation. PMID:27043286

  13. Short-term alpha- or gamma-delta-enriched tocopherol oil supplementation differentially effects the expression of proinflammatory mediators: selective impacts on characteristics of protein tyrosine nitration in vivo¿.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein 3’-nitrotyrosine (pNT) is an established biomarker of nitrosative cell stress in animals challenged with proinflammatory mediators like endotoxin (LPS). We determined that short-term feeding of diets supplemented with a-tocopherol- (a-T -96% a-isomer) or '- and d-enriched mixed tocopherol o...

  14. Dietary Vitamin D Deficiency in Rats from Middle- to Old-age Leads to Elevated Tyrosine Nitration and Proteomics Changes in Levels of Key Proteins in Brain: Implications for Low Vitamin D-dependent Age-Related Cognitive Decline

    PubMed Central

    Keeney, Jeriel T. R.; Förster, Sarah; Sultana, Rukhsana; Brewer, Lawrence D.; Latimer, Caitlin S.; Cai, Jian; Klein, Jon B.; Porter, Nada M.; Butterfield, D. Allan

    2013-01-01

    In addition to the well-known effects of vitamin D (VitD) in maintaining bone health, there is increasing appreciation that this vitamin may serve important roles in other organs and tissues, including the brain. Given that VitD deficiency is especially widespread among the elderly, it is important to understand how the range of serum VitD levels that mimic those found in humans (from low to high) affects the brain during aging from middle-age to old-age. To address this issue, twenty-seven male F344 rats were split into three groups and fed isocaloric diets containing low (100 IU/kg food), control (1000 IU/kg food), or high (10000 IU/kg food) VitD beginning at middle-age (12 months) and continued for a period of 4–5 months. We compared the effects of these dietary VitD manipulations on oxidative and nitrosative stress measures in posterior brain cortices. The low VitD group showed global elevation of 3-nitrotyrosine (3-NT) compared to control and high VitD treated groups. Further investigation showed that this elevation may involve dysregulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and NF-κB mediated transcription of inducible nitric oxide synthase (iNOS) as indicated by translocation of NF-κB to the nucleus and elevation of iNOS levels. Proteomic techniques were used to provide insights into potential mechanisms underlying these effects. Several brain proteins were found at significantly elevated levels in low VitD group compared to the control and high VitD groups. Three of these proteins, 6-phosphofructokinase, triosephosphate isomerase, and pyruvate kinase, are involved directly in glycolysis. Two others, peroxiredoxin-3 and DJ-1/PARK7, have peroxidase activity and are found in mitochondria. Peptidyl-prolyl cis-trans isomerase A (PPIA or cyclophilin A) has been shown to have multiple roles including protein folding, regulation of protein kinases and phosphatases, immunoregulation, cell signaling, and redox

  15. Dietary vitamin D deficiency in rats from middle to old age leads to elevated tyrosine nitration and proteomics changes in levels of key proteins in brain: implications for low vitamin D-dependent age-related cognitive decline.

    PubMed

    Keeney, Jeriel T R; Förster, Sarah; Sultana, Rukhsana; Brewer, Lawrence D; Latimer, Caitlin S; Cai, Jian; Klein, Jon B; Porter, Nada M; Butterfield, D Allan

    2013-12-01

    In addition to the well-known effects of vitamin D (VitD) in maintaining bone health, there is increasing appreciation that this vitamin may serve important roles in other organs and tissues, including the brain. Given that VitD deficiency is especially widespread among the elderly, it is important to understand how the range of serum VitD levels that mimic those found in humans (from low to high) affects the brain during aging from middle age to old age. To address this issue, 27 male F344 rats were split into three groups and fed isocaloric diets containing low (100 IU/kg food), control (1000 IU/kg food), or high (10,000 IU/kg food) VitD beginning at middle age (12 months) and continued for a period of 4-5 months. We compared the effects of these dietary VitD manipulations on oxidative and nitrosative stress measures in posterior brain cortices. The low-VitD group showed global elevation of 3-nitrotyrosine compared to control and high-VitD-treated groups. Further investigation showed that this elevation may involve dysregulation of the nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) pathway and NF-κB-mediated transcription of inducible nitric oxide synthase (iNOS) as indicated by translocation of NF-κB to the nucleus and elevation of iNOS levels. Proteomics techniques were used to provide insight into potential mechanisms underlying these effects. Several brain proteins were found at significantly elevated levels in the low-VitD group compared to the control and high-VitD groups. Three of these proteins, 6-phosphofructokinase, triose phosphate isomerase, and pyruvate kinase, are involved directly in glycolysis. Two others, peroxiredoxin-3 and DJ-1/PARK7, have peroxidase activity and are found in mitochondria. Peptidyl-prolyl cis-trans isomerase A (cyclophilin A) has been shown to have multiple roles, including protein folding, regulation of protein kinases and phosphatases, immunoregulation, cell signaling, and redox status. Together, these

  16. Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B.

    PubMed

    Ali, Md Yousof; Jannat, Susoma; Jung, Hyun Ah; Jeong, Hyong Oh; Chung, Hae Young; Choi, Jae Sue

    2016-05-25

    In the present study, we investigated the anti-diabetic potential of six natural coumarins, 4-hydroxy Pd-C-III (1), 4'-methoxy Pd-C-I (2), decursinol (3), decursidin (4), umbelliferone 6-carboxylic acid (5), and 2'-isopropyl psoralene (6) isolated from Angelica decursiva and evaluated their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, and ONOO(-)-mediated protein tyrosine nitration. Coumarins 1-6 showed potent PTP1B and α-glucosidase inhibitory activities with ranges of IC50 values of 5.39-58.90 μM and 65.29-172.10 μM, respectively. In the kinetic study for PTP1B enzyme inhibition, compounds 1, 5, and 6 were competitive, whereas 2 and 4 showed mixed type, and 3 displayed noncompetitive type inhibition. For α-glucosidase enzyme inhibition, compounds 1 and 3 exhibited good mixed-type, while 2, 5, and 6 showed noncompetitive and 4 displayed competitive type inhibition. Furthermore, these coumarins also effectively suppressed ONOO(-)-mediated tyrosine nitration in a dose-dependent manner. To further investigate PTP1B inhibition, we generated a 3D structure of PTP1B using Autodock 4.2 and simulated the binding of compounds 1-6. Docking simulations showed that different residues of PTP1B interacted with different functional groups of compounds 1-6 through hydrogen and hydrophobic interactions. In addition, the binding energies of compounds 1-6 were negative, suggesting that hydrogen bonding may stabilize the open form of the enzyme and potentiate tight binding of the active site of PTP1B, thereby resulting in more effective PTP1B inhibition. These results demonstrate that the whole plant of A. decursiva and its coumarins are useful as potential functional food ingredients for the prevention and treatment of type 2 diabetes. PMID:27085377

  17. Tyrosine-selective protein alkylation using pi-allylpalladium complexes.

    PubMed

    Tilley, S David; Francis, Matthew B

    2006-02-01

    A new protein modification reaction has been developed based on a palladium-catalyzed allylic alkylation of tyrosine residues. This technique employs electrophilic pi-allyl intermediates derived from allylic acetate and carbamate precursors and can be used to modify proteins in aqueous solution at room temperature. To facilitate the detection of modified proteins using SDS-PAGE analysis, a fluorescent allyl acetate was synthesized and coupled to chymotrypsinogen A and bacteriophage MS2. The tyrosine selectivity of the reaction was confirmed through trypsin digest analysis. The utility of the reaction was demonstrated by using taurine-derived carbamates as water solubilizing groups that are cleaved upon protein functionalization. This solubility switching technique was used to install hydrophobic farnesyl and C(17) chains on chymotrypsinogen A in water using little or no cosolvent. Following this, the C(17) alkylated proteins were found to associate with lipid vesicles. In addition to providing a new protein modification strategy targeting an under-utilized amino acid side chain, this method provides convenient access to synthetic lipoproteins. PMID:16433516

  18. Phospho-tyrosine dependent protein–protein interaction network

    PubMed Central

    Grossmann, Arndt; Benlasfer, Nouhad; Birth, Petra; Hegele, Anna; Wachsmuth, Franziska; Apelt, Luise; Stelzl, Ulrich

    2015-01-01

    Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes. PMID:25814554

  19. Endogenous 3, 4- Dihydroxyphenylalanine and Dopaquinone Modifications on Protein Tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical

    SciTech Connect

    Zhang, Xu; Monroe, Matthew E.; Chen, Baowei; Chin, Mark H.; Heibeck, Tyler H.; Schepmoes, Athena A.; Yang, Feng; Petritis, Brianne O.; Camp, David G.; Pounds, Joel G.; Jacobs, Jon M.; Smith, Desmond J.; Bigelow, Diana J.; Smith, Richard D.; Qian, Weijun

    2010-06-02

    Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3, 4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first global proteome survey of endogenous site-specific modifications, i.e, DOPA and its further oxidation product dopaquinone (DQ) in mouse brain and heart tissues. Results from LC-MS/MS analyses included 203 and 71 DOPA-modified tyrosine sites identified from brain and heart, respectively, with a false discovery rate of ~1%; while only a few nitrotyrosine containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 57 and 29 DQ modified peptides were observed from brain and heart, respectively; nearly half of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal-binding properties, consistent with metal catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondria-associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation suggesting potential disruption of signaling pathways. Structural aspects of DOPA-modified tyrosine sequences are distinct from those of nitrotyrosines suggesting that each type of modifications provides a marker for different in vivo reactive chemistries and can be used to predict sensitive protein targets. Collectively, the results suggest that these modifications are linked with mitochondrially-derived oxidative stress, and may serve as sensitive markers for disease pathologies.

  20. Characterization of the protein tyrosine phosphatase PRL from Entamoeba histolytica.

    PubMed

    Ramírez-Tapia, Ana Lilia; Baylón-Pacheco, Lidia; Espíritu-Gordillo, Patricia; Rosales-Encina, José Luis

    2015-12-01

    Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host-pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite Entamoeba histolytica. Here, we reported that the EhPRL protein possessed the classical HCX5R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31-32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3-o-methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o-vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism. PMID:26431820

  1. Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

    SciTech Connect

    Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M. )

    1989-04-01

    The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.

  2. Protein-Tyrosine Phosphatase 1B Substrates and Metabolic Regulation

    PubMed Central

    Bakke, Jesse; Haj, Fawaz G.

    2014-01-01

    Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell-cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes. PMID:25263014

  3. Methods to monitor classical protein-tyrosine phosphatase oxidation

    PubMed Central

    Karisch, Robert; Neel, Benjamin G.

    2012-01-01

    SUMMARY Reactive oxygen species (ROS), particularly H2O2, act as intracellular second messengers in many signaling pathways. Protein-tyrosine phosphatases (PTPs) are now believed to be important targets of ROS. PTPs contain a conserved catalytic cysteine with an unusually low pKa. This property allows PTPs to execute nucleophilic attack on substrate phosphotyrosyl residues, but also renders them highly susceptible to oxidation. Reversible oxidation, which inactivates PTPs, is emerging as an important cellular regulatory mechanism and might contribute to human diseases, including cancer. Given their potential toxicity, it seems likely that ROS generation is highly controlled within cells to restrict oxidation to those PTPs that must be inactivated for signaling to proceed. Thus, identifying ROS-inactivated PTPs could be tantamount to finding the PTP(s) that critically regulate a specific signaling pathway. This article provides an overview of the methods currently available to identify and quantify PTP oxidation and outlines future challenges in redox signaling. PMID:22577968

  4. Induction of protein tyrosine phosphorylation in macrophages incubated with tumor cells.

    PubMed

    Sodhi, A; Shrivastava, A; Kumar, R

    1995-03-01

    The cellular and molecular interaction between monocyte/macrophage and tumor cells leading to macrophage activation is not clearly understood. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event, we checked whether the tumor cells alter tyrosine phosphorylation of proteins in macrophages. We found that both L929 and Yac-1 tumor cells induced increased tyrosine phosphorylation of several polypeptides in peritoneal as well as P388D-1 and IC-21 macrophages. Macrophages co-cultured with tumor cells also showed increased fluorescence with anti-phosphotyrosine-FITC antibody. These observations suggest that increased tyrosine phosphorylation plays a role in tumor cell-induced activation of macrophages. PMID:7539664

  5. Nitration of JAK-2 at the 1007Y-1008Y activation epitope impedes phosphorylation at this site: defining a GH, AKT/protein kinase B and nitric oxide synthase axis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Generalized liver protein tyrosine nitration (3’-nitrotyrosine, 3’-NT) increases in vivo after GH injection with immunohistocellular patterns strikingly similar to those we observed for a specific nitration of JAK2 at its 1007Y-1008Y regulatory phosphorylation epitope following proinflammatory chall...

  6. Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement.

    PubMed

    Vattemi, Gaetano; Mechref, Yehia; Marini, Matteo; Tonin, Paola; Minuz, Pietro; Grigoli, Laura; Guglielmi, Valeria; Klouckova, Iveta; Chiamulera, Cristiano; Meneguzzi, Alessandra; Di Chio, Marzia; Tedesco, Vincenzo; Lovato, Laura; Degan, Maurizio; Arcaro, Guido; Lechi, Alessandro; Novotny, Milos V; Tomelleri, Giuliano

    2011-04-01

    Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress. PMID:21156839

  7. Increased Protein Nitration in Mitochondrial Diseases: Evidence for Vessel Wall Involvement

    PubMed Central

    Vattemi, Gaetano; Mechref, Yehia; Marini, Matteo; Tonin, Paola; Minuz, Pietro; Grigoli, Laura; Guglielmi, Valeria; Klouckova, Iveta; Chiamulera, Cristiano; Meneguzzi, Alessandra; Di Chio, Marzia; Tedesco, Vincenzo; Lovato, Laura; Degan, Maurizio; Arcaro, Guido; Lechi, Alessandro; Novotny, Milos V.; Tomelleri, Giuliano

    2011-01-01

    Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress. PMID:21156839

  8. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders. PMID:26975317

  9. Related proteins are phosphorylated at tyrosine in response to mitogenic stimuli and at meiosis.

    PubMed Central

    Cooper, J A

    1989-01-01

    Forty-two-kilodalton proteins that contain phosphotyrosine in metaphase-arrested Xenopus laevis eggs are closely related to p42, a protein that is phosphorylated at tyrosine when somatic cells are exposed to mitogenic stimuli. Images PMID:2779558

  10. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    PubMed

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  11. Effects of short-term tocopherol (T) feeding on nitric oxide production and protein nitration following endotoxin (LPS) challenge in beef calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Posttranslational protein tyrosine nitration (pNT) contributes to functional tissue damage during pro-inflammatory stress. With regard to chemical reactivity, a-T has a greater antioxidant potential while '-T has greater ability to inactivate reactive oxynitrogen species potentially involved in pTN ...

  12. Protein tyrosine phosphatases expression during development of mouse superior colliculus.

    PubMed

    Reinhard, Jacqueline; Horvat-Bröcker, Andrea; Illes, Sebastian; Zaremba, Angelika; Knyazev, Piotr; Ullrich, Axel; Faissner, Andreas

    2009-12-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis. PMID:19727691

  13. Redox and zinc signalling pathways converging on protein tyrosine phosphatases.

    PubMed

    Bellomo, Elisa; Hogstrand, Christer; Maret, Wolfgang

    2014-10-01

    Zinc ions, though redox-inert, have either pro-antioxidant or pro-oxidant functions at critical junctures in redox metabolism and redox signalling. They are released from cells and in cells, e.g. from metallothionein, a protein that transduces redox signals into zinc signals (1). The released zinc ions inhibit enzymes such as protein tyrosine phosphatases (PTPs), key regulatory enzymes of cellular phosphorylation signalling. The Ki(Zn) value for inhibition of receptor PTPB is 21pM (2). The binding is about as tight as the binding of zinc to zinc metalloenzymes and suggests tonic zinc inhibition. PTP1-B (PTPN1), an enzyme regulating the insulin and leptin receptors and involved in cancer and diabetes pathobiochemistry, has a Ki(Zn) value of about 5nM (3). Zinc ions bind to the enzyme in the closed conformation when additional metal-binding ligands are brought into the vicinity of the active site. In contrast, redox reactions target cysteines in the active sites of PTPs in the open conformation. This work provides a molecular basis how hydrogen peroxide and free zinc ions generated by growth factor signalling stimulate phosphorylation signalling differentially. (Supported by the Biotechnology and Biological Sciences Research Council UK, grant BB/K001442/1.). PMID:26461422

  14. Mechanism of the Reaction of Human Manganese Superoxide Dismutase with Peroxynitrite: Nitration of Critical Tyrosine 34.

    PubMed

    Demicheli, Verónica; Moreno, Diego M; Jara, Gabriel E; Lima, Analía; Carballal, Sebastián; Ríos, Natalia; Batthyany, Carlos; Ferrer-Sueta, Gerardo; Quijano, Celia; Estrı́n, Darío A; Martí, Marcelo A; Radi, Rafael

    2016-06-21

    Human Mn-containing superoxide dismutase (hMnSOD) is a mitochondrial enzyme that metabolizes superoxide radical (O2(•-)). O2(•-) reacts at diffusional rates with nitric oxide to yield a potent nitrating species, peroxynitrite anion (ONOO(-)). MnSOD is nitrated and inactivated in vivo, with active site Tyr34 as the key oxidatively modified residue. We previously reported a k of ∼1.0 × 10(5) M(-1) s(-1) for the reaction of hMnSOD with ONOO(-) by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In this study, we further established the mechanism of the reaction of hMnSOD with ONOO(-), including the necessary re-examination of the second-order rate constant by an independent method and the delineation of the microscopic steps that lead to the regio-specific nitration of Tyr34. The redetermination of k was performed by competition kinetics utilizing coumarin boronic acid, which reacts with ONOO(-) at a rate of ∼1 × 10(6) M(-1) s(-1) to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a k of ∼1.0 × 10(5) M(-1) s(-1), fully consistent with the direct method. Proteomic analysis indicated that ONOO(-), but not other nitrating agents, mediates the selective modification of active site Tyr34. Hybrid quantum-classical (quantum mechanics/molecular mechanics) simulations supported a series of steps that involve the initial reaction of ONOO(-) with Mn(III) to yield Mn(IV) and intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis for rationalizing how MnSOD constitutes an intramitochondrial target for ONOO(-) and the microscopic events, with atomic level resolution, that lead to selective and efficient nitration of critical Tyr34. PMID:27227512

  15. Phosphonate derivatives of tetraazamacrocycles as new inhibitors of protein tyrosine phosphatases.

    PubMed

    Kobzar, Oleksandr L; Shevchuk, Michael V; Lyashenko, Alesya N; Tanchuk, Vsevolod Yu; Romanenko, Vadim D; Kobelev, Sergei M; Averin, Alexei D; Beletskaya, Irina P; Vovk, Andriy I; Kukhar, Valery P

    2015-07-21

    α,α-Difluoro-β-ketophosphonated derivatives of tetraazamacrocycles were synthesized and found to be potential inhibitors of protein tyrosine phosphatases. N-Substituted conjugates of cyclam and cyclen with bioisosteric phosphonate groups displayed good activities toward T-cell protein tyrosine phosphatase with IC50 values in the micromolar to nanomolar range and showed selectivity over PTP1B, CD45, SHP2, and PTPβ. Kinetic studies indicated that the inhibitors can occupy the region of the active site of TC-PTP. This study demonstrates a new approach which employs tetraazamacrocycles as a molecular platform for designing inhibitors of protein tyrosine phosphatases. PMID:26058329

  16. New functional aspects of the atypical protein tyrosine phosphatase VHZ

    PubMed Central

    Kuznetsov, Vyacheslav I.; Hengge, Alvan C.

    2013-01-01

    LDP3 (VHZ) is the smallest classical protein tyrosine phosphatase (PTP) known to date, and was originally misclassified as an atypical dual specificity phosphatase (DSP). Kinetic isotope effects with steady state and pre-steady state kinetics of VHZ and mutants with para-nitrophenol phosphate (pNPP) have revealed several unusual properties. VHZ is significantly more active than previously reported, but remains one of the least active PTPs. Highly unusual for a PTP, VHZ possesses two acidic residues (E134 and D65) in the active site. D65 occupies the position corresponding to the typical general acid in the PTP family. However, VHZ primarily utilizes E134 as the general acid, with D65 taking over this role when E134 is mutated. This unusual behavior is facilitated by two coexisting, but unequally populated, substrate binding modes. Unlike most classical PTPs, VHZ exhibits phosphotransferase activity. Despite the presence of the Q-loop that normally prevents alcoholysis of the phosphoenzyme intermediate in other classical PTPs, VHZ readily phosphorylates ethylene glycol. Although mutations to Q-loop residues affect this phosphotransferase activity, mutations on the IPD-loop that contains the general acid exert more control over this process. A single P68V substitution on this loop completely abolishes phosphotransferase activity. The ability of native VHZ to catalyze transphosphorylation may lead to an imbalance of intracellular phosphorylation, which could explain the correlation of its overexpression with several types of cancer. PMID:24073992

  17. Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana.

    PubMed

    Shah, Muhammad Raza; Ishtiaq; Hizbullah, Syed Muhammad; Habtemariam, Solomon; Zarrelli, Armando; Muhammad, Akhtar; Collina, Simona; Khan, Inamulllah

    2016-08-01

    Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC50 values 3.49 ± 0.02, 4.17 ± 0.03 and 87.52 ± 0.03 µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds. PMID:26118418

  18. Protein Tyrosine Phosphatase φ Regulates Paxillin Tyrosine Phosphorylation and Mediates Colony-Stimulating Factor 1-Induced Morphological Changes in Macrophages

    PubMed Central

    Pixley, Fiona J.; Lee, Pierre S. W.; Condeelis, John S.; Stanley, E. Richard

    2001-01-01

    Removal of colony-stimulating factor 1 (CSF-1) causes macrophages to round up and to increase their expression of protein tyrosine phosphatase φ (PTPφ). This is accompanied by the disruption of focal complexes and the formation of ruffles. Here we have overexpressed wild-type (WT) PTPφ and a phosphatase-inactive (C325S) mutant in a macrophage cell line in the presence and absence of CSF-1. In the presence of CSF-1, WT PTPφ induces cell rounding and ruffle formation, while C325S PTPφ has no effect. In contrast, in CSF-1-starved cells, C325S PTPφ behaves in a dominant negative fashion, preventing rounding and ruffling. Furthermore, C325S PTPφ increases adhesion in cycling cells, while WT PTPφ enhances motility. In WT PTPφ-overexpressing cells, the focal contact protein paxillin is selectively depleted from focal complexes and specifically dephosphorylated on tyrosine. In contrast, paxillin is hyperphosphorylated in C325S PTPφ-expressing cells. Moreover, a complex containing PTPφ, paxillin, and a paxillin-associated tyrosine kinase, Pyk2, can be immunoprecipitated from macrophage lysates, and the catalytic domain of PTPφ selectively binds paxillin and Pyk2 in vitro. Although PTPφ and Pyk2 do not colocalize with paxillin in focal complexes, all three proteins are colocalized in dorsal ruffles. The results suggest that paxillin is dephosphorylated by PTPφ in dorsal ruffles, using Pyk2 as a bridging molecule, resulting in a reduced pool of tyrosine-phosphorylated paxillin available for incorporation into focal complexes, thereby mediating CSF-1 regulation of macrophage morphology, adhesion, and motility. PMID:11238916

  19. Pharmacophore modeling for protein tyrosine phosphatase 1B inhibitors.

    PubMed

    Bharatham, Kavitha; Bharatham, Nagakumar; Lee, Keun Woo

    2007-05-01

    A three dimensional chemical feature based pharmacophore model was developed for the inhibitors of protein tyrosine phosphatase 1B (PTP1B) using the CATALYST software, which would provide useful knowledge for performing virtual screening to identify new inhibitors targeted toward type II diabetes and obesity. A dataset of 27 inhibitors, with diverse structural properties, and activities ranging from 0.026 to 600 microM, was selected as a training set. Hypol, the most reliable quantitative four featured pharmacophore hypothesis, was generated from a training set composed of compounds with two H-bond acceptors, one hydrophobic aromatic and one ring aromatic features. It has a correlation coefficient, RMSD and cost difference (null cost-total cost) of 0.946, 0.840 and 65.731, respectively. The best hypothesis (Hypol) was validated using four different methods. Firstly, a cross validation was performed by randomizing the data using the Cat-Scramble technique. The results confirmed that the pharmacophore models generated from the training set were valid. Secondly, a test set of 281 molecules was scored, with a correlation of 0.882 obtained between the experimental and predicted activities. Hypol performed well in correctly discriminating the active and inactive molecules. Thirdly, the model was investigated by mapping on two PTP1B inhibitors identified by different pharmaceutical companies. The Hypol model correctly predicted these compounds as being highly active. Finally, docking simulations were performed on few compounds to substantiate the role of the pharmacophore features at the binding site of the protein by analyzing their binding conformations. These multiple validation approaches provided confidence in the utility of this pharmacophore model as a 3D query for virtual screening to retrieve new chemical entities showing potential as potent PTP1B inhibitors. PMID:17615669

  20. Cellular Biochemistry Methods for Investigating Protein Tyrosine Phosphatases

    PubMed Central

    Stanford, Stephanie M.; Ahmed, Vanessa

    2014-01-01

    Abstract Significance: The protein tyrosine phosphatases (PTPs) are a family of proteins that play critical roles in cellular signaling and influence many aspects of human health and disease. Although a wealth of information has been collected about PTPs since their discovery, many questions regarding their regulation and function still remain. Critical Issues: Of particular importance are the elucidation of the biological substrates of individual PTPs and understanding of the chemical and biological basis for temporal and spatial resolution of PTP activity within a cell. Recent Advances: Drawing from recent advances in both biology and chemistry, innovative approaches have been developed to study the intracellular biochemistry and physiology of PTPs. We provide a summary of PTP-tailored techniques and approaches, emphasizing methodologies to study PTP activity within a cellular context. We first provide a discussion of methods for identifying PTP substrates, including substrate-trapping mutants and synthetic peptide libraries for substrate selectivity profiling. We next provide an overview of approaches for monitoring intracellular PTP activity, including a discussion of mechanistic-based probes, gel-based assays, substrates that can be used intracellularly, and assays tied to cell growth. Finally, we review approaches used for monitoring PTP oxidation, a key regulatory pathway for these enzymes, discussing the biotin switch method and variants of this approach, along with affinity trapping techniques and probes designed to detect PTP oxidation. Future Directions: Further development of approaches to investigate the intracellular PTP activity and functions will provide specific insight into their mechanisms of action and control of diverse signaling pathways. Antioxid. Redox Signal. 20, 2160–2178. PMID:24294920

  1. Cementum attachment protein/protein-tyrosine phosphotase-like member A is not expressed in teeth.

    PubMed

    Schild, Christof; Beyeler, Michael; Lang, Niklaus P; Trueb, Beat

    2009-02-01

    Cementum is a highly specialized connective tissue that covers tooth roots. The only cementum-specific protein described to date is the cementum attachment protein (CAP). A putative sequence for CAP was established from a cDNA clone isolated from a human cementifying fibroma cDNA library. This sequence overlaps with a phosphatase-like protein in muscle termed the protein-tyrosine phosphatase-like member A (PTPLA). To clarify the nature of CAP/PTPLA, we cloned the homologous rat protein and determined its sequence. The rat protein shared 94% sequence identity with the human protein. On Northern blots containing RNA from various rat tissues of different developmental stages, the cDNA hybridized to an mRNA expressed in heart and skeletal muscle but not in teeth. These results were confirmed by real-time PCR. Thus, the sequence deposited in public databanks under the name 'cementum attachment protein' does not represent genuine CAP. PMID:19148556

  2. Cadmium inhibits mouse sperm motility through inducing tyrosine phosphorylation in a specific subset of proteins.

    PubMed

    Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Zhao, Na; Yang, Qiangzhen; Li, Sisi; Li, Xinhong

    2016-08-01

    Cadmium (Cd) has been reported to impair male fertility, primarily by disrupting sperm motility, but the underlying molecular mechanism remains unclear. Here we investigated the effects of Cd on sperm motility, tyrosine phosphorylation, AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, and ATP levels in vitro. Our results demonstrated that Cd inhibited sperm motility, GAPDH activity, AMPK activity and ATP production, and induced tyrosine phosphorylation of 55-57KDa proteins. Importantly, all the parameters affected by Cd were restored to normal levels when incubated with 10μM Cd in the presence of 30μM ethylene diamine tetraacetic acid (EDTA). Interestingly, changes of tyrosine phosphorylation levels of 55-57KDa proteins are completely contrary to that of other parameters. These results suggest that Cd-induced tyrosine phosphorylation of 55-57KDa proteins might act as an engine to block intracellular energy metabolism and thus decrease sperm motility. PMID:27233480

  3. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  4. Fluorogenic tagging of protein 3-nitrotyrosine with 4-(aminomethyl)benzene sulfonate in tissues: a useful alternative to Immunohistochemistry for fluorescence microscopy imaging of protein nitration.

    PubMed

    Sharov, V S; Pal, R; Dremina, E S; Michaelis, E K; Schöneich, C

    2012-11-15

    Protein tyrosine nitration is a common biomarker of biological aging and diverse pathologies associated with the excessive formation of reactive oxygen and nitrogen species. Recently, we suggested a novel fluorogenic derivatization procedure for the detection of 3-nitrotyrosine (3-NT) using benzylamine derivatives to convert specifically protein- or peptide-bound 3-NT to a highly fluorescent benzoxazole product. In this study, we applied this procedure to fluorogenic derivatization of protein 3-NT in sections from adult rat cerebellum to: (i) test this method for imaging nitrated proteins in fixed brain tissue sections and (ii) compare the chemical approach to immunohistochemical labeling with anti-3-NT antibodies. Immunofluorescence analysis of cerebellar sections using anti-3-NT antibodies showed differential levels of immunostaining in the molecular, Purkinje, and granule cell layers of the cerebellar cortex; in agreement with previous reports, the Purkinje cells were most highly labeled. Importantly, fluorogenic derivatization reactions of cerebellar proteins with 4-(aminomethyl)benzene sulfonic acid (ABS) and K(3)Fe(CN)(6) at pH 9, after sodium dithionite reduction of 3-NT to 3-aminotyrosine, showed a very similar pattern of relative intensity of cell labeling and improved resolution compared with antibody labeling. Our data demonstrate that ABS derivatization may be either a useful alternative to or a complementary approach to immunolabeling in imaging protein nitration in cells and tissues, including under conditions of dual labeling with antibodies to cell proteins, thus allowing for cellular colocalization of nitrated proteins and any protein of interest. PMID:22995636

  5. Analysis of free and protein-bound nitrotyrosine in human plasma by a gas chromatography/mass spectrometry method that avoids nitration artifacts.

    PubMed Central

    Frost, M T; Halliwell, B; Moore, K P

    2000-01-01

    Measurement of nitrotyrosine in biological fluids and tissues is increasingly being used to monitor the production of reactive nitrogen species in vivo. The detection of nitrotyrosine in vivo has been reported with the use of a variety of methods including immunoassay, HPLC and GLC/MS. The validity of HPLC and immunoassays have been questioned with regard to their selectivity and sensitivity limits. In principle, the measurement of nitrotyrosine by GLC/MS permits a highly specific, highly sensitive and fully quantitative assay. The nitration of tyrosine under acidic conditions in the presence of nitrite is well documented. Derivatization for the full quantification of nitrotyrosine by using GLC/MS can lead to the artifactual nitration of tyrosine if performed under acidic conditions in the presence of nitrite. We describe a novel alkaline method for the hydrolysis and derivatization of nitrotyrosine and tyrosine, and demonstrate its applicability to the measurement of plasma concentrations of both free and protein-bound nitrotyrosine and tyrosine. A detection limit of 1 pg for nitrotyrosine and 100 pg for tyrosine has been achieved. Our method allows, for the first time, the analysis of free and protein-bound nitrotyrosine and tyrosine in biological samples. The plasma concentrations (means+/-S.E.M.) of free tyrosine and nitrotyrosine in eight normal subjects were 12+/-0.6 microg/ml and 14+/-0.7 ng/ml respectively. Plasma proteins contained tyrosine and nitrotyrosine at 60.7+/-1.7 microg/mg and 2.7+/-0.4 ng/mg respectively. PMID:10642501

  6. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    PubMed Central

    Mijakovic, Ivan; Petranovic, Dina; Macek, Boris; Cepo, Tina; Mann, Matthias; Davies, Julian; Jensen, Peter R.; Vujaklija, Dusica

    2006-01-01

    Single-stranded DNA-binding proteins (SSBs) are required for repair, recombination and replication in all organisms. Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues. To our knowledge, phosphorylation of SSBs in bacteria has not been reported. A systematic search for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (ScSSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation of SSBs is a conserved process of post-translational modification in taxonomically distant bacteria. PMID:16549871

  7. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase.

    PubMed

    Schumacher, Dominik; Helma, Jonas; Mann, Florian A; Pichler, Garwin; Natale, Francesco; Krause, Eberhard; Cardoso, M Cristina; Hackenberger, Christian P R; Leonhardt, Heinrich

    2015-11-01

    A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin. PMID:26404067

  8. The role of non-receptor protein tyrosine kinases in the excitotoxicity induced by the overactivation of NMDA receptors.

    PubMed

    Sun, Yongjun; Chen, You; Zhan, Liying; Zhang, Linan; Hu, Jie; Gao, Zibin

    2016-04-01

    Protein tyrosine phosphorylation is one of the primary modes of regulation of N-methyl-d-aspartate (NMDA) receptors. The non-receptor tyrosine kinases are one of the two types of protein tyrosine kinases that are involved in this process. The overactivation of NMDA receptors is a primary reason for neuron death following cerebral ischemia. Many studies have illustrated the important role of non-receptor tyrosine kinases in ischemia insults. This review introduces the roles of Src, Fyn, focal adhesion kinase, and proline-rich tyrosine kinase 2 in the excitotoxicity induced by the overactivation of NMDA receptors following cerebral ischemia. PMID:26540220

  9. Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability

    PubMed Central

    Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela

    2014-01-01

    Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ−/− mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ−/− mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ−/− mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper–tyrosine-phosphorylated in the PTPσ−/− mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ−/− mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD. PMID:24385580

  10. A Multifeatures Fusion and Discrete Firefly Optimization Method for Prediction of Protein Tyrosine Sulfation Residues.

    PubMed

    Guo, Song; Liu, Chunhua; Zhou, Peng; Li, Yanling

    2016-01-01

    Tyrosine sulfation is one of the ubiquitous protein posttranslational modifications, where some sulfate groups are added to the tyrosine residues. It plays significant roles in various physiological processes in eukaryotic cells. To explore the molecular mechanism of tyrosine sulfation, one of the prerequisites is to correctly identify possible protein tyrosine sulfation residues. In this paper, a novel method was presented to predict protein tyrosine sulfation residues from primary sequences. By means of informative feature construction and elaborate feature selection and parameter optimization scheme, the proposed predictor achieved promising results and outperformed many other state-of-the-art predictors. Using the optimal features subset, the proposed method achieved mean MCC of 94.41% on the benchmark dataset, and a MCC of 90.09% on the independent dataset. The experimental performance indicated that our new proposed method could be effective in identifying the important protein posttranslational modifications and the feature selection scheme would be powerful in protein functional residues prediction research fields. PMID:27034949

  11. A Multifeatures Fusion and Discrete Firefly Optimization Method for Prediction of Protein Tyrosine Sulfation Residues

    PubMed Central

    Liu, Chunhua; Zhou, Peng; Li, Yanling

    2016-01-01

    Tyrosine sulfation is one of the ubiquitous protein posttranslational modifications, where some sulfate groups are added to the tyrosine residues. It plays significant roles in various physiological processes in eukaryotic cells. To explore the molecular mechanism of tyrosine sulfation, one of the prerequisites is to correctly identify possible protein tyrosine sulfation residues. In this paper, a novel method was presented to predict protein tyrosine sulfation residues from primary sequences. By means of informative feature construction and elaborate feature selection and parameter optimization scheme, the proposed predictor achieved promising results and outperformed many other state-of-the-art predictors. Using the optimal features subset, the proposed method achieved mean MCC of 94.41% on the benchmark dataset, and a MCC of 90.09% on the independent dataset. The experimental performance indicated that our new proposed method could be effective in identifying the important protein posttranslational modifications and the feature selection scheme would be powerful in protein functional residues prediction research fields. PMID:27034949

  12. 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...

  13. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells

    PubMed Central

    Xie, Yajun; Wang, Jinlong; Zhang, Yuanya; Liu, Xiaofei; Wang, Xiaorong; Liu, Kehui; Huang, Xiahe; Wang, Yingchun

    2016-01-01

    Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading. PMID:27554326

  14. Detection and characterization of in vivo nitration and oxidation of tryptophan residues in proteins.

    PubMed

    Bregere, Catherine; Rebrin, Igor; Sohal, Rajindar S

    2008-01-01

    Oxygen and nitrogen centered reactive species can cause specific structural modifications in amino acids and proteins, such as the addition of a nitro group onto aromatic residues. Heretofore, studies on protein nitration have mainly focused on the in vitro and in vivo nitro addition to tyrosine residues (3-nitrotyrosine or 3NT), whereas the formation of nitrotryptophan in proteins in vivo and/or its functional significance has remained quite obscure. A novel structural modification, involving the addition of nitro and hydroxy groups to tryptophan, has been detected in the mitochondrial protein succinyl-CoA:3-oxoacid CoA transferase (SCOT) in rat heart. Modified SCOT accumulated progressively with age, which was associated with an elevation of its activity. The specific biochemical properties of this novel amino acid were characterized by a combination of HPLC-electrochemical detection and mass spectrometric analysis. This chapter describes the experimental steps involved in the characterizations and a procedure for the synthesis of nitrohydroxytryptophan. Similar methodology can be applied to the identification of nitrohydroxytryptophan in other proteins. PMID:18554544

  15. Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

    SciTech Connect

    Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

    1986-05-01

    A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn/sup 2 +/ and (..gamma..-/sup 32/P)ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes.

  16. DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

    PubMed Central

    Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

    2011-01-01

    The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

  17. Tyrosine phosphorylation of band 3 protein in Ca2+/A23187-treated human erythrocytes.

    PubMed Central

    Minetti, G; Piccinini, G; Balduini, C; Seppi, C; Brovelli, A

    1996-01-01

    Human erythrocytes were induced to release membrane vesicles by treatment with Ca2+ and ionophore A23187. In addition to the biochemical changes already known to accompany loading of human erythrocytes with Ca2+, the present study reveals that tyrosine phosphorylation of the anion exchanger band 3 protein also occurs. The relationship between tyrosine phosphorylation of band 3 and membrane vesiculation was analysed using quinine (a non-specific inhibitor of the Ca(2+)-activated K+ channel, and the only known inhibitor of Ca(2+)-induced vesiculation) and charybdotoxin, a specific inhibitor of the apamin-insensitive K(+)-channel. Both inhibitors suppressed tyrosine phosphorylation of band 3. In the presence of quinine, membrane vesiculation was also suppressed. In contrast, at the concentration of charybdotoxin required to suppress tyrosine phosphorylation of band 3, membrane vesiculation was only mildly inhibited (16-23% inhibition), suggesting that tyrosine phosphorylation of band 3 is not necessary for membrane vesiculation. Phosphorylation of band 3 was in fact observed when erythrocytes were induced to shrink in a Ca(2+)-independent manner, e.g. by treatment with the K+ ionophore valinomycin or with hypertonic solutions. These observations suggest that band 3 tyrosine phosphorylation occurs when cell volume regulation is required. PMID:8973551

  18. Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction

    PubMed Central

    Teramoto, Takamasa; Fujikawa, Yukari; Kawaguchi, Yoshirou; Kurogi, Katsuhisa; Soejima, Masayuki; Adachi, Rumi; Nakanishi, Yuichi; Mishiro-Sato, Emi; Liu, Ming-Cheh; Sakakibara, Yoichi; Suiko, Masahito; Kimura, Makoto; Kakuta, Yoshimitsu

    2013-01-01

    Post-translational protein modification by tyrosine-sulfation plays an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalyzed by the Golgi-enzyme called the tyrosylprotein sulfotransferase (TPST). To date, no crystal structure is available for TPST. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human TPST isoform 2 (TPST2) complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3’-phosphoadenosine-5’-phosphate (PAP) at 1.9Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. TPST2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel β-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the TPST2 structure resembles that observed for the receptor type tyrosine kinases. PMID:23481380

  19. Thrombin Ca(2+)-dependently stimulates protein tyrosine phosphorylation in BC3H1 muscle cells.

    PubMed Central

    Offermanns, S; Bombien, E; Schultz, G

    1993-01-01

    The proteinase thrombin, known to act via heptahelical G-protein-coupled receptors, is a mitogenic agent for different cell types, including the mouse muscle cell line BC3H1. In this study, the effect of thrombin on tyrosine phosphorylation was examined using anti-phosphotyrosine antibodies. Thrombin was found to induce phosphorylation of 65-70 and 110-120 kDa proteins in BC3H1 cells. The effect of thrombin was concentration-dependent, being half-maximal and maximal at concentrations of 0.03 and 1 unit/ml respectively. The thrombin-induced increase in phosphorylation was rapid (< or = 10 s) and transient, with a peak response after about 1-2 min. The effect of thrombin could be mimicked by the thrombin receptor agonist peptide SFLLRN-NH2. Preincubation of cells with pertussis toxin (PT) had no effect on thrombin-induced tyrosine phosphorylation. Epidermal growth factor, platelet-derived growth factor and insulin stimulated tyrosine phosphorylation of different proteins, among which were 65-70 and 110-120 kDa proteins. The phorbol ester 12-myristate 13-acetate (PMA) as well as the Ca2+ ionophore A23187 both stimulated tyrosine phosphorylation of proteins identical to those phosphorylated by thrombin, suggesting that activation of protein kinase C (PKC) and elevation of the cytosolic Ca2+ concentration alone are sufficient to induce tyrosine phosphorylation. However, calphostin C and other PKC inhibitors, which completely inhibited tyrosine phosphorylation induced by PMA, had no influence on the effect of thrombin, whereas loading of cells with the intracellular Ca2+ chelator bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid totally blocked thrombin-stimulated tyrosine phosphorylation. Thus tyrosine phosphorylation stimulated by thrombin is an early PT-insensitive cellular response which is either directly mediated by elevation of cytosolic Ca2+ concentration or by a presently unknown mechanism that requires an elevated cytosolic Ca2+ concentration. Images Figure 1

  20. Tissue protein nitration and peripheral blood endotoxin activity are indicative of the severity of systemic organ compromise in naturally-occurring clinical cases of bacterial mastitis in Holstein dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this survey study was to determine a relationship between the intensity of tissue protein tyrosine nitration measured in samples of mammary gland, liver, pancreas and lung compared to estimated blood endotoxin (LPS) activity. Blood was collected from nine multiparous Holstein cows...

  1. Activation of a protein tyrosine phosphatase and inactivation of Raf-1 by somatostatin.

    PubMed Central

    Reardon, D B; Wood, S L; Brautigan, D L; Bell, G I; Dent, P; Sturgill, T W

    1996-01-01

    Human somatostatin receptor 3 ('hsstr3') was transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras (G12V). Somatostatin activated a protein tyrosine phosphatase and inactivated the constitutively active, membrane-associated form of the Raf-1 serine kinase present in these cells in vivo and in vitro. PMID:8670047

  2. MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    EPA Science Inventory

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

  3. Hydroxyindole Carboxylic Acid-Based Inhibitors for Receptor-Type Protein Tyrosine Protein Phosphatase Beta

    PubMed Central

    Zeng, Li-Fan; Zhang, Ruo-Yu; Bai, Yunpeng; Wu, Li; Gunawan, Andrea M.

    2014-01-01

    Abstract Aims: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. Results: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPβ), a potential target that is implicated in blood vessel development. The representative RPTPβ inhibitor 8b-1 (L87B44) has an IC50 of 0.38 μM and at least 14-fold selectivity for RPTPβ over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. Innovation: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Conclusion: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity. Antioxid. Redox Signal. 20, 2130–2140. PMID:24180557

  4. Nitrate

    Integrated Risk Information System (IRIS)

    Nitrate ; CASRN 14797 - 55 - 8 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effects

  5. Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

    PubMed Central

    Novotna, Jitka; Olsovska, Jana; Novak, Petr; Mojzes, Peter; Chaloupkova, Radka; Kamenik, Zdenek; Spizek, Jaroslav; Kutejova, Eva; Mareckova, Marketa; Tichy, Pavel; Damborsky, Jiri; Janata, Jiri

    2013-01-01

    The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis. PMID:24324587

  6. Mushroom tyrosinase oxidizes tyrosine-rich sequences to allow selective protein functionalization.

    PubMed

    Long, Marcus J C; Hedstrom, Lizbeth

    2012-08-13

    We show that mushroom tyrosinase catalyzes the formation of reactive o-quinones on unstructured, tyrosine-rich sequences such as hemagglutinin (HA) tags (YPYDVPDYA). In the absence of exogenous nucleophiles and at low protein concentrations, the o-quinone decomposes with fragmentation of the HA tag. At higher protein concentrations (>5 mg mL⁻¹), crosslinking is observed. Besthorn's reagent intercepts the o-quinone to give a characteristic pink complex that can be observed directly on a denaturing SDS-PAGE gel. Similar labeled species can be formed by using other nucleophiles such as Cy5-hydrazide. These reactions are selective for proteins bearing HA and other unstructured poly-tyrosine-containing tags and can be performed in lysates to create specifically tagged proteins. PMID:22807021

  7. Mushroom Tyrosinase Oxidizes Tyrosine-rich Sequences, Allowing Selective Protein Functionalization

    PubMed Central

    Long, Marcus J. C.

    2012-01-01

    We show that mushroom tyrosinase catalyzes formation of reactive o-quinones on unstructured, tyrosine-rich sequences such as hemagglutinin (HA)-tags (YPYDVPDYA). In the absence of exogenous nucleophiles and at low protein concentrations, the o-quinone decomposes with fragmentation of the HA-tag. At higher protein concentrations (>5 mg/ml), cross-linking is observed. Besthorn’s reagent intercepts the o-quinone to give a characteristic pink complex, which can be observed directly on a denaturing SDS-PAGE gel. Similar labeled species can be formed using other nucleophiles such as Cy5-hydrazide. These reactions are selective for proteins bearing HA- and other unstructured poly-tyrosine-containing tags and can be performed in lysates to create specifically tagged proteins. PMID:22807021

  8. Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis.

    PubMed

    Dixelius, J; Larsson, H; Sasaki, T; Holmqvist, K; Lu, L; Engström, A; Timpl, R; Welsh, M; Claesson-Welsh, L

    2000-06-01

    Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)-induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells. PMID:10828022

  9. An immunochemical approach to detect oxidized protein tyrosine phosphatases using a selective C-nucleophile tag.

    PubMed

    Garcia, Francisco J; Carroll, Kate S

    2016-05-24

    Protein tyrosine phosphatases are crucial regulators of signal transduction and function as antagonists towards protein tyrosine kinases to control reversible tyrosine phosphorylation, thereby regulating fundamental physiological processes. Growing evidence has supported the notion that reversible oxidative inactivation of the catalytic cysteine residue in protein tyrosine phosphatases serves as an oxidative post-translational modification that regulates its activity to influence downstream signaling by promoting phosphorylation and induction of the signaling cascade. The oxidation of cysteine to the sulfenic acid is often transient and difficult to detect, thus making it problematic in understanding the role that this oxidative post-translational modification plays in redox-biology and pathogenesis. Several methods to detect cysteine oxidation in biological systems have been developed, though targeted approaches to directly detect oxidized phosphatases are still lacking. Herein we describe the development of a novel immunochemical approach to directly profile oxidized phosphatases. This immunochemical approach consists of an antibody designed to recognize the conserved sequence of the PTP active site (VHCDMDSAG) harboring the catalytic cysteine modified with dimedone (CDMD), a nucleophile that chemoselectively reacts with cysteine sulfenic acids to form a stable thioether adduct. Additionally, we provide biochemical and mass spectrometry workflows to be used in conjugation with this newly developed immunochemical approach to assist in the identification and quantification of basal and oxidized phosphatases. PMID:26757830

  10. Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

    PubMed

    Uhrig, R Glen; Labandera, Anne-Marie; Muhammad, Jamshed; Samuel, Marcus; Moorhead, Greg B

    2016-03-11

    Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants. PMID:26742850

  11. MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase.

    PubMed

    Rai, Reeta; Singh, Neetu; Elesela, Srikanth; Tiwari, Savitri; Rathaur, Sushma

    2013-01-01

    A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP. PMID:23052758

  12. Robust protein nitration contributes to acetaminophen-induced mitochondrial dysfunction and acute liver injury

    PubMed Central

    Abdelmegeed, Mohamed A.; Jang, Sehwan; Banerjee, Atrayee; Hardwick, James P.; Song, Byoung-Joon

    2013-01-01

    Acetaminophen (APAP), a widely-used analgesic agent, can cause liver injury through increased nitrative stress, leading to protein nitration. However, the identities of nitrated proteins and their roles in hepatotoxicity are poorly understood. Thus, we aimed at studying the mechanism of APAP-induced hepatotoxicity by systematic identification and characterization of nitrated proteins in the absence or presence of an anti-oxidant N-acetylcysteine (NAC). The levels of nitrated proteins markedly increased at 2 h in mice exposed to a single APAP dose (350 mg/kg ip), which caused severe liver necrosis at 24 h. Protein nitration and liver necrosis were minimal in mice exposed to nontoxic 3-hydroxyacetanilide or animals co-treated with APAP and NAC. Mass-spectral analysis of the affinity-purified nitrated proteins identified numerous mitochondrial and cytosolic proteins including mitochondrial aldehyde dehydrogenase, Mn-superoxide dismutase, glutathione peroxidase, ATP synthase, and 3-ketoacyl-CoA thiolase involved in anti-oxidant defense, energy supply, and fatty acid metabolism, respectively. Immunoprecipitation followed by immunoblot with anti-3-NT antibody confirmed that the aforementioned proteins were nitrated in APAP-exposed mice but not in NAC-co-treated mice. Consistently, NAC co-treatment significantly restored the suppressed activities of these enzymes. Thus, we demonstrate a new mechanism by which many nitrated proteins with concomitantly suppressed activities promotes APAP-induced mitochondrial dysfunction and hepatotoxicity. PMID:23454065

  13. Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis.

    PubMed

    Rahmouni, Souad; Hego, Alexandre; Delierneux, Céline; Wéra, Odile; Musumeci, Lucia; Tautz, Lutz; Oury, Cécile

    2016-01-01

    Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets. PMID:27514813

  14. Protein nitration and nitrosylation by NO-donating aspirin in colon cancer cells: Relevance to its mechanism of action

    SciTech Connect

    Williams, Jennie L.; Ji, Ping; Ouyang, Nengtai; Kopelovich, Levy; Rigas, Basil

    2011-06-10

    Nitric oxide-donating aspirin (NO-ASA) is a promising agent for cancer prevention. Although studied extensively, its molecular targets and mechanism of action are still unclear. S-nitrosylation of signaling proteins is emerging as an important regulatory mechanism by NO. Here, we examined whether S-nitrosylation of the NF-{kappa}B, p53, and Wnt signaling proteins by NO-ASA might explain, in part, its mechanism of action in colon cancer. NO-ASA releases significant amounts of NO detected intracellularly in HCT116 and HT-29 colon cells. Using a modified biotin switch assay we demonstrated that NO-ASA S-nitrosylates the signaling proteins p53, {beta}-catenin, and NF-{kappa}B, in colon cancer cells in a time- and concentration-dependent manner. NO-ASA suppresses NF-{kappa}B binding to its cognate DNA oligonucleotide, which occurs without changes in the nuclear levels of the NF-{kappa}B subunits p65 and p50 and is reversed by dithiothreitol that reduces -S-NO to -SH. In addition to S-nitrosylation, we documented both in vitro and in vivo widespread nitration of tyrosine residues of cellular proteins in response to NO-ASA. Our results suggest that the increased intracellular NO levels following treatment with NO-ASA modulate cell signaling by chemically modifying key protein members of signaling cascades. We speculate that S-nitrosylation and tyrosine nitration are responsible, at least in part, for the inhibitory growth effect of NO-ASA on cancer cell growth and that this may represent a general mechanism of action of NO-releasing agents.

  15. Molecular cloning of a docking protein, BRDG1, that acts downstream of the Tec tyrosine kinase

    PubMed Central

    Ohya, Ken-ichi; Kajigaya, Sachiko; Kitanaka, Akira; Yoshida, Koji; Miyazato, Akira; Yamashita, Yoshihiro; Yamanaka, Takeo; Ikeda, Uichi; Shimada, Kazuyuki; Ozawa, Keiya; Mano, Hiroyuki

    1999-01-01

    Tec, Btk, Itk, Bmx, and Txk constitute the Tec family of protein tyrosine kinases (PTKs), a family with the distinct feature of containing a pleckstrin homology (PH) domain. Tec acts in signaling pathways triggered by the B cell antigen receptor (BCR), cytokine receptors, integrins, and receptor-type PTKs. Although upstream regulators of Tec family kinases are relatively well characterized, little is known of the downstream effectors of these enzymes. The yeast two-hybrid system has identified several proteins that interact with the kinase domain of Tec, one of which is now revealed to be a previously unknown docking protein termed BRDG1 (BCR downstream signaling 1). BRDG1 contains a proline-rich motif, a PH domain, and multiple tyrosine residues that are potential target sites for Src homology 2 domains. In 293 cells expressing recombinant BRDG1 and various PTKs, Tec and Pyk2, but not Btk, Bmx, Lyn, Syk, or c-Abl, induced marked phosphorylation of BRDG1 on tyrosine residues. BRDG1 was also phosphorylated by Tec directly in vitro. Efficient phosphorylation of BRDG1 by Tec required the PH and SH2 domains as well as the kinase domain of the latter. Furthermore, BRDG1 was shown to participate in a positive feedback loop by increasing the activity of Tec. BRDG1 transcripts are abundant in the human B cell line Ramos, and the endogenous protein underwent tyrosine phosphorylation in response to BCR stimulation. BRDG1 thus appears to function as a docking protein acting downstream of Tec in BCR signaling. PMID:10518561

  16. LACK OF PROTEIN-TYROSINE SULFATION DISRUPTS PHOTORECEPTOR OUTER SEGMENT MORPHOGENESIS, RETINAL FUNCTION AND RETINAL ANATOMY

    PubMed Central

    Sherry, David M.; Murray, Anne R.; Kanan, Yogita; Arbogast, Kelsey L.; Hamilton, Robert A.; Fliesler, Steven J.; Burns, Marie E.; Moore, Kevin L.; Al-Ubaidi, Muayyad R.

    2010-01-01

    To investigate the role(s) of protein-tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1−/−/Tpst2−/−) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing, and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein-tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance. PMID:21039965

  17. Expression, purification, and characterization of human osteoclastic protein-tyrosine phosphatase catalytic domain in Escherichia coli.

    PubMed

    Jiang, Huan; Sui, Yuan; Cui, Yue; Lin, Peng; Li, Wannan; Xing, Shu; Wang, Deli; Hu, Min; Fu, Xueqi

    2015-03-01

    Osteoclastic protein tyrosine phosphatase (PTP-oc) is a structurally unique transmembrane protein tyrosine phosphatase (PTP) that contains only a relatively small intracellular PTP catalytic domain, does not have an extracellular domain, and lacks a signal peptide proximal to the NH2 terminus. The present study reports the expression, purification, and characterization of the intracellular catalytic domain of PTP-oc (ΔPTP-oc). ΔPTP-oc was expressed in Escherichia coli cells as a fusion with a six-histidine tag and was purified via nickel affinity chromatography. When with para-nitrophenylphosphate (p-NPP) as a substrate, ΔPTP-oc exhibited classical Michaelis-Menten kinetics. Its responses to temperature and ionic strength were similar to those of other PTPs. The optimal pH value of ΔPTP-oc is approximately 7.0, unlike other PTPs, whose optimal pH values are approximately 5.0. PMID:25462809

  18. Protein tyrosine phosphatase variants in human hereditary disorders and disease susceptibilities.

    PubMed

    Hendriks, Wiljan J A J; Pulido, Rafael

    2013-10-01

    Reversible tyrosine phosphorylation of proteins is a key regulatory mechanism to steer normal development and physiological functioning of multicellular organisms. Phosphotyrosine dephosphorylation is exerted by members of the super-family of protein tyrosine phosphatase (PTP) enzymes and many play such essential roles that a wide variety of hereditary disorders and disease susceptibilities in man are caused by PTP alleles. More than two decades of PTP research has resulted in a collection of PTP genetic variants with corresponding consequences at the molecular, cellular and physiological level. Here we present a comprehensive overview of these PTP gene variants that have been linked to disease states in man. Although the findings have direct bearing for disease diagnostics and for research on disease etiology, more work is necessary to translate this into therapies that alleviate the burden of these hereditary disorders and disease susceptibilities in man. PMID:23707412

  19. Disruption of striatal-enriched protein tyrosine phosphatase (STEP) function in neuropsychiatric disorders

    PubMed Central

    Karasawa, Takatoshi; Lombroso, Paul J.

    2014-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific tyrosine phosphatase that plays a major role in the development of synaptic plasticity. Recent findings have implicated STEP in several psychiatric and neurological disorders, including Alzheimer’s disease, schizophrenia, fragile X syndrome, Huntington’s disease, stroke/ischemia, and stress-related psychiatric disorders. In these disorders, STEP protein expression levels and activity are dysregulated, contributing to the cognitive deficits that are present. In this review, we focus on the most recent findings on STEP, discuss how STEP expression and activity are maintained during normal cognitive function, and how disruptions in STEP activity contribute to a number of illnesses. PMID:25218562

  20. Role of protein-tyrosine phosphatases in regulation of osteoclastic activity.

    PubMed

    Sheng, M H-C; Lau, K-H W

    2009-06-01

    Osteoclasts, the primary cell type mediating bone resorption, are multinucleated, giant cells derived from hematopoietic cells of monocyte-macrophage lineage. Osteoclast activity is, in a large part, regulated by protein-tyrosine phosphorylation. While information about functional roles of several protein-tyrosine kinases (PTK), including c-Src, in osteoclastic resorption has been accumulated, little is known about the roles of protein-tyrosine phosphatases (PTPs) in regulation of osteoclast activity. Recent evidence implicates important regulatory roles for four PTPs (SHP-1, cyt-PTP-epsilon, PTP-PEST, and PTPoc) in osteoclasts. Cyt-PTP-epsilon, PTP-PEST, and PTP-oc are positive regulators of osteoclast activity, while SHP-1 is a negative regulator. Of these PTPs in osteoclasts, only PTP-oc is a positive regulator of c-Src PTK through dephosphorylation of the inhibitory phosphotyrosine-527 residue. Although some information about mechanisms of action of these PTPs to regulate osteoclast activity is reviewed in this article, much additional work is required to provide more comprehensive details about their functions in osteoclasts. PMID:19189046

  1. 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...

  2. Phosphonate monoesters on a thiacalix[4]arene framework as potential inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Trush, Viacheslav V; Kharchenko, Sergiy G; Tanchuk, Vsevolod Yu; Kalchenko, Vitaly I; Vovk, Andriy I

    2015-09-01

    Monoester derivatives of thiacalix[4]arene tetrakis(methylphosphonic) acid were found to be capable of inhibiting protein tyrosine phosphatase 1B. In addition, these compounds can strongly bind to human serum albumin. PMID:26205135

  3. 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

  4. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

    PubMed

    González-Fernández, Lauro; Macías-García, Beatriz; Loux, Shavahn C; Varner, Dickson D; Hinrichs, Katrin

    2013-06-01

    Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways. PMID:23595906

  5. Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

    PubMed Central

    Meconi, Sonia; Capo, Christian; Remacle-Bonnet, Maryse; Pommier, Gilbert; Raoult, Didier; Mege, Jean-Louis

    2001-01-01

    Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetii virulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization. PMID:11254615

  6. Tyrosine analogues for probing proton-coupled electron transfer processes in peptides and proteins.

    PubMed

    Nara, Susheel J; Valgimigli, Luca; Pedulli, Gian Franco; Pratt, Derek A

    2010-01-20

    A series of amino acids analogous to tyrosine, but differing in the physicochemical properties of the aryl alcohol side chain, have been prepared and characterized. These compounds are expected to be useful in understanding the relationships between structure, thermodynamics, and kinetics in long-range proton-coupled electron transfer processes in peptides and proteins. Systematic changes in the acidity, redox potential, and O-H bond strength of the tyrosine side chain could be induced upon substituting the phenol for pyridinol and pyrimidinol moieties. Further modulation was possible by introducing methyl and t-butyl substitution in the position ortho to the phenolic hydroxyl. The unnatural amino acids were prepared by Pd-catalyzed cross-coupling of the corresponding halogenated aryl alcohol protected as their benzyl ethers with an organozinc reagent derived from N-Boc L-serine carboxymethyl ester. Subsequent debenzylation by catalytic hydrogenation yielded the tyrosine analogues in good yield. Spectrophotometric titrations revealed a decrease in tyrosine pK(a) of ca. 1.5 log units per included nitrogen atom, along with a corresponding increase in the oxidation (peak) potentials of ca. 200 mV, respectively. All told, the six novel amino acids described here have phenol-like side chains with pK(a)'s that span a range of 7.0 to greater than 10, and an oxidation (peak) potential range of greater than 600 mV at and around physiological pH. Radical equilibration EPR experiments were carried out to reveal that the O-H bond strengths increase systematically upon nitrogen incorporation (by ca. 0.5-1.0 kcal/mol), and radical stability and persistence increase systematically upon introduction of alkyl substitution in the ortho positions. The EPR spectra of the aryloxyl radicals derived from tyrosine and each of the analogues could be determined at room temperature, and each featured distinct spectral properties. The uniqueness of their spectra will be helpful in discerning

  7. Family of receptor-linked protein tyrosine phosphatases in humans and Drosophila

    SciTech Connect

    Streuli, M.; Krueger, N.X.; Saito, H. ); Tsai, A.Y.M. )

    1989-11-01

    To understand the regulation of cell proliferation by tyrosine phosphorylation, characterization of protein tyrosine phosphatases is essential. The human genes LCA (leukocyte common antigen) and LAR encode putative receptor-linked PTPases. By using consensus sequence probes, two additional receptor-linked PTPase genes, DLAR and DPTP, were isolated from Drosophila melanogaster. The extra-cellular segments of both DLAR and DPTP are composed of multiple immunoglobulin-like domains and fibronectin type III-like domains. The cytoplasmic region of DLAR and DPTP, as well as human LCA and LAR, are composed of two tandemly repeated PTPase domains. PTPase activities of immunoprecipitated LCA and LAR were demonstrated by measuring the release of phosphate from a {sup 32}P-labeled (Tyr(P))peptide. Furthermore, the cytoplasmic domains of LCA, LAR, DLAR, and DPTP, expressed in Escherichia coli, have PTPase activity. Site-directed mutagenesis showed that a conserved cysteine residue is essential for PTPase activity.

  8. Regulation by nitrate of protein synthesis and translation of RNA in maize roots

    SciTech Connect

    McClure, P.R.; Bouthyette, P.Y.

    1986-04-01

    Roots of maize seedlings were exposed to /sup 35/S-methionine in the presence or absence of nitrate. Using SDS-PAGE, nitrate-induced changes in labeled polypeptides were noted in the soluble (at 92, 63 and 21kD) and organellar(at 14kD) fractions, as well as in a membrane fraction of putative tonoplast origin (at 31kD). No nitrate-induced changes were noted in a plasmamembrane-enriched fraction or in a membrane fraction of mixed origin. Total RNA from nitrate-treated and control roots was translated in a rabbit reticulocyte system. Five translation products (94, 63, 41, 39 and 21kD) were identified as nitrate-inducible by comparative gel electrophoresis. Changes in protein synthesis and translation of mRNA were apparent within 2-3 h after introduction of nitrate. Within 4-6 h after removal of nitrate, the level of nitrate-inducible translation products diminished to that of control roots. In contrast, the 31kD tonoplast polypeptide was still labeled 26 h after removal of external nitrate and /sup 35/S-methionine. The results will be discussed in relation to the nitrate induction of nitrate reductase, nitrite reductase, and the nitrate uptake system.

  9. Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

    PubMed Central

    Ko, Han Seok; Lee, Yunjong; Shin, Joo-Ho; Karuppagounder, Senthilkumar S.; Gadad, Bharathi Shrikanth; Koleske, Anthony J.; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.

    2010-01-01

    Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571’s protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD. PMID:20823226

  10. Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

    PubMed

    Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

    2015-08-01

    Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity. PMID:26195794

  11. Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium

    PubMed Central

    Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

    2015-01-01

    Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer–associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10–deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1–deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity. PMID:26195794

  12. Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.

    PubMed Central

    Koyasu, S; Tse, A G; Moingeon, P; Hussey, R E; Mildonian, A; Hannisian, J; Clayton, L K; Reinherz, E L

    1994-01-01

    To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains. Images PMID:7517560

  13. Phenotypic characteristics of expressed tyrosine hydroxylase protein in the adult rat nucleus tractus solitarius: plasticity revealed by RU24722 treatment.

    PubMed

    Garcia, C; Marcel, D; Le Cavorsin, M; Pujol, J F; Weissmann, D

    1994-10-01

    The phenotypic characteristics of expressed tyrosine hydroxylase protein have been precisely analysed in the rat nucleus tractus solitarius, which contains the majority of A2 noradrenergic and C2 adrenergic neurons of the medulla oblongata. This study was based upon quantitative analysis of immunohistochemical and immunoradioautographic staining of tyrosine hydroxylase protein in serial coronal sections. In control rats, there were few tyrosine hydroxylase-expressing cell bodies which express less than 2% of the immunoradiolabeled tyrosine hydroxylase protein measured in the structure. These cell bodies were scattered throughout an extensive immunopositive neuropile, which precisely delimited the topological space of the nucleus tractus solitarius quantiatively reconstructed using a polar coordinate system. The quantification of tyrosine hydroxylase tissue concentration from immunoradioautograms allowed us to subdivide the structure into two distinct regions. The posterior region of the nucleus tractus solitarius, which mainly corresponds to the A2 cell group, contains a relatively high tissue concentration of tyrosine hydroxylase protein (18.56 +/- 0.154 units per mg of tissue). The anterior region, which mainly corresponds to the C2 cell group, exhibits a relatively low concentration (12.09 +/- 0.81) of this protein. Three days after an intraperitoneal injection of RU24722, there was a strong increase (90 +/- 17%) in tyrosine hydroxylase protein content only in the anterior region of the nucleus tractus solitarius. This increase was associated with a dramatic elevation (142 +/- 20%) in the number of tyrosine hydroxylase-expressing cell bodies. The additional cell bodies were mainly located inside the initial perikarya-containing area.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7845594

  14. Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis).

    PubMed

    Tulake, Kuerban; Wang, Xuguang; Chen, Yong; Yu, Chucai; Jing, Binyu; Li, Heping

    2015-03-01

    High efficiency of in vitro capacitation of deer sperm has not yet been achieved as low sperm penetration rates were reported in in vitro fertilization studies. Our main goal in this study was to identify the changes of frozen-thawed sperm of the rare red deer Tarim wapiti (Cervus elaphus yarkandensis) and detect the effect of bovine serum albumin (BSA), serum, and heparin on the protein tyrosine phosphorylation of frozen-thawed sperm. The frozen-thawed sperm of Tarim wapiti was suspended in improved modified tyrode-albumin-lactate-pyruvate medium and cultured in 5% CO2 at 38.5°C, and the status of protein tyrosine phosphorylation of sperm was detected by Western blotting. Although the results showed that the type number and expression of protein tyrosine phosphorylation of frozen-thawed wapiti sperm were decreased, the tyrosine-phosphorylated proteins such as 10, 14, 40, 47, and 55kDa were increased significantly during the process of capacitation culture (1-2h). In addition, tyrosine-phosphorylated proteins were promoted by BSA rather than serum, and estrus sheep serum (ESS) rather than estrus deer serum. When ESS and heparin were used together at 4h after capacitation, four main tyrosine phosphorylation proteins (10±2, 14±2, 25±3, and 47±3kDa) had a significantly higher expression than that at 2h after capacitation. We demonstrated that these proteins were involved in wapiti sperm in vitro capacitation, heparin in the incubation media was necessary for the capacitation and tyrosine phosphorylation protein was promoted by ESS. PMID:25638741

  15. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ravikumar, Vaishnavi; Dobrinic, Paula; Macek, Boris; Franjevic, Damjan; Noirot-Gros, Marie-Francoise; Mijakovic, Ivan

    2014-01-01

    Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells. PMID:25278935

  16. Protein tyrosine kinase 6 mediates TNFα-induced endothelial barrier dysfunction

    PubMed Central

    Haines, RJ; Beard, RS; Wu, MH

    2014-01-01

    A key event in the progression of systemic inflammation resulting from severe trauma or shock involves microvascular hyperpermeability, which leads to excessive plasma fluid and proteins accumulating in extravascular space resulting in tissue edema. The precise molecular mechanism of the hyperpermeability response is not completely understood. Protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) is a non-receptor tyrosine kinase related to Src-family proteins. Although it has also been shown that PTK6 participates in regulating epithelial barrier function, the role of PTK6 in endothelial barrier function has not been reported. In this study, we hypothesized that PTK6 is 1) expressed in vascular endothelial cells, and 2) contributes to vascular endothelial hyperpermeability in response to TNFα. Results showed that PTK6 was detected in mouse endothelial cells at the level of protein and mRNA. In addition, PTK6 knockdown attenuated TNFα induced decrease in endothelial barrier function as measured by electric cell-substrate impedance sensing (ECIS) and in vitro transwell albumin-flux assays. Furthermore, we showed that TNFα treatment of endothelial cells increased active PTK6 association with p120-catenin at endothelial cell-cell junctions. Further analysis using immunocytochemistry and immunoprecipitation demonstrated that PTK6 knockdown attenuated TNFα induced VE-cadherin internalization as well as promoting its association with p120-catenin. Our study demonstrates a novel role of PTK6 in mediating endothelial barrier dysfunction. PMID:25446122

  17. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    SciTech Connect

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M.; Iuliano, Rodolfo; Fusco, Alfredo; Polikarpov, Igor

    2006-09-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit.

  18. Protein-tyrosine phosphorylation interaction network in Bacillus subtilis reveals new substrates, kinase activators and kinase cross-talk

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ventroux, Magali; Derouiche, Abderahmane; Bidnenko, Vladimir; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise

    2014-01-01

    Signal transduction in eukaryotes is generally transmitted through phosphorylation cascades that involve a complex interplay of transmembrane receptors, protein kinases, phosphatases and their targets. Our previous work indicated that bacterial protein-tyrosine kinases and phosphatases may exhibit similar properties, since they act on many different substrates. To capture the complexity of this phosphorylation-based network, we performed a comprehensive interactome study focused on the protein-tyrosine kinases and phosphatases in the model bacterium Bacillus subtilis. The resulting network identified many potential new substrates of kinases and phosphatases, some of which were experimentally validated. Our study highlighted the role of tyrosine and serine/threonine kinases and phosphatases in DNA metabolism, transcriptional control and cell division. This interaction network reveals significant crosstalk among different classes of kinases. We found that tyrosine kinases can bind to several modulators, transmembrane or cytosolic, consistent with a branching of signaling pathways. Most particularly, we found that the division site regulator MinD can form a complex with the tyrosine kinase PtkA and modulate its activity in vitro. In vivo, it acts as a scaffold protein which anchors the kinase at the cell pole. This network highlighted a role of tyrosine phosphorylation in the spatial regulation of the Z-ring during cytokinesis. PMID:25374563

  19. Unbiased identification of substrates of protein tyrosine phosphatase ptp-3 in C. elegans.

    PubMed

    Mitchell, Christopher J; Kim, Min-Sik; Zhong, Jun; Nirujogi, Raja Sekhar; Bose, Anjun K; Pandey, Akhilesh

    2016-06-01

    The leukocyte antigen related (LAR) family of receptor-like protein tyrosine phosphatases has three members in humans - PTPRF, PTPRD and PTPRS - that have been implicated in diverse processes including embryonic development, inhibition of cell growth and axonal guidance. Mutations in the LAR family are associated with developmental defects such as cleft palate as well as various cancers including breast, neck, lung, colon and brain. Although this family of tyrosine phosphatases is important for many developmental processes, little is known of their substrates. This is partially due to functional redundancy within the LAR family, as deletion of a single gene in the LAR family does not have an appreciable phenotype, but a dual knockout is embryonically lethal in mouse models. To circumvent the inability to knockout multiple members of the LAR family in mouse models, we used a knockout of ptp-3, which is the only known ortholog of the LAR family in Caenorhabditis elegans and allows for the study of the LAR family at the organismal level. Using SILAC-based quantitative phosphoproteomics, we identified 255 putative substrates of ptp-3, which included four of the nine known annotated substrates of the LAR family. A motif analysis of the identified phosphopeptides allowed for the determination of sequences that appear to be preferentially dephosphorylated. Finally, we discovered that kinases were overrepresented in the list of identified putative substrates and tyrosine residues whose phosphorylation is known to increase kinase activity were dephosphorylated by ptp-3. These data are suggestive of ptp-3 as a potential negative regulator of several kinase families, such as the mitogen activated kinases (MAPKs), and multiple tyrosine kinases including FER, MET, and NTRK2. PMID:27067626

  20. PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue

    SciTech Connect

    Lim, So-Hee; Moon, Jeonghee; Lee, Myungkyu; Lee, Jae-Ran

    2013-09-13

    Highlights: •PTPRT is a brain-specific, expressed, protein tyrosine phosphatase. •PTPRT regulated the interaction of Syntaxin-binding protein 1 with Syntaxin 1. •PTPRT dephosphorylated the specific tyrosine residue of Syntaxin-binding protein 1. •Dephosphorylation of Syntaxin-binding protein 1 enhanced the interaction with Syntaxin 1. •PTPRT appears to regulate the fusion of synaptic vesicle through dephosphorylation. -- Abstract: PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified as a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1.

  1. Comparison of nitrotyrosine antibodies and development of immunoassays for the detection of nitrated proteins.

    PubMed

    Franze, Thomas; Weller, Michael G; Niessner, Reinhard; Poschl, Ulrich

    2004-07-01

    Three monoclonal antibodies (mAb) and three polyclonal antibodies (pAb) have been characterized and compared with respect to their cross-reactivities and affinities for 3-nitrotyrosine, eight aromatic compounds with similar chemical structures, a peptide containing a single nitrotyrosine residue, and fourteen nitrated protein standards (bovine serum albumin, BSA) containing different numbers of nitrotyrosine residues per protein molecule (0.2 to 16.8). In indirect competitive immunoassays, mAb Alexis 39B6 exhibited the highest affinity for free 3-nitrotyrosine (10(6) L mol(-1)), while the pAb Oxis 24312 from sheep exhibited the highest affinities for nitrated proteins (up to 10(8) L mol(-1)). The apparent affinities determined in the indirect competitive assays were inversely correlated with the limits of detection (LOD) determined in one-sided immunoassays. With the sheep pAb minimum LOD on the order of 10 pmol L(-1) were achieved for highly nitrated proteins, corresponding to effective LOD on the order of 100 pmol L(-1) for nitrotyrosine residues. In the one-sided assays, however, the LOD for nitrated proteins increased proportionally with increasing background concentrations of native proteins in the investigated samples. Sandwich immunoassays combining pAb and mAb for selective enrichment and detection of nitrated proteins allowed to eliminate this native protein matrix effect and to achieve LOD on the order of 300 pmol L(-1) for highly nitrated proteins independent of native protein background concentrations. PMID:15213824

  2. 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

  3. Phosphacan and Receptor Protein Tyrosine Phosphatase β Expression Mediates Deafferentation-Induced Synaptogenesis

    PubMed Central

    Harris, Janna L.; Reeves, Thomas M.; Phillips, Linda L.

    2009-01-01

    This study documents the spatial and temporal expression of three structurally related chondroitin sulfated proteoglycans (CSPGs) during synaptic regeneration induced by brain injury. Using the unilateral entorhinal cortex lesion model of adaptive synaptogenesis, we documented mRNA and protein profiles of phosphacan and its two splice variants, full length receptor protein tyrosine phosphatase β (RPTPβ) and the short transmembrane receptor form (sRPTPβ), at 2, 7, and 15 d postlesion. We report that whole hippocampal sRPTPβ protein and mRNA are persistently elevated over the first two weeks after UEC. As predicted, this transmembrane family member was localized adjacent to synaptic sites in the deafferented neuropil and showed increased distribution over that zone following lesion. By contrast, whole hippocampal phosphacan protein was not elevated with deafferentation, however, its mRNA was increased during the period of sprouting and synapse formation (7d). When the zone of synaptic reorganization was sampled using molecular layer/granule cell (ML/GCL) enriched dissections, we observed an increase in phosphacan protein at 7d, concurrent with the observed hippocampal mRNA elevation. Immunohistochemistry also showed a shift in phosphacan distribution from granule cell bodies to the deafferented ML at 2 and 7d postlesion. Phosphacan and sRPTPβ were not co-localized with glial fibrillary acid protein (GFAP), suggesting that reactive astrocytes were not a major source of either proteoglycan. While transcript for the developmentally prominent full length RPTPβ was also increased at 2 and 15d, its protein was not detected in our adult samples. These results indicate that phosphacan and RPTPβ splice variants participate in both the acute degenerative and long-term regenerative phases of reactive synaptogenesis. These results suggest that increase in the transmembrane sRPTPβ tyrosine phosphatase activity is critical to this plasticity, and that local elevation of

  4. Elevated intracellular calcium concentration increases secretory processing of the amyloid precursor protein by a tyrosine phosphorylation-dependent mechanism.

    PubMed Central

    Petryniak, M A; Wurtman, R J; Slack, B E

    1996-01-01

    Secretory cleavage of the amyloid precursor protein (APP), a process that releases soluble APP derivatives (APPs) into the extracellular space, is stimulated by the activation of muscarinic receptors coupled to phosphoinositide hydrolysis. The signalling pathways involved in the release process exhibit both protein kinase C- and protein tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337-8344]. The possibility that elevations in intracellular Ca2+ concentration initiate the tyrosine phosphorylation-dependent release of APPs was examined in human embryonic kidney cells expressing muscarinic m3 receptors. Inhibition of protein kinase C with the bisindolylmaleimide GF 109203X decreased the carbachol-evoked release of APPs by approx. 30%, as shown previously. The residual response was further decreased, in an additive manner, by the Ca2+ chelator EGTA, or by the tyrosine kinase inhibitor tyrphostin A25. The Ca2+ ionophore, ionomycin, like carbachol, stimulated both the release of APPs and the tyrosine phosphorylation of several proteins, one of which was identified as paxillin, a component of focal adhesions. The effects of ionomycin on APPs release and on protein tyrosine phosphorylation were concentration-dependent, and occurred over similar concentration ranges; both effects were inhibited only partly by GF 109203X, but were abolished by EGTA or by tyrosine kinase inhibitors. The results demonstrate for the first time that ionophore-induced elevations in intracellular Ca2+ levels elicit APPs release via increased tyrosine phosphorylation. Part of the increase in APPs release evoked by muscarinic receptor activation might be attributable to a similar mechanism. PMID:9003386

  5. Expression of receptor protein tyrosine phosphatase δ, PTPδ, in mouse central nervous system.

    PubMed

    Shishikura, Maria; Nakamura, Fumio; Yamashita, Naoya; Uetani, Noriko; Iwakura, Yoichiro; Goshima, Yoshio

    2016-07-01

    Protein tyrosine phosphate δ (PTPδ), one of the receptor type IIa protein tyrosine phosphates, is known for its roles in axon guidance, synapse formation, cell adhesion, and tumor suppression. Alternative splicing of this gene generates at least four (A-D) isoforms; however, the major isoform in vivo is yet to be determined. The protein localization has neither been revealed. We have generated anti-mouse PTPδ-specific monoclonal antibody and analyzed the protein expression in wild-type and Ptpδ knockout mice. Immunoblot analysis of various organs revealed that neuronal tissues express both C-and D-isoforms of PTPδ, whereas non-neuronal tissues express only C-isoform. Immunohistochemistry of wild-type or Ptpδ heterozygous sections showed that olfactory bulb, cerebral cortex, hippocampus, cerebellum, and several nuclei in brain stem exhibit moderate to strong positive signals. These signals were absent in Ptpδ knockout specimens. Higher magnification revealed differences between expression patterns of PTPδ mRNA and its protein product. In hippocampus, weak mRNA expression in CA1 stratum pyramidale but strong immunostaining in the stratum lacunosum moleculare was observed, suggesting the axonal expression of PTPδ in the entorhinal cortical afferents. Olfactory mitral cells exhibited mRNA expression in cell bodies and protein localization in their dendritic fields, glomerular and external plexiform layers. Nissl staining showed that the external plexiform layer was reduced in Ptpδ knockout mice. Golgi-impregnation confirmed the poor dendritic growth of homozygous mitral cells. These results suggest that PTPδ may localize in axons as well as in dendrites to regulate their elaboration in the central nervous system. PMID:27026654

  6. Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

    SciTech Connect

    Rehman, Kanwal; Chen, Zhe; Wang, Wen Wen; Wang, Yan Wei; Sakamoto, Akira; Zhang, Yan Fang; Naranmandura, Hua; Suzuki, Noriyuki

    2012-09-15

    Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involved in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs{sup III}) and its intermediate metabolites such as monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA{sup III} and DMA{sup III}) but not by iAs{sup III}. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA{sup III} directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation. Highlights: ► This study focused on the interaction of PTPs with trivalent arsenicals in vitro. ► We for the first time confirmed that DMA{sup III} strongly inhibited activity of PTP1B. ► DMA{sup III} directly

  7. Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase (STEP)

    PubMed Central

    Li, Hui; Li, Kang-shuai; Su, Jing; Chen, Lai-Zhong; Xu, Yun-Fei; Wang, Hong-Mei; Gong, Zheng; Cui, Guo-Ying; Yu, Xiao; Wang, Kai; Yao, Wei; Xin, Tao; Li, Min-Yong; Xiao, Kun-Hong; An, Xiao-fei; Huo, Yuqing; Xu, Zhi-gang; Sun, Jin-Peng; Pang, Qi

    2013-01-01

    Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward pNPP, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both KIM and KIS of STEP were required for ERK interaction. In addition to the N-terminal KIS region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. PMID:24117863

  8. Tyrosine Coupling Creates a Hyperbranched Multivalent Protein Polymer Using Horseradish Peroxidase via Bipolar Conjugation Points.

    PubMed

    Minamihata, Kosuke; Yamaguchi, Sou; Nakajima, Kei; Nagamune, Teruyuki

    2016-05-18

    Protein polymers of covalently cross-linked protein monomers are highly attractive biomaterials because each monomer unit possesses distinct protein functions. Protein polymers often show enhancement effects on the function by integrating a large number of molecules into one macromolecule. The cross-linking site of component proteins should be precisely controlled to avoid diminishing the protein function. However, preparing protein polymers that are cross-linked site-specifically with a high cross-linking degree is a challenge. Here, we demonstrate the preparation of a site-specifically cross-linked protein polymer that has a hyperbranched polymer-like structure with a high cross-linking degree. A horseradish peroxidase (HRP) reaction was used to achieve the protein polymerization through a peptide tag containing a tyrosine residue (Y-tag). Y-tag sequences were introduced to both N- and C-termini of a model protein, protein G. The dual Y-tagged protein G (Y-pG-Y) was treated with HRP to form a Y-pG-Y polymer possessing average and maximum cross-linking degree of approximately 70-mer and 150-mer, respectively. The Y-pG-Y polymer shows the highest cross-linking degree among the protein polymers reported, which are completely soluble in water and cross-linked via covalent bonding. The Y-pG-Y was cross-linked site-specifically at the Tyr residue in the Y-tag, retaining its function, and the Y-pG-Y polymer showed extremely strong avidity against immunoglobulin G. The reactivities of N- and C-terminal Y-tags were evaluated, and we revealed that the difference in the radical formation rate by HRP was the key for yielding highly cross-linked protein polymers. PMID:27093089

  9. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B.

    PubMed

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  10. Intersecting roles of protein tyrosine kinase and calcium signaling during fertilization.

    PubMed

    Kinsey, William H

    2013-01-01

    The oocyte is a highly specialized cell that must respond to fertilization with a preprogrammed series of signal transduction events that establish a block to polyspermy, trigger resumption of the cell cycle and execution of a developmental program. The fertilization-induced calcium transient is a key signal that initiates the process of oocyte activation and studies over the last several years have examined the signaling pathways that act upstream and downstream of this calcium transient. Protein tyrosine kinase signaling was found to be an important component of the upstream pathways that stimulated calcium release at fertilization in oocytes from animals that fertilize externally, but a similar pathway has not been found in mammals which fertilize internally. The following review will examine the diversity of signaling in oocytes from marine invertebrates, amphibians, fish and mammals in an attempt to understand the basis for the observed differences. In addition to the pathways upstream of the fertilization-induced calcium transient, recent studies are beginning to unravel the role of protein tyrosine kinase signaling downstream of the calcium transient. The PYK2 kinase was found to respond to fertilization in the zebrafish system and seems to represent a novel component of the response of the oocyte to fertilization. The potential impact of impaired PTK signaling in oocyte quality will also be discussed. PMID:23201334

  11. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B

    PubMed Central

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  12. Phosphorylation of Mycobacterium tuberculosis protein tyrosine kinase A PtkA by Ser/Thr protein kinases.

    PubMed

    Zhou, Peifu; Wong, Dennis; Li, Wu; Xie, Jianping; Av-Gay, Yossef

    2015-11-13

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has inflicted about one third of mankind and claims millions of deaths worldwide annually. Signalling plays an important role in Mtb pathogenesis and persistence, and thus represents attractive resource for drug target candidates. Here, we show that protein tyrosine kinase A (PtkA) can be phosphorylated by Mtb endogenous eukaryotic-like Ser/Thr protein kinases (eSTPKs). Kinase assays showed that PknA, PknD, PknF, and PknK can phosphorylate PtkA in dose- and time-dependent manner. Enzyme kinetics suggests that PknA has the highest affinity and enzymatic efficiency towards PtkA. Furthermore, protein-protein interaction assay in surrogate host showed that PtkA interacts with multi-eSTPKs in vivo, including PknA. Lastly, we show that PtkA phosphorylation by eSTPKs occurs on threonine residues and may effect tyrosine phosphorylation levels and thus PtkA activity in vitro. These results demonstrate that PtkA can serve as a substrate to many eSTPKs and suggests that's its activity can be regulated. PMID:26417687

  13. A RP-UFLC Assay for Protein Tyrosine Phosphatases: Focus on Protein Tyrosine Phosphatase Non-Receptor Type 2 (PTPN2).

    PubMed

    Duval, Romain; Bui, Linh-Chi; Berthelet, Jérémy; Dairou, Julien; Mathieu, Cécile; Guidez, Fabien; Dupret, Jean-Marie; Cools, Jan; Chomienne, Christine; Rodrigues-Lima, Fernando

    2015-01-01

    Protein tyrosine phosphatases (PTPs) are involved in numerous signaling pathways and dysfunctions of certain of these enzymes have been linked to several human diseases including cancer and autoimmune diseases. PTPN2 is a PTP mainly expressed in hematopoietic cells and involved in growth factor and JAK/STAT signaling pathways. Loss of function analyses in patients with mutation/deletion of the PTPN2 gene and knock-out mouse models indicate that PTPN2 acts as a tumor suppressor in T-cell malignancies and as a regulator of inflammation and immunity. The use of sensitive and quantitative assays is of prime importance to better characterize the biochemical properties of PTPN2 and its biological roles. We report a highly sensitive non-radioactive assay that allows the measurement of the activity of purified PTPN2 and of endogenous PTPN2 immunoprecipitated on agarose beads. The assay relies on separation and quantitation by reverse-phase ultra fast liquid chromatography (RP-UFLC) of a fluorescein-labeled phosphotyrosine peptide substrate derived from the sequence of STAT1. The applicability and reliability of this approach is supported by kinetic and mechanistic studies using PTP inhibitors. More broadly, our PTPN2 assay provides the basis for the design of flexible methods for the measurement of other PTPs. PMID:26040922

  14. Protein Tyrosine Nitration in Chronic Intramuscular Parasitism: Immunohistochemical evaluation of Relationships Between Nitration, Fiber Types, and Ubiquitin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies from our laboratory demonstrated that the catabolic processes associated with the proinflammatory impact of protozoan parasitic infection in Holstein calves were significantly more evident in red postural muscle such as psoas major (PM) than locomotor muscles typified by white rectu...

  15. QSAR Study of p56lck Protein Tyrosine Kinase Inhibitory Activity of Flavonoid Derivatives Using MLR and GA-PLS

    PubMed Central

    Fassihi, Afshin; Sabet, Razieh

    2008-01-01

    Quantitative relationships between molecular structure and p56lck protein tyrosine kinase inhibitory activity of 50 flavonoid derivatives are discovered by MLR and GA-PLS methods. Different QSAR models revealed that substituent electronic descriptors (SED) parameters have significant impact on protein tyrosine kinase inhibitory activity of the compounds. Between the two statistical methods employed, GA-PLS gave superior results. The resultant GA-PLS model had a high statistical quality (R2 = 0.74 and Q2 = 0.61) for predicting the activity of the inhibitors. The models proposed in the present work are more useful in describing QSAR of flavonoid derivatives as p56lck protein tyrosine kinase inhibitors than those provided previously. PMID:19325836

  16. Prediction and verification of novel peptide targets of protein tyrosine phosphatase 1B.

    PubMed

    Li, Xun; Köhn, Maja

    2016-08-01

    Phosphotyrosine peptides are useful starting points for inhibitor design and for the search for protein tyrosine phosphatase (PTP) phosphoprotein substrates. To identify novel phosphopeptide substrates of PTP1B, we developed a computational prediction protocol based on a virtual library of protein sequences with known phosphotyrosine sites. To these we applied sequence-based methods, biologically meaningful filters and molecular docking. Five peptides were selected for biochemical testing of their potential as PTP1B substrates. All five peptides were equally good substrates for PTP1B compared to a known peptide substrate whereas appropriate control peptides were not recognized, showing that our protocol can be used to identify novel peptide substrates of PTP1B. PMID:27025565

  17. Protein Tyrosine Phosphatases: From Housekeeping Enzymes to Master-Regulators of Signal Transduction

    PubMed Central

    Tonks, Nicholas K.

    2013-01-01

    There are many misconceptions surrounding the roles of protein phosphatases in the regulation of signal transduction, perhaps the most damaging of which is the erroneous view that these enzymes exert their effects merely as constitutively active housekeeping enzymes. On the contrary, the phosphatases are critical, specific regulators of signaling in their own right and serve an essential function, in a coordinated manner with the kinases, to determine the response to a physiological stimulus. This review is a personal perspective on the development of our understanding of the protein tyrosine phosphatase (PTP) family of enzymes. I have discussed various aspects of the structure, regulation and function of the PTP family, which I hope will illustrate the fundamental importance of these enzymes to the control of signal transduction. PMID:23176256

  18. TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

    SciTech Connect

    Li, Feifei; Jiang, Yinan; Zheng, Qiping; Yang, Xiaoming; Wang, Siying

    2011-01-07

    Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.

  19. Striatal-enriched protein tyrosine phosphatase modulates nociception: evidence from genetic deletion and pharmacological inhibition.

    PubMed

    Azkona, Garikoitz; Saavedra, Ana; Aira, Zigor; Aluja, David; Xifró, Xavier; Baguley, Tyler; Alberch, Jordi; Ellman, Jonathan A; Lombroso, Paul J; Azkue, Jon J; Pérez-Navarro, Esther

    2016-02-01

    The information from nociceptors is processed in the dorsal horn of the spinal cord by complex circuits involving excitatory and inhibitory interneurons. It is well documented that GluN2B and ERK1/2 phosphorylation contributes to central sensitization. Striatal-enriched protein tyrosine phosphatase (STEP) dephosphorylates GluN2B and ERK1/2, promoting internalization of GluN2B and inactivation of ERK1/2. The activity of STEP was modulated by genetic (STEP knockout mice) and pharmacological (recently synthesized STEP inhibitor, TC-2153) approaches. STEP(61) protein levels in the lumbar spinal cord were determined in male and female mice of different ages. Inflammatory pain was induced by complete Freund's adjuvant injection. Behavioral tests, immunoblotting, and electrophysiology were used to analyze the effect of STEP on nociception. Our results show that both genetic deletion and pharmacological inhibition of STEP induced thermal hyperalgesia and mechanical allodynia, which were accompanied by increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Striatal-enriched protein tyrosine phosphatase heterozygous and knockout mice presented a similar phenotype. Furthermore, electrophysiological experiments showed that TC-2153 increased C fiber-evoked spinal field potentials. Interestingly, we found that STEP(61) protein levels in the lumbar spinal cord inversely correlated with thermal hyperalgesia associated with age and female gender in mice. Consistently, STEP knockout mice failed to show age-related thermal hyperalgesia, although gender-related differences were preserved. Moreover, in a model of inflammatory pain, hyperalgesia was associated with increased phosphorylation-mediated STEP(61) inactivation and increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Collectively, the present results underscore an important role of spinal STEP activity in the modulation of nociception. PMID:26270590

  20. The Novel Smad Protein Expansion Regulates Receptor Tyrosine Kinase Pathway to Control Drosophila Tracheal Tube Size

    PubMed Central

    Iordanou, Ekaterini; Chandran, Rachana R.; Yang, Yonghua; Essak, Mina; Blackstone, Nicholas; Jiang, Lan

    2014-01-01

    Tubes with distinct shapes and sizes are critical for the proper function of many tubular organs. Here we describe a unique phenotype caused by the loss of a novel, evolutionarily-conserved, Drosophila Smad-like protein, Expansion. In expansion mutants, unicellular and intracellular tracheal branches develop bubble-like cysts with enlarged apical membranes. Cysts in unicellular tubes are enlargements of the apical lumen, whereas cysts in intracellular tubes are cytoplasmic vacuole-like compartments. The cyst phenotype in expansion mutants is similar to, but weaker than, that observed in double mutants of Drosophila type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. Ptp4E and Ptp10D negatively regulate the receptor tyrosine kinase (RTK) pathways, especially epithelial growth factor receptor (EGFR) and fibroblast growth factor receptor/breathless (FGFR, Btl) signaling to maintain the proper size of unicellular and intracellular tubes. We show Exp genetically interacts with RTK signaling, the downstream targets of RPTPs. Cyst size and number in expansion mutants is enhanced by increased RTK signaling and suppressed by reduced RTK signaling. Genetic interaction studies strongly suggest that Exp negatively regulates RTK (EGFR, Btl) signaling to ensure proper tube sizes. Smad proteins generally function as intermediate components of the transforming growth factor-β (TGF-β, DPP) signaling pathway. However, no obvious genetic interaction between expansion and TGF-β (DPP) signaling was observed. Therefore, Expansion does not function as a typical Smad protein. The expansion phenotype demonstrates a novel role for Smad-like proteins in epithelial tube formation. PMID:24973580

  1. Serine/Threonine/Tyrosine Protein Kinase Phosphorylates Oleosin, a Regulator of Lipid Metabolic Functions1[OA

    PubMed Central

    Parthibane, Velayoudame; Iyappan, Ramachandiran; Vijayakumar, Anitha; Venkateshwari, Varadarajan; Rajasekharan, Ram

    2012-01-01

    Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A2 activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A2 activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca2+ inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation. PMID:22434039

  2. Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

    PubMed Central

    Primo, María E.; Jakoncic, Jean; Noguera, Martín E.; Risso, Valeria A.; Sosa, Laura; Sica, Mauricio P.; Solimena, Michele; Poskus, Edgardo; Ermácora, Mario R.

    2011-01-01

    ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo. PMID:21935384

  3. Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

    SciTech Connect

    Primo M. E.; Jakoncic J.; Noguera M.E.; Risso V.A.; Sosa L.; Sica M.P.; Solimena M.; Poskus E. and Ermacora M.

    2011-09-15

    ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

  4. A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium.

    PubMed

    Vijayaraghavan, S; Trautman, K D; Goueli, S A; Carr, D W

    1997-06-01

    Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1gamma2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or alpha-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways. PMID:9166697

  5. Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking

    PubMed Central

    Hellmuth, Klaus; Grosskopf, Stefanie; Lum, Ching Tung; Würtele, Martin; Röder, Nadine; von Kries, Jens Peter; Rosario, Marta; Rademann, Jörg; Birchmeier, Walter

    2008-01-01

    The protein tyrosine phosphatase Shp2 is a positive regulator of growth factor signaling. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. We performed a high-throughput in silico screen for small-molecular-weight compounds that bind the catalytic site of Shp2. We have identified the phenylhydrazonopyrazolone sulfonate PHPS1 as a potent and cell-permeable inhibitor, which is specific for Shp2 over the closely related tyrosine phosphatases Shp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocks Shp2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of the Erk1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibits activation of Erk1/2 by the leukemia-associated Shp2 mutant, Shp2-E76K, and blocks the anchorage-independent growth of a variety of human tumor cell lines. The PHPS compound class is therefore suitable for further development of therapeutics for the treatment of Shp2-dependent diseases. PMID:18480264

  6. Role of Receptor Protein Tyrosine Phosphatase γ in Sensing Extracellular CO2 and HCO3.

    PubMed

    Zhou, Yuehan; Skelton, Lara A; Xu, Lumei; Chandler, Margaret P; Berthiaume, Jessica M; Boron, Walter F

    2016-09-01

    Regulation of blood pH-critical for virtually every facet of life-requires that the renal proximal tubule (PT) adjust its rate of H(+) secretion (nearly the same as the rate of HCO3 (-) reabsorption, JHCO3 ) in response to changes in blood [CO2] and [HCO3 (-)]. Yet CO2/HCO3 (-) sensing mechanisms remain poorly characterized. Because receptor tyrosine kinase inhibitors render JHCO3 in the PT insensitive to changes in CO2 concentration, we hypothesized that the structural features of receptor protein tyrosine phosphatase-γ (RPTPγ) that are consistent with binding of extracellular CO2 or HCO3 (-) facilitate monitoring of blood CO2/HCO3 (-) concentrations. We now report that PTs express RPTPγ on blood-facing membranes. Moreover, RPTPγ deletion in mice eliminated the CO2 and HCO3 (-) sensitivities of JHCO3 as well as the normal defense of blood pH during whole-body acidosis. Thus, RPTPγ appears to be a novel extracellular CO2/HCO3 (-) sensor critical for pH homeostasis. PMID:26839367

  7. Nitropropenyl Benzodioxole, An Anti-Infective Agent with Action as a Protein Tyrosine Phosphatase Inhibitor

    PubMed Central

    White, Kylie S; Nicoletti, Gina; Borland, Robert

    2014-01-01

    We report on the activities of a broad spectrum antimicrobial compound,nitropropenyl benzodioxole (NPBD) which are of relevance to its potential as an anti-infective drug. These investigations support the proposal that a major mechanism of NPBD is action as a tyrosine mimetic, competitively inhibiting bacterial and fungal protein tyrosine phosphatases (PTP). NPBD did not affect major anti-bacterial drug targets, namely, ATP production, cell wall or cell membrane integrity, or transcription and translation of RNA. NPBD inhibited bacterial YopH and human PTP1B and not human CD45 in enzyme assays. NPBD inhibited PTP-associated bacterial virulence factors, namely, endospore formation in Bacillus cereus, prodigiosin secretion in Serratia marcescens, motility in Proteus spp., and adherence and invasion of mammalian cells by Yersinia enterocolitica. NPBD acts intracellularly to inhibit the early development stages of the Chlamydia trachomatis infection cycle in mammalian cells known to involve sequestration of host cell PTPs. NPBD thus both kills pathogens and inhibits virulence factors relevant to early infection, making it a suitable candidate for development as an anti-infective agent, particularly for pathogens that enter through, or cause infections at, mucosal surfaces. Though much is yet to be understood about bacterial PTPs, they are proposed as suitable anti-infective targets and have been linked to agents similar to NPBD. The structural and functional diversity and heterogeneous distribution of PTPs across microbial species make them suitably selective targets for the development of both broadly active and pathogen-specific drugs. PMID:24976873

  8. Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function.

    PubMed

    Paz, Cristina; Cornejo Maciel, Fabiana; Gorostizaga, Alejandra; Castillo, Ana F; Mori Sequeiros García, M Mercedes; Maloberti, Paula M; Orlando, Ulises D; Mele, Pablo G; Poderoso, Cecilia; Podesta, Ernesto J

    2016-01-01

    In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the "classical" protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed. PMID:27375556

  9. The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice.

    PubMed

    Baldauf, Christina; Jeschke, Anke; Kanbach, Vincent; Catala-Lehnen, Philip; Baumhoer, Daniel; Gerull, Helwe; Buhs, Sophia; Amling, Michael; Nollau, Peter; Harroch, Sheila; Schinke, Thorsten

    2015-01-01

    Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation in vitro. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development in vivo. We therefore analyzed the skeletal phenotype of mice lacking Ptprz1, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. Trp53-heterozygosity. By screening a large number of 52 week old Trp53-heterozygous mice by contact radiography we found that Ptprz1-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in Ptprz1-deficient Trp53-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation ex vivo. A comparison between Ptprz1-heterozygous and Ptprz1-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells. PMID:26360410

  10. Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke.

    PubMed

    Lee, Heung M; Reed, Jason; Greeley, George H; Englander, Ella W

    2009-03-01

    Survivors of massive inhalation of combustion smoke endure critical injuries, including lasting neurological complications. We have previously reported that acute inhalation of combustion smoke disrupts the nitric oxide homeostasis in the rat brain. In this study, we extend our findings and report that a 30-minute exposure of awake rats to ambient wood combustion smoke induces protein nitration in the rat hippocampus and that mitochondrial proteins are a sensitive nitration target in this setting. Mitochondria are central to energy metabolism and cellular signaling and are critical to proper cell function. Here, analyses of the mitochondrial proteome showed elevated protein nitration in the course of a 24-hour recovery following exposure to smoke. Mass spectrometry identification of several significantly nitrated mitochondrial proteins revealed diverse functions and involvement in central aspects of mitochondrial physiology. The nitrated proteins include the ubiquitous mitochondrial creatine kinase, F1-ATP synthase alpha subunit, dihydrolipoamide dehydrogenase (E3), succinate dehydrogenase Fp subunit, and voltage-dependent anion channel (VDAC1) protein. Furthermore, acute exposure to combustion smoke significantly compromised the respiratory capacity of hippocampal mitochondria. Importantly, elevated protein nitration and reduced mitochondrial respiration in the hippocampus persisted beyond the time required for restoration of normal oxygen and carboxyhemoglobin blood levels after the cessation of exposure to smoke. Thus, the time frame for intensification of the various smoke-induced effects differs between blood and brain tissues. Taken together, our findings suggest that nitration of essential mitochondrial proteins may contribute to the reduction in mitochondrial respiratory capacity and underlie, in part, the brain pathophysiology after acute inhalation of combustion smoke. PMID:19133281

  11. Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke

    SciTech Connect

    Lee, Heung M.; Reed, Jason; Greeley, George H.; Englander, Ella W.

    2009-03-01

    Survivors of massive inhalation of combustion smoke endure critical injuries, including lasting neurological complications. We have previously reported that acute inhalation of combustion smoke disrupts the nitric oxide homeostasis in the rat brain. In this study, we extend our findings and report that a 30-minute exposure of awake rats to ambient wood combustion smoke induces protein nitration in the rat hippocampus and that mitochondrial proteins are a sensitive nitration target in this setting. Mitochondria are central to energy metabolism and cellular signaling and are critical to proper cell function. Here, analyses of the mitochondrial proteome showed elevated protein nitration in the course of a 24-hour recovery following exposure to smoke. Mass spectrometry identification of several significantly nitrated mitochondrial proteins revealed diverse functions and involvement in central aspects of mitochondrial physiology. The nitrated proteins include the ubiquitous mitochondrial creatine kinase, F1-ATP synthase {alpha} subunit, dihydrolipoamide dehydrogenase (E3), succinate dehydrogenase Fp subunit, and voltage-dependent anion channel (VDAC1) protein. Furthermore, acute exposure to combustion smoke significantly compromised the respiratory capacity of hippocampal mitochondria. Importantly, elevated protein nitration and reduced mitochondrial respiration in the hippocampus persisted beyond the time required for restoration of normal oxygen and carboxyhemoglobin blood levels after the cessation of exposure to smoke. Thus, the time frame for intensification of the various smoke-induced effects differs between blood and brain tissues. Taken together, our findings suggest that nitration of essential mitochondrial proteins may contribute to the reduction in mitochondrial respiratory capacity and underlie, in part, the brain pathophysiology after acute inhalation of combustion smoke.

  12. Inhibition of human neutrophil responses by alpha-cyano-3,4-dihydroxythiocinnamamide; a protein-tyrosine kinase inhibitor.

    PubMed Central

    Dryden, P.; Duronio, V.; Martin, L.; Hudson, A. T.; Salari, H.

    1992-01-01

    1. Activation of neutrophils results in increased tyrosine phosphorylation of several proteins that may have important roles in receptor/effector coupling. In this study, the effect of a protein tyrosine kinase inhibitor on receptor-mediated neutrophil activation by platelet-activating factor (PAF), leukotriene, B4 (LTB4) and N-formylmethionylleucylphenylalanine (FMLP) is investigated. 2. alpha-Cyano-3,4-dihydroxythiocinnamamide dose-dependently inhibited intracellular calcium release and superoxide generation from human neutrophils activated by 1 microM LTB4, PAF, and FMLP. 3. In the presence of cytochalasin B, FMLP stimulated elastase release from neutrophils was also inhibited to unstimulated levels by 5 min pretreatment with alpha-cyano-3,4-dihydroxythiocinnamamide. 4. The inhibitory action of alpha-cyano-3,4-dihydroxythiocinnamamide was found to be at or upstream of phospholipase C activation, blocking both phosphatidylinositol hydrolysis and protein kinase C activation. alpha-Cyano-3,4-dihydroxythiocinnamamide did not affect agonist receptor binding sites or receptor affinity in neutrophils. 5. Immunoblot analysis demonstrated the tyrosine phosphorylation of proteins of 41, 56, 66, and 104 kDa in neutrophils treated with agonists. Treatment of neutrophils with alpha-cyano-3,4-dihydroxythiocinnamamide prior to stimulation with chemoattractants reduced tyrosine phosphorylation of the above phosphoproteins. 6. These results indicate that alpha-cyano-3,4-dihydroxythiocinnamamide might be a useful agent in characterizing the essential proteins and biochemical pathways that regulate neutrophil activation. PMID:1504749

  13. The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method.

    PubMed

    Kumaresan, A; Johannisson, A; Saravia, F; Bergqvist, A S

    2012-02-01

    Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO(2). Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study

  14. Protein Tyrosine Phosphatase N2 Is a Positive Regulator of Lipopolysaccharide Signaling in Raw264.7 Cell through Derepression of Src Tyrosine Kinase.

    PubMed

    Ha Thi, Huyen Trang; Choi, Seo-Won; Kim, Young-Mi; Kim, Hye-Youn; Hong, Suntaek

    2016-01-01

    T cell protein tyrosine phosphatase N2 (PTPN2) is a phosphotyrosine-specific nonreceptor phosphatase and is ubiquitously expressed in tissues. Although PTPN2 functions as an important regulator in different signaling pathways, it is still unclear what is specific target protein of PTPN2 and how is regulated in lipopolysaccharide (LPS)-induced inflammatory signaling pathway. Here, we found that PTPN2 deficiency downregulated the expression of LPS-mediated pro-inflammtory cytokine genes. Conversely, overexpression of PTPN2 in Raw264.7 cells enhanced the expression and secretion of those cytokines. The activation of MAPK and NF-κB signaling pathways by LPS was reduced in PTPN2-knockdowned cells and ectopic expression of PTPN2 reversed these effects. Furthermore, we found that PTNP2 directly interacted with Src and removed the inhibitory Tyr527 phosphorylation of Src to enhance the activatory phosphorylation of Tyr416 residue. These results suggested that PTPN2 is a positive regulator of LPS-induced inflammatory response by enhancing the activity of Src through targeting the inhibitory phosphor-tyrosine527 of Src. PMID:27611995

  15. Dephosphorylation of Tyrosine 393 in Argonaute 2 by Protein Tyrosine Phosphatase 1B Regulates Gene Silencing in Oncogenic RAS-Induced Senescence

    PubMed Central

    Yang, Ming; Haase, Astrid D.; Huang, Fang-Ke; Coulis, Gérald; Rivera, Keith D.; Dickinson, Bryan C.; Chang, Christopher J.; Pappin, Darryl J.; Neubert, Thomas A.; Hannon, Gregory J.; Boivin, Benoit; Tonks, Nicholas K.

    2014-01-01

    SUMMARY Oncogenic RAS (H-RASV12) induces premature senescence in primary cells by triggering production of reactive oxygen species (ROS), but the molecular role of ROS in senescence remains elusive. We investigated whether inhibition of protein tyrosine phosphatases by ROS contributed to H-RASV12-induced senescence. We identified protein tyrosine phosphatase 1B (PTP1B) as a major target of H-RASV12-induced ROS. Inactivation of PTP1B was necessary and sufficient to induce premature senescence in H-RASV12-expressing IMR90 fibroblasts. We identified phospho-Tyr 393 of argonaute 2 (AGO2) as a direct substrate of PTP1B. Phosphorylation of AGO2 at Tyr 393 inhibited loading with microRNAs (miRNA) and thus miRNA-mediated gene silencing, which counteracted the function of H-RASV12-induced oncogenic miRNAs. Overall, our data illustrate that premature senescence in H-RASV12-transformed primary cells is a consequence of oxidative inactivation of PTP1B and inhibition of miRNA-mediated gene silencing. PMID:25175024

  16. Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation

    PubMed Central

    Pierce, Andrew; Williamson, Andrew; Jaworska, Ewa; Griffiths, John R.; Taylor, Sam; Walker, Michael; O’Dea, Mark Aspinall; Spooncer, Elaine; Unwin, Richard D.; Poolman, Toryn; Ray, David; Whetton, Anthony D.

    2012-01-01

    Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases. PMID:22745689

  17. INHIBITORY POTENTIAL OF POLYHYDROXYLATED FULLERENES AGAINST PROTEIN TYROSINE PHOSPHATASE 1B.

    PubMed

    Kobzar, O L; Trush, V V; Tanchuk, V Yu; Vovk, A I

    2015-01-01

    Inhibition of PTP1B by polyhydroxylated fullerenes was studied in silico and in vitro. The enzyme kinetics in the presence of polyhydroxy small gap fullerenes showed that reciprocal value of maximum velocity non-linearly increases with increasing the inhibitor concentration. Analysis of the dose-dependent curve of PTP1B inhibition suggests an apparent positive cooperativity with involvement of at least two binding sites for the hydroxylated fullerene cages. Molecular docking calculations indicated that highly hydroxylated fullerene C60 may occupy the active site and additional allosteric binding site with similar affinity. In silico analysis of a number of fullerenols with 6, 12, 18, 24, 30, and 36 hydroxyl groups showed that the inhibitory activity may depend on the degree of hydroxylation of the nanoparticles surface. These data provide some understanding of the mechanisms of inhibitory action of fullerenols on activity of protein tyrosine phosphatases. PMID:26547960

  18. Implication of protein tyrosine phosphatase SHP-1 in cancer-related signaling pathways.

    PubMed

    Sharma, Yadhu; Ahmad, Altaf; Bashir, Samina; Elahi, Asif; Khan, Farah

    2016-05-01

    The altered expression of SHP-1 (SH2 domain-containing protein tyrosine phosphatase) as a consequence of promoter hypermethylation or mutations has evidently been linked to cancer development. The notion of being a cancer drug target is conceivable as SHP-1 negatively regulates cell cycle and inflammatory pathways which are an inevitable part of oncogenic transformation. In the present review, we try to critically analyze the role of SHP-1 in cancer progression via regulating the above mentioned pathways with the major emphasis on cell cycle components and JAK/STAT pathway, commencing with the SHP-1 biology in immune cell signaling. Lastly, we have provided the future directions for researchers to encourage SHP-1 as a prognostic marker and curative target for this debilitating disease called as cancer. PMID:26987952

  19. Controlled targeting of tyrosine hydroxylase protein toward processes of locus coeruleus neurons during postnatal development.

    PubMed

    Bezin, L; Diaz, J J; Marcel, D; Le Cavorsin, M; Madjar, J J; Pujol, J F; Weissmann, D

    1997-10-15

    Dendrites of locus coeruleus (LC) neurons laying within the pericoerulean neuropil (PCA) organize the major site where tyrosine hydroxylase (TH) is present throughout postnatal development. Those dendrites constitute the neuronal compartment in which TH levels increase beyond postnatal day (P) 21 or after RU24722-induced TH expression. Distal LC dendrites are present in the PCA by at least P20 but are devoid of TH and can rapidly accumulate TH protein when gene induction is triggered. Contrasting with the increase in TH levels within LC perikarya and dendrites, TH-mRNA concentration remains constant in LC perikarya from P4 to P42. Thus, supposing TH synthesis and degradation are also constant, any change in TH levels targeted toward axons might be balanced by a shift in the TH deposition within LC dendrites. This mechanism may be crucial in functions that the different processes of LC neurons have at critical steps of postnatal ontogeny. PMID:9406914

  20. Overexpression, purification, and characterization of SHPTP1, a Src homology 2-containing protein-tyrosine-phosphatase.

    PubMed Central

    Pei, D; Neel, B G; Walsh, C T

    1993-01-01

    A protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) containing two Src homology 2 (SH2) domains, SHPTP1, was previously identified in hematopoietic and epithelial cells. By placing the coding sequence of the PTPase behind a bacteriophage T7 promoter, we have overexpressed both the full-length enzyme and a truncated PTPase domain in Escherichia coli. In each case, the soluble enzyme was expressed at levels of 3-4% of total soluble E. coli protein. The recombinant proteins had molecular weights of 63,000 and 45,000 for the full-length protein and the truncated PTPase domain, respectively, as determined by SDS/PAGE. The recombinant enzymes dephosphorylated p-nitrophenyl phosphate, phosphotyrosine, and phosphotyrosyl peptides but not phosphoserine, phosphothreonine, or phosphoseryl peptides. The enzymes showed a strong dependence on pH and ionic strength for their activity, with pH optima of 5.5 and 6.3 for the full-length enzyme and the catalytic domain, respectively, and an optimal NaCl concentration of 250-300 mM. The recombinant PTPases had high Km values for p-nitrophenyl phosphate and exhibited non-Michaelis-Menten kinetics for phosphotyrosyl peptides. Images PMID:8430079

  1. A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells

    PubMed Central

    Chakedis, Jeffery; French, Randall; Babicky, Michele; Jaquish, Dawn; Howard, Haleigh; Mose, Evangeline; Lam, Raymond; Holman, Patrick; Miyamoto, Jaclyn; Walterscheid, Zakk; Lowy, Andrew M.

    2015-01-01

    The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. The splice variant is detected in 73% of pancreatic adenocarcinoma patient derived xenografts and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirms translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing (HPNE) cells activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target. PMID:26477314

  2. A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells.

    PubMed

    Chakedis, J; French, R; Babicky, M; Jaquish, D; Howard, H; Mose, E; Lam, R; Holman, P; Miyamoto, J; Walterscheid, Z; Lowy, A M

    2016-06-23

    The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription domain. The splice variant is detected in 73% of xenografts derived from pancreatic adenocarcinoma patients and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirm translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing cells, activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target. PMID:26477314

  3. The Potent Inhibitors of Protein Tyrosine Phosphatase 1B from the Fruits of Melaleuca leucadendron

    PubMed Central

    Saifudin, Azis; Lallo, Subehan Ab; Tezuka, Yasuhiro

    2016-01-01

    Background: Melaleuca leucadendron (Myrtaceae) is a kind of fruit used as Indonesian medicinal component and recorded in Jamu (tonic made of medical herbs) prescription records for the diabetes treatment. Its methanol extract exhibited a strong inhibitory activity with the half maximal inhibitory concentration (IC50) value of 2.05 μg/mL, while it is the same value with positive control RK-682. Objective: To isolate the chemical constituents of M. leucadendron and to evaluate their activity against protein tyrosine phosphatase 1B (PTP1B). Further, determine their toxicity potential against T-cell protein tyrosine phosphatase (TCPTP). Materials and Methods: Methanol extract was fractionated using silica column chromatography, and the obtained fraction was purified using Sephadex 20-LH. The structure of isolated compounds was identified based on 1H and 13Nuclear Magnetic Resonance Spectrometry. Furthermore, the compounds were examined against PTP1B and TCPTP. Results: Methanol extract of M. leucadendron (Myrtaceae) afforded two triterpenes: Betulinic acid and ursolic acid in high quantities. Both compounds exhibited a strong inhibitory activity against PTP1B inhibition with IC50 value of 1.5 and 2.3 μg/mL, respectively (positive control RK-682, IC50 = 2.05 μg/mL). Their activity toward TCPTP, on the other hand, were at 2.4 and 3.1 μg/mL, respectively. Based on this purification work, betulinic acid and ursolic acid presented 7.6% and 2.4%, respectively, as markedly M. leucadendron most potential for betulinic acid source among Indonesian plants. The result should have demonstrated that the antidiabetes of M. dendron could be through the inhibition of PTP1B. SUMMARY Melaleuca leucadendron is a good source for ursolic acid.Confirming traditional use for type II diabetes via PTP1B inhibition. PMID:27114690

  4. Identification and Expression of the Family of Classical Protein-Tyrosine Phosphatases in Zebrafish

    PubMed Central

    van Eekelen, Mark; Overvoorde, John; van Rooijen, Carina; den Hertog, Jeroen

    2010-01-01

    Protein-tyrosine phosphatases (PTPs) have an important role in cell survival, differentiation, proliferation, migration and other cellular processes in conjunction with protein-tyrosine kinases. Still relatively little is known about the function of PTPs in vivo. We set out to systematically identify all classical PTPs in the zebrafish genome and characterize their expression patterns during zebrafish development. We identified 48 PTP genes in the zebrafish genome by BLASTing of human PTP sequences. We verified all in silico hits by sequencing and established the spatio-temporal expression patterns of all PTPs by in situ hybridization of zebrafish embryos at six distinct developmental stages. The zebrafish genome encodes 48 PTP genes. 14 human orthologs are duplicated in the zebrafish genome and 3 human orthologs were not identified. Based on sequence conservation, most zebrafish orthologues of human PTP genes were readily assigned. Interestingly, the duplicated form of ptpn23, a catalytically inactive PTP, has lost its PTP domain, indicating that PTP activity is not required for its function, or that ptpn23b has lost its PTP domain in the course of evolution. All 48 PTPs are expressed in zebrafish embryos. Most PTPs are maternally provided and are broadly expressed early on. PTP expression becomes progressively restricted during development. Interestingly, some duplicated genes retained their expression pattern, whereas expression of other duplicated genes was distinct or even mutually exclusive, suggesting that the function of the latter PTPs has diverged. In conclusion, we have identified all members of the family of classical PTPs in the zebrafish genome and established their expression patterns. This is the first time the expression patterns of all members of the large family of PTP genes have been established in a vertebrate. Our results provide the first step towards elucidation of the function of the family of classical PTPs. PMID:20838449

  5. Role of protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation.

    PubMed

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2015-03-01

    Current hypothesis suggests that genetic, immunological, and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease. Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of inflammatory bowel disease. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signaling processes. Evidence emerges that expression levels of PTPN2, PTPN11, and PTPN22 are altered in actively inflamed intestinal tissue. PTPN2 seems to be critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses and finally for maintaining intestinal homeostasis. These observations have been confirmed in PTPN2 knockout mice in vivo. Those animals are clearly more susceptible to intestinal and systemic inflammation and feature alterations in innate and adaptive immune responses. PTPN22 controls inflammatory signaling in lymphocytes and mononuclear cells resulting in aberrant cytokine secretion pattern and autophagosome formation. PTPN22 deficiency in vivo results in more severe colitis demonstrating the relevance of PTPN22 for intestinal homeostasis in vivo. Of note, loss of PTPN22 promotes mitogen-activated protein kinase-induced cytokine secretion but limits secretion of nuclear factor κB-associated cytokines and autophagy in mononuclear cells. Loss of PTPN11 is also associated with increased colitis severity in vivo. In summary, dysfunction of those PTPs results in aberrant and uncontrolled immune responses that result in chronic inflammatory conditions. This way, it becomes more and more evident that dysfunction of PTPs displays an important factor in the pathogenesis of chronic intestinal inflammation, in particular inflammatory bowel disease. PMID:25581833

  6. Detection and kinetics of protein nitration in aerosols by NO2 and O3

    NASA Astrophysics Data System (ADS)

    Yang, H.; Zhang, Y.; Pöschl, U.

    2009-04-01

    The effects of air pollution on allergic diseases are not yet well-understood, but recent studies have shown that proteins are efficiently nitrated by polluted air (Franze et al., 2005) and that nitration enhances the allergenic potential of proteins such as the prominent birch pollen allergen Bet v 1 (Gruijthuijsen et al., 2006). Accordingly, the nitration of proteins in bioaerosol particles such as pollen and spores by NO2 and O3 might be a reason why allergies are on the increase in areas with traffic-related air pollution such as mega-cities and city clusters. In this study we have developed a method to determine the nitrotyrosine residue number per molecule in nitrated model proteins (bovine serum albumin, BSA; ovalbumin, OVA) by liquid chromatography coupled to UV-Vis photometry and mass spectrometry detectors (LC-DAD and LC-ESI-MS). Nitration experiments were carried out by exposing proteins to synthetic gas mixtures of nitrogen dioxide, ozone, nitrogen, synthetic air and water vapor. Reaction rates were measured at different concentration levels of NO2 and O3, and rate coefficients for the heterogeneous chemical reaction were determined. The implications for atmospheric aging and chemical transformation of bioaerosol particles and their potential effects on public health will be discussed. References: Franze, T., Weller, M.G., Niessner, R., Pöschl, U., Protein nitration by polluted air, Environ. Sci. Technol. 39, 1673-1678, 2005. Gruijthuijsen, Y.K., Grieshuber, I., Stöcklinger, A., Tischler, U., Fehrenbach, T., Weller, M.G., Vogel, L., Vieths, S., Pöschl, U., Duschl, A., Nitration enhances the allergenic potential of proteins, Int. Arch. Allergy Immunol. 141, 265-275, 2006.

  7. Tyrosine Phosphorylation Based Homo-dimerization of Arabidopsis RACK1A Proteins Regulates Oxidative Stress Signaling Pathways in Yeast

    PubMed Central

    Sabila, Mercy; Kundu, Nabanita; Smalls, Deana; Ullah, Hemayet

    2016-01-01

    Scaffold proteins are known as important cellular regulators that can interact with multiple proteins to modulate diverse signal transduction pathways. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, plays regulatory roles in diverse signal transduction and stress response pathways. RACK1 in humans has been implicated in myriads of neuropathological diseases including Alzheimer and alcohol addictions. Model plant Arabidopsis thaliana genome maintains three different RACK1 genes termed RACK1A, RACK1B, and RACK1C with a very high (85–93%) sequence identity among them. Loss of function mutation in Arabidopsis indicates that RACK1 proteins regulate diverse environmental stress signaling pathways including drought and salt stress resistance pathway. Recently deduced crystal structure of Arabidopsis RACK1A- very first among all of the RACK1 proteins, indicates that it can potentially be regulated by post-translational modifications, like tyrosine phosphorylations and sumoylation at key residues. Here we show evidence that RACK1A proteins, depending on diverse environmental stresses, are tyrosine phosphorylated. Utilizing site-directed mutagenesis of key tyrosine residues, it is found that tyrosine phosphorylation can potentially dictate the homo-dimerization of RACK1A proteins. The homo-dimerized RACK1A proteins play a role in providing UV-B induced oxidative stress resistance. It is proposed that RACK1A proteins ability to function as scaffold protein may potentially be regulated by the homo-dimerized RACK1A proteins to mediate diverse stress signaling pathways. PMID:26941753

  8. Tyrosine Phosphorylation Based Homo-dimerization of Arabidopsis RACK1A Proteins Regulates Oxidative Stress Signaling Pathways in Yeast.

    PubMed

    Sabila, Mercy; Kundu, Nabanita; Smalls, Deana; Ullah, Hemayet

    2016-01-01

    Scaffold proteins are known as important cellular regulators that can interact with multiple proteins to modulate diverse signal transduction pathways. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, plays regulatory roles in diverse signal transduction and stress response pathways. RACK1 in humans has been implicated in myriads of neuropathological diseases including Alzheimer and alcohol addictions. Model plant Arabidopsis thaliana genome maintains three different RACK1 genes termed RACK1A, RACK1B, and RACK1C with a very high (85-93%) sequence identity among them. Loss of function mutation in Arabidopsis indicates that RACK1 proteins regulate diverse environmental stress signaling pathways including drought and salt stress resistance pathway. Recently deduced crystal structure of Arabidopsis RACK1A- very first among all of the RACK1 proteins, indicates that it can potentially be regulated by post-translational modifications, like tyrosine phosphorylations and sumoylation at key residues. Here we show evidence that RACK1A proteins, depending on diverse environmental stresses, are tyrosine phosphorylated. Utilizing site-directed mutagenesis of key tyrosine residues, it is found that tyrosine phosphorylation can potentially dictate the homo-dimerization of RACK1A proteins. The homo-dimerized RACK1A proteins play a role in providing UV-B induced oxidative stress resistance. It is proposed that RACK1A proteins ability to function as scaffold protein may potentially be regulated by the homo-dimerized RACK1A proteins to mediate diverse stress signaling pathways. PMID:26941753

  9. Spinach 14-3-3 protein interacts with the plasma membrane H(+)-ATPase and nitrate reductase in response to excess nitrate stress.

    PubMed

    Xu, Huini; Zhao, Xiuling; Guo, Chuanlong; Chen, Limei; Li, Kunzhi

    2016-09-01

    To investigate the function of 14-3-3 protein in response to excess nitrate stress, a 14-3-3 protein, designated as So14-3-3, was isolated from spinach. Phylogenetic analysis demonstrated that So14-3-3 belongs to non-ε group of 14-3-3 superfamily. Real time-quantitative RT-PCR and western blot analysis showed that So14-3-3 was induced by excess nitrate stress in spinach roots and leaves. After nitrate treatment, the phosphorylated H(+)-ATPase and nitrate reductase (NR) increased and decreased respectively. Co-Immunoprecipitation (Co-IP) suggested that the interaction of So14-3-3 with the phosphorylated H(+)-ATPase enhanced, but reduced with phosphorylated NR in spinach roots after nitrate treatment. Besides, 5 proteins interacted with So14-3-3 were found by Co-IP and LC-MS/MS analysis. So14-3-3 overexpressing transgenic tobacco plants showed enhanced tolerance to nitrate treatment at the germination and young seedlings stage. The transgenic plants showed longer root length, lower malondialdehyde (MDA), H2O2, protein carbonyl contents, relatively higher soluble sugar and protein contents, than the WT plants after nitrate treatment. The phosphorylation levels of H(+)-ATPase in transgenic plants were higher than the WT plants after nitrate treatment, whereas NR were lower. Additionally, in transgenic plants, the interaction of So14-3-3 with phosphorylated H(+)-ATPase and NR increased and decreased more than the WT plants under nitrate stress, leading to higher H(+)-ATPase and NR activities in transgenic plants. These data suggested that So14-3-3 might be involved in nitrate stress response by interacting with H(+)-ATPase and NR. PMID:27161584

  10. The role of small adaptor proteins in the control of oncogenic signaling driven by tyrosine kinases in human cancer

    PubMed Central

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-01-01

    Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology. PMID:26788993

  11. UBC9-dependent Association between Calnexin and Protein Tyrosine Phosphatase 1B (PTP1B) at the Endoplasmic Reticulum*

    PubMed Central

    Lee, Dukgyu; Kraus, Allison; Prins, Daniel; Groenendyk, Jody; Aubry, Isabelle; Liu, Wen-Xin; Li, Hao-Dong; Julien, Olivier; Touret, Nicolas; Sykes, Brian D.; Tremblay, Michel L.; Michalak, Marek

    2015-01-01

    Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism. PMID:25586181

  12. Nitration of succinyl-CoA:3-oxoacid CoA-transferase in rats after endotoxin administration

    PubMed Central

    Marcondes, Sisi; Turko, Illarion V.; Murad, Ferid

    2001-01-01

    The tyrosine nitration of proteins has been observed in diverse inflammatory conditions and has been linked to the presence of reactive nitrogen species. From many in vitro experiments, it is apparent that tyrosine nitration may alter the function of proteins. A limited number of experiments under in vivo conditions also demonstrate that protein nitration is associated with altered cellular processes. To understand the association of protein nitration with the pathogenic mechanism of the disease, it is essential to identify specific protein targets of nitration with in vivo or intact tissue models. Using anti-nitrotyrosine antibodies, we demonstrated the accumulation of nitrotyrosine in a 52-kDa protein in rat kidney after lipopolysaccharide treatment. The 52-kDa protein was purified and identified with partial sequence as succinyl-CoA:3-oxoacid CoA-transferase (SCOT; EC 2.8.3.5). Western blot analysis revealed that the nitration of this mitochondrial enzyme increased in the kidneys and hearts of lipopolysaccharide-treated rats, whereas its catalytic activity decreased. These data suggest that tyrosine nitration may be a mechanism for the inhibition of SCOT activity in inflammatory conditions. SCOT is a key enzyme for ketone body utilization. Thus, tyrosine nitration of the enzyme with sepsis or inflammation may explain the altered metabolism of ketone bodies present in these disorders. PMID:11416199

  13. Receptor protein-tyrosine phosphatase. gamma. is a candidate tumor suppressor gene at human chromosome region 3p21

    SciTech Connect

    LaForgia, S.; Cannizzaro, L.A.; Boghosian-Sell, L.; Croce, C.M.; Huebner, K. ); Morse, B. ); Levy, J.; Barnea, G.; Schlessinger, J. ); Li, F. ); Nowell, P.C.; Glick, J. ); Weston, A.; Harris, C.C. ); Drabkin, H. ); Patterson, D. )

    1991-06-01

    PTPG, the gene for protein-tyrosine phosphatase {gamma} (PTP{gamma}), maps to a region of human chromosome 3, 3p21, that is frequently deleted in renal cell carcinoma and lung carcinoma. One of the functions of protein-tyrosine phosphatases is to reverse the effect of protein-tyrosine kinases, many of which are oncogenes, suggesting that some protein-tyrosine phosphatase genes may act as tumor suppressor genes. A hallmark of tumor suppressor genes is that they are deleted in tumors in which their inactivation contributes to the malignant phenotype. In this study, one PTP {gamma} allele was lost in 3 of 5 renal carcinoma cell lines and 5 of 10 lung carcinoma tumor samples tested. Importantly, one PTP {gamma} allele was lost in three lung tumors that had not lost flanking loci. PTP {gamma} mRNA was expressed in kidney cell lines and lung cell lines but not expressed in several hematopoietic cell lines tested. Thus, the PTP {gamma} gene has characteristics that suggest it as a candidate tumor suppressor gene at 3p21.

  14. Structure and Configuration of Phosphoeleganin, a Protein Tyrosine Phosphatase 1B Inhibitor from the Mediterranean Ascidian Sidnyum elegans.

    PubMed

    Imperatore, Concetta; Luciano, Paolo; Aiello, Anna; Vitalone, Rocco; Irace, Carlo; Santamaria, Rita; Li, Jia; Guo, Yue-W; Menna, Marialuisa

    2016-04-22

    A new phosphorylated polyketide, phosphoeleganin (1), has been isolated from the Mediterranean ascidian Sidnyum elegans. Its structure and configuration have been determined by extensive use of 2D NMR and microscale chemical degradation and/or derivatization. Phosphoeleganin (1) inhibited the protein tyrosine phosphatase 1B (PTP1B) activity. PMID:27064611

  15. Inhibitory evaluation of oligonol on α-glucosidase, protein tyrosine phosphatase 1B, cholinesterase, and β-secretase 1 related to diabetes and Alzheimer's disease.

    PubMed

    Choi, Jae Sue; Bhakta, Himanshu Kumar; Fujii, Hajime; Min, Byung-Sun; Park, Chan Hum; Yokozawa, Takako; Jung, Hyun Ah

    2016-03-01

    Oligonol is a low-molecular-weight form of polyphenol that is derived from lychee fruit extract and contains catechin-type monomers and oligomers of proanthocyanidins. This study investigates the anti-diabetic activities of oligonol via α-glucosidase and human recombinant protein tyrosine phosphatase 1B (PTP1B) assays, as well as its anti-Alzheimer activities by evaluating the ability of this compound to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1). Oligonol exhibited potent concentration-dependent anti-diabetic activities by inhibiting α-glucosidase and PTP1B with IC50 values of 23.14 µg/mL and 1.02 µg/mL, respectively. Moreover, a kinetics study revealed that oligonol inhibited α-glucosidase (K i = 22.36) and PTP1B (K i = 8.51) with characteristics typical of a mixed inhibitor. Oligonol also displayed potent concentration-dependent inhibitory activity against AChE and BChE with IC50 values of 4.34 µg/mL and 2.07 µg/mL, respectively. However, oligonol exhibited only marginal concentration-dependent BACE1 inhibitory activity with an IC50 value of 130.45 µg/mL. A kinetics study revealed mixed-type inhibition against AChE (K i = 4.65) and BACE1 (K i = 58.80), and noncompetitive-type inhibition against BChE (K i = 9.80). Furthermore, oligonol exhibited dose-dependent inhibitory activity against peroxynitrite (ONOO(-))-mediated protein tyrosine nitration. These results indicate that oligonol has strong preventative potential in diabetes mellitus and in Alzheimer's disease. PMID:26724817

  16. Low molecular weight protein tyrosine phosphatase: Multifaceted functions of an evolutionarily conserved enzyme.

    PubMed

    Caselli, Anna; Paoli, Paolo; Santi, Alice; Mugnaioni, Camilla; Toti, Alessandra; Camici, Guido; Cirri, Paolo

    2016-10-01

    Originally identified as a low molecular weight acid phosphatase, LMW-PTP is actually a protein tyrosine phosphatase that acts on many phosphotyrosine-containing cellular proteins that are primarily involved in signal transduction. Differences in sequence, structure, and substrate recognition as well as in subcellular localization in different organisms enable LMW-PTP to exert many different functions. In fact, during evolution, the LMW-PTP structure adapted to perform different catalytic actions depending on the organism type. In bacteria, this enzyme is involved in the biosynthesis of group 1 and 4 capsules, but it is also a virulence factor in pathogenic strains. In yeast, LMW-PTPs dephosphorylate immunophilin Fpr3, a peptidyl-prolyl-cis-trans isomerase member of the protein chaperone family. In humans, LMW-PTP is encoded by the ACP1 gene, which is composed of three different alleles, each encoding two active enzymes produced by alternative RNA splicing. In animals, LMW-PTP dephosphorylates a number of growth factor receptors and modulates their signalling processes. The involvement of LMW-PTP in cancer progression and in insulin receptor regulation as well as its actions as a virulence factor in a number of pathogenic bacterial strains may promote the search for potent, selective and bioavailable LMW-PTP inhibitors. PMID:27421795

  17. The Atlantic salmon protein tyrosine kinase Tyk2: molecular cloning, modulation of expression and function.

    PubMed

    Sobhkhez, Mehrdad; Hansen, Tom; Iliev, Dimitar B; Skjesol, Astrid; Jørgensen, Jorunn B

    2013-12-01

    Tyk2, a member of the Janus Kinase (JAK) family of protein tyrosine kinases, is required for interferon-α/β binding and signaling in higher vertebrates. Currently, little is known about the role of the different JAKs in signaling responses to interferon (IFN) in lower vertebrates including fish. In this paper we report the identification and characterization of Atlantic salmon (Salmo salar) Tyk2. Four cDNA sequences, two containing an open reading frame encoding full-length Tyk protein and two with an up-stream in frame stop codon, were identified. The deduced amino acid sequences of the salmon full-length Tyk2 proteins showed highest identity with Tyk2 from other species and their transcripts were ubiquitously expressed. Like in mammals the presented data suggests that salmon Tyk2 is auto-phosporylated when ectopically expressed in cells. In our experiments, full-length salmon Tyk2 overexpressed in CHSE-cells phosphorylated itself, while both a kinase-deficient mutant and the truncated Tyk2 (Tyk-short) were inactive. Interestingly, the overexpression of full length Tyk2 was shown to up-regulate the transcript levels of the IFN induced gene Mx, thus indicating the involvement of salmon Tyk2 in the salmon IFN I pathway. PMID:23872231

  18. Kinetic isotope effects in the characterization of catalysis by protein tyrosine phosphatases.

    PubMed

    Hengge, Alvan C

    2015-11-01

    Although thermodynamically favorable, the uncatalyzed hydrolysis of phosphate monoesters is extraordinarily slow, making phosphatases among the most catalytically efficient enzymes known. Protein-tyrosine phosphatases (PTPs) are ubiquitous in biology, and kinetic isotope effects were one of the key mechanistic tools used to discern molecular details of their catalytic mechanism and the transition state for phosphoryl transfer. Later, the unique level of detail KIEs provided led to deeper questions about the potential role of protein motions in PTP catalysis. The recent discovery that such motions are responsible for different catalytic rates between PTPs arose from questions originating from KIE data showing that the transition states and chemical mechanisms are identical, combined with structural data demonstrating superimposable active sites. KIEs also reveal perturbations to the transition state as mutations are made to residues directly involved in chemistry, and to residues that affect protein motions essential for catalysis. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment. PMID:25840000

  19. Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study.

    PubMed

    Singh, Neetu; Wadhawan, Mohit; Tiwari, Savitri; Kumar, Ranjeet; Rathaur, Sushma

    2016-07-01

    Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis. PMID:26965172

  20. Probing the target-specific inhibition of sensitized protein tyrosine phosphatases with biarsenical probes

    PubMed Central

    Pomorski, Adam; Adamczyk, Justyna; Bishop, Anthony C.; Krężel, Artur

    2014-01-01

    Selective control of enzyme activity is critical for elucidating the roles of specific proteins in signaling pathways. One potential means for developing truly target-specific inhibitors involves the use of protein engineering to sensitize a target enzyme to inhibition by a small molecule that does not inhibit homologous wild-type enzymes. Previously, it has been shown that protein tyrosine phosphatases (PTPs) can be sensitized to inhibition by a biarsenical probe, FlAsH-EDT2, which inhibits PTP activity by specifically binding to cysteine residues that have been introduced into catalytically important regions. In the present study, we developed an array of biarsenical probes, some newly synthesized and some previously reported, to investigate for the first time the structure-activity relationships for PTP inhibition by biarsenicals. Our data show that biarsenical probes which contain substitutions at the 2′ and 7′ positions are more effective than FlAsH-EDT2 at inhibiting sensitized PTPs. The increased potency of 2′,7′-substituted probes was observed when PTPs were assayed with both para-nitrophenylphosphate and phosphopeptide PTP substrates and at multiple probe concentrations. The data further indicate that the enhanced inhibitory properties are the result of increased binding affinity between the 2′,7′-substituted biarsenical probes and sensitized PTPs. In addition we provide previously unknown physicochemical and stability data for various biarsenical probes. PMID:25460004

  1. Decreased expression of receptor tyrosine kinase of EphB1 protein in renal cell carcinomas

    PubMed Central

    Zhou, Shuigen; Wang, Longxin; Li, Guimei; Zhang, Zhengyu; Wang, Jiandong

    2014-01-01

    Receptors tyrosine kinase of Eph superfamily plays an important role in human cancers. We previously found that EphB1 subtype is down-regulated in gastric cancer, colorectal cancer and ovary serous carcinoma. Fore the more, the decreased expression of EphB1 is related to invasion and metastasis in cancers. Although EphB1 has been revealed as an important receptor in cancers, our understanding of its roles in renal cell carcinoma (RCC) is limited. In the present study, using specific anit-EphB1 polyclonal antibody and immunohistochemistry, we evaluated EphB1 protein expression levels in RCC specimens surgically resected from 82 patients (including 62 conventional clear-cell RCC, 10 papillary, and 10 chromophobic RCC cases). We found EphB1 protein is positively expressed in the epithelium of renal tubules. Decreased expression of EphB1 was found in all RCC carcinomas compared with expression in the normal epithelium of renal tubules. EphB1 protein moderately expressed in chromophobic RCC, weakly expressed in clear-cell RCC and negatively expressed in papillary RCC. Our results indicate that EphB1 may be involved in carcinogenesis of RCC, the molecular mechanisms of down-regulation of EphB1 including genetic and epigenetic alterations and the dedicated roles of EphB1 in occurrence and progress of RCC need to be explicated in next step. PMID:25120806

  2. Are tyrosine residues involved in the photoconversion of the water-soluble chlorophyll-binding protein of Chenopodium album?

    PubMed

    Takahashi, S; Seki, Y; Uchida, A; Nakayama, K; Satoh, H

    2015-05-01

    Non-photosynthetic and hydrophilic chlorophyll (Chl) proteins, called water-soluble Chl-binding proteins (WSCPs), are distributed in various species of Chenopodiaceae, Amaranthaceae, Polygonaceae and Brassicaceae. Based on their photoconvertibility, WSCPs are categorised into two classes: Class I (photoconvertible) and Class II (non-photoconvertible). Chenopodium album WSCP (CaWSCP; Class I) is able to convert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton under light in the presence of molecular oxygen. Potassium iodide (KI) is a strong inhibitor of the photoconversion. Because KI attacks tyrosine residues in proteins, tyrosine residues in CaWSCP are considered to be important amino acid residues for the photoconversion. Recently, we identified the gene encoding CaWSCP and found that the mature region of CaWSCP contained four tyrosine residues: Tyr13, Tyr14, Tyr87 and Tyr134. To gain insight into the effect of the tyrosine residues on the photoconversion, we constructed 15 mutant proteins (Y13A, Y14A, Y87A, Y134A, Y13-14A, Y13-87A, Y13-134A, Y14-87A, Y14-134A, Y87-134A, Y13-14-87A, Y13-14-134A, Y13-87-134A, Y14-87-134A and Y13-14-87-134A) using site-directed mutagenesis. Amazingly, all the mutant proteins retained not only chlorophyll-binding activity, but also photoconvertibility. Furthermore, we found that KI strongly inhibited the photoconversion of Y13-14-87-134A. These findings indicated that the four tyrosine residues are not essential for the photoconversion. PMID:25287526

  3. Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity

    SciTech Connect

    Telerman, A.; Amson, R.; Zakut-Houri, R.; Givol, D.

    1988-04-01

    The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work the authors characterized the pim protein product. In vitro translation of positively selected poly(A)/sup +/ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo (/sup 35/S)methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33/sup pim/-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrphoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

  4. Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A.

    PubMed Central

    Hu, D E; Fan, T P

    1995-01-01

    1. Vascular endothelial growth factor (VEGF) is a heparin-binding angiogenic factor which specifically acts on endothelial cells via distinct membrane-spanning tyrosine kinase receptors. Here we used the rat sponge implant model to test the hypothesis that the angiogenic activity of VEGF can be suppressed by protein tyrosine kinase (PTK) inhibitors. 2. Neovascular responses in subcutaneous sponge implants were determined by measurements of relative sponge blood flow by use of a 133Xe clearance technique, and confirmed by histological studies and morphometric analysis. 3. Daily local administration of 250 ng VEGF165 accelerated the rate of 133Xe clearance from the sponges and induced an intense neovascularisation. This VEGF165-induced angiogenesis was inhibited by daily co-administration of the selective PTK inhibitor, lavendustin A (10 micrograms), but not its negative control, lavendustin B (10 micrograms). Blood flow measurements and morphometric analysis of 8-day-old sponges showed that lavendustin A reduced the 133Xe clearance of VEGF165-treated sponges from 32.9 +/- 1.5% to 20.9 +/- 1.6% and the total fibrovascular growth area from 62.4 +/- 6.1% to 21.6 +/- 6.8% (n = 12, P < 0.05). 4. Co-injection of suramin (3 mg), an inhibitor of heparin-binding growth factors, also suppressed the VEGF165-elicited neovascular response. In contrast, neither lavendustin A nor suramin produced any effect on the basal sponge-induced angiogenesis. 5. When given alone, low doses of VEGF165 (25 ng) or basic fibroblast growth factor (bFGF; 10 ng) did not modify the basal sponge-induced neovascularisation.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2 Figure 2 PMID:7533611

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

    PubMed

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

    2004-04-01

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

  6. 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

  7. Glutamate signaling proteins and tyrosine hydroxylase in the locus coeruleus of alcoholics

    PubMed Central

    Karolewicz, Beata; Johnson, Laurel; Szebeni, Katalin; Stockmeier, Craig A.; Ordway, Gregory A.

    2008-01-01

    It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and 8 psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (−30%) and middle (−41%)portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism. PMID:17481661

  8. Protein Tyrosine Phosphatase-1B Negatively Impacts Host Defense against Pseudomonas aeruginosa Infection.

    PubMed

    Yue, Lei; Xie, Zhongping; Li, Hua; Pang, Zheng; Junkins, Robert D; Tremblay, Michel L; Chen, Xiaochun; Lin, Tong-Jun

    2016-05-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance and reduced disease scores, which are accompanied by increased neutrophil infiltration and cytokine production. Interestingly, PTP1B deficiency mainly up-regulates the production of interferon-stimulated response elements-regulated cytokines and chemokines, including chemokine ligand 5 (regulated on activation normal T cell expressed and secreted), CXCL10 (interferon γ-inducible protein 10), and interferon-β production. Further studies reveal that PTP1B deficiency leads to increased interferon regulatory factor 7 (IRF7) expression and activation. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-interferon-stimulated response elements pathway may have broader implications in Toll-like receptor-mediated innate immunity. PMID:27105736

  9. Inactivation of Protein Tyrosine Phosphatases Enhances Interferon Signaling in Pancreatic Islets.

    PubMed

    Stanley, William J; Litwak, Sara A; Quah, Hong Sheng; Tan, Sih Min; Kay, Thomas W H; Tiganis, Tony; de Haan, Judy B; Thomas, Helen E; Gurzov, Esteban N

    2015-07-01

    Type 1 diabetes (T1D) is the result of an autoimmune assault against the insulin-producing pancreatic β-cells, where chronic local inflammation (insulitis) leads to β-cell destruction. T cells and macrophages infiltrate into islets early in T1D pathogenesis. These immune cells secrete cytokines that lead to the production of reactive oxygen species (ROS) and T-cell invasion and activation. Cytokine-signaling pathways are very tightly regulated by protein tyrosine phosphatases (PTPs) to prevent excessive activation. Here, we demonstrate that pancreata from NOD mice with islet infiltration have enhanced oxidation/inactivation of PTPs and STAT1 signaling compared with NOD mice that do not have insulitis. Inactivation of PTPs with sodium orthovanadate in human and rodent islets and β-cells leads to increased activation of interferon signaling and chemokine production mediated by STAT1 phosphorylation. Furthermore, this exacerbated STAT1 activation-induced cell death in islets was prevented by overexpression of the suppressor of cytokine signaling-1 or inactivation of the BH3-only protein Bim. Together our data provide a mechanism by which PTP inactivation induces signaling in pancreatic islets that results in increased expression of inflammatory genes and exacerbated insulitis. PMID:25732191

  10. An Enzyme Cascade for Selective Modification of Tyrosine Residues in Structurally Diverse Peptides and Proteins.

    PubMed

    Struck, Anna-Winona; Bennett, Matthew R; Shepherd, Sarah A; Law, Brian J C; Zhuo, Ying; Wong, Lu Shin; Micklefield, Jason

    2016-03-01

    Bioorthogonal chemistry enables a specific moiety in a complex biomolecule to be selectively modified in the presence of many reactive functional groups and other cellular entities. Such selectivity has become indispensable in biology, enabling biomolecules to be derivatized, conjugated, labeled, or immobilized for imaging, biochemical assays, or therapeutic applications. Methyltransferase enzymes (MTase) that accept analogues of the cofactor S-adenosyl methionine have been widely deployed for alkyl-diversification and bioorthogonal labeling. However, MTases typically possess tight substrate specificity. Here we introduce a more flexible methodology for selective derivatization of phenolic moieties in complex biomolecules. Our approach relies on the tandem enzymatic reaction of a fungal tyrosinase and the mammalian catechol-O-methyltransferase (COMT), which can effect the sequential hydroxylation of the phenolic group to give an intermediate catechol moiety that is subsequently O-alkylated. When used in this combination, the alkoxylation is highly selective for tyrosine residues in peptides and proteins, yet remarkably tolerant to changes in the peptide sequence. Tyrosinase-COMT are shown to provide highly versatile and regioselective modification of a diverse range of substrates including peptide antitumor agents, hormones, cyclic peptide antibiotics, and model proteins. PMID:26867114

  11. Protein tyrosine phosphatase mu regulates glioblastoma cell growth and survival in vivo

    PubMed Central

    Kaur, Harpreet; Burden-Gulley, Susan M.; Phillips-Mason, Polly J.; Basilion, James P.; Sloan, Andrew E.; Brady-Kalnay, Susann M.

    2012-01-01

    Glioblastoma multiforme (GBM) is the most lethal primary brain tumor. Extensive proliferation and dispersal of GBM tumor cells within the brain limits patient survival to approximately 1 year. Hence, there is a great need for the development of better means to treat GBM. Receptor protein tyrosine phosphatase (PTP)µ is proteolytically cleaved in GBM to yield fragments that promote dispersal of GBM cells. While normal brain tissue retains expression of full-length PTPµ, low-grade human astrocytoma samples have varying amounts of full-length PTPµ and cleaved PTPµ. In the highest-grade astrocytomas (i.e., GBM), PTPµ is completely proteolyzed into fragments. We demonstrate that short hairpin RNA mediated knockdown of full-length PTPµ and PTPµ fragments reduces glioma cell growth and survival in vitro. The reduction in growth and survival following PTPµ knockdown is enhanced when cells are grown in the absence of serum, suggesting that PTPµ may regulate autocrine signaling. Furthermore, we show for the first time that reduction of PTPµ protein expression decreases the growth and survival of glioma cells in vivo using mouse xenograft flank and i.c. tumor models. Inhibitors of PTPµ could be used to reduce the growth and survival of GBM cells in the brain, representing a promising therapeutic target for GBM. PMID:22505657

  12. 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

  13. Hole hopping through tyrosine/tryptophan chains protects proteins from oxidative damage

    PubMed Central

    Gray, Harry B.; Winkler, Jay R.

    2015-01-01

    Living organisms have adapted to atmospheric dioxygen by exploiting its oxidizing power while protecting themselves against toxic side effects. Reactive oxygen and nitrogen species formed during oxidative stress, as well as high-potential reactive intermediates formed during enzymatic catalysis, could rapidly and irreversibly damage polypeptides were protective mechanisms not available. Chains of redox-active tyrosine and tryptophan residues can transport potentially damaging oxidizing equivalents (holes) away from fragile active sites and toward protein surfaces where they can be scavenged by cellular reductants. Precise positioning of these chains is required to provide effective protection without inhibiting normal function. A search of the structural database reveals that about one third of all proteins contain Tyr/Trp chains composed of three or more residues. Although these chains are distributed among all enzyme classes, they appear with greatest frequency in the oxidoreductases and hydrolases. Consistent with a redox-protective role, approximately half of the dioxygen-using oxidoreductases have Tyr/Trp chain lengths ≥3 residues. Among the hydrolases, long Tyr/Trp chains appear almost exclusively in the glycoside hydrolases. These chains likely are important for substrate binding and positioning, but a secondary redox role also is a possibility. PMID:26195784

  14. Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120

    PubMed Central

    Frías, José E.

    2015-01-01

    ABSTRACT Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. IMPORTANCE Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many

  15. Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line.

    PubMed Central

    Casnellie, J E; Harrison, M L; Pike, L J; Hellström, K E; Krebs, E G

    1982-01-01

    The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [gamma-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70--80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the Mr 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60src; the Km for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The Km for this peptide was 3--4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide. Images PMID:6804939

  16. The Arabidopsis NRG2 Protein Mediates Nitrate Signaling and Interacts with and Regulates Key Nitrate Regulators[OPEN

    PubMed Central

    Zhao, Lufei; Zhang, Chengfei; Li, Zehui; Lei, Zhao; Liu, Fei; Guan, Peizhu; Crawford, Nigel M.

    2016-01-01

    We show that NITRATE REGULATORY GENE2 (NRG2), which we identified using forward genetics, mediates nitrate signaling in Arabidopsis thaliana. A mutation in NRG2 disrupted the induction of nitrate-responsive genes after nitrate treatment by an ammonium-independent mechanism. The nitrate content in roots was lower in the mutants than in the wild type, which may have resulted from reduced expression of NRT1.1 (also called NPF6.3, encoding a nitrate transporter/receptor) and upregulation of NRT1.8 (also called NPF7.2, encoding a xylem nitrate transporter). Genetic and molecular data suggest that NRG2 functions upstream of NRT1.1 in nitrate signaling. Furthermore, NRG2 directly interacts with the nitrate regulator NLP7 in the nucleus, but nuclear retention of NLP7 in response to nitrate is not dependent on NRG2. Transcriptomic analysis revealed that genes involved in four nitrogen-related clusters including nitrate transport and response to nitrate were differentially expressed in the nrg2 mutants. A nitrogen compound transport cluster containing some members of the NRT/PTR family was regulated by both NRG2 and NRT1.1, while no nitrogen-related clusters showed regulation by both NRG2 and NLP7. Thus, NRG2 plays a key role in nitrate regulation in part through modulating NRT1.1 expression and may function with NLP7 via their physical interaction. PMID:26744214

  17. Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function

    PubMed Central

    Paz, Cristina; Cornejo Maciel, Fabiana; Gorostizaga, Alejandra; Castillo, Ana F.; Mori Sequeiros García, M. Mercedes; Maloberti, Paula M.; Orlando, Ulises D.; Mele, Pablo G.; Poderoso, Cecilia; Podesta, Ernesto J.

    2016-01-01

    In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the “classical” protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed. PMID:27375556

  18. Tailoring a low-molecular weight protein tyrosine phosphatase into an efficient reporting protein

    SciTech Connect

    Liu, Xiao-Yan; Li, Lan-Fen; Su, Xiao-Dong; Shenzhen Graduate School of Peking University, Shenzhen 518055

    2009-05-15

    Fusion reporter methods are important tools for biology and biotechnology. An ideal reporter protein in a fusion system should have little effects on its fusion partner and provide an easy and accurate readout. Therefore, a small monomeric protein with high activity for detection assays often has advantages as a reporter protein. For this purpose, we have tailored the human B-form low-molecular-weight phosphotyrosyl phosphatase (HPTP-B) to increase its general applicability as a potent reporter protein. With the aim to eliminate interference from cysteine residues in the native HPTP-B, combined with a systematic survey of N- and C-terminal truncated variants, a series of cysteine to serine mutations were introduced, which allowed isolation of an engineered soluble protein with suitable biophysical properties. When we deleted both the first six residues and the last two residues, we still obtained a soluble mutant protein with correct folding and similar activity with wild-type protein. This mutant with two cysteine to serine mutations, HPTP-B{sup N{sub {Delta}}6-C{sub {Delta}}2-C90S-C109S}, has good potential as an optimal reporter.

  19. 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

  20. Tyrosine-rich acidic matrix protein (TRAMP) accelerates collagen fibril formation in vitro.

    PubMed

    MacBeath, J R; Shackleton, D R; Hulmes, D J

    1993-09-15

    Tyrosine-rich acidic matrix protein (TRAMP) is a recently discovered protein that co-purifies with porcine skin lysyl oxidase and is equivalent to the M(r) 22,000 extracellular matrix protein from bovine skin that co-purifies with dermatan sulfate proteoglycans (Cronshaw, A. D., MacBeath, J. R. E., Shackleton, D. R., Collins, J. F., Fothergill-Gilmore, L. A., and Hulmes, D. J. S. (1993) Matrix 13, 255-266; Neame, P. J., Choi, H. U., and Rosenberg, L. C. (1989) J. Biol. Chem. 264, 5474-5479). The effect of TRAMP on collagen fibril formation was studied in vitro by reconstitution of fibrils from lathyritic rat skin collagen I. Fibril formation was initiated by the warm start procedure, in which acidic collagen solutions and double strength neutral buffer, both preincubated separately at 34 degrees C, were mixed. When monitored by turbidimetry, TRAMP was found to accelerate collagen fibril formation. Acceleration occurred at sub-stoichiometric molar ratios of TRAMP collagen, and the presence of TRAMP stabilized the fibrils against low temperature dissociation. It was confirmed by centrifugation that the amount of fibrillar collagen formed in the presence of TRAMP was greater than in its absence. By SDS-polyacrylamide gel electrophoresis and scanning densitometry, binding of TRAMP to collagen was detected that approached saturation with a molar ratio of TRAMP to collagen of approximately 1:2. Fibrils formed in the presence of TRAMP were normal when observed by electron microscopy, although fibril diameters were smaller than the controls. TRAMP was found to partially reverse the inhibitory effects of urea and increased ionic strength on the kinetics of fibril formation, although inhibition by glucose was unaffected. TRAMP also accelerated the assembly of pepsin-treated collagen, where the non-helical, telopeptide regions were partially removed. Acceleration of collagen fibril formation by TRAMP is discussed in the light of the known effects of other extracellular matrix

  1. Pancreatic Protein Tyrosine Phosphatase 1B Deficiency Exacerbates Acute Pancreatitis in Mice.

    PubMed

    Bettaieb, Ahmed; Koike, Shinichiro; Chahed, Samah; Bachaalany, Santana; Griffey, Stephen; Sastre, Juan; Haj, Fawaz G

    2016-08-01

    Acute pancreatitis (AP) is a common and devastating gastrointestinal disorder that causes significant morbidity. The disease starts as local inflammation in the pancreas that may progress to systemic inflammation and complications. Protein tyrosine phosphatase 1B (PTP1B) is implicated in inflammatory signaling, but its significance in AP remains unclear. To investigate whether PTP1B may have a role in AP, we used pancreas PTP1B knockout (panc-PTP1B KO) mice and determined the effects of pancreatic PTP1B deficiency on cerulein- and arginine-induced acute pancreatitis. We report that PTP1B protein expression was increased in the early phase of AP in mice and rats. In addition, histological analyses of pancreas samples revealed enhanced features of AP in cerulein-treated panc-PTP1B KO mice compared with controls. Moreover, cerulein- and arginine-induced serum amylase and lipase were significantly higher in panc-PTP1B KO mice compared with controls. Similarly, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B, IL-6, and tumor necrosis factor-α were increased in panc-PTP1B KO mice compared with controls. Furthermore, panc-PTP1B KO mice exhibited enhanced cerulein- and arginine-induced NF-κB inflammatory response accompanied with increased mitogen-activated protein kinases activation and elevated endoplasmic reticulum stress. Notably, these effects were recapitulated in acinar cells treated with a pharmacological inhibitor of PTP1B. These findings reveal a novel role for pancreatic PTP1B in cerulein- and arginine-induced acute pancreatitis. PMID:27461362

  2. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene.

    PubMed

    Norris, M L; Millhorn, D E

    1995-10-01

    We reported recently that the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is regulated by hypoxia in the dopaminergic cells of the mammalian carotid body (Czyzyk-Krzeska, M. F., Bayliss, D. A., Lawson, E. E. & Millhorn, D. E. (1992) J. Neurochem. 58, 1538-1546) and in pheochromocytoma (PC12) cells (Czyzyk-Krzeska, M. F., Furnari, B. A., Lawson, E. E. & Millhorn, D. E. (1994) J. Biol. Chem. 269, 760-764). Regulation of this gene during low O2 conditions occurs at both the level of transcription and RNA stability. Increased transcription during hypoxia is regulated by a region of the proximal promoter that extends from -284 to + 27 bases, relative to transcription start site. The present study was undertaken to further characterize the sequences that confer O2 responsiveness of the TH gene and to identify hypoxia-induced protein interactions with these sequences. Results from chloramphenicol acetyltransferase assays identified a region between bases -284 and -150 that contains the essential sequences for O2 regulation. This region contains a number of regulatory elements including AP1, AP2, and HIF-1. Gel shift assays revealed enhanced protein interactions at the AP1 and HIF-1 elements of the native gene. Further investigations using supershift and shift-Western analysis showed that c-Fos and JunB bind to the AP1 element during hypoxia and that these protein levels are stimulated by hypoxia. Mutation of the AP1 sequence prevented stimulation of transcription of the TH-chloramphenicol acetyltransferase reporter gene by hypoxia. PMID:7559551

  3. 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

  4. Atomic structure of nitrate-binding protein crucial for photosynthetic productivity

    SciTech Connect

    Koropatkin, Nicole M.; Pakrasi, Himadri B.; Smith, Thomas J.

    2006-06-27

    Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of all aquatic food chains by fixing carbon and nitrogen into cellular biomass. The single most important nutrient for photosynthesis and growth is nitrate, which is severely limiting in many aquatic environments particularly the open ocean (1, 2). It is therefore not surprising that NrtA, the solute-binding component of the high-affinity nitrate ABC transporter, is the single-most abundant protein in the plasma membrane of these bacteria (3). Here we describe the first structure of a nitratespecific receptor, NrtA from Synechocystis sp. PCC 6803, complexed with nitrate and determined to a resolution of 1.5Å. NrtA is significantly larger than other oxyanionbinding proteins, representing a new class of transport proteins. From sequence alignments, the only other solute-binding protein in this class is CmpA, a bicarbonatebinding protein. Therefore, these organisms created a novel solute-binding protein for two of the most important nutrients; inorganic nitrogen and carbon. The electrostatic charge distribution of NrtA appears to force the protein off of the membrane while the flexible tether facilitates the delivery of nitrate to the membrane pore. The structure not only details the determinants for nitrate selectivity in NrtA, but also the bicarbonate specificity in CmpA. Nitrate and bicarbonate transport are regulated by the cytoplasmic proteins NrtC and CmpC, respectively. Interestingly, the residues lining the ligand binding pockets suggest that they both bind nitrate. This implies that the nitrogen and carbon uptake pathways are synchronized by intracellular nitrate and nitrite.3 The nitrate ABC transporter of cyanobacteria is composed of four polypeptides (Figure 1): a high-affinity periplasmic solute-binding lipoprotein (NrtA), an integral membrane permease (NrtB), a cytoplasmic ATPase (NrtD), and a unique ATPase/solute-binding fusion protein (Nrt

  5. Nitric oxide attenuates hydrogen peroxide-induced barrier disruption and protein tyrosine phosphorylation in monolayers of intestinal epithelial cell.

    PubMed

    Katsube, Takanori; Tsuji, Hideo; Onoda, Makoto

    2007-06-01

    The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H(2)O(2), Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and beta-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H(2)O(2) and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H(2)O(2) alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells. PMID:17451824

  6. Negative regulation of HER2 signaling by the PEST-type protein-tyrosine phosphatase BDP1.

    PubMed

    Gensler, Miriam; Buschbeck, Marcus; Ullrich, Axel

    2004-03-26

    Signaling by receptor tyrosine kinases (RTK) mediates a variety of complex cellular functions and in case of deregulation can contribute to pathophysiological processes. A tight and finely tuned control of RTK activity is therefore critical for the cell. We investigated the role of the PEST-type protein-tyrosine phosphatase BDP1 in the regulation of HER2, a member of the epidermal growth factor receptor (EGFR) family of RTKs. Here we demonstrate that HER2 signaling is highly sensitive to BDP1 activity. Overexpression of BDP1 inhibited ligand-induced activation of HER2 but not that of the closely related EGFR. On the other hand, suppression of endogenous BDP1 expression increased the phosphorylation state of HER2. In addition, BDP1 was able to interfere with downstream signaling events by inhibiting the phosphorylation of the adaptor protein Gab1 and reducing mitogen-activated protein kinase activation. Supported by the finding that BDP1 is coexpressed with HER2 in breast cancer cells, we suggest that BDP1 is an important regulator of HER2 activity and thus the first protein-tyrosine phosphatase shown to be involved in HER2 signal attenuation. PMID:14660651

  7. Net Increase of platelet membrane tyrosine specific-protein kinase activity by phorbol myristate acetate

    SciTech Connect

    Ishihara, Noriko; Sakamoto, Hikaru; Iwama, Minako; Kobayashi, Bonro )

    1990-01-01

    Tyrosine protein kinase (TPK) activity in rabbit platelets after stimulation by phorbol myristate acetate (PMA) or thrombin was directly estimated by {sup 32}P incorporation from ({gamma}-{sup 32})ATP into synthetic peptide angiotensin II. By PMA-treatment a net increase of TPK activity was obtained, while thrombin acted on the TPK quickly but stimulation was limited within the range attained by the control after lengthy incubation. The responsive TPK to these stimulators was localized mainly in membrane but much less in cytosol. The specific activity of the particulate TPK was low in the sonicate of control ice cold platelets but increased about 6-fold when the platelets were incubated at 37{degree}C. On a brief contact of platelets with PMA at 37{degrees}C the TPK was fully activated and reached a maximum value about 130% of the control. Determination of phosphotyrosine phosphatase in the stimulated platelet sonicate revealed that its participation in the above described increase of {sup 32}P-incorporation was meagre. The quick response suggested a possible role of TPK in the signal transduction through the platelet cell membrane.

  8. Protein tyrosine phosphatase SHP2 promotes invadopodia formation through suppression of Rho signaling

    PubMed Central

    Tsai, Wan-Chen; Chen, Chien-Lin; Chen, Hong-Chen

    2015-01-01

    Invadopodia are actin-enriched membrane protrusions that are important for extracellular matrix degradation and invasive cell motility. Src homolog domain-containing phosphatase 2 (SHP2), a non-receptor protein tyrosine phosphatase, has been shown to play an important role in promoting cancer metastasis, but the underlying mechanism is unclear. In this study, we found that depletion of SHP2 by short-hairpin RNA suppressed invadopodia formation in several cancer cell lines, particularly in the SAS head and neck squamous cell line. In contrast, overexpression of SHP2 promoted invadopodia formation in the CAL27 head and neck squamous cell line, which expresses low levels of endogenous SHP2. The depletion of SHP2 in SAS cells significantly decreased their invasive motility. The suppression of invadopodia formation by SHP2 depletion was restored by the Clostridium botulinum C3 exoenzyme (a Rho GTPase inhibitor) or Y27632 (a specific inhibitor for Rho-associated kinase). Together, our results suggest that SHP2 may promote invadopodia formation through inhibition of Rho signaling in cancer cells. PMID:26204488

  9. The Mechanism of Allosteric Inhibition of Protein Tyrosine Phosphatase 1B

    PubMed Central

    Lu, Shaoyong; Huang, Wenkang; Geng, Lv; Shen, Qiancheng; Zhang, Jian

    2014-01-01

    As the prototypical member of the PTP family, protein tyrosine phosphatase 1B (PTP1B) is an attractive target for therapeutic interventions in type 2 diabetes. The extremely conserved catalytic site of PTP1B renders the design of selective PTP1B inhibitors intractable. Although discovered allosteric inhibitors containing a benzofuran sulfonamide scaffold offer fascinating opportunities to overcome selectivity issues, the allosteric inhibitory mechanism of PTP1B has remained elusive. Here, molecular dynamics (MD) simulations, coupled with a dynamic weighted community analysis, were performed to unveil the potential allosteric signal propagation pathway from the allosteric site to the catalytic site in PTP1B. This result revealed that the allosteric inhibitor compound-3 induces a conformational rearrangement in helix α7, disrupting the triangular interaction among helix α7, helix α3, and loop11. Helix α7 then produces a force, pulling helix α3 outward, and promotes Ser190 to interact with Tyr176. As a result, the deviation of Tyr176 abrogates the hydrophobic interactions with Trp179 and leads to the downward movement of the WPD loop, which forms an H-bond between Asp181 and Glu115. The formation of this H-bond constrains the WPD loop to its open conformation and thus inactivates PTP1B. The discovery of this allosteric mechanism provides an overall view of the regulation of PTP1B, which is an important insight for the design of potent allosteric PTP1B inhibitors. PMID:24831294

  10. Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction.

    PubMed Central

    Wilks, A F

    1989-01-01

    The pivotal role that protein-tyrosine kinases (PTKs) play in the growth regulation of eukaryotic cells is manifest in the frequent appearance of members of the PTK family as growth factor receptors or as the transforming agents of acutely transforming retroviruses. A feature common to all members of the PTK family is a highly conserved catalytic domain which is characteristic of the group as a whole and whose activity appears to be tightly regulated within the cell by other domains of the PTK. Degenerate oligonucleotide probes corresponding to two invariant amino acid sequence motifs within the catalytic domains of all PTK family members were synthesized and employed in the polymerase chain reaction (PCR) to amplify cDNA sequences between them. An M13 PCR library was produced in this way from cDNA prepared against mRNA from the murine hemopoietic cell line FDC-P1. The PCR library was then screened by DNA sequencing for PTK-related sequences. Two sequences were identified that, on the basis of sequence comparison with known PTKs, may encode representatives of a new class of PTK. Images PMID:2466296

  11. Discovery of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B (PtpB) Inhibitors from Natural Products

    PubMed Central

    Chiaradia-Delatorre, Louise Domeneghini; Menegatti, Angela Camila Orbem; Monache, Franco Delle; Ferrari, Franco; Yunes, Rosendo Augusto; Nunes, Ricardo José; Terenzi, Hernán; Botta, Bruno; Botta, Maurizio

    2013-01-01

    Protein tyrosine phosphatase B (PtpB) is one of the virulence factors secreted into the host cell by Mycobacterium tuberculosis. PtpB attenuates host immune defenses by interfering with signal transduction pathways in macrophages and, therefore, it is considered a promising target for the development of novel anti-tuberculosis drugs. Here we report the discovery of natural compound inhibitors of PtpB among an in house library of more than 800 natural substances by means of a multidisciplinary approach, mixing in silico screening with enzymatic and kinetics studies and MS assays. Six natural compounds proved to inhibit PtpB at low micromolar concentrations (< 30 µM) with Kuwanol E being the most potent with Ki = 1.6 ± 0.1 µM. To the best of our knowledge, Kuwanol E is the most potent natural compound PtpB inhibitor reported so far, as well as it is the first non-peptidic PtpB inhibitor discovered from natural sources. Compounds herein identified may inspire the design of novel specific PtpB inhibitors. PMID:24155919

  12. Comprehensive protein tyrosine phosphatase mRNA profiling identifies new regulators in the progression of glioma.

    PubMed

    Bourgonje, Annika M; Verrijp, Kiek; Schepens, Jan T G; Navis, Anna C; Piepers, Jolanda A F; Palmen, Chantal B C; van den Eijnden, Monique; Hooft van Huijsduijnen, Rob; Wesseling, Pieter; Leenders, William P J; Hendriks, Wiljan J A J

    2016-01-01

    The infiltrative behavior of diffuse gliomas severely reduces therapeutic potential of surgical resection and radiotherapy, and urges for the identification of new drug-targets affecting glioma growth and migration. To address the potential role of protein tyrosine phosphatases (PTPs), we performed mRNA expression profiling for 91 of the 109 known human PTP genes on a series of clinical diffuse glioma samples of different grades and compared our findings with in silico knowledge from REMBRANDT and TCGA databases. Overall PTP family expression levels appeared independent of characteristic genetic aberrations associated with lower grade or high grade gliomas. Notably, seven PTP genes (DUSP26, MTMR4, PTEN, PTPRM, PTPRN2, PTPRT and PTPRZ1) were differentially expressed between grade II-III gliomas and (grade IV) glioblastomas. For DUSP26, PTEN, PTPRM and PTPRT, lower expression levels correlated with poor prognosis, and overexpression of DUSP26 or PTPRT in E98 glioblastoma cells reduced tumorigenicity. Our study represents the first in-depth analysis of PTP family expression in diffuse glioma subtypes and warrants further investigations into PTP-dependent signaling events as new entry points for improved therapy. PMID:27586084

  13. Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells.

    PubMed

    Bunin, Anna; Sisirak, Vanja; Ghosh, Hiyaa S; Grajkowska, Lucja T; Hou, Z Esther; Miron, Michelle; Yang, Cliff; Ceribelli, Michele; Uetani, Noriko; Chaperot, Laurence; Plumas, Joel; Hendriks, Wiljan; Tremblay, Michel L; Häcker, Hans; Staudt, Louis M; Green, Peter H; Bhagat, Govind; Reizis, Boris

    2015-08-18

    Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS(-) pDCs produced IFN-α. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation. PMID:26231120

  14. [Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases.

    PubMed Central

    Desmarais, S; Friesen, R W; Zamboni, R; Ramachandran, C

    1999-01-01

    Peptides containing the non-hydrolysable phosphotyrosine analogue 4-[difluro(phosphono)methyl]phenylalanine [Phe(CF2P)] were synthesized and tested as inhibitors of the protein tyrosine phosphatases (PTPs) PTP1B, CD45, PTPbeta, LAR and SHP-1. We have identified peptides containing two adjacent Phe(CF2P) residues as potent inhibitors of PTPs. The tripeptide having the sequence Glu-Phe(CF2P)-Phe(CF2P) is a potent and selective inhibitor of PTP1B. This peptide inhibits PTP1B with an IC50 of 40 nM, which is at least 100-fold lower than with other PTPs. A second tripeptide, Pro-Phe(CF2P)-Phe(CF2P), is most potent against PTPbeta, with an IC50 of 200 nM, and inhibits PTP1B with an IC50 of 300 nM. These data suggest that it is possible to develop selective, active-site-directed, reversible, potent inhibitors of PTPs. PMID:9882618

  15. Protein Tyrosine Phosphatase α in the Dorsomedial Striatum Promotes Excessive Ethanol-Drinking Behaviors

    PubMed Central

    Ben Hamida, Sami; Darcq, Emmanuel; Wang, Jun; Wu, Su; Phamluong, Khanhky; Kharazia, Viktor

    2013-01-01

    We previously found that excessive ethanol drinking activates Fyn in the dorsomedial striatum (DMS) (Wang et al., 2010; Gibb et al., 2011). Ethanol-mediated Fyn activation in the DMS leads to the phosphorylation of the GluN2B subunit of the NMDA receptor, to the enhancement of the channel's activity, and to the development and/or maintenance of ethanol drinking behaviors (Wang et al., 2007, 2010). Protein tyrosine phosphatase α (PTPα) is essential for Fyn kinase activation (Bhandari et al., 1998), and we showed that ethanol-mediated Fyn activation is facilitated by the recruitment of PTPα to synaptic membranes, the compartment where Fyn resides (Gibb et al., 2011). Here we tested the hypothesis that PTPα in the DMS is part of the Fyn/GluN2B pathway and is thus a major contributor to the neuroadaptations underlying excessive ethanol intake behaviors. We found that RNA interference (RNAi)-mediated PTPα knockdown in the DMS reduces excessive ethanol intake and preference in rodents. Importantly, no alterations in water, saccharine/sucrose, or quinine intake were observed. Furthermore, downregulation of PTPα in the DMS of mice significantly reduces ethanol-mediated Fyn activation, GluN2B phosphorylation, and ethanol withdrawal-induced long-term facilitation of NMDAR activity without altering the intrinsic features of DMS neurons. Together, these results position PTPα upstream of Fyn within the DMS and demonstrate the important contribution of the phosphatase to the maladaptive synaptic changes that lead to excessive ethanol intake. PMID:24005290

  16. An Affinity-Based Fluorescence Polarization Assay for Protein Tyrosine Phosphatases

    PubMed Central

    Zhang, Sheng; Chen, Lan; Kumar, Sanjai; Wu, Li; Lawrence, David S.; Zhang, Zhong-Yin

    2007-01-01

    Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate “false” positives due to modification of the active site Cys that destroy the phosphatase activity. PMID:17532513

  17. Protection from impulse noise-induced hearing loss with novel Src-protein tyrosine kinase inhibitors

    PubMed Central

    Bielefeld, Eric C.; Hangauer, David; Henderson, Donald

    2011-01-01

    Apoptosis is a significant mechanism of cochlear hair cell loss from noise. Molecules that inhibit apoptotic intracellular signaling reduce cochlear damage and hearing loss from noise. The current study is an extension of a previous study of the protective value of Src-protein tyrosine kinase inhibitors against noise (Harris et al., 2005). The current study tested three Src-inhibitors: the indole-based KX1-141, the biaryl-based KX2-329, and the ATP-competitive KX2-328. Each of the three drugs was delivered into the chinchillas’ cochleae by allowing the solutions to diffuse across the round window membrane thirty minutes prior to exposure to impulse noise. Hearing thresholds were measured using auditory evoked responses from electrodes in the inferior colliculi. Ears treated with KX2-329 showed significantly lower threshold shifts and outer hair cell losses than the control group. The cochleae treated with KX1-141 and KX2-328 did not show statistically significant protection from the impulse noise. The finding of protection with KX2-329 demonstrates that a biaryl-based Src inhibitor has protective capacity against noise-induced hearing loss that is as good as that demonstrated by KX1-004, a Src inhibitor drug that has been studied extensively as an otoprotectant against noise, and suggests that KX2-329 could be useful for protection against noise. PMID:21840347

  18. Protein tyrosine phosphatase SHP-1: resurgence as new drug target for human autoimmune disorders.

    PubMed

    Sharma, Yadhu; Bashir, Samina; Bhardwaj, Puja; Ahmad, Altaf; Khan, Farah

    2016-08-01

    Recognition of self-antigen and its destruction by the immune system is the hallmark of autoimmune diseases. During the developmental stages, immune cells are introduced to the self-antigen, for which tolerance develops. The inflammatory insults that break the immune tolerance provoke immune system against self-antigen, progressively leading to autoimmune diseases. SH2 domain containing protein tyrosine phosphatase (PTP), SHP-1, was identified as hematopoietic cell-specific PTP that regulates immune function from developing immune tolerance to mediating cell signaling post-immunoreceptor activation. The extensive research on SHP-1-deficient mice elucidated the diversified role of SHP-1 in immune regulation, and inflammatory process and related disorders such as cancer, autoimmunity, and neurodegenerative diseases. The present review focalizes upon the implication of SHP-1 in the pathogenesis of autoimmune disorders, such as allergic asthma, neutrophilic dermatosis, atopic dermatitis, rheumatoid arthritis, and multiple sclerosis, so as to lay the background in pursuance of developing therapeutic strategies targeting SHP-1. Also, new SHP-1 molecular targets have been suggested like SIRP-α, PIPKIγ, and RIP-1 that may prove to be the focal point for the development of therapeutic strategies. PMID:27216862

  19. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    NASA Astrophysics Data System (ADS)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  20. Protein tyrosine phosphatase non-receptor type 2 and inflammatory bowel disease

    PubMed Central

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2016-01-01

    Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) with the onset of inflammatory bowel disease (IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and adaptive immune responses. In particular, PTPN2 is involved in the regulation of inflammatory signalling cascades, and critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses, and finally for maintaining intestinal homeostasis. On one hand, dysfunction of PTPN2 has drastic effects on innate host defence mechanisms, including increased secretion of pro-inflammatory cytokines, limited autophagosome formation in response to invading pathogens, and disruption of the intestinal epithelial barrier. On the other hand, PTPN2 function is crucial for controlling adaptive immune functions, by regulating T cell proliferation and differentiation as well as maintaining T cell tolerance. In this way, dysfunction of PTPN2 contributes to the manifestation of IBD. The aim of this review is to present an overview of recent findings on the role of PTPN2 in intestinal homeostasis and the impact of dysfunctional PTPN2 on intestinal inflammation. PMID:26811645

  1. Expression, prognostic significance and mutational analysis of protein tyrosine phosphatase SHP-1 in chronic myeloid leukemia.

    PubMed

    Papadopoulou, Vasiliki; Kontandreopoulou, Elina; Panayiotidis, Panayiotis; Roumelioti, Maria; Angelopoulou, Maria; Kyriazopoulou, Lydia; Diamantopoulos, Panagiotis T; Vaiopoulos, George; Variami, Eleni; Kotsianidis, Ioannis; Athina Viniou, Nora

    2016-05-01

    The protein tyrosine phosphatase SHP-1 dephosphorylates BCR-ABL1, thereby serving as a potential control mechanism of BCR-ABL1 kinase activity. Pathways regulating SHP-1 expression, which could be exploited in the therapeutics of TKI-resistant chronic myeloid leukemia (CML), remain unknown. Moreover, the questions of whether there is any kind of SHP-1 deregulation in CML, contributing to disease initiation or evolution, as well as the question of prognostic significance of SHP-1, have not been definitively answered. This study shows moderately lower SHP-1 mRNA expression in chronic phase CML patients in comparison to healthy individuals and no change in SHP-1 mRNA levels after successful TKI treatment. Mutational analysis of the aminoterminal and phosphatase domains of SHP-1 in patients did not reveal genetic lesions. This study also found no correlation of SHP-1 expression at diagnosis with response to treatment, although a trend for lower SHP-1 expression was noted in the very small non-responders' group of the 3-month therapeutic milestone. PMID:26373709

  2. JAK2 Tyrosine Kinase Phosphorylates and Is Negatively Regulated by Centrosomal Protein Ninein

    PubMed Central

    Jay, Jennifer; Hammer, Alan; Nestor-Kalinoski, Andrea

    2014-01-01

    JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways. PMID:25332239

  3. Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling

    PubMed Central

    Holmes, Derek A.; Suto, Eric; Lee, Wyne P.; Ou, Qinglin; Gong, Qian; Smith, Hamish R.C.; Caplazi, Patrick

    2015-01-01

    The protein tyrosine phosphatase PTPN22(C1858T) allelic polymorphism is associated with increased susceptibility for development of systemic lupus erythematosus (SLE) and other autoimmune diseases. PTPN22 (also known as LYP) and its mouse orthologue PEP play important roles in antigen and Toll-like receptor signaling in immune cell functions. We demonstrate here that PEP also plays an important inhibitory role in interferon-α receptor (IFNAR) signaling in mice. PEP co-immunoprecipitates with components of the IFNAR signaling complex. Pep−/− hematopoietic progenitors demonstrate increased IFNAR signaling, increased IFN-inducible gene expression, and enhanced proliferation and activation compared to Pep+/+ progenitors in response to IFN-α. In addition, Pep−/− mice treated with IFN-α display a profound defect in hematopoiesis, resulting in anemia, thrombocytopenia, and neutropenia when compared to IFN-α–treated Pep+/+ mice. As SLE patients carrying the PTPN22(C1858T) risk variant have higher serum IFN-α activity, these data provide a molecular basis for how type I IFNs and PTPN22 may cooperate to contribute to lupus-associated cytopenias. PMID:26077719

  4. Surface-tuned electron transfer and electrocatalysis of hexameric tyrosine-coordinated heme protein.

    PubMed

    Peng, Lei; Utesch, Tillmann; Yarman, Aysu; Jeoung, Jae-Hun; Steinborn, Silke; Dobbek, Holger; Mroginski, Maria Andrea; Tanne, Johannes; Wollenberger, Ulla; Scheller, Frieder W

    2015-05-11

    Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant ks values between 0.93 and 2.86 s(-1) and apparent formal potentials ${E{{0{^{\\prime }}\\hfill \\atop {\\rm app}\\hfill}}}$ between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH. PMID:25825040

  5. Protein tyrosine phosphatase non-receptor type 2 and inflammatory bowel disease.

    PubMed

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2016-01-21

    Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) with the onset of inflammatory bowel disease (IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and adaptive immune responses. In particular, PTPN2 is involved in the regulation of inflammatory signalling cascades, and critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses, and finally for maintaining intestinal homeostasis. On one hand, dysfunction of PTPN2 has drastic effects on innate host defence mechanisms, including increased secretion of pro-inflammatory cytokines, limited autophagosome formation in response to invading pathogens, and disruption of the intestinal epithelial barrier. On the other hand, PTPN2 function is crucial for controlling adaptive immune functions, by regulating T cell proliferation and differentiation as well as maintaining T cell tolerance. In this way, dysfunction of PTPN2 contributes to the manifestation of IBD. The aim of this review is to present an overview of recent findings on the role of PTPN2 in intestinal homeostasis and the impact of dysfunctional PTPN2 on intestinal inflammation. PMID:26811645

  6. Protein tyrosine phosphatase SHP2 promotes invadopodia formation through suppression of Rho signaling.

    PubMed

    Tsai, Wan-Chen; Chen, Chien-Lin; Chen, Hong-Chen

    2015-09-15

    Invadopodia are actin-enriched membrane protrusions that are important for extracellular matrix degradation and invasive cell motility. Src homolog domain-containing phosphatase 2 (SHP2), a non-receptor protein tyrosine phosphatase, has been shown to play an important role in promoting cancer metastasis, but the underlying mechanism is unclear. In this study, we found that depletion of SHP2 by short-hairpin RNA suppressed invadopodia formation in several cancer cell lines, particularly in the SAS head and neck squamous cell line. In contrast, overexpression of SHP2 promoted invadopodia formation in the CAL27 head and neck squamous cell line, which expresses low levels of endogenous SHP2. The depletion of SHP2 in SAS cells significantly decreased their invasive motility. The suppression of invadopodia formation by SHP2 depletion was restored by the Clostridium botulinum C3 exoenzyme (a Rho GTPase inhibitor) or Y27632 (a specific inhibitor for Rho-associated kinase). Together, our results suggest that SHP2 may promote invadopodia formation through inhibition of Rho signaling in cancer cells. PMID:26204488

  7. Potent inhibition of protein-tyrosine phosphatase by phosphotyrosine-mimic containing cyclic peptides.

    PubMed

    Akamatsu, M; Roller, P P; Chen, L; Zhang, Z Y; Ye, B; Burke, T R

    1997-01-01

    In an effort to derive potent and bioavailable protein-tyrosine phosphatase inhibitors, we have previously reported hexameric peptides based on the epidermal growth factor receptor sequence EGFR988-993 (Asp-Ala-Asp-Glu-Xxx-Leu, where Xxx = Tyr), in which the tyrosyl residue has been replaced by the non-hydrolyzable phosphotyrosyl mimics phosphonomethylphenylalanine (Pmp), difluorophosphonomethylphenylalanine (F2Pmp) and O-malonyltyrosine (OMT). Inhibitory potencies (IC50 values) of these peptides against the tyrosine phosphatase PTP IB were 200, 0.2 and 10 microM, respectively. Since cellular penetration of peptides containing highly charged phosphonate residues is compromised, and good bioreversible protection strategies for the F2Pmp residue have not yet been reported, the OMT residue is of particular interest in that it affords potential new prodrug approaches. In the current study we have prepared cyclized versions of the OMT-containing EGFR988-993 peptide in order to increase its proteolytic stability and restrain conformational flexibility. Three different cyclic analogues were synthesized. Two of these were cyclized through the peptide backbone ('head to tail') using in one case a single glycine spacer (heptamer peptide) and in the second instance, two glycines (octamer peptide). In a PTPI-based assay the cyclic heptamer experienced a two-fold loss of potency (Ki = 25.2 +/- 3.9 microM) relative to the linear hexamer parent (Ki = 13 +/- 0.9 microM), while the cyclic octamer demonstrated a live-fold increase in potency (Ki = 2.60 +/- 0.11 microM). The third peptide was cyclized by means of a sulfide bridge between the side chain of a C-terminally added cysteine residue and the beta-carbon of a N-terminal acetyl residue. Although the overall size of this ring was identical to that exhibited by the preceding backbone-cyclized octamer, it displayed a three-fold enhancement in potency (Ki = 0.73 +/- 0.03 microM). The structural basis for the observed results are

  8. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  9. Formation of 3-nitrotyrosine by riboflavin photosensitized oxidation of tyrosine in the presence of nitrite.

    PubMed

    Fontana, Mario; Blarzino, Carla; Pecci, Laura

    2012-05-01

    The results of the present investigation show the susceptibility of tyrosine to undergo visible light-induced photomodification to 3-nitrotyrosine in the presence of nitrite and riboflavin, as sensitizer. By changing H2O by D2O, it could be established that singlet oxygen has a minor role in the reaction. The finding that nitration of tyrosine still occurred to a large extent under anaerobic conditions indicates that the process proceeds mainly through a type I mechanism, which involves the direct interaction of the excited state of riboflavin with tyrosine and nitrite to give tyrosyl radical and nitrogen dioxide radical, respectively. The tyrosyl radicals can either dimerize to yield 3,3'-dityrosine or combine with nitrogen dioxide radical to form 3-nitrotyrosine. The formation of 3-nitrotyrosine was found to increase with the concentration of nitrite added and was accompanied by a decrease in the recovery of 3,3'-dityrosine, suggesting that tyrosine nitration competes with dimerization reaction. The riboflavin photosensitizing reaction in the presence of nitrite was also able to induce nitration of tyrosine residues in proteins as revealed by the spectral changes at 430 nm, a characteristic absorbance of 3-nitrotyrosine, and by immunoreactivity using 3-nitrotyrosine antibodies. Since riboflavin and nitrite are both present endogenously in living organism, it is suggested that this pathway of tyrosine nitration may potentially occur in tissues and organs exposed to sunlight such as skin and eye. PMID:21479936

  10. Substrate Specificity of Protein Tyrosine Phosphatases 1B, RPTPα, SHP-1, and SHP-2†

    PubMed Central

    Ren, Lige; Chen, Xianwen; Luechapanichkul, Rinrada; Selner, Nicholas G.; Meyer, Tiffany M.; Wavreille, Anne-Sophie; Chan, Richard; Iorio, Caterina; Zhou, Xiang; Neel, Benjamin G.; Pei, Dehua

    2011-01-01

    We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solution-phase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately two orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active towards multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY−1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residues on the C-terminal side of pY, but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY−1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme’s in vivo substrate specificity. PMID:21291263

  11. The anti-inflammatory compound BAY 11-7082 is a potent inhibitor of Protein Tyrosine Phosphatases

    PubMed Central

    Krishnan, Navasona; Bencze, Gyula; Cohen, Philip; Tonks, Nicholas K.

    2013-01-01

    Summary The families of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) function in a coordinated manner to regulate signal transduction events that are critical for cellular homeostasis. Aberrant tyrosine phosphorylation, resulting from disruption of either PTP or PTK function, has been shown to be the cause of major human diseases, including cancer and diabetes. Consequently, the characterization of small molecule inhibitors of these kinases and phosphatases may not only provide molecular probes with which to define the significance of particular signalling events, but also may have therapeutic implications. BAY 11-7082 is an anti-inflammatory compound that has been reported to inhibit IκB kinase activity. The compound has an α,β-unsaturated electrophilic center, which confers the property of being a Michael acceptor; this suggests that it may react with nucleophilic cysteine-containing proteins, such as PTPs. In this study, we demonstrated that BAY 11-7082 was a potent, irreversible inhibitor of PTPs. Using mass spectrometry, we have shown that BAY 11-7082 inactivated PTPs by forming a covalent adduct with the active site cysteine. Administration of the compound caused an increase in protein tyrosine phosphorylation in RAW 264 macrophages, similar to the effects of the generic PTP inhibitor sodium orthovanadate. These data illustrate that BAY 11-7082 is an effective pan-PTP inhibitor with cell permeability, revealing its potential as a new probe for chemical biology approaches to the study of PTP function. Furthermore, the data suggest that inhibition of PTP function may contribute to the many biological effects of BAY 11-7082 that have been reported to date. PMID:23578302

  12. The anti-inflammatory compound BAY-11-7082 is a potent inhibitor of protein tyrosine phosphatases.

    PubMed

    Krishnan, Navasona; Bencze, Gyula; Cohen, Philip; Tonks, Nicholas K

    2013-06-01

    The families of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) function in a coordinated manner to regulate signal transduction events that are critical for cellular homeostasis. Aberrant tyrosine phosphorylation, resulting from disruption of either PTP or PTK function, has been shown to be the cause of major human diseases, including cancer and diabetes. Consequently, the characterization of small-molecule inhibitors of these kinases and phosphatases may not only provide molecular probes with which to define the significance of particular signaling events, but also may have therapeutic implications. BAY-11-7082 is an anti-inflammatory compound that has been reported to inhibit IκB kinase activity. The compound has an α,β-unsaturated electrophilic center, which confers the property of being a Michael acceptor; this suggests that it may react with nucleophilic cysteine-containing proteins, such as PTPs. In this study, we demonstrated that BAY-11-7082 was a potent, irreversible inhibitor of PTPs. Using mass spectrometry, we have shown that BAY-11-7082 inactivated PTPs by forming a covalent adduct with the active-site cysteine. Administration of the compound caused an increase in protein tyrosine phosphorylation in RAW 264 macrophages, similar to the effects of the generic PTP inhibitor sodium orthovanadate. These data illustrate that BAY-11-7082 is an effective pan-PTP inhibitor with cell permeability, revealing its potential as a new probe for chemical biology approaches to the study of PTP function. Furthermore, the data suggest that inhibition of PTP function may contribute to the many biological effects of BAY-11-7082 that have been reported to date. PMID:23578302

  13. Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

    SciTech Connect

    Kim, Rae-Kwon; Yoon, Chang-Hwan; Hyun, Kyung-Hwan; Lee, Hyejin; An, Sungkwan; Park, Myung-Jin; Kim, Min-Jung; Lee, Su-Jae

    2010-11-26

    Research highlights: {yields} Activation of Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the fractionated radiation-induced expansion of glioma stem-like cells. {yields} Inhibition of LCK prevents acquisition of fractionated radiation-induced resistance to chemotherapeutic treatment. {yields} LCK activity is critical for the maintenance of self-renewal in glioma stem-like cells. -- Abstract: Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In this study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133{sup +} cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and

  14. Growth hormone (GH) induces tyrosine-phosphorylated proteins in mouse L cells that express recombinant GH receptors.

    PubMed Central

    Wang, X; Xu, B; Souza, S C; Kopchick, J J

    1994-01-01

    Porcine and bovine GH receptor (GHR) cDNAs were stably expressed in mouse L cells, which normally do not possess detectable levels of mouse GHR. Expression of the GHR cDNAs resulted in specific binding of 125I-labeled GH by these cell lines. To study GHR-related signaling events in these cells, protein tyrosine phosphorylation was examined. In GH-treated cells, a tyrosine-phosphorylated protein with a molecular mass of approximately 95 kDa (pp95) was increased dramatically (approximately 100-fold) relative to non-GH-treated cells. The amount of pp95 within the cells after GH treatment was positively correlated with the number of GHRs on the cells. Tyrosine phosphorylation of pp95 could not be induced by prolactin, insulin, insulin-like growth factor I, interleukin 2, epidermal growth factor, platelet-derived growth factor, or fibroblast growth factor. Phosphorylation of pp95 was found to be a rapid event that could be observed 60 sec after GH treatment. Also, pp95 appears to exist as a complex of two proteins, i.e., pp95 and pp96. The GH-induced response by these cells may be of use in screening GH analogs for biological activity. Images PMID:7509070

  15. Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

    PubMed

    Singh, Neetu; Yadav, Smita; Rathaur, Sushma

    2015-11-01

    A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis. PMID:26341797

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

    SciTech Connect

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

    1988-04-01

    It has been hypothesized that bombesin-like peptides produced by small cell lung carcinomas may sustain deregulated proliferation through an autocrine mechanism. The authors 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. They 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 Mn{sup 2+}. 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.

  17. Structure­based drug design and AutoDock study of potential protein tyrosine kinase inhibitors.

    PubMed Central

    Ali, Hamed Ismail; Nagamatsu, Tomofumi; Akaho, Eiichi

    2011-01-01

    Different classes of compounds were investigated for their binding affinities into different protein tyrosine kinases (PTKs) employing a novel flexible ligand docking approach by using AutoDock 3.05 and 4. These compounds include many flavin analogs, which were developed in our group with varying degrees of cytotoxic activity (comparable or moderately superior to cisplatin and ara-c), and database selected analogs. They were docked onto twelve different families of PTKs retrieved from the Protein Data Bank. These proteins are representatives of plausible models of interactions with chemotherapeutic agents. A comparative study of the intact co-crystallized ligands of various types of PTKs was carried out. Results revealed that the new class of 5-deazapteridine and steroid hybrid compounds VIa,b, and d, and the vertical-type bispyridodipyrimidine with n-hexyl chain junction between its N-10 and N-10 atoms Xa, exhibited non-selective PTK binding capacities, with the lowest (Gb). On the other hand, 2-amino benzoic acid analog IIa, phenoxypyrido [3, 4-d]pyrimidine derivative IVc, tyrosine containing tripeptide Vd, and the one from Sumisho data base 831 are proposed to have selective PTK binding affinities to certain classes of tyrosine kinases, namely, HGFR (c-met), ZAP-70, insulin receptor kinase, EGFR, respectively. All These compounds of highest affinities were docked within the binding sites of PTKs with reasonable RMSD and 1-5 hydrogen bonds. PMID:21383902

  18. NasFED proteins mediate assimilatory nitrate and nitrite transport in Klebsiella oxytoca (pneumoniae) M5al.

    PubMed

    Wu, Q; Stewart, V

    1998-03-01

    Klebsiella oxytoca can use nitrate and nitrite as sole nitrogen sources. The enzymes required for nitrate and nitrite assimilation are encoded by the nasFEDCBA operon. We report here the complete nasFED sequence. Sequence comparisons indicate that the nasFED genes encode components of a conventional periplasmic binding protein-dependent transport system consisting of a periplasmic binding protein (NasF), a homodimeric intrinsic membrane protein (NasE), and a homodimeric ATP-binding cassette (ABC) protein (NasD). The NasF protein and the related NrtA and CmpA proteins of cyanobacteria contain leader (signal) sequences with the double-arginine motif that is hypothesized to direct prefolded proteins to an alternate protein export pathway. The NasE protein and the related NrtB and CmpB proteins of cyanobacteria contain unusual variants of the EAA loop sequence that defines membrane-intrinsic proteins of ABC transporters. To characterize nitrate and nitrite transport, we constructed in-frame nonpolar deletions of the chromosomal nasFED genes. Growth tests coupled with nitrate and nitrite uptake assays revealed that the nasFED genes are essential for nitrate transport and participate in nitrite transport as well. Interestingly, the delta nasF strain exhibited leaky phenotypes, particularly at elevated nitrate concentrations, suggesting that the NasED proteins are not fully dependent on the NasF protein. PMID:9495773

  19. NasFED Proteins Mediate Assimilatory Nitrate and Nitrite Transport in Klebsiella oxytoca (pneumoniae) M5al

    PubMed Central

    Wu, Qitu; Stewart, Valley

    1998-01-01

    Klebsiella oxytoca can use nitrate and nitrite as sole nitrogen sources. The enzymes required for nitrate and nitrite assimilation are encoded by the nasFEDCBA operon. We report here the complete nasFED sequence. Sequence comparisons indicate that the nasFED genes encode components of a conventional periplasmic binding protein-dependent transport system consisting of a periplasmic binding protein (NasF), a homodimeric intrinsic membrane protein (NasE), and a homodimeric ATP-binding cassette (ABC) protein (NasD). The NasF protein and the related NrtA and CmpA proteins of cyanobacteria contain leader (signal) sequences with the double-arginine motif that is hypothesized to direct prefolded proteins to an alternate protein export pathway. The NasE protein and the related NrtB and CmpB proteins of cyanobacteria contain unusual variants of the EAA loop sequence that defines membrane-intrinsic proteins of ABC transporters. To characterize nitrate and nitrite transport, we constructed in-frame nonpolar deletions of the chromosomal nasFED genes. Growth tests coupled with nitrate and nitrite uptake assays revealed that the nasFED genes are essential for nitrate transport and participate in nitrite transport as well. Interestingly, the ΔnasF strain exhibited leaky phenotypes, particularly at elevated nitrate concentrations, suggesting that the NasED proteins are not fully dependent on the NasF protein. PMID:9495773

  20. Evidence for an Inactivating System of Nitrate Reductase in Hordeum vulgare L. during Darkness That Requires Protein Synthesis 1

    PubMed Central

    Travis, R. L.; Jordan, W. R.; Huffaker, R. C.

    1969-01-01

    The disappearance of nitrate reductase activity in leaves of Hordeum vulgare L. during darkness was inhibited by cycloheximide, actinomycin D, and low temperature. Thus, protein synthesis was probably required for the disappearance of nitrate reductase in the dark. Since chloramphenicol did not affect the rate of loss of activity, the degradation or inactivation apparently required protein synthesis by the cytoplasmic ribosomal system. Consistent with this observation, nitrate reductase is also reportedly located in the cytoplasm. Thus, the amount of nitrate reductase activity present in leaves of barley may be controlled by a balance between activating and inactivating systems. PMID:16657182

  1. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells

    SciTech Connect

    Haering, H.U.; White, M.F.; Machicao, F.; Ermel, B.; Schleicher, E.; Obermaier, B.

    1987-01-01

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, the authors labeled intact rat fat cells with (/sup 32/P)orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, they found, in addition to the 95-kDa ..beta..-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor ..beta..-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. Insulin (0.5 nM, 100 nM) stimulated within 2 min the /sup 32/P incorporation into a 116-kDa band, a 62 kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. They suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission.

  2. Protein tyrosine kinase regulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking induced by acute hypoxia in cultured brainstem neurons.

    PubMed

    Wang, H; Yu, L C; Li, Y C

    2016-01-01

    This study was performed to investigate the modulation effect of protein tyrosine kinase on postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking induced by acute hypoxia in cultured brainstem neurons. The cultured neurons were exposed to 1% O2 and the expression of AMPA receptor subunit GluR2 on the cell surface was significantly increased, while total GluR2 was not markedly changed. Furthermore, the hypoxia-induced increase in GluR2 expression on the cell surface was partially blocked by the protein tyrosine kinase membrane-permeable inhibitor genistein. In contrast, both the protein tyrosine kinase agonist nerve growth factor and protein tyrosine phosphatase inhibitor vanadate promoted the hypoxia-induced increase of GluR2 expression on cell surface. Moreover, GluR2 could be phosphorylated by tyrosine under normoxia and hypoxia conditions in vitro on brainstem neurons, and tyrosine phosphorylation of GluR2 was significantly stronger under hypoxia conditions. Our results indicate that acute hypoxia induces the AMPA receptor subunit GluR2 to rapidly migrate to the cell membrane to modify the strength of the synapse. This study indicates that tyrosine phosphorylation of the receptor is an important pathway regulating the rapid migration of GluR2 in the postsynaptic domain induced by hypoxia. PMID:27525851

  3. Photochemical Tyrosine Oxidation in the Structurally Well-Defined α3Y Protein: Proton-Coupled Electron Transfer and a Long-Lived Tyrosine Radical

    PubMed Central

    2014-01-01

    Tyrosine oxidation–reduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The α3Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of α3Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in α3Y. Time-resolved spectroscopy using a soluble flash-quench generated [Ru(2,2′-bipyridine)3]3+ oxidant provides high-quality Y32–O• absorption spectra. The rate constant of Y32 oxidation (kPCET) is pH dependent: 1.4 × 104 M–1 s–1 (pH 5.5), 1.8 × 105 M–1 s–1 (pH 8.5), 5.4 × 103 M–1 s–1 (pD 5.5), and 4.0 × 104 M–1 s–1 (pD 8.5). kH/kD of Y32 oxidation is 2.5 ± 0.5 and 4.5 ± 0.9 at pH(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32–O• is 28–58% versus the concentration of [Ru(2,2′-bipyridine)3]3+. Y32–O• decays slowly, t1/2 in the range of 2–10 s, at both pH 5.5 and 8.5, via radical–radical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32–O• is discussed relative to the structural properties of the Y32 site. Finally, the static α3Y NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32–O• come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of α3Y. PMID:25121576

  4. Regulation of interleukin-3-induced substrate phosphorylation and cell survival by SHP-2 (Src-homology protein tyrosine phosphatase 2).

    PubMed

    Wheadon, Helen; Edmead, Christine; Welham, Melanie J

    2003-11-15

    The cytosolic SHP-2 (Src homology protein tyrosine phosphatase 2) has previously been implicated in IL-3 (interleukin-3) signalling [Bone, Dechert, Jirik, Schrader and Welham (1997) J. Biol. Chem. 272, 14470 -14476; Craddock and Welham (1997) J. Biol. Chem. 272, 29281-29289; Welham, Dechert, Leslie, Jirik and Schrader (1994) J. Biol. Chem. 269, 23764-23768; Qu, Nguyen, Chen and Feng (2001) Blood 97, 911-914]. To investigate the role of SHP-2 in IL-3 signalling in greater detail, we have inducibly expressed WT (wild-type) or two potentially substrate-trapping mutant forms of SHP-2, generated by mutation of Asp-425 to Ala (D425A) or Cyst-459 to Ser (C459S), in IL-3-dependent BaF/3 cells. Effects on IL-3-induced tyrosine phosphorylation, signal transduction and functional responses were examined. Expression of C459S SHP-2 protected the beta-chain of the murine IL-3R (IL-3 receptor), the adaptor protein Gab2 (Grb2-associated binder 2), and a cytosolic protein of 48 kDa from tyrosine dephosphorylation, consistent with them being bona fide substrates of SHP-2 in IL-3 signalling. The tyrosine phosphorylation of a 135 kDa transmembrane protein was also protected upon expression of C459S SHP-2. We have identified the inhibitory immunoreceptor PECAM-1 (platelet endothelial cell adhesion molecule-1)/CD31 (cluster determinant 31) as a component of this 135 kDa substrate and also show that IL-3 can induce tyrosine phosphorylation of PECAM-1. Expression of WT, C459S and D425A forms of SHP-2 had little effect on IL-3-driven proliferation or STAT5 (signal transduction and activators of transcription) phosphorylation or activation of protein kinase B. However, expression of WT SHP-2 increased ERK (extracellular-signal-regulated kinase) activation. Interestingly, expression of C459S SHP-2 decreased ERK activation at later times after IL-3 stimulation, but potentiated IL-3-induced activation of Jun N-terminal kinases. In addition, expression of C459S SHP-2 decreased cell survival in

  5. Regulation of interleukin-3-induced substrate phosphorylation and cell survival by SHP-2 (Src-homology protein tyrosine phosphatase 2).

    PubMed Central

    Wheadon, Helen; Edmead, Christine; Welham, Melanie J

    2003-01-01

    The cytosolic SHP-2 (Src homology protein tyrosine phosphatase 2) has previously been implicated in IL-3 (interleukin-3) signalling [Bone, Dechert, Jirik, Schrader and Welham (1997) J. Biol. Chem. 272, 14470 -14476; Craddock and Welham (1997) J. Biol. Chem. 272, 29281-29289; Welham, Dechert, Leslie, Jirik and Schrader (1994) J. Biol. Chem. 269, 23764-23768; Qu, Nguyen, Chen and Feng (2001) Blood 97, 911-914]. To investigate the role of SHP-2 in IL-3 signalling in greater detail, we have inducibly expressed WT (wild-type) or two potentially substrate-trapping mutant forms of SHP-2, generated by mutation of Asp-425 to Ala (D425A) or Cyst-459 to Ser (C459S), in IL-3-dependent BaF/3 cells. Effects on IL-3-induced tyrosine phosphorylation, signal transduction and functional responses were examined. Expression of C459S SHP-2 protected the beta-chain of the murine IL-3R (IL-3 receptor), the adaptor protein Gab2 (Grb2-associated binder 2), and a cytosolic protein of 48 kDa from tyrosine dephosphorylation, consistent with them being bona fide substrates of SHP-2 in IL-3 signalling. The tyrosine phosphorylation of a 135 kDa transmembrane protein was also protected upon expression of C459S SHP-2. We have identified the inhibitory immunoreceptor PECAM-1 (platelet endothelial cell adhesion molecule-1)/CD31 (cluster determinant 31) as a component of this 135 kDa substrate and also show that IL-3 can induce tyrosine phosphorylation of PECAM-1. Expression of WT, C459S and D425A forms of SHP-2 had little effect on IL-3-driven proliferation or STAT5 (signal transduction and activators of transcription) phosphorylation or activation of protein kinase B. However, expression of WT SHP-2 increased ERK (extracellular-signal-regulated kinase) activation. Interestingly, expression of C459S SHP-2 decreased ERK activation at later times after IL-3 stimulation, but potentiated IL-3-induced activation of Jun N-terminal kinases. In addition, expression of C459S SHP-2 decreased cell survival in

  6. Protein tyrosine phosphatase PTPN22 +1858C/T polymorphism is associated with active vitiligo

    PubMed Central

    GARCIA-MELENDEZ, MARTHA ELENA; SALINAS-SANTANDER, MAURICIO; SANCHEZ-DOMINGUEZ, CELIA; GONZALEZ-CARDENAS, HUGO; CERDA-FLORES, RICARDO M.; OCAMPO-CANDIANI, JORGE; ORTIZ-LÓPEZ, ROCÍO

    2014-01-01

    Vitiligo is characterized by a skin depigmentation disorder resulting from an autoimmune response targeting melanocytes. Within the genetic factors involved in the development of the vitiligo immune response, various genes in the major histocompatibility complex (MHC) and non-MHC loci have been considered to be risk factors. The PTPN22 gene encodes for a lymphoid protein tyrosine phosphatase, a regulator of the activation and development of T-cells. The +1858C/T polymorphism has been associated to autoimmune disease susceptibility in different populations and could be implicated in the onset of vitiligo. To assess the possible association between the presence of PTPN22 +1858C/T and vitiligo, 187 patients with vitiligo and 223 control subjects were analyzed in the study. Genomic DNA was isolated using the salting-out method and samples were subjected to polymerase chain reaction-restriction fragment length polymorphism in order to detect the PTPN22 +1858C/T polymorphism. Causal associations were determined by χ2 test and their respective odds ratio (OR) was assessed in a 2×2 contingency table. The results showed an association between active vitiligo and the allele T load [P=0.0418; OR, 2.5706; 95% confidence interval (CI), 1.0040–6.5816], and active vitiligo-CT genotype (P=0.0389, OR, 2.6548; 95% CI, 1.0191–6.9156). In conclusion, the present data indicates a possible association between the PTPN22 +1858C/T genotype and a significant susceptibility of developing an active form of vitiligo. PMID:25289035

  7. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase* ♦

    PubMed Central

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W. Todd; Tonks, Nicholas K.

    2015-01-01

    Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. PMID:25897081

  8. Ex Vivo Chemical Cytometric Analysis of Protein Tyrosine Phosphatase Activity in Single Human Airway Epithelial Cells

    PubMed Central

    Phillips, Ryan M.; Dailey, Lisa A.; Bair, Eric; Samet, James M.; Allbritton, Nancy L.

    2014-01-01

    We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min−1 mg−1) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn2+, and 1,2-naphthoquinone (76%, 69%, 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min−1 mg−1, and was also effectively inhibited by pre-incubation of the cells with the inhibitors pervanadate, Zn2+, and 1,2- naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min−1 mg−1 resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34–36 pmol min−1 mg−1 (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues. PMID:24380370

  9. Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis

    PubMed Central

    Phillips, Ryan M.; Bair, Eric; Lawrence, David S.; Sims, Christopher E.; Allbritton, Nancy L.

    2013-01-01

    A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates, and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 nM and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn2+ and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min−1 mg−1) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min−1 mg−1). Zn2+, 1,2-naphthoquinone and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 μM, and 100 nM, respectively. Dephosphorylation of the reporter following loading into living single cells occurred at rates of at least 2 pmol min−1 mg−1. When single cells were exposed to 1,2-naphthoquinone (50 μM), Zn2+ (100 μM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens. PMID:23682679

  10. Striatal-Enriched Protein Tyrosine Phosphatase Controls Responses to Aversive Stimuli: Implication for Ethanol Drinking

    PubMed Central

    Legastelois, Rémi; Darcq, Emmanuel; Wegner, Scott A.; Lombroso, Paul J.; Ron, Dorit

    2015-01-01

    The STriatal-Enriched protein tyrosine Phosphatase (STEP) is a brain-specific phosphatase whose dysregulation in expression and/or activity is associated with several neuropsychiatric disorders. We recently showed that long-term excessive consumption of ethanol induces a sustained inhibition of STEP activity in the dorsomedial striatum (DMS) of mice. We further showed that down-regulation of STEP expression in the DMS, and not in the adjacent dorsolateral striatum, increases ethanol intake, suggesting that the inactivation of STEP in the DMS contributes to the development of ethanol drinking behaviors. Here, we compared the consequence of global deletion of the STEP gene on voluntary ethanol intake to the consumption of an appetitive rewarding substance (saccharin) or an aversive solution (quinine or denatonium). Whereas saccharin intake was similar in STEP knockout (KO) and wild type (WT) littermate mice, the consumption of ethanol as well as quinine and denatonium was increased in STEP KO mice. These results suggested that the aversive taste of these substances was masked upon deletion of the STEP gene. We therefore hypothesized that STEP contributes to the physiological avoidance towards aversive stimuli. To further test this hypothesis, we measured the responses of STEP KO and WT mice to lithium-induced conditioned place aversion (CPA) and found that whereas WT mice developed lithium place aversion, STEP KO mice did not. In contrast, conditioned place preference (CPP) to ethanol was similar in both genotypes. Together, our results indicate that STEP contributes, at least in part, to the protection against the ingestion of aversive agents. PMID:25992601

  11. c-Jun and Ets2 proteins regulate expression of spleen tyrosine kinase in T cells.

    PubMed

    Ghosh, Debjani; Tsokos, George C; Kyttaris, Vasileios C

    2012-04-01

    Effector T cells and T cells from patients with systemic lupus erythematosus (SLE) express increased levels of the spleen tyrosine kinase (Syk). Syk binds to the T cell receptor (TCR)-CD3 complex and transduces the TCR-mediated signal in the cell more efficiently than the canonical CD3ζ chain. The reasons for the increased expression of Syk are unclear. In the present study, we found that Syk is regulated by the transcription factor c-Jun in cooperation with Ets2. c-Jun and Ets2 bound to the SYK promoter in close proximity and increased the promoter activity in a specific manner. Disruption of c-Jun and Ets2 expression by siRNA resulted in decreased expression of Syk. Overexpression of c-Jun but not Ets2 resulted in increase in Syk protein. c-Jun and Ets2 co-immunoprecipitated and had an additive effect on Syk expression. c-Jun-driven SYK promoter activation showed a similar pattern in B cells; however, as expected, basal promoter activity was much higher in B cells as compared with T cells. Overexpression of c-Jun led to increase in intracytoplasmic calcium flux following TCR stimulation. Moreover, we found that SLE T cells had increased levels of c-Jun at baseline and phosphorylated c-Jun upon activation. Finally, disruption of c-Jun and Ets2 in SLE T cells resulted in a decrease in calcium flux upon TCR stimulation. In conclusion, c-Jun in cooperation with Ets2 increases the expression of Syk and contributes to Syk-mediated heightened calcium responses in SLE T cells. PMID:22354960

  12. Transgenic mice expressing yellow fluorescent protein under control of the human tyrosine hydroxylase promoter.

    PubMed

    Choi, Eun Yang; Yang, Jae Won; Park, Myung Sun; Sun, Woong; Kim, Hyun; Kim, Seung U; Lee, Myung Ae

    2012-10-01

    Pathogenesis of Parkinson's disease and related catecholaminergic neurological disorders is closely associated with changes in the levels of tyrosine hydroxylase (TH). Therefore, investigation of the regulation of the TH gene system should assist in understanding the pathomechanisms involved in these neurological disorders. To identify regulatory domains that direct human TH expression in the central nervous system (CNS), we generated two transgenic mouse lines in which enhanced yellow fluorescent protein (EYFP) is expressed under the control of either 3.2-kb (hTHP-EYFP construct) human TH promoter or 3.2-kb promoter with 2-kb 3'-flanking regions (hTHP-ex3-EYFP construct) of the TH gene. In the adult transgenic mouse brain, the hTHP-EYFP construct directs neuron-specific EYFP expression in various CNS areas, such as olfactory bulb, striatum, interpeduncular nucleus, cerebral cortex, hippocampus, and particularly dentate gyrus. Although these EYFP-positive cells were identified as mature neurons, few EYFP-positive cells were TH-positive neurons. On the other hand, we could detect the EYFP mRNA expression in a subset of neurons in the olfactory bulb, midbrain, and cerebellum, in which expression of endogenous TH is enriched, with hTHP-ex3-EYFP transgenic mice. These results indicate that the 3.2-kb sequence upstream of the TH gene is not sufficient for proper expression and that the 2-kb sequence from the translation start site to exon 3 is necessary for expression of EYFP in a subset of catecholaminergic neurons. PMID:22714400

  13. Protein modification in the post-mating spermatophore of the signal crayfish Pacifastacus leniusculus: insight into the tyrosine phosphorylation in a non-motile spermatozoon.

    PubMed

    Niksirat, Hamid; Vancová, Marie; Andersson, Liselotte; James, Peter; Kouba, Antonín; Kozák, Pavel

    2016-09-01

    After mating, spermatophores of signal crayfish are stored on the body of the female for a period before fertilization. This study compared the post-mating protein profile and pattern of protein tyrosine phosphorylation of the signal crayfish spermatophore to that of the freshly ejaculated spermatophore and found substantial differences. Two major bands of tyrosine-phosphorylated proteins of molecular weights 10 and 50kDa were observed in the freshly ejaculated spermatophore of the signal crayfish. While the tyrosine-phosphorylated protein band with molecular weight 10kDa was formed by protein(s) of similar pH, the band with molecular weight of 50kDa consisted of proteins of varying pH. In the post-mating spermatophore, the band with molecular weight of 50kDa was not detected, and an increase in the level of protein tyrosine phosphorylation was observed in the 10kDa band. The microtubular radial arms of the spermatozoon showed a positive reaction to an anti-tyrosine antibody conjugated with gold particles in both the freshly ejaculated and post-mating spermatophores. In conclusion, the male gamete of the signal crayfish undergoes molecular modification during post-mating storage on the body of the female including changes in the level of protein expression and protein tyrosine phosphorylation. Structural similarity of the radial arms in the crayfish immotile spermatozoon with flagellum, which is the main site of protein tyrosine phosphorylation in the mammalian motile spermatozoa, raises questions regarding evolution and function of such organelles across the animal kingdom that must be addressed in the future studies. PMID:27481552

  14. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis

    PubMed Central

    Gerwig, Jan; Kiley, Taryn B.; Gunka, Katrin; Stanley-Wall, Nicola

    2014-01-01

    The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA. PMID:24493247

  15. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis.

    PubMed

    Gerwig, Jan; Kiley, Taryn B; Gunka, Katrin; Stanley-Wall, Nicola; Stülke, Jörg

    2014-04-01

    The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA. PMID:24493247

  16. The Streptococcus pyogenes orphan protein tyrosine phosphatase, SP-PTP, possesses dual specificity and essential virulence regulatory functions.

    PubMed

    Kant, Sashi; Agarwal, Shivani; Pancholi, Preeti; Pancholi, Vijay

    2015-08-01

    Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP-PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP-PTP is, therefore, questionable. Here, we demonstrate that SP-PTP possesses dual phosphatase specificity for Tyr- and Ser/Thr-phosphorylated GAS proteins, such as Ser/Thr kinase (SP-STK) and the SP-STK-phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP-PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP-PTP displayed increased Ser-/Thr-/Tyr-phosphorylation. SP-PTP also differentially regulates the expression of ∼50% of the total GAS genes, including several virulence genes potentially through the two-component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP-PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr-phosphorylation in GAS and the relevance of SP-PTP as an important therapeutic target. PMID:25939957

  17. Taxol shares the ability of bacterial lipopolysaccharide to induce tyrosine phosphorylation of microtubule-associated protein kinase.

    PubMed

    Ding, A; Sanchez, E; Nathan, C F

    1993-11-15

    Microtubule-associated proteins may mediate the activation of macrophages by bacterial LPS. Three lines of evidence support this hypothesis: a) Taxol, a microtubule-binding diterpene, mimics the ability of LPS to induce cytokines and down-regulate receptors for TNF-alpha. In recombinant inbred mouse strains differing at the Lps gene, responsiveness to these effects of Taxol co-segregates with responsiveness to LPS. b) In vitro, LPS binds specifically to MT and preferentially to beta-tubulin. c) LPS activates microtubule-associated protein-2 kinase (MAPK). The present studies bring together and extend these lines of evidence. a) Taxol, like LPS, rapidly induces tyrosine phosphorylation of MAPK in mouse macrophages, and triggers MAPK to phosphorylate an exogenous substrate. b) Tyrosine phosphorylation of MAPK is an extremely rapid cellular response both to taxol and LPS. c) Macrophages from C3H/HeJ mice, which carry a defective Lps gene, fail to activate MAPK in response to taxol or LPS, although they activate MAPK in response to insulin or IFN-gamma. These results suggest that tyrosine phosphorylation of MAPK is among the earliest known response of macrophages to LPS. Taxol mimics LPS with respect to immediate MAPK activation, later transcriptional events, and the genetic control of both sets of responses. LPS and taxol thus appear to share an early step in a functionally important signal transduction pathway that may involve MT. PMID:7901279

  18. Transcriptional regulation of tyrosine phenol-lyase gene mediated through TyrR and cAMP receptor protein.

    PubMed

    Katayama, T; Suzuki, H; Yamamoto, K; Kumagai, H

    1999-10-01

    Using a lac reporter system in Escherichia coli, we showed that the expression of E. herbicola tpl was regulated through TyrR and cAMP receptor protein. Three TyrR boxes upstream of tpl were essential for full expression. The results suggested that the tyrosine-mediated TyrR hexamerization was an important process. The DNA bending between two TyrR boxes, which is triggered by the binding of cAMP receptor protein, may facilitate the conformational change of TyrRs. PMID:10586511

  19. 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

  20. Nitration of the Birch Pollen Allergen Bet v 1.0101: Efficiency and Site-Selectivity of Liquid and Gaseous Nitrating Agents

    PubMed Central

    2014-01-01

    Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO–), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O3/NO2). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO–, and Y83 and Y158 for O3/NO2. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O3/NO2 was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein. PMID:24517313

  1. Nitration of the birch pollen allergen Bet v 1.0101: efficiency and site-selectivity of liquid and gaseous nitrating agents.

    PubMed

    Reinmuth-Selzle, Kathrin; Ackaert, Chloé; Kampf, Christopher J; Samonig, Martin; Shiraiwa, Manabu; Kofler, Stefan; Yang, Hong; Gadermaier, Gabriele; Brandstetter, Hans; Huber, Christian G; Duschl, Albert; Oostingh, Gertie J; Pöschl, Ulrich

    2014-03-01

    Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO(-)), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O₃/NO₂). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO(-), and Y83 and Y158 for O₃/NO₂. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O₃/NO₂ was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein. PMID:24517313

  2. Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

    PubMed Central

    Chung, Heesung; Jung, Hyejung; Lee, Jung-hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-01-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. PMID:24951591

  3. Frequent mutation of receptor protein tyrosine phosphatases provides a mechanism for STAT3 hyperactivation in head and neck cancer

    PubMed Central

    Lui, Vivian Wai Yan; Peyser, Noah D.; Ng, Patrick Kwok-Shing; Hritz, Jozef; Zeng, Yan; Lu, Yiling; Li, Hua; Wang, Lin; Gilbert, Breean R.; General, Ignacio J.; Bahar, Ivet; Ju, Zhenlin; Wang, Zhenghe; Pendleton, Kelsey P.; Xiao, Xiao; Du, Yu; Vries, John K.; Hammerman, Peter S.; Garraway, Levi A.; Mills, Gordon B.; Johnson, Daniel E.; Grandis, Jennifer R.

    2014-01-01

    The underpinnings of STAT3 hyperphosphorylation resulting in enhanced signaling and cancer progression are incompletely understood. Loss-of-function mutations of enzymes that dephosphorylate STAT3, such as receptor protein tyrosine phosphatases, which are encoded by the PTPR gene family, represent a plausible mechanism of STAT3 hyperactivation. We analyzed whole exome sequencing (n = 374) and reverse-phase protein array data (n = 212) from head and neck squamous cell carcinomas (HNSCCs). PTPR mutations are most common and are associated with significantly increased phospho-STAT3 expression in HNSCC tumors. Expression of receptor-like protein tyrosine phosphatase T (PTPRT) mutant proteins induces STAT3 phosphorylation and cell survival, consistent with a “driver” phenotype. Computational modeling reveals functional consequences of PTPRT mutations on phospho-tyrosine–substrate interactions. A high mutation rate (30%) of PTPRs was found in HNSCC and 14 other solid tumors, suggesting that PTPR alterations, in particular PTPRT mutations, may define a subset of patients where STAT3 pathway inhibitors hold particular promise as effective therapeutic agents. PMID:24395800

  4. The Interaction of Protein-tyrosine Phosphatase α (PTPα) and RACK1 Protein Enables Insulin-like Growth Factor 1 (IGF-1)-stimulated Abl-dependent and -independent Tyrosine Phosphorylation of PTPα*

    PubMed Central

    Khanna, Ranvikram S.; Le, Hoa T.; Wang, Jing; Fung, Thomas C. H.; Pallen, Catherine J.

    2015-01-01

    Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration. PMID:25694432

  5. The interaction of protein-tyrosine phosphatase α (PTPα) and RACK1 protein enables insulin-like growth factor 1 (IGF-1)-stimulated Abl-dependent and -independent tyrosine phosphorylation of PTPα.

    PubMed

    Khanna, Ranvikram S; Le, Hoa T; Wang, Jing; Fung, Thomas C H; Pallen, Catherine J

    2015-04-10

    Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration. PMID:25694432

  6. Quantitative Profiling of Protein Tyrosine Kinases in Human Cancer Cell Lines by Multiplexed Parallel Reaction Monitoring Assays.

    PubMed

    Kim, Hye-Jung; Lin, De; Lee, Hyoung-Joo; Li, Ming; Liebler, Daniel C

    2016-02-01

    Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported up-regulation of MET, but also with up-regulation of FLK2 and down-regulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with parallel reaction monitoring data. Multiplexed parallel reaction monitoring assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706. PMID:26631510

  7. The protein tyrosine phosphatase DEP-1/PTPRJ promotes breast cancer cell invasion and metastasis.

    PubMed

    Spring, K; Fournier, P; Lapointe, L; Chabot, C; Roussy, J; Pommey, S; Stagg, J; Royal, I

    2015-10-29

    DEP-1/PTPRJ is a receptor-like protein tyrosine phosphatase mainly known for its antiproliferative and tumor-suppressive functions. Many identified substrates are growth factor receptors, and DEP-1 is deleted and/or mutated in human cancers including that of the breast. However, DEP-1 was also identified as a promoter of Src activation and proinvasive functions in the endothelium, suggesting it could perhaps mediate breast cancer invasiveness that is likewise driven by Src family kinases. We show here that DEP-1 expression was greater in highly invasive breast cancer cells (MDA-MB-231, Hs578T, BT-549) than in the less invasive or untransformed cell lines tested (MCF-7, T47D, SK-BR3 and MCF10A). DEP-1 silencing experiments in invasive cells demonstrated that moderately expressed and catalytically active DEP-1 was required, in collaboration with basal epidermal growth factor receptor activity, for Src activation and the phosphorylation of its substrate Cortactin, and for their colocalization at the cell's leading edge. This correlated with an increased number of cell protrusions, and an enhanced capacity of the cells to migrate and invade. Similarly, moderate overexpression of DEP-1 in the low-invasive cells resulted in the promotion of their invasiveness in an Src-dependent manner. Consistent with these data, the expression of endogenous DEP-1 was elevated in a bone metastatic cell line derived from MDA-MB-231 cells, and promoted increased Src Y418 and Cortactin Y421 phosphorylation, as well as pro-MMP9 secretion and Matrigel invasion. Importantly, the silencing of DEP-1 in MDA-MB-231 cells greatly decreased their ability to metastasize, despite having no effect on tumor growth or angiogenesis. Hence, we found that moderate expression of DEP-1 was associated with the increased relapse and decreased survival of breast cancer patients. These results therefore identify a new and unsuspected role for DEP-1 as a mediator of an invasive cell program implicating Src

  8. The role of a conserved tyrosine residue in high-potential iron sulfur proteins.

    PubMed Central

    Iwagami, S. G.; Creagh, A. L.; Haynes, C. A.; Borsari, M.; Felli, I. C.; Piccioli, M.; Eltis, L. D.

    1995-01-01

    Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring. PMID:8580847

  9. Protein tyrosine phosphatase κ and SHP-1 are involved in the regulation of cell-cell contacts at adherens junctions in the exocrine pancreas

    PubMed Central

    Schnekenburger, J; Mayerle, J; Krüger, B; Buchwalow, I; Weiss, F U; Albrecht, E; Samoilova, V E; Domschke, W; Lerch, M M

    2005-01-01

    Background: We have previously shown that cell contacts between pancreatic acinar cells dissociate early in pancreatitis and that this is a prerequisite for the development of pancreatic oedema. Here we studied the underlying mechanism. Methods: Employing experimental caerulein induced pancreatitis in vivo and isolated pancreatic acini ex vivo, in conjunction with protein chemistry, morphology, and electron microscopy, we determined whether cell contact regulation in the pancreas requires or involves: (1) changes in cadherin-catenin protein expression, (2) tyrosine phosphorylation of adhesion proteins, or (3) alterations in the actin cytoskeleton. Results: During initial cell-cell contact dissociation at adherens junctions, expression of adhesion proteins remained stable. At time points of dissociated adherens junctions, the cadherin-catenin complex was found to be tyrosine phosphorylated and internalised. The receptor type protein tyrosine phosphatase (PTP)κ was constitutively associated with the cadherin-catenin complex at intact cell contacts whereas following the dissociation of adherens junctions, the internalised components of the cadherin-catenin complex were tyrosine phosphorylated and associated with the cytosolic PTP SHP-1. In isolated acini, inhibition of endogenous protein tyrosine phosphatases alone was sufficient to induce dissociation of adherens junctions analogous to that found with supramaximal caerulein stimulation. Dissociation of actin microfilaments had no effect on adherens junction integrity. Conclusions: These data identify tyrosine phosphorylation as the key regulator for cell contacts at adherens junctions and suggest a definitive role for the protein tyrosine phosphatases PTPκ and SHP-1 in the regulation, maintenance, and restitution of cell adhesions in a complex epithelial organ such as the pancreas. PMID:15987791

  10. 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

  11. The Biological Buffer, Bicarbonate/CO2, Potentiates H2O2-Mediated Inactivation of Protein Tyrosine Phosphatases

    PubMed Central

    Zhou, Haiying; Singh, Harkewal; Parsons, Zachary D.; Lewis, Sarah M.; Bhattacharya, Sanjib; Seiner, Derrick R.; LaButti, Jason N.; Reilly, Thomas J.; Tanner, John J.; Gates, Kent S.

    2012-01-01

    Hydrogen peroxide is a cell signaling agent that inactivates protein tyrosine phosphatases (PTPs) via oxidation of their catalytic cysteine residue. PTPs are inactivated rapidly during H2O2-mediated cellular signal transduction processes but, paradoxically, hydrogen peroxide is a rather sluggish PTP inactivator in vitro. Here we present evidence that the biological buffer, bicarbonate/CO2, potentiates the ability of H2O2 to inactivate PTPs. The results of biochemical experiments and high resolution crystallographic analysis are consistent with a mechanism involving oxidation of the catalytic cysteine residue by peroxymonocarbonate generated via the reaction of H2O2 with HCO3 −/CO2. PMID:21913686

  12. New Hippolide Derivatives with Protein Tyrosine Phosphatase 1B Inhibitory Activity from the Marine Sponge Hippospongia lachne

    PubMed Central

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

    2014-01-01

    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. PMID:25007159

  13. High-performance liquid chromatography with fluorescence detection for quantitation of tryptophan and tyrosine in a shrimp waste protein concentrate.

    PubMed

    Sánchez-Machado, D I; Chavira-Willys, B; López-Cervantes, J

    2008-02-15

    A new, simple, and reproducible isocratic high-performance liquid chromatography (HPLC) method has been developed for the determination of free and total tyrosine and tryptophan in a protein concentrate. To determine total amino acids, the method involves alkaline hydrolysis of the proteins with sodium hydroxide at 120 degrees C for 4h in the absence of air. Best results were achieved with a SS Exil ODS column 5microm (25cmx0.46cm i.d.), with an eluent of methanol: 40mM sodium acetate buffer (adjusted to pH 4.5 with acetic acid; 20:80, v/v), a flow rate of 0.80mL/min at 26 degrees C, and with programmable fluorescence detection. Under optimum conditions excellent linearity was obtained, and the overall recovery was 90.5, and 95.9% for total tryptophan and tyrosine, respectively. The precision results showed that the relative standard deviation of the repeatability and reproducibility were < or =4.78 and < or =4.65, respectively. This method was used to quantify the cited analytes in the protein concentrate obtained during the lactic acid fermentation of shrimp waste. PMID:18243075

  14. Colocalisation of the protein tyrosine phosphatases PTP-SL and PTPBR7 with beta4-adaptin in neuronal cells.

    PubMed

    Dilaver, Gönül; Schepens, Jan; van den Maagdenberg, Arn; Wijers, Mietske; Pepers, Barry; Fransen, Jack; Hendriks, Wiljan

    2003-01-01

    The mouse gene Ptprr encodes the neuronal protein tyrosine phosphatases PTP-SL and PTPBR7. These proteins differ in their N-terminal domains, with PTP-SL being a cytosolic, membrane-associated phosphatase and PTPBR7 a type I transmembrane protein. In this study, we further explored the nature of the PTP-SL-associated vesicles in neuronal cells using a panel of organelle markers and noted a comparable subcellular distribution for PTP-SL and the beta4-adaptin subunit of the AP4 complex. PTP-SL, PTPBR7 and beta4-adaptin are localised at the Golgi apparatus and at vesicles throughout the cytoplasm. Immunohistochemical analysis demonstrated that PTP-SL, PTPBR7 and beta4-adaptin are all endogenously expressed in brain. Interestingly, coexpression of PTP-SL and beta4-adaptin leads to an altered subcellular localisation for PTP-SL. Instead of the Golgi and vesicle-type staining pattern, still observable for beta4-adaptin, PTP-SL is now distributed throughout the cytoplasm. Although beta4-adaptin was found to interact with the phosphatase domain of PTP-SL and PTPBR7 in the yeast two-hybrid system, it failed to do so in transfected neuronal cells. Our data suggest that the tyrosine phosphatases PTP-SL and PTPBR7 may be involved in the formation and transport of AP4-coated vesicles or in the dephosphorylation of their transmembrane cargo molecules at or near the Golgi apparatus. PMID:12548400

  15. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity.

    PubMed Central

    Lee, S K; Su, B; Maher, S E; Bothwell, A L

    1994-01-01

    To characterize the function of the Ly-6A antigen in T cell activation, antisense Ly-6 RNA was expressed in a stably transfected antigen-specific T cell clone. Reduced Ly-6A expression results in inhibition of responses to antigen, anti-TCR (anti-T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly-6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly-6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity. Images PMID:8187770

  16. Integrin α1β1 Promotes Caveolin-1 Dephosphorylation by Activating T Cell Protein-tyrosine Phosphatase*

    PubMed Central

    Borza, Corina M.; Chen, Xiwu; Mathew, Sijo; Mont, Stacey; Sanders, Charles R.; Zent, Roy; Pozzi, Ambra

    2010-01-01

    Integrin α1β1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1β1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1β1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1β1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1β1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1β1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1β1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1β1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury. PMID:20940300

  17. Integrin {alpha}1{beta}1 promotes caveolin-1 dephosphorylation by activating T cell protein-tyrosine phosphatase.

    PubMed

    Borza, Corina M; Chen, Xiwu; Mathew, Sijo; Mont, Stacey; Sanders, Charles R; Zent, Roy; Pozzi, Ambra

    2010-12-17

    Integrin α1β1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1β1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1β1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1β1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1β1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1β1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1β1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1β1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury. PMID:20940300

  18. Neurotrophin-3 Enhances the Synaptic Organizing Function of TrkC-Protein Tyrosine Phosphatase σ in Rat Hippocampal Neurons.

    PubMed

    Ammendrup-Johnsen, Ina; Naito, Yusuke; Craig, Ann Marie; Takahashi, Hideto

    2015-09-01

    Neurotrophin-3 (NT-3) and its high-affinity receptor TrkC play crucial trophic roles in neuronal differentiation, axon outgrowth, and synapse development and plasticity in the nervous system. We demonstrated previously that postsynaptic TrkC functions as a glutamatergic synapse-inducing (synaptogenic) cell adhesion molecule trans-interacting with presynaptic protein tyrosine phosphatase σ (PTPσ). Given that NT-3 and PTPσ bind distinct domains of the TrkC extracellular region, here we tested the hypothesis that NT-3 modulates TrkC/PTPσ binding and synaptogenic activity. NT-3 enhanced PTPσ binding to cell surface-expressed TrkC and facilitated the presynapse-inducing activity of TrkC in rat hippocampal neurons. Imaging of recycling presynaptic vesicles combined with TrkC knockdown and rescue approaches demonstrated that NT-3 rapidly potentiates presynaptic function via binding endogenous postsynaptic TrkC in a tyrosine kinase-independent manner. Thus, NT-3 positively modulates the TrkC-PTPσ complex for glutamatergic presynaptic assembly and function independently from TrkC kinase activation. Our findings provide new insight into synaptic roles of neurotrophin signaling and mechanisms controlling synaptic organizing complexes. Significance statement: Although many synaptogenic adhesion complexes have been identified in recent years, little is known about modulatory mechanisms. Here, we demonstrate a novel role of neurotrophin-3 in synaptic assembly and function as a positive modulator of the TrkC-protein tyrosine phosphatase σ complex. This study provides new insight into the involvement of neurotrophin signaling in synapse development and plasticity, presenting a molecular mechanism that may underlie previous observations of short- and long-term enhancement of presynaptic function by neurotrophin. Given the links of synaptogenic adhesion molecules to autism and schizophrenia, this study might also contribute to a better understanding of the pathogenesis of

  19. Looking for a needle in a haystack: Cellular proteins that may interact with the tyrosine-based sorting signal of the TGEV S protein.

    PubMed

    Trincone, Anna; Schwegmann-Weßels, Christel

    2015-04-16

    The spike protein S of transmissible gastroenteritis virus, an Alphacoronavirus, contains a tyrosine-based sorting signal that is responsible for ERGIC retention and may be important for a correct viral assembly process. To find out whether the S protein interacts with cellular proteins via this sorting signal, a pulldown assay with GST fusion proteins was performed. Filamin A has been identified as a putative interaction candidate. Immunofluorescence assays confirmed a co-localization between the TGEV S protein and filamin A. Further experiments have to be performed to prove a significant impact of filamin A on TGEV infection. Different approaches of several researchers for the identification of cellular interaction candidates relevant for coronavirus replication are summarized. These results may help in the future to identify the role of cellular proteins during coronavirus assembly at the ER-Golgi intermediate compartment. PMID:25481285

  20. Saccharomyces cerevisiae Yak1p protein kinase autophosphorylates on tyrosine residues and phosphorylates myelin basic protein on a C-terminal serine residue.

    PubMed Central

    Kassis, S; Melhuish, T; Annan, R S; Chen, S L; Lee, J C; Livi, G P; Creasy, C L

    2000-01-01

    The serine/threonine protein kinase, Yak1p, functions as a negative regulator of the cell cycle in Saccharomyces cerevisiae, acting downstream of the cAMP-dependent protein kinase. In the present work we report that overexpression of haemagglutinin-tagged full-lengthYak1p and an N-terminally truncated form (residues 148-807) lead to growth arrest in PKA compromised yak1 null yeast cells. Both forms of recombinant Yak1p kinase were catalytically active and preferred myelin basic protein (MBP) as a substrate over several other proteins. Phosphopeptide analysis of bovine MBP by tandem MS revealed two major Yak1p phosphorylation sites, Thr-97 and Ser-164. Peptides containing each site were obtained and tested as Yak1p substrates. Both forms of Yak1p phosphorylated a peptide containing the Ser-164 residue with far more efficient kinetics than MBP. The maximal velocity (V(max)) values of the full-length Yak1p reaction were 110+/-21 (Ser-164) and 8.7+/-1.7 (MBP), and those of N-terminally truncated Yak1p were 560.7+/-74.8 (Ser-164) and 34. 4+/-2.2 (MBP) pmol/min per mg of protein. Although neither form of Yak1p was able to phosphorylate two generic protein tyrosine kinase substrates, both were phosphorylated on tyrosine residues in vivo and underwent tyrosine autophosphorylation when reacted with ATP in vitro. Tandem MS showed that Tyr-530 was phosphorylated both in vivo and in vitro after reaction with ATP. Pre-treatment with protein tyrosine phosphatase 1B removed all of Yak1p phosphotyrosine content and drastically reduced Yak1p activity against exogenous substrates, suggesting that the phosphotyrosine content of the enzyme is essential for its catalytic activity. Although the N-terminally truncated Yak1p was expressed at a lower level than the full-length protein, its catalytic activity and phosphotyrosine content were significantly higher than those of the full-length enzyme. Taken together, our results suggest that Yak1p is a dual specificity protein kinase which

  1. Tyrosine 129 of the Murine Gammaherpesvirus M2 Protein Is Critical for M2 Function In Vivo

    PubMed Central

    Rangaswamy, Udaya S.; O’Flaherty, Brigid M.; Speck, Samuel H.

    2014-01-01

    A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes – predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo. PMID:25122496

  2. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

    SciTech Connect

    Blanquart, Christophe; Karouri, Salah-Eddine; Issad, Tarik

    2009-10-02

    The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.

  3. Isothiazolidinone (IZD) as a phosphoryl mimetic in inhibitors of the Yersinia pestis protein tyrosine phosphatase YopH

    SciTech Connect

    Kim, Sung-Eun; Bahta, Medhanit; Lountos, George T.; Ulrich, Robert G.; Burke, Terrence R. Jr Waugh, David S.

    2011-07-01

    The first X-ray crystal structure of the Y. pestis protein tyrosine phosphatase YopH in complex with an isothiazolidinone-based lead-fragment compound is reported. Isothiazolidinone (IZD) heterocycles can act as effective components of protein tyrosine phosphatase (PTP) inhibitors by simultaneously replicating the binding interactions of both a phosphoryl group and a highly conserved water molecule, as exemplified by the structures of several PTP1B–inhibitor complexes. In the first unambiguous demonstration of IZD interactions with a PTP other than PTP1B, it is shown by X-ray crystallography that the IZD motif binds within the catalytic site of the Yersinia pestis PTP YopH by similarly displacing a highly conserved water molecule. It is also shown that IZD-based bidentate ligands can inhibit YopH in a nonpromiscuous fashion at low micromolar concentrations. Hence, the IZD moiety may represent a useful starting point for the development of YopH inhibitors.

  4. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  5. Increased nitration and carbonylation of proteins in MRL +/+ mice exposed to trichloroethene: Potential role of protein oxidation in autoimmunity

    SciTech Connect

    Wang Gangduo; Wang Jianling; Ma Huaxian; Khan, M. Firoze

    2009-06-01

    Even though reactive oxygen and nitrogen species (RONS) are implicated as mediators of autoimmune diseases (ADs), little is known about contribution of protein oxidation (carbonylation and nitration) in the pathogenesis of such diseases. The focus of this study was, therefore, to establish a link between protein oxidation and induction and/or exacerbation of autoimmunity. To achieve this, female MRL +/+ mice were treated with trichloroethene (TCE), an environmental contaminant known to induce autoimmune response, for 6 or 12 weeks (10 mmol/kg, i.p., every 4{sup th} day). TCE treatment resulted in significantly increased formation of nitrotyrosine (NT) and induction of iNOS in the serum at both 6 and 12 weeks of treatment, but the response was greater at 12 weeks. Likewise, TCE treatment led to greater NT formation, and iNOS protein and mRNA expression in the livers and kidneys. Moreover, TCE treatment also caused significant increases ({approx}3 fold) in serum protein carbonyls (a marker of protein oxidation) at both 6 and 12 weeks. Significantly increased protein carbonyls were also observed in the livers and kidneys (2.1 and 1.3 fold, respectively) at 6 weeks, and to a greater extent at 12 weeks (3.5 and 2.1 fold, respectively) following TCE treatment. The increases in TCE-induced protein oxidation (carbonylation and nitration) were associated with significant increases in Th1 specific cytokine (IL-2, IFN-{gamma}) release into splenocyte cultures. These results suggest an association between protein oxidation and induction/exacerbation of autoimmune response. The results present a potential mechanism by which oxidatively modified proteins could contribute to TCE-induced autoimmune response and necessitates further investigations for clearly establishing the role of protein oxidation in the pathogenesis of ADs.

  6. Presence of ecto-protein tyrosine phosphatase activity is vital for survival of Setaria cervi, a bovine filarial parasite.

    PubMed

    Singh, Neetu; Heneberg, Petr; Rathaur, Sushma

    2014-10-01

    The ecto protein tyrosine phosphatases (PTP) are known to play a crucial role in the pathogenesis and survival of the intracellular parasites. However, their presence and role in filarial parasites is still unknown. We found a significant amount of tyrosine phosphatase activity in the surface antigen fraction extracted from Setaria cervi (S. cervi), a bovine filarial parasite. An antibody designed against the conserved catalytic core of human protein tyrosine phosphatases, PTP1B cross reacted with a 63 kDa band in the surface antigen. We detected a significant amount of PTP activity in the intact S. cervi adult parasites as well as microfilariae in this study for the first time. This PTP may be localized on the surface of the parasite with an exposed active site available for the external substrates. The PTP activity was also inhibited by sodium orthovanadate and phenyl arsine oxide, specific inhibitors of PTP in both the life stages. The Km and Vmax for PTP in the adult parasites and microfilariae were determined to be 2.574 ± 0.14 mM; 206.3 ± 2.75 μM Pi/h/two parasites and 5.510 ± 0.59 mM; 62.27 ± 2.27 μM Pi/h/10(6) parasites respectively using O-P-L-Tyrosine as substrate. Interestingly, a positive correlation was observed between the inhibition in PTP activity and reduction in the motility/ viability of the parasites when they were subjected to the specific PTP inhibitors (Orthovanadate and Phenyl arsine oxide) for 4 h in the KRB maintenance medium. The activity was also significantly inhibited in the parasites exposed to antifilarial drug/compounds for e.g. Diethylcarbamazine, Acetylsalicylic Acid and SK7, a methyl chalcone. Therefore suggesting a possible role played by PTP in the survival of the parasite, its interaction with the host as well as in the screening of newly synthesized antifilarials/drugs. PMID:25028209

  7. The HPV16 E6 Oncoprotein Causes Prolonged Receptor Protein Tyrosine Kinase Signaling and Enhances Internalization of Phosphorylated Receptor Species

    PubMed Central

    Spangle, Jennifer M.; Munger, Karl

    2013-01-01

    The high-risk human papillomavirus (HPV) E6 proteins are consistently expressed in HPV-associated lesions and cancers. HPV16 E6 sustains the activity of the mTORC1 and mTORC2 signaling cascades under conditions of growth factor deprivation. Here we report that HPV16 E6 activated mTORC1 by enhanced signaling through receptor protein tyrosine kinases, including epidermal growth factor receptor and insulin receptor and insulin-like growth factor receptors. This is evidenced by sustained signaling through these receptors for several hours after growth factor withdrawal. HPV16 E6 increased the internalization of activated receptor species, and the signaling adaptor protein GRB2 was shown to be critical for HPV16 E6 mediated enhanced EGFR internalization and mTORC1 activation. As a consequence of receptor protein kinase mediated mTORC1 activation, HPV16 E6 expression increased cellular migration of primary human epithelial cells. This study identifies a previously unappreciated mechanism by which HPV E6 proteins perturb host-signaling pathways presumably to sustain protein synthesis during the viral life cycle that may also contribute to cellular transforming activities of high-risk HPV E6 proteins. PMID:23516367

  8. Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

    PubMed

    Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M Michael; Taguchi, Y-H; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A Mary; Velmurugan, D; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

    2015-01-01

    A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective. PMID:26607293

  9. Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target

    PubMed Central

    Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M. Michael; Taguchi, Y-h.; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A. Mary; Velmurugan, D.; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

    2015-01-01

    A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective. PMID:26607293

  10. Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B.

    PubMed

    Luna, Sandra; Mingo, Janire; Aurtenetxe, Olaia; Blanco, Lorena; Amo, Laura; Schepens, Jan; Hendriks, Wiljan J; Pulido, Rafael

    2016-01-01

    In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the tumor-suppressor PTEN and the receptor-type PTPRZ-B (short isoform from PTPRZ1 gene) phosphatases as examples, we provide a brief insight into the utility of specific mutations in the experimental analysis of PTP functions. We describe a standardized, rapid, and simple method of mutagenesis to perform single and multiple amino acid substitutions, as well as deletions of short nucleotide sequences, based on one-step inverse PCR and DpnI restriction enzyme treatment. This method of SDM is generally applicable to any other protein of interest. PMID:27514801

  11. A homogeneous fluorescence polarization assay adaptable for a range of protein serine/threonine and tyrosine kinases.

    PubMed

    Gaudet, Elizabeth A; Huang, Kuo-Sen; Zhang, Yan; Huang, Wei; Mark, David; Sportsman, J Richard

    2003-04-01

    Recently, a new technology for high-throughput screening has been developed, called IMAP(patent pending). IMAP technology has previously been implemented in an assay for cyclic nucleotide phosphodiesterases (PDE). The authors describe the development of a homogeneous, non-antibody-based fluorescence polarization (FP) assay for a variety of protein kinases. In this assay, fluorescently labeled peptide substrate phosphorylated by the kinase is captured on modified nanoparticles through interactions with immobilized metal (M(III)) coordination complexes, resulting in a change from low to high polarization values. This assay is applicable to protein kinases that phosphorylate serine, threonine, or tyrosine residues. The IMAP platform is very compatible with high-throughput robotics and can be applied to the 1536-well format. As there are hundreds of different kinases coded for in the human genome, the assay platform described in this report is a valuable new tool in drug discovery. PMID:12844437

  12. Histidine-domain-containing protein tyrosine phosphatase regulates platelet-derived growth factor receptor intracellular sorting and degradation.

    PubMed

    Ma, Haisha; Wardega, Piotr; Mazaud, David; Klosowska-Wardega, Agnieszka; Jurek, Aleksandra; Engström, Ulla; Lennartsson, Johan; Heldin, Carl-Henrik

    2015-11-01

    Histidine domain-containing protein tyrosine phosphatase (HD-PTP) is a putative phosphatase that has been shown to affect the signaling and downregulation of certain receptor tyrosine kinases. To investigate if HD-PTP affects platelet-derived growth factor receptor β (PDGFRβ) signaling, we employed the overexpression of HA-tagged HD-PTP, as well as siRNA-mediated and lentivirus shRNA-mediated silencing of HD-PTP in NIH3T3 cells. We found that HD-PTP was recruited to the PDGFRβ in a ligand-dependent manner. Depletion of HD-PTP resulted in an inability of PDGF-BB to promote tyrosine phosphorylation of the ubiquitin ligases c-Cbl and Cbl-b, with a concomitant missorting and reduction of the degradation of activated PDGFRβ. In contrast, ligand-induced internalization of PDGFRβ was unaffected by HD-PTP silencing. Furthermore, the levels of STAM and Hrs of the ESCRT0 machinery were decreased, and immunofluorescence staining showed that in HD-PTP-depleted cells, PDGFRβ accumulated in large aberrant intracellular structures. After the reduction of HD-PTP expression, an NIH3T3-derived cell line that has autocrine PDGF-BB signaling (sis-3T3) showed increased ability of anchorage-independent growth. However, exogenously added PDGF-BB promoted efficient additional colony formation in control cells, but was not able to do so in HD-PTP-depleted cells. Furthermore, cells depleted of HD-PTP migrated faster than control cells. In summary, HD-PTP affects the intracellular sorting of activated PDGFRβ and the migration, proliferation and tumorigenicity of cells stimulated by PDGF. PMID:26232618

  13. A Tyrosine-Rich Cell Surface Protein in the Diatom Amphora coffeaeformis Identified through Transcriptome Analysis and Genetic Transformation

    PubMed Central

    Buhmann, Matthias T.; Poulsen, Nicole; Klemm, Jennifer; Kennedy, Matthew R.; Sherrill, C. David; Kröger, Nils

    2014-01-01

    Diatoms are single-celled eukaryotic microalgae that are ubiquitously found in almost all aquatic ecosystems, and are characterized by their intricately structured SiO2 (silica)-based cell walls. Diatoms with a benthic life style are capable of attaching to any natural or man-made submerged surface, thus contributing substantially to both microbial biofilm communities and economic losses through biofouling. Surface attachment of diatoms is mediated by a carbohydrate- and protein- based glue, yet no protein involved in diatom underwater adhesion has been identified so far. In the present work, we have generated a normalized transcriptome database from the model adhesion diatom Amphora coffeaeformis. Using an unconventional bioinformatics analysis we have identified five proteins that exhibit unique amino acid sequences resembling the amino acid composition of the tyrosine-rich adhesion proteins from mussel footpads. Establishing the first method for the molecular genetic transformation of A. coffeaeformis has enabled investigations into the function of one of these proteins, AC3362, through expression as YFP fusion protein. Biochemical analysis and imaging by fluorescence microscopy revealed that AC3362 is not involved in adhesion, but rather plays a role in biosynthesis and/or structural stability of the cell wall. The methods established in the present study have paved the way for further molecular studies on the mechanisms of underwater adhesion and biological silica formation in the diatom A. coffeaeformis. PMID:25372470

  14. Protein tyrosine phosphatase PTPN14 is a regulator of lymphatic function and choanal development in humans.

    PubMed

    Au, Audrey C; Hernandez, Paolo A; Lieber, Ernest; Nadroo, Ali M; Shen, Yu-Ming; Kelley, Kevin A; Gelb, Bruce D; Diaz, George A

    2010-09-10

    The lymphatic vasculature is essential for the recirculation of extracellular fluid, fat absorption, and immune function and as a route of tumor metastasis. The dissection of molecular mechanisms underlying lymphangiogenesis has been accelerated by the identification of tissue-specific lymphatic endothelial markers and the study of congenital lymphedema syndromes. We report the results of genetic analyses of a kindred inheriting a unique autosomal-recessive lymphedema-choanal atresia syndrome. These studies establish linkage of the trait to chromosome 1q32-q41 and identify a loss-of-function mutation in PTPN14, which encodes a nonreceptor tyrosine phosphatase. The causal role of PTPN14 deficiency was confirmed by the generation of a murine Ptpn14 gene trap model that manifested lymphatic hyperplasia with lymphedema. Biochemical studies revealed a potential interaction between PTPN14 and the vascular endothelial growth factor receptor 3 (VEGFR3), a receptor tyrosine kinase essential for lymphangiogenesis. These results suggest a unique and conserved role for PTPN14 in the regulation of lymphatic development in mammals and a nonconserved role in choanal development in humans. PMID:20826270

  15. Composition and variation in vitamin C, carotenoids, protein, nitrate and oxalate contents in Celosia leaves.

    PubMed

    Prakash, D; Nath, P; Pal, M

    1995-04-01

    Twenty two lines/accessions belonging to 5 species of Celosia were analysed for their nutritional and antinutritional composition. The nutrient contents, vitamin C varied from 18.8 to 53.6 mg/100 g, carotenoids 9.1 to 15.1 mg/100g, protein 2.1 to 5.9%, while the antinutritional factors, nitrate 0.18 to 0.46% and oxalate 0.98 to 3.93%. Moisture ranged 81 to 89%. Variation of these parameters with leaf position and the amino acids profile of some promising lines was also evaluated. PMID:7659699

  16. Biochemical characteristics of cytosolic and particulate forms of protein tyrosine kinases from methyl nitrosourea (MNU)-induced rat mammary carcinoma

    SciTech Connect

    Srivastava, A.K.; Chiasson, J.C.; Chiasson, J.L.; Lacroix, A.; Windisch, L. )

    1991-03-11

    Protein tyrosine kinase (PTK) activities in MNU-induced rat mammary carcinoma has been investigated by using poly (glu: tyr; 4:1) as an exogenous substrate. The PTK activity of the mammary carcinoma was about equally distributed between the particulate and cytosolic fractions at 110 000 x g. Both particulate and cytosolic PTKs catalyzed the phosphorylation of several tyrosine containing synthetic substrates to various degrees, however, poly (glu: tyr; 4:1) was the best substrate. Both the forms utilized ATP as the phosphoryl group donor. Among various divalent cations tested, Co{sup 2+}, Mn{sup 2+} and Mg{sup 2+} were able to fulfill the divalent cation requirement. Poly-lysine exerted a stimulatory effect on the particulate, but not on the cytosolic form. On the other hand, though heparin and quercetin inhibited both the forms in a concentration dependent manner, the particulate form was more sensitive to inhibition. These data indicate that MNU-induced rat mammary carcinoma expresses both particulate and cytosolic forms of PTKs and that there are significant differences in the properties of the two forms. Differential differences in the properties of the two forms. Differential effects of some agents on mammary carcinoma PTKs suggest that these enzymes may be acutely regulated in vivo and could play important role in mammary carcinogenesis.

  17. The trkB Tyrosine Protein Kinase Is a Receptor for Brain-Derived Neurotrophic Factor and Neurotrophin-3

    PubMed Central

    Klein, Rüdiger; Nanduri, Venkata; Jing, Shuqian; Lamballe, Fabienne; Tapley, Peter; Bryant, Sherri; Cordon-Cardo, Carlos; Jones, Kevin R.; Reichardt, Louis F.; Barbacid, Mariano

    2009-01-01

    Summary trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3. PMID:1649702

  18. Neural stem cells from protein tyrosine phosphatase sigma knockout mice generate an altered neuronal phenotype in culture

    PubMed Central

    Kirkham, David L; Pacey, Laura KK; Axford, Michelle M; Siu, Roberta; Rotin, Daniela; Doering, Laurie C

    2006-01-01

    Background The LAR family Protein Tyrosine Phosphatase sigma (PTPσ) has been implicated in neuroendocrine and neuronal development, and shows strong expression in specific regions within the CNS, including the subventricular zone (SVZ). We established neural stem cell cultures, grown as neurospheres, from the SVZ of PTPσ knockout mice and sibling controls to determine if PTPσ influences the generation and the phenotype of the neuronal, astrocyte and oligodendrocyte cell lineages. Results The neurospheres from the knockout mice acquired heterogeneous developmental characteristics and they showed similar morphological characteristics to the age matched siblings. Although Ptprs expression decreases as a function of developmental age in vivo, it remains high with the continual renewal and passage of the neurospheres. Stem cells, progenitors and differentiated neurons, astrocytes and oligodendrocytes all express the gene. While no apparent differences were observed in developing neurospheres or in the astrocytes and oligodendrocytes from the PTPσ knockout mice, the neuronal migration patterns and neurites were altered when studied in culture. In particular, neurons migrated farther from the neurosphere centers and the neurite outgrowth exceeded the length of the neuronal processes from age matched sibling controls. Conclusion Our results imply a specific role for PTPσ in the neuronal lineage, particularly in the form of inhibitory influences on neurite outgrowth, and demonstrate a role for tyrosine phosphatases in neuronal stem cell differentiation. PMID:16784531

  19. Platelet-activating factor attenuation of long-term potentiation in rat hippocampal slices via protein tyrosine kinase signaling.

    PubMed

    Reiner, Benjamin; Wang, Wenwei; Liu, Jianuo; Xiong, Huangui

    2016-02-26

    It is well established that HIV-1-infected mononuclear phagocytes release platelet activating factor (PAF) and elevated levels of PAF have been detected in blood and in the cerebrospinal fluid (CSF) of acquired immunodeficiency syndrome (AIDS) patients with HIV-associated neurocognitive disorders (HAND). It is our hypothesis that the elevated levels of PAF alter long-term potentiation (LTP) in the hippocampus, leading to neurocognitive dysfunction. To test this hypothesis, we studied the effects of PAF on LTP in the CA1 region of rat hippocampal slices. Our results showed incubation of hippocampal slices with PAF attenuated LTP. The PAF-mediated attenuation was blocked by ginkgolide B, a PAF receptor antagonist, suggesting PAF attenuation of LTP via PAF receptors. Application of lyso-PAF, an inactive PAF analog, had no apparent effect on LTP. Further investigation revealed an involvement of tyrosine kinase in PAF attenuation of LTP, which was demonstrated by lavendustin A (a specific protein tyrosine kinase inhibitor) blockage of PAF attenuation of LTP. As LTP is widely considered as the cellular and synaptic basis for learning and memory, the attenuation of LTP by PAF may contribute at least in part to the HAND pathogenesis. PMID:26808643

  20. 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

  1. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    PubMed

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases. PMID:27079226

  2. The receptor protein tyrosine phosphatase (RPTP){beta}/{zeta} is expressed in different subtypes of human breast cancer

    SciTech Connect

    Perez-Pinera, Pablo; Garcia-Suarez, Olivia; Menendez-Rodriguez, Primitiva; Mortimer, J.; Chang, Y.; Astudillo, A.; Deuel, T.F.

    2007-10-12

    Increasing evidence suggests mutations in human breast cancer cells that induce inappropriate expression of the 18-kDa cytokine pleiotrophin (PTN, Ptn) initiate progression of breast cancers to a more malignant phenotype. Pleiotrophin signals through inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, leading to increased tyrosine phosphorylation of different substrate proteins of RPTP{beta}/{zeta}, including {beta}-catenin, {beta}-adducin, Fyn, GIT1/Cat-1, and P190RhoGAP. PTN signaling thus has wide impact on different important cellular systems. Recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway; this discovery potentially is very important, since constitutive ALK activity of nucleophosmin (NPM)-ALK fusion protein is causative of anaplastic large cell lymphomas, and, activated ALK is found in other malignant cancers. Recently ALK was identified in each of 63 human breast cancers from 22 subjects. We now demonstrate that RPTP{beta}/{zeta} is expressed in each of these same 63 human breast cancers that previously were found to express ALK and in 10 additional samples of human breast cancer. RPTP{beta}/{zeta} furthermore was localized not only in its normal association with the cell membrane but also scattered in cytoplasm and in nuclei in different breast cancer cells and, in the case of infiltrating ductal carcinomas, the distribution of RPTP{beta}/{zeta} changes as the breast cancer become more malignant. The data suggest that the PTN/RPTP{beta}/{zeta} signaling pathway may be constitutively activated and potentially function to constitutively activate ALK in human breast cancer.

  3. The ER structural protein Rtn4A stabilizes and enhances signaling through the receptor tyrosine kinase ErbB3.

    PubMed

    Hatakeyama, Jason; Wald, Jessica H; Rafidi, Hanine; Cuevas, Antonio; Sweeney, Colleen; Carraway, Kermit L

    2016-01-01

    ErbB3 and ErbB4 are receptor tyrosine kinases that are activated by the neuregulin (NRG) family of growth factors. These receptors govern various developmental processes, and their dysregulation contributes to several human disease states. The abundance of ErbB3 and ErbB4, and thus signaling through these receptors, is limited by the E3 ubiquitin ligase Nrdp1, which targets ErbB3 and ErbB4 for degradation. Reticulons are proteins that influence the morphology of the endoplasmic reticulum (ER) by promoting the formation of tubules, a response of cells to some stressors. We found that the ER structural protein reticulon 4A (Rtn4A, also known as Nogo-A) increased ErbB3 abundance and proliferative signaling by suppressing Nrdp1 function. Rtn4A interacted with Nrdp1 and stabilized ErbB3 in an Nrdp1-dependent manner. Rtn4A overexpression induced the redistribution of Nrdp1 from a cytosolic or perinuclear localization to ER tubules. Rtn4A knockdown in human breast tumor cells decreased ErbB3 abundance, NRG-stimulated signaling, and cellular proliferation and migration. Because proteins destined for the plasma membrane are primarily synthesized in the sheet portions of the ER, our observations suggest that Rtn4A counteracts the Nrdp1-mediated degradation of ErbB3 by sequestering the ubiquitin ligase into ER tubules. The involvement of a reticulon suggests a molecular link between ER structure and the sensitivity of cells to receptor tyrosine kinase-mediated survival signals at the cell surface. PMID:27353365

  4. The Src-family tyrosine kinase inhibitor PP1 interferes with the activation of ribosomal protein S6 kinases.

    PubMed Central

    Shah, O Jameel; Kimball, Scot R; Jefferson, Leonard S

    2002-01-01

    Considerable biochemical and pharmacological evidence suggests that the activation of ribosomal protein S6 kinases (S6Ks) by activated receptor tyrosine kinases involves multiple co-ordinated input signals. However, the identities of many of these inputs remain poorly described, and their precise involvement in S6K activation has been the subject of great investigative effort. In the present study, we have shown that 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), a selective inhibitor of the Src family of non-receptor tyrosine kinases, interferes with the activation of 70 and 85 kDa S6K gene products (p70S6K1 and p85S6K1) by insulin, insulin-like growth factor 1, sodium orthovanadate and activated alleles of phosphoinositide 3-kinase and H-Ras. PP1 also impedes the activation of AKT/protein kinase B and the extracellular signal-regulated protein kinases 1 and 2 by these various stimuli. Insulin-like growth factor 1 was observed to induce a sustained increase in c-Src autophosphorylation as revealed using anti-phospho-Y416 antisera, but this effect was absent from the cells treated with PP1. To conclude, an activated allele of p70S6K1 is compared with the wild-type allele, resistant to inhibition by PP1 when co-expressed with phosphoinositide-dependent kinase 1 (PDK1), suggesting that PP1 affects p70S6K1 via a PDK1-independent pathway. Thus activation of Src may supply a necessary signal for the activation of p70S6K1 and possibly other S6Ks. PMID:12014987

  5. Catalytic and substrate promiscuity: distinct multiple chemistries catalysed by the phosphatase domain of receptor protein tyrosine phosphatase.

    PubMed

    Srinivasan, Bharath; Marks, Hanna; Mitra, Sreyoshi; Smalley, David M; Skolnick, Jeffrey

    2016-07-15

    The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. In the present study, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalysed by the catalytic domain of receptor protein tyrosine phosphatase isoform δ (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C─O─P) with high catalytic efficiency, whereas the secondary activity is the hydrolysis of a glycosidic bond (C─O─C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolysing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of protein tyrosine phosphatase Ω (PTPRΩ), a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain (PD) of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbour a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete. PMID:27208174

  6. ANSID: A Solid-Phase Proteomic Approach for Identification and Relative Quantification of Aromatic Nitration Sites

    PubMed Central

    Nuriel, Tal; Whitehouse, Julia; Ma, Yuliang; Mercer, Emily J.; Brown, Neil; Gross, Steven S.

    2016-01-01

    Nitration of tyrosine and other aromatic amino acid residues in proteins occurs in the setting of inflammatory, neurodegenerative, and cardiovascular diseases—importantly, this modification has been implicated in the pathogenesis of diverse diseases and the physiological process of aging. To understand the biological consequences of aromatic nitration in both health and disease, it is critical to molecularly identify the proteins that undergo nitration, specify their cognate modification sites and quantify their extent of nitration. To date, unbiased identification of nitrated proteins has often involved painstaking 2D-gel electrophoresis followed by Western Blotting with an anti-nitrotyrosine antibody for detection. Apart from being relatively slow and laborious, this method suffers from limited coverage, the potential for false-positive identifications, and failure to reveal specific amino acid modification sites. To overcome these shortcomings, we have developed a solid-phase, chemical-capture approach for unbiased and high-throughput discovery of nitrotyrosine and nitrotryptophan sites in proteins. Utilizing this method, we have successfully identified several endogenously nitrated proteins in rat brain and a total of 244 nitrated peptides from 145 proteins following in vitro exposure of rat brain homogenates to the nitrating agent peroxynitrite (1 mM). As expected, Tyr residues constituted the great majority of peroxynitrite-mediated protein nitration sites; however, we were surprised to discover several brain proteins that contain nitrated Trp residues. By incorporating a stable-isotope labeling step, this new Aromatic Nitration Site IDentification (ANSID) method was also adapted for relative quantification of nitration site abundances in proteins. Application of the ANSID method offers great potential to advance our understanding of the role of protein nitration in disease pathogenesis and normal physiology. PMID:26779476

  7. Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter.

    PubMed

    Bagchi, Rammyani; Salehin, Mohammad; Adeyemo, O Sarah; Salazar, Carolina; Shulaev, Vladimir; Sherrier, D Janine; Dickstein, Rebecca

    2012-10-01

    The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function. PMID:22858636

  8. Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein.

    PubMed

    Simon, Jörg; Sänger, Monica; Schuster, Stephan C; Gross, Roland

    2003-07-01

    The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor. Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined. We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities. A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced. The napA gene was inactivated by inserting a kanamycin resistance gene cassette. The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type. An antigen was detected in wild-type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus. This antigen was absent in the W. succinogenes napA mutant. It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W. succinogenes, although a second nitrate-reducing enzyme is apparently induced in the napA mutant. The nap cluster of W. succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria. The W. succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC. Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration. A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild-type properties with respect to nitrate respiration. A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex. Inspection of the W. succinogenes genome sequence suggests that ammonia formation from

  9. Verruculides A and B, two new protein tyrosine phosphatase 1B inhibitors from an Indonesian ascidian-derived Penicillium verruculosum.

    PubMed

    Yamazaki, Hiroyuki; Nakayama, Wataru; Takahashi, Ohgi; Kirikoshi, Ryota; Izumikawa, Yuta; Iwasaki, Kohei; Toraiwa, Kengo; Ukai, Kazuyo; Rotinsulu, Henki; Wewengkang, Defny S; Sumilat, Deiske A; Mangindaan, Remy E P; Namikoshi, Michio

    2015-08-15

    Two new merosesquiterpenes, verruculides A (1) and B (2), were isolated from a culture broth of the Indonesian ascidian-derived Penicillium verruculosum TPU1311, together with three known congeners, chrodrimanins A (3), B (4), and H (5). The structures of 1 and 2 were assigned on the basis of their spectroscopic data (1D and 2D NMR, HRMS, UV, CD, and IR). Compound 2 had a linear sesquiterpene moiety and was considered to be the derivative of the biosynthetic precursor for 1 and 3-5. Compounds 1, 3, and 5 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 8.4, 8.5, and 14.9 μM, respectively. Compound 2 showed 40% inhibition at 23.1 μM, while 4 was not active at 20.7 μM. PMID:26115570

  10. Trichoketides A and B, two new protein tyrosine phosphatase 1B inhibitors from the marine-derived fungus Trichoderma sp.

    PubMed

    Yamazaki, Hiroyuki; Saito, Ryo; Takahashi, Ohgi; Kirikoshi, Ryota; Toraiwa, Kengo; Iwasaki, Kohei; Izumikawa, Yuta; Nakayama, Wataru; Namikoshi, Michio

    2015-10-01

    Two new octaketides, trichoketides A (1) and B (2), were isolated from a culture broth of the seawater-derived fungus Trichoderma sp. TPU1237 together with two known analogs, trichodermaketones C (3) and D (4), by ODS column chromatography followed by preparative ODS and chiral HPLC. The structures of 1 and 2 were elucidated on the basis of their spectroscopic data, and absolute configurations were assigned by comparing their experimental electronic circular dichroism (ECD) spectra with the calculated ECD spectra. Compounds 1 and 2 were epimers at the C-8 position (α-position of dihydrofuran ring). The IC50 values of compounds 1-4 against protein tyrosine phosphatase 1B were 53.1, 65.1, 68.0 and 55.9 μM, respectively. PMID:25899128

  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. PMID:27336796

  12. Novel chromenedione derivatives displaying inhibition of protein tyrosine phosphatase 1B (PTP1B) from Flemingia philippinensis.

    PubMed

    Wang, Yan; Yuk, Heung Joo; Kim, Jeong Yoon; Kim, Dae Wook; Song, Yeong Hun; Tan, Xue Fei; Curtis-Long, Marcus J; Park, Ki Hun

    2016-01-15

    Protein tyrosine phosphatase 1B (PTP1B) is an important target to treat obesity and diabetes due to its key roles in insulin and leptin signaling. The MeOH extracts of the root bark of Flemingia philippinensis yielded eight inhibitory molecules (1-8) capable of targeting PTP1B. Three of them were identified to be novel compounds, philippin A (1), philippin B (2), and philippin C (3) which have a rare 3-phenylpropanoyl chromenedione skeleton. The other compounds (4-8) were known prenylated isoflavones. All compounds (1-8) inhibited PTP1B in a dose dependent manner with IC50s ranging between 2.4 and 29.4μM. The most potent compound emerged to be prenylated isoflavone 5 (IC50=2.4μM). In kinetic studies, chromenedione derivatives (1-3) emerged to be reversible, competitive inhibitors, whereas prenylated isoflavones (5-8) were noncompetitive inhibitors. PMID:26704263

  13. Protein tyrosine phosphatase 1B (PTP1B)-inhibiting constituents from the leaves of Syzygium polyanthum.

    PubMed

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

    2012-08-01

    A methanol extract of the leaves of Syzygium polyanthum (Wight) Walp. afforded four new acylbenzene derivatives (1-4) together with seven known compounds (5-11). The structures of 1-11 were elucidated by extensive spectroscopic methods and comparison with the literature data. The new compounds 1-3 and a known compound, campest-4-en-3-one (10), exhibited a significant protein tyrosine phosphatase 1B inhibitory activity with IC₅₀ values of 13.1 ± 0.1, 5.77 ± 0.15, 4.01 ± 0.26, and 10.4 ± 0.5 µM, respectively. The inhibitory potency of the new compounds 2 and 3 was comparable to that of a positive control RK-682 (IC₅₀, 5.51 ± 0.04 µM). PMID:22763740

  14. Discovery of new potent human protein tyrosine phosphatase inhibitors via pharmacophore and QSAR analysis followed by in silico screening.

    PubMed

    Taha, Mutasem O; Bustanji, Yasser; Al-Bakri, Amal G; Yousef, Al-Motassem; Zalloum, Waleed A; Al-Masri, Ihab M; Atallah, Naji

    2007-03-01

    A pharmacophoric model was developed for human protein tyrosine phosphatase 1B (h-PTP 1B) inhibitors utilizing the HipHop-REFINE module of CATALYST software. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select an optimal combination of physicochemical descriptors and pharmacophore hypothesis that yield consistent QSAR equation of good predictive potential (r = 0.87,F-statistic = 69.13,r(BS)2 = 0.76,r(LOO)2 = 0.68). The validity of the QSAR equation and the associated pharmacophoric hypothesis was experimentally established by the identification of five new h-PTP 1B inhibitors retrieved from the National Cancer Institute (NCI) database. PMID:17035054

  15. Nature of sulphated macromolecules in mouse Reichert's membrane. Evidence for tyrosine O-sulphate in basement-membrane proteins.

    PubMed Central

    Paulsson, M; Dziadek, M; Suchanek, C; Huttner, W B; Timpl, R

    1985-01-01

    Seven different sulphated macromolecules were detected in 6 M-guanidinium chloride extracts of metabolically [35S]sulphate-labelled mouse Reichert's membrane and were partially separated. Polypeptide bands of apparent Mr 50 000, 150 000 (tentatively identified as entactin) and 170 000 contained essentially tyrosine O-sulphate as the labelled component. Most of the radioactive sulphate was incorporated into three different proteoglycans, which could be separated by chromatography and density-gradient centrifugation before and after enzymic degradation. Enzymic analysis of glycosaminoglycans and of protein cores by immunoassays identified these components as low-density and high-density forms of heparan sulphate proteoglycan and a high-density form of chondroitin sulphate or dermatan sulphate proteoglycan. Images Fig. 2. PMID:4074325

  16. Antagonistic regulation of swelling-activated Cl− current in rabbit ventricle by Src and EGFR protein tyrosine kinases

    PubMed Central

    Ren, Zuojun; Baumgarten, Clive M.

    2005-01-01

    Regulation of swelling-activated Cl− current (ICl,swell) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates ICl,swell and to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). ICl,swell evoked by hyposmotic swelling was increased 181 ± 17% by 100 μM genistein, and the genistein-induced current was blocked by the selective ICl,swell blocker tamoxifen (10 μM). Block of Src with PP2 (10 μM) stimulated tamoxifen-sensitive ICl,swell by 234 ± 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 μM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited ICl,swell and prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited ICl,swell. Several lines of evidence argue that the PP2-stimulated current was ICl,swell: 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl− gradients, and 4) the current reversed near the Cl− equilibrium potential. To rule out contributions of other currents, Cd2+ (0.2 mM) and Ba2+ (1 mM) were added to the bath. Surprisingly, Cd2+ suppressed the decay of Cd2+ plus Ba2+ eliminated time-dependent ICl,swell, and currents between −100 and −100 mV. Nevertheless, these divalent ions did not eliminate ICl,swell or prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls ICl,swell, and regulation of ICl,swell by the Src and EGFR kinase families of PTK is antagonistic. PMID:15681694

  17. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

    PubMed Central

    2012-01-01

    Background In nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in

  18. Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate.

    PubMed

    Kumaresan, A; Siqueira, A P; Hossain, M S; Bergqvist, A S

    2011-12-01

    Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P<0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca(2+)]i between P1 and SRF-P1 in fresh as well as in frozen-thawed semen. A higher (P<0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF. PMID:21893053

  19. Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK)

    PubMed Central

    Lin, Dao-Hong; Yue, Peng; Yarborough, Orlando; Scholl, Ute I.; Giebisch, Gerhard; Lifton, Richard P.; Rinehart, Jesse; Wang, Wen-Hui

    2015-01-01

    With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr1092, Tyr1094, and Tyr1143, and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr1092 or Tyr1143 to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4Y1092/1094F. Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser1196, an effect that was abrogated in the double mutant. WNK4Y1143F inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4Y1143F could not be reversed by SGK1. The failure to reverse WNK4Y1143F-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4–PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4Y1143F-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr1092 and Tyr1143 plays an important role in modulating the inhibitory effect of WNK4 on ROMK. PMID:25805816

  20. Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK).

    PubMed

    Lin, Dao-Hong; Yue, Peng; Yarborough, Orlando; Scholl, Ute I; Giebisch, Gerhard; Lifton, Richard P; Rinehart, Jesse; Wang, Wen-Hui

    2015-04-01

    With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr(1092), Tyr(1094), and Tyr(1143), and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr(1092) or Tyr(1143) to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4(Y1092/1094F). Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser(1196), an effect that was abrogated in the double mutant. WNK4(Y1143F) inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4(Y1143F) could not be reversed by SGK1. The failure to reverse WNK4(Y1143F)-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4-PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4(Y1143F)-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr(1092) and Tyr(1143) plays an important role in modulating the inhibitory effect of WNK4 on ROMK. PMID:25805816

  1. Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

    PubMed Central

    Zheng, L; Nibbering, P H; Zomerdijk, T P; van Furth, R

    1994-01-01

    Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes. Images PMID:7927687

  2. Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal

    PubMed Central

    Almela, Pilar; García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

    2013-01-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK) pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signaling, resulting in high expression of protein kinase (PK) A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK), one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR) can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH) at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies. PMID:24409147

  3. Site-specific Incorporation of 3-Iodo-L-tyrosine into Proteins and Single-wavelength Anomalous Dispersion Phasing with Soft X-ray in Protein Crystallography

    NASA Astrophysics Data System (ADS)

    Murayama, Kazutaka; Sakamoto, Kensaku

    Iodine is a good anomalous scatter for radiations from in-house X-ray generators (Cu/CrKα). Non-natural amino acid, 3-iodo-L-tyrosine, is able to be site-specifically incorporated into proteins with amber suppresser tRNA and mutated TyrRS from M. jannaschii in the E. coli expression system. To determine the crystal structure of acetyl transferase from T. thermophilus, iodotyrosine-containing proteins were prepared and crystallized. Structure determination was successfully conducted with the protein variant with iodotyrosine at position 111. Anomalous signals from iodotyrosine with Cu/CrKα radiations were both sufficient to calculate clear electron density map. In the crystal structure, iodotyrosine did not significantly disturb the native structure.

  4. Gi-mediated tyrosine phosphorylation of Grb2 (growth-factor-receptor-bound protein 2)-bound dynamin-II by lysophosphatidic acid.

    PubMed Central

    Kranenburg, O; Verlaan, I; Moolenaar, W H

    1999-01-01

    Lysophosphatidic acid (LPA) is the prototypic G-protein-coupled receptor agonist that activates the Ras-mitogen-activated protein (MAP) kinase cascade through pertussis toxin (PTX)-sensitive Gi and enhanced tyrosine kinase activity. We recently detected a 100 kDa protein (p100) that binds to the C-terminal SH3 domain of growth-factor-receptor-bound protein 2 (Grb2) and becomes tyrosine phosphorylated in a PTX-sensitive manner in LPA-treated Rat-1 cells [Kranenburg, Verlaan, Hordijk and Moolenaar (1997) EMBO J. 16, 3097-3105]. Through glutathione S-transferase-Grb2 affinity purification and microsequencing, we have now identified p100 as dynamin-II, a GTPase that regulates clathrin-mediated endocytosis. We show that in Rat-1 cells, Grb2-bound dynamin-II is rapidly tyrosine phosphorylated in response to LPA in a PTX-sensitive manner. Thus, tyrosine phosphorylation of Grb2-bound dynamin-II may be a critical event in Gi-mediated activation of the Ras-MAP kinase cascade in fibroblasts. PMID:10085221

  5. Purification and characterization of a rat liver membrane tyrosine-protein kinase, the possible protooncogene c-yes product, p60c-yes.

    PubMed

    Azuma, K; Ariki, M; Miyauchi, T; Usui, H; Takeda, M; Semba, K; Matsuzawa, Y; Yamamoto, T; Toyoshima, K

    1991-03-15

    A tyrosine-protein kinase was purified more than 270-fold from the rat liver plasma membrane fraction by successive column chromatographies on Sephacryl S-300, wheat germ agglutinin-agarose, casein-Sepharose, and hydroxylapatite, followed by isoelectrofocusing electrophoresis. The enzyme with pI of 6.2 was a 60-kDa single polypeptide which represented 42% of total protein. The enzyme reacted quantitatively with a monoclonal antibody to the amino-terminal sequence (Cys-3 to Ser-66) specific to the human c-yes protein, but not with antibodies to the specific amino-terminal sequences of the c-src, fyn, and lck proteins. The purified enzyme contained almost no phosphotyrosine residue but was autophosphorylated with Mg.ATP exclusively at tyrosine residues with concomitant increase in the kinase activity. The rates of autophosphorylation of the enzyme and phosphorylation of tyrosine-glutamate (1:4) copolymers, catalyzed by the enzyme were proportional to the square of enzyme concentration, suggesting that p60c-yes undergoes autophosphorylation through intermolecular catalysis, resulting in stimulation of the enzyme activity. Although the enzyme reaction showed an essential requirement for Mg2+ or Mn2+ with optimal concentrations of 20 and 3 mM, respectively, autophosphorylation significantly activated the enzyme only in the presence of Mg2+. Autophosphorylation of the enzyme reduced the Km for tyrosine-glutamate copolymers and tubulin, but not for ATP, and increased the Vmax of copolymer and tubulin phosphorylation. PMID:2002030

  6. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  7. The Counteradhesive Proteins, Thrombospondin 1 and SPARC/Osteonectin, Open the Tyrosine Phosphorylation-Responsive Paracellular Pathway in Pulmonary Vascular Endothelia

    PubMed Central

    Liu, Anguo; Mosher, Deane F.; Murphy-Ullrich, Joanne E.

    2009-01-01

    The counteradhesive proteins are a group of genetically and structurally distinct multidomain proteins that have been grouped together for their ability to inhibit cell-substrate interactions. Three counteradhesive proteins that influence endothelial cell behavior include thrombospondin (TSP)1, SPARC (Secreted Protein Acidic and Rich in Cysteine), also known as osteonectin, and tenascin. More recently, these proteins have been shown to not only regulate cell-matrix interactions but cell-cell interactions as well. TSP1 increases tyrosine phosphorylation of components of the cell-cell adherens junctions or zonula adherens (ZA) and opens the paracellular pathway in human lung microvascular endothelia. The EGF-like repeats of TSP1 activate the epidermal growth factor receptor (EGFR) and ErbB2 and these two receptor protein tyrosine kinase (PTK)s participate in ZA protein tyrosine phosphorylation and barrier disruption in response to the TSP1 stimulus. For the barrier response to TSP1, EGFR/ErbB2 activation is necessary but insufficient. Protein tyrosine phosphatase (PTP)µ counter-regulates phosphorylation of selected tyrosine residues within the cytoplasmic domain of EGFR. Although tenascin, like TSP1, also contains EGF-like repeats and is known to activate EGFR, whether it too opens the paracellular pathway is unknown. In addition to TSP1, tenascin, and the other TSP family members, there are numerous other proteins that also contain EGF-like repeats and participate in hemostasis, wound healing, and tissue remodeling. EGFR not only responds to direct binding of EGF motif-containing ligands but can be transactivated by a wide range of diverse stimuli. In fact, several established mediators of increased vascular permeability and/or lung injury, including thrombin, tumor necrosis factor-α, platelet-activating factor, bradykinin, angiopoietin, and H2O2, each transactivate EGFR. It is conceivable that EGFR serves a pivotal signaling role in a final common pathway for the

  8. Different zinc sensitivity of Brassica organs is accompanied by distinct responses in protein nitration level and pattern.

    PubMed

    Feigl, Gábor; Kolbert, Zsuzsanna; Lehotai, Nóra; Molnár, Árpád; Ördög, Attila; Bordé, Ádám; Laskay, Gábor; Erdei, László

    2016-03-01

    Zinc is an essential microelement, but its excess exerts toxic effects in plants. Heavy metal stress can alter the metabolism of reactive oxygen (ROS) and nitrogen species (RNS) leading to oxidative and nitrosative damages; although the participation of these processes in Zn toxicity and tolerance is not yet known. Therefore this study aimed to evaluate the zinc tolerance of Brassica organs and the putative correspondence of it with protein nitration as a relevant marker for nitrosative stress. Both examined Brassica species (B. juncea and B. napus) proved to be moderate Zn accumulators; however B. napus accumulated more from this metal in its organs. The zinc-induced damages (growth diminution, altered morphology, necrosis, chlorosis, and the decrease of photosynthetic activity) were slighter in the shoot system of B. napus than in B. juncea. The relative zinc tolerance of B. napus shoot was accompanied by moderate changes of the nitration pattern. In contrast, the root system of B. napus suffered more severe damages (growth reduction, altered morphology, viability loss) and slighter increase in nitration level compared to B. juncea. Based on these, the organs of Brassica species reacted differentially to excess zinc, since in the shoot system modification of the nitration pattern occurred (with newly appeared nitrated protein bands), while in the roots, a general increment in the nitroproteome could be observed (the intensification of the same protein bands being present in the control samples). It can be assumed that the significant alteration of nitration pattern is coupled with enhanced zinc sensitivity of the Brassica shoot system and the general intensification of protein nitration in the roots is attached to relative zinc endurance. PMID:26685787

  9. ANSID: a Solid-Phase Proteomic Approach for Identification and Relative Quantification of Aromatic Nitration Sites

    NASA Astrophysics Data System (ADS)

    Nuriel, Tal; Whitehouse, Julia; Ma, Yuliang; Mercer, Emily; Brown, Neil; Gross, Steven

    2015-12-01

    Nitration of tyrosine and other aromatic amino acid residues in proteins occurs in the setting of inflammatory, neurodegenerative and cardiovascular diseases – importantly, this modification has been implicated in the pathogenesis of diverse diseases and the physiological process of tissue aging. To understand the biological consequences of aromatic nitration in both health and disease, it is critical to molecularly identify the proteins that undergo nitration, specify their cognate modification sites and quantify their extent of nitration. To date, unbiased identification of nitrated proteins has painstakingly employed 2D-gel electrophoresis followed by Western Blotting with an anti-nitrotyrosine antibody for detection. Apart from being relatively slow and laborious, this method suffers from limited coverage, the potential for false-positive identifications and failure to reveal specific amino acid modification sites. To overcome these shortcomings, we have developed a solid-phase, chemical-capture approach for unbiased and high-throughput discovery of nitrotyrosine and nitrotryptophan sites in proteins. Utilizing this method, we have successfully identified several endogenously nitrated proteins in rat brain and a total of 244 nitrated peptides from 145 proteins following in vitro exposure of rat brain homogenates to the nitrating agent peroxynitrite (1 mM). As expected, Tyr residues constituted the great majority of peroxynitrite-mediated protein nitration sites; however, we were surprised to discover several brain proteins that contain nitrated Trp residues. By incorporating a stable-isotope labeling step, this new Aromatic Nitrtion Site IDentification (ANSID) method was also adapted for relative quantification of nitration site abundances in proteins. Application of the quantitative ANSID method offers great potential to advance our understanding of the role of protein nitration in disease pathogenesis and normal physiology.

  10. Interaction of the protein tyrosine phosphatase PTPL1 with the PtdIns(3,4)P2-binding adaptor protein TAPP1.

    PubMed Central

    Kimber, Wendy A; Deak, Maria; Prescott, Alan R; Alessi, Dario R

    2003-01-01

    It has been postulated that PtdIns(3,4) P (2), one of the immediate breakdown products of PtdIns(3,4,5) P (3), functions as a signalling molecule in insulin- and growth-factor-stimulated pathways. To date, the t andem- P H-domain-containing p rotein- 1 (TAPP1) and related TAPP2 are still the only known PH-domain-containing proteins that interact strongly and specifically with PtdIns(3,4) P (2). In this study we demonstrate that endogenously expressed TAPP1, is constitutively associated with the protein-tyrosine-phosphatase-like protein-1 (PTPL1 also known as FAP-1). We show that PTPL1 binds to TAPP1 and TAPP2, principally though its first PDZ domain [where PDZ is postsynaptic density protein ( P SD-95)/ Drosophila disc large tumour suppressor ( d lg)/tight junction protein ( Z O1)] and show that this renders PTPL1 capable of associating with PtdIns(3,4) P (2) in vitro. Our data suggest that the binding of TAPP1 to PTPL1 does not influence PTPL1 phosphatase activity, but instead functions to maintain PTPL1 in the cytoplasm. Following stimulation of cells with hydrogen peroxide to induce PtdIns(3,4) P (2) production, PTPL1, complexed to TAPP1, translocates to the plasma membrane. This study provides the first evidence that TAPP1 and PtdIns(3,4) P (2) could function to regulate the membrane localization of PTPL1. We speculate that if PTPL1 was recruited to the plasma membrane by increasing levels of PtdIns(3,4) P (2), it could trigger a negative feedback loop in which phosphoinositide-3-kinase-dependent or other signalling pathways could be switched off by the phosphatase-catalysed dephosphorylation of receptor tyrosine kinases or tyrosine phosphorylated adaptor proteins such as IRS1 or IRS2. Consistent with this notion we observed RNA-interference-mediated knock-down of TAPP1 in HEK-293 cells, enhanced activation and phosphorylation of PKB following IGF1 stimulation. PMID:14516276

  11. Human protein S inhibits the uptake of AcLDL and expression of SR-A through Mer receptor tyrosine kinase in human macrophages

    PubMed Central

    Liao, Dan; Wang, Xinwen; Li, Min; Lin, Peter H.; Yao, Qizhi

    2009-01-01

    Human protein S is an anticoagulation protein. However, it is unknown whether protein S could regulate the expression and function of macrophage scavenger receptor A (SR-A) in macrophages. Human THP-1 monocytes and peripheral blood monocytes were differentiated into macrophages and then treated with physiological concentrations of human protein S. We found that protein S significantly reduced acetylated low-density lipoprotein (AcLDL) uptake and binding by macrophages and decreased the intracellular cholesteryl ester content. Protein S suppressed the expression of the SR-A at both mRNA and protein levels. Protein S reduced the SR-A promoter activity primarily through inhibition in the binding of transcription factors to the AP-1 promoter element in macrophages. Furthermore, human protein S could bind and induce phosphorylation of Mer receptor tyrosine kinase (Mer RTK). Soluble Mer protein or tyrosine kinase inhibitor herbimycin A effectively blocked the effects of protein S on AcLDL uptake. Immunohistochemical analysis revealed that the level of protein S was substantially increased in human atherosclerotic arteries. Thus, human protein S can inhibit the expression and activity of SR-A through Mer RTK in macrophages, suggesting that human protein S is a modulator for macrophage functions in uptaking of modified lipoproteins. PMID:18922854

  12. Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output

    PubMed Central

    Reyes, Gloria; Niederst, Matt; Cohen-Katsenelson, Ksenya; Stender, Joshua D.; Kunkel, Maya T.; Chen, Muhan; Brognard, John; Sierecki, Emma; Gao, Tianyan; Nowak, Dawid G.; Trotman, Lloyd C.; Glass, Christopher K.; Newton, Alexandra C.

    2014-01-01

    Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs. PMID:25201979

  13. Inactivation and unfolding of protein tyrosine phosphatase from Thermus thermophilus HB27 during urea and guanidine hydrochloride denaturation.

    PubMed

    Wang, Yejing; He, Huawei; Liu, Lina; Gao, Chunyan; Xu, Shui; Zhao, Ping; Xia, Qingyou

    2014-01-01

    The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase. PMID:25255086

  14. Light-switched inhibitors of protein tyrosine phosphatase PTP1B based on phosphonocarbonyl phenylalanine as photoactive phosphotyrosine mimetic.

    PubMed

    Wagner, Stefan; Schütz, Anja; Rademann, Jörg

    2015-06-15

    Phosphopeptide mimetics containing the 4-phosphonocarbonyl phenylalanine (pcF) as a photo-active phosphotyrosine isoster are developed as potent, light-switchable inhibitors of the protein tyrosine phosphatase PTP1B. The photo-active inhibitors 6-10 are derived from phosphopeptide substrates and are prepared from the suitably protected pcF building block 12 by Fmoc-based solid phase peptide synthesis. All pcF-containing peptides are moderate inhibitors of PTP1B with KI values between 10 and 50μM. Irradiation of the inhibitors at 365nm in the presence of the protein PTP1B amplify the inhibitory activity of pcF-peptides up to 120-fold, switching the KI values of the best inhibitors to the sub-micromolar range. Photo-activation of the inhibitors results in the formation of triplet intermediates of the benzoylphosphonate moiety, which deactivate PTP1B following an oxidative radical mechanism. Deactivation of PTP1B proceeds without covalent crosslinking of the protein target with the photo-switched inhibitors and can be reverted by subsequent addition of reducing agent dithiothreitol (DTT). PMID:25907367

  15. Protein Tyrosine Phosphatases and Type 1 Diabetes: Genetic and Functional Implications of PTPN2 and PTPN22

    PubMed Central

    Cerosaletti, Karen; Buckner, Jane H.

    2012-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in modulating the transduction of cellular signals, including the cells of the immune system. Several PTPs, PTPN22, PTPN2, and UBASH3A, have been associated with risk of type 1 diabetes (T1D) by genome wide association studies. Based on the current understanding of PTPs, it is clear that these variants impact antigen receptor signaling and cytokine signaling. This impact likely contributes to the development and progression of autoimmunity through multiple mechanisms, including failures of central and peripheral tolerance and the promotion of proinflammatory T cell responses. In this review, we discuss the genetic and functional implications of two of these PTPs, PTPN22 and PTPN2, in the development of T1D. We describe the known roles of these proteins in immune function, and how the expression and function of these proteins is altered by the genetic variants associated with T1D. Yet, there are still controversies in the field that require further study and the development of new approaches to extend our understanding of these PTP variants, with the goal of using the information gained to improve our ability to predict and cure T1D. PMID:23804260

  16. Protein tyrosine and serine–threonine phosphatases in the sea urchin, Strongylocentrotus purpuratus: Identification and potential functions

    PubMed Central

    Byrum, C.A.; Walton, K.D.; Robertson, A.J.; Carbonneau, S.; Thomason, R.T.; Coffman, J.A.; McClay, D.R.

    2011-01-01

    Protein phosphatases, in coordination with protein kinases, play crucial roles in regulation of signaling pathways. To identify protein tyrosine phosphatases (PTPs) and serine–threonine (ser–thr) phosphatases in the Strongylocentrotus purpuratus genome, 179 annotated sequences were studied (122 PTPs, 57 ser–thr phosphatases). Sequence analysis identified 91 phosphatases (33 conventional PTPs, 31 dual specificity phosphatases, 1 Class III Cysteine-based PTP, 1 Asp-based PTP, and 25 ser–thr phosphatases). Using catalytic sites, levels of conservation and constraint in amino acid sequence were examined. Nine of 25 receptor PTPs (RPTPs) corresponded to human, nematode, or fly homologues. Domain structure revealed that sea urchin-specific RPTPs including two, PTPRLec and PTPRscav, may act in immune defense. Embryonic transcription of each phosphatase was recorded from a high-density oligonucleotide tiling microarray experiment. Most RPTPs are expressed at very low levels, whereas nonreceptor PTPs (NRPTPs) are generally expressed at moderate levels. High expression was detected in MAP kinase phosphatases (MKPs) and numerous ser–thr phosphatases. For several expressed NRPTPs, MKPs, and ser–thr phosphatases, morpholino antisense-mediated knockdowns were performed and phenotypes obtained. Finally, to assess roles of annotated phosphatases in endomesoderm formation, a literature review of phosphatase functions in model organisms was superimposed on sea urchin developmental pathways to predict areas of functional activity. PMID:17087928

  17. Fibulin 2, a tyrosine O-sulfated protein, is up-regulated following retinal detachment.

    PubMed

    Kanan, Yogita; Brobst, Daniel; Han, Zongchao; Naash, Muna I; Al-Ubaidi, Muayyad R

    2014-05-01

    Retinal detachment is the physical separation of the retina from the retinal pigment epithelium. It occurs during aging, trauma, or during a variety of retinal disorders such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or as a complication following cataract surgery. This report investigates the role of fibulin 2, an extracellular component, in retinal detachment. A major mechanism for detachment resolution is enhancement of cellular adhesion between the retina and the retinal pigment epithelium and prevention of its cellular migration. This report shows that fibulin 2 is mainly present in the retinal pigment epithelium, Bruch membrane, choriocapillary, and to a lesser degree in the retina. In vitro studies revealed the presence of two isoforms for fibulin 2. The small isoform is located inside the cell, and the large isoform is present inside and outside the cells. Furthermore, fibulin 2 is post-translationally modified by tyrosine sulfation, and the sulfated isoform is present outside the cell, whereas the unsulfated pool is internally located. Interestingly, sulfated fibulin 2 significantly reduced the rate of cellular growth and migration. Finally, levels of fibulin 2 dramatically increased in the retinal pigment epithelium following retinal detachment, suggesting a direct role for fibulin 2 in the re-attachment of the retina to the retinal pigment epithelium. Understanding the role of fibulin 2 in enhancing retinal attachment is likely to help improve the current therapies or allow the development of new strategies for the treatment of this sight-threatening condition. PMID:24692557

  18. Tyrosine hydroxylase protein expression in ventral nerve cord of Neotropical freshwater crab.

    PubMed

    Ponzoni, Silvia

    2014-12-01

    Given the importance of catecholamines in coordinating physiological and behavioral responses in brachyurans, the present study was designed to investigate the distribution of tyrosine hydroxylase (TH)-positive cells and fibers in the ventral nerve cord of Dilocarcinus pagei the Neotropical freshwater crab. TH immunoreactivity was visualized in adult crabs of both sexes, during the intermolt period. We found TH-positive cells that have not been previously described in brachyurans. Specifically, we found a pair of TH-positive cells in the ventral region of the thoracic ganglion, and in ventral and dorsal regions of the abdominal (pleonic) ganglion, suggesting catecholaminergic modulation of claws' function and abdominal structures. In addition, great population of TH-positive cells was observed in the subesophageal ganglion, indicating conservation during evolution of catecholamines in this ganglion of decapods. Dopamine is present in cells and fiber tracts of brachyuran ventral nerve cord, projecting to endocrine, cardiac and digestive structures, suggesting widespread modulation and control of physiological functions and behavior. Dopamine plays a central role in movement and psychiatric disorders in humans. Information on dopaminergic function in the nervous system of invertebrates should improve the understanding of its function in more complex systems, such as human beings. PMID:25217291

  19. Heat shock protein 90 and tyrosine kinase regulate eNOS NO* generation but not NO* bioactivity.

    PubMed

    Ou, Jingsong; Fontana, Jason T; Ou, Zhijun; Jones, Deron W; Ackerman, Allan W; Oldham, Keith T; Yu, Jun; Sessa, William C; Pritchard, Kirkwood A

    2004-02-01

    An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO*) production. Despite the progress in this field, the mechanisms by which HSP90 modulates eNOS remain unclear due, in part, to the fact that geldanamycin (GA) redox cycles to generate superoxide anion (O(2)(-*) and the fact that inhibiting HSP90 with GA or radicicol (RAD) destabilizes tyrosine kinases that rely on the chaperone for maturation. In this report, we determine the extent to which these side effects alter vascular and endothelial cell function in physiologically relevant systems and in cultured endothelial cells. Vascular endothelial growth factor (VEGF)-stimulated vascular permeability, as measured by Evans blue leakage in the ears of male Swiss mice in vivo, and acetylcholine-induced vasodilation of isolated, pressurized mandibular arterioles from male C57BL6 mice ex vivo were attenuated by N(omega)-nitro-L-arginine methyl ester (L-NAME), GA, and RAD. Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), a slow releasing NO. donor, increased vasodilation of arterioles pretreated with GA, RAD, and L-NAME equally well except at 10(-5) M, the highest concentration used, where vasodilation was greater in pressurized arterioles treated with L-NAME than in arterioles pretreated with GA or RAD alone. Both GA and RAD reduced NO* release from stimulated endothelial cell cultures and increased O(2)(-*) production in the endothelium of isolated aortas by an L-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated O(2)(-*) production from eNOS, whereas pretreatment with genistein (GE), a broad-spectrum tyrosine kinase inhibitor, did not; however, pretreatment with GE + RAD resulted in a super-induced state of uncoupled eNOS activity upon stimulation. These data suggest that the tyrosine kinases, either directly or indirectly, and HSP90-dependent signaling pathways

  20. Transforming properties and substrate specificities of the protein tyrosine kinase oncogenes ros and src and their recombinants.

    PubMed Central

    Jong, S M; Zong, C S; Dorai, T; Wang, L H

    1992-01-01

    To determine the sequences of the oncogenes src (encoded by Rous sarcoma virus [RSV]) and ros (encoded by UR2) that are responsible for causing different transformation phenotypes and to correlate those sequences with differences in substrate recognition, we constructed recombinants of the two transforming protein tyrosine kinases (PTKs) and studied their biological and biochemical properties. A recombinant with a 5' end from src and a 3' end from ros, called SRC x ROS, transformed chicken embryo fibroblasts (CEF) to a spindle shape morphology, mimicking that of UR2. Neither of the two reverse constructs, ROS x SRC I and ROS x SRC II, could transform CEF. However, a transforming variant of ROS x SRC II appeared during passages of the transfected cells and was called ROS x SRC (R). ROS x SRC (R) contains a 16-amino-acid deletion that includes the 3' half of the transmembrane domain of ros. Unlike RSV, ROS x SRC (R) also transformed CEF to an elongated shape similar to that of UR2. We conclude that distinct phenotypic changes of RSV- and UR2-infected cells do not depend solely on the kinase domains of their oncogenes. We next examined cellular proteins phosphorylated by the tyrosine kinases of UR2, RSV, and their recombinants as well as a number of other avian sarcoma viruses including Fujinami sarcoma virus Y73, and some ros-derived variants. Our results indicate that the UR2-encoded receptorlike PTK P68gag-ros and its derivatives have a very restricted substrate specificity in comparison with the nonreceptor PTKs encoded by the rest of the avian sarcoma viruses. Data from ros and src recombinants indicate that sequences both inside and outside the catalytic domains of ros and src exert a significant effect on the substrate specificity of the two recombinant proteins. Phosphorylation of most of the proteins in the 100- to 200-kDa range correlated with the presence of the 5' src domain, including the SH2 region, but not with the kinase domain in the recombinants

  1. Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal.

    PubMed

    Maeda, A; Kurosaki, M; Ono, M; Takai, T; Kurosaki, T

    1998-04-20

    Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B-mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5'-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B-mediated inhibition requires redundant functions of SHP-1 and SHP-2. PMID:9547347

  2. Identification of genomic regions that interact with a viable allele of the Drosophila protein tyrosine phosphatase corkscrew.

    PubMed Central

    Firth, L; Manchester, J; Lorenzen, J A; Baron, M; Perkins, L A

    2000-01-01

    Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Previously, Csw has been demonstrated to function throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unambiguously elucidated, current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question. The isolation and characterization of a new, spontaneously arising, viable allele of csw, csw(lf), has allowed us to undertake a genetic approach to identify loci required for Csw function. The rough eye and wing vein gap phenotypes exhibited by adult flies homo- or hemizygous for csw(lf) has provided a sensitized background from which we have screened a collection of second and third chromosome deficiencies to identify 33 intervals that enhance and 21 intervals that suppress these phenotypes. We have identified intervals encoding known positive mediators of RTK signaling, e.g., drk, dos, Egfr, E(Egfr)B56, pnt, Ras1, rolled/MAPK, sina, spen, Src64B, Star, Su(Raf)3C, and vein, as well as known negative mediators of RTK signaling, e.g., aos, ed, net, Src42A, sty, and su(ve). Of particular interest are the 5 lethal enhancing intervals and 14 suppressing intervals for which no candidate genes have been identified. PMID:11014820

  3. Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca²⁺ concentration in boar spermatozoa during cryopreservation.

    PubMed

    Kumaresan, A; Siqueira, A P; Hossain, M S; Johannisson, A; Eriksson, I; Wallgren, M; Bergqvist, A S

    2012-01-01

    Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation. PMID:22541541

  4. Steady state protein levels in Geobacter metallireducens grown with Iron (III) citrate or nitrate as terminal electron acceptor.

    SciTech Connect

    Ahrendt, A. J.; Tollaksen, S. L.; Lindberg, C.; Zhu, W.; Yates, J. R., III; Nevin, K. P.; Lovley, D.; Giometti, C. S.; Biosciences Division; The Scripps Research Inst.; Univ. of Massachusetts

    2007-01-01

    Geobacter species predominate in aquatic sediments and submerged soils where organic carbon sources are oxidized with the reduction of Fe(III). The natural occurrence of Geobacter in some waste sites suggests this microorganism could be useful for bioremediation if growth and metabolic activity can be regulated. 2-DE was used to monitor the steady state protein levels of Geobacter metallireducens grown with either Fe(III) citrate or nitrate to elucidate metabolic differences in response to different terminal electron acceptors present in natural environments populated by Geobacter. Forty-six protein spots varied significantly in abundance (p<0.05) between the two growth conditions; proteins were identified by tryptic peptide mass and peptide sequence determined by MS/MS. Enzymes involved in pyruvate metabolism and the tricarboxylic acid (TCA) cycle were more abundant in cells grown with Fe(III) citrate, while proteins associated with nitrate metabolism and sensing cellular redox status along with several proteins of unknown function were more abundant in cells grown with nitrate. These results indicate a higher level of flux through the TCA cycle in the presence of Fe(III) compared to nitrate. The oxidative stress response observed in previous studies of Geobacter sulfurreducens grown with Fe(III) citrate was not seen in G. metallireducens.

  5. The neural receptor protein tyrosine phosphatase DPTP69D is required during periods of axon outgrowth in Drosophila.

    PubMed Central

    Desai, Chand; Purdy, Joy

    2003-01-01

    We have isolated and characterized a series of 18 chemically induced alleles of Ptp69D ranging in strength from viable to worse than null, which represent unique tools for probing the structure, function, and signaling pathway of DPTP69D. Three alleles are strongly temperature sensitive and were used to define the developmental periods requiring DPTP69D function; adult health requires DPTP69D during the mid- to late-pupal stage, eclosion requires DPTP69D during the early to mid-larval stage, and larval survival requires DPTP69D during embryogenesis. Mutations predicted to abolish the phosphatase activity of the membrane proximal D1 domain severely reduce but do not abolish DPTP69D function. Six alleles appear null; only 20% of null homozygotes pupate and <5% eclose, only to fall into the food and drown. One allele, Ptp69D(7), confers axon and viability defects more severe than those of the null phenotype. Sequence analysis predicts that Ptp69D(7) encodes a mutant protein that may bind but not release substrate. Like mutations in the protein tyrosine phosphatase gene Dlar, strong Ptp69D alleles cause the ISNb nerve to bypass its muscle targets. Genetic analysis reveals that the bypass defect in Dlar and Ptp69D mutants is dependent upon DPTP99A function, consistent with the hypothesis that DPTP69D and DLAR both counteract DPTP99A, allowing ISNb axons to enter their target muscle field. PMID:12807778

  6. Loss of the Protein Tyrosine Phosphatase PTPN22 Reduces Mannan-Induced Autoimmune Arthritis in SKG Mice

    PubMed Central

    Sood, Shatakshi; Brownlie, Rebecca J.; Garcia, Celine; Cowan, Graeme; Salmond, Robert J.; Sakaguchi, Shimon

    2016-01-01

    The cytoplasmic phosphatase, protein tyrosine phosphatase nonreceptor type 22 (PTPN22), is a negative regulator of T cell signaling. Genome-wide association studies have shown that single-nucleotide polymorphisms in PTPN22 confer an increased risk of developing multiple autoimmune diseases in humans. The precise function of PTPN22 and how the variant protein contributes to autoimmunity is not well understood. To address this issue, we investigated the effect of PTPN22 deficiency on disease susceptibility in a mouse model of autoimmune arthritis. The SKG mouse expresses a hypomorphic mutant allele of ZAP70, which, upon exposure to fungal Ags, predisposes the mice to a CD4+ T cell–mediated autoimmune arthritis that closely resembles rheumatoid arthritis in humans. Surprisingly, SKG Ptpn22−/− mice developed less severe mannan-induced arthritis compared with SKG mice. Diminution of disease was not due to significant alterations in thymocyte development or repertoire selection in SKG Ptpn22−/− mice, even though T cell–mediated signal transduction was improved. Instead, Ptpn22 deficiency appeared to bias CD4 Th cell differentiation away from the Th17 lineage, which is pathogenic in this setting, to a more Th1/T regulatory–focused response. These data show that even small perturbations in TCR signal transduction pathways can have profound consequences on the differentiation of T cell lineages and thus for the development of autoimmune diseases. PMID:27288531

  7. CCAAT/enhancer-binding protein β: its role in breast cancer and associations with receptor tyrosine kinases

    PubMed Central

    Zahnow, Cynthia A.

    2011-01-01

    The CCAAT/enhancer-binding proteins (C/EBPs) are a family of leucine-zipper transcription factors that regulate gene expression to control cellular proliferation, differentiation, inflammation and metabolism. Encoded by an intronless gene, C/EBPβ is expressed as several distinct protein isoforms (LAP1, LAP2, LIP) whose expression is regulated by the differential use of several in-frame translation start sites. LAP1 and LAP2 are transcriptional activators and are associated with differentiation, whereas LIP is frequently elevated in proliferative tissue and acts as a dominant-negative inhibitor of transcription. However, emerging evidence suggests that LIP can serve as a transcriptional activator in some cellular contexts, and that LAP1 and LAP2 might also have unique actions. The LIP:LAP ratio is crucial for the maintenance of normal growth and development, and increases in this ratio lead to aggressive forms of breast cancer. This review discusses the regulation of C/EBPβ activity by post-translational modification, the individual actions of LAP1, LAP2 and LIP, and the functions and downstream targets that are unique to each isoform. The role of the C/EBPβ isoforms in breast cancer is discussed and emphasis is placed on their interactions with receptor tyrosine kinases. PMID:19351437

  8. Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins

    PubMed Central

    Lopez-Sanchez, Inmaculada; Kalogriopoulos, Nicholas; Lo, I-Chung; Kabir, Firooz; Midde, Krishna K.; Wang, Honghui; Ghosh, Pradipta

    2015-01-01

    GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via β1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated β1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell–ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression. PMID:26446841

  9. Loss of the Protein Tyrosine Phosphatase PTPN22 Reduces Mannan-Induced Autoimmune Arthritis in SKG Mice.

    PubMed

    Sood, Shatakshi; Brownlie, Rebecca J; Garcia, Celine; Cowan, Graeme; Salmond, Robert J; Sakaguchi, Shimon; Zamoyska, Rose

    2016-07-15

    The cytoplasmic phosphatase, protein tyrosine phosphatase nonreceptor type 22 (PTPN22), is a negative regulator of T cell signaling. Genome-wide association studies have shown that single-nucleotide polymorphisms in PTPN22 confer an increased risk of developing multiple autoimmune diseases in humans. The precise function of PTPN22 and how the variant protein contributes to autoimmunity is not well understood. To address this issue, we investigated the effect of PTPN22 deficiency on disease susceptibility in a mouse model of autoimmune arthritis. The SKG mouse expresses a hypomorphic mutant allele of ZAP70, which, upon exposure to fungal Ags, predisposes the mice to a CD4(+) T cell-mediated autoimmune arthritis that closely resembles rheumatoid arthritis in humans. Surprisingly, SKG Ptpn22(-/-) mice developed less severe mannan-induced arthritis compared with SKG mice. Diminution of disease was not due to significant alterations in thymocyte development or repertoire selection in SKG Ptpn22(-/-) mice, even though T cell-mediated signal transduction was improved. Instead, Ptpn22 deficiency appeared to bias CD4 Th cell differentiation away from the Th17 lineage, which is pathogenic in this setting, to a more Th1/T regulatory-focused response. These data show that even small perturbations in TCR signal transduction pathways can have profound consequences on the differentiation of T cell lineages and thus for the development of autoimmune diseases. PMID:27288531

  10. Deficiency of the protein-tyrosine phosphatase DEP-1/PTPRJ promotes matrix metalloproteinase-9 expression in meningioma cells.

    PubMed

    Petermann, Astrid; Stampnik, Yvonn; Cui, Yan; Morrison, Helen; Pachow, Doreen; Kliese, Nadine; Mawrin, Christian; Böhmer, Frank-D

    2015-05-01

    Brain-invasive growth of a subset of meningiomas is associated with less favorable prognosis. The molecular mechanisms causing invasiveness are only partially understood, however, the expression of matrix metalloproteinases (MMPs) has been identified as a contributing factor. We have previously found that loss of density enhanced phosphatase-1 (DEP-1, also designated PTPRJ), a transmembrane protein-tyrosine phosphatase, promotes meningioma cell motility and invasive growth in an orthotopic xenotransplantation model. We have now analyzed potential alterations of the expression of genes involved in motility control, caused by DEP-1 loss in meningioma cell lines. DEP-1 depleted cells exhibited increased expression of mRNA encoding MMP-9, and the growth factors EGF and FGF-2. The increase of MMP-9 expression in DEP-1 depleted cells was also readily detectable at the protein level by zymography. MMP-9 upregulation was sensitive to chemical inhibitors of growth factor signal transduction. Conversely, MMP-9 mRNA levels could be stimulated with growth factors (e.g. EGF) and inflammatory cytokines (e.g. TNFα). Increase of MMP-9 expression by DEP-1 depletion, or growth factor/cytokine stimulation qualitatively correlated with increased invasiveness in vitro scored as transmigration through matrigel-coated membranes. The studies suggest induction of MMP-9 expression promoted by DEP-1 deficiency, or potentially by growth factors and inflammatory cytokines, as a mechanism contributing to meningioma brain invasiveness. PMID:25672645

  11. A novel homolog of protein tyrosine kinase Fyn identified in Lampetra japonica with roles in the immune response.

    PubMed

    Zhang, Qiong; Song, Xueying; Su, Peng; Li, Ranran; Liu, Chang; Gou, Meng; Wang, Hao; Liu, Xin; Li, Qingwei

    2016-04-01

    The non-receptor protein tyrosine kinase (nrPTK) Fyn, a member of the avian sarcoma virus transforming gene (Src) kinase family, plays a very significant role in cell growth, survival, apoptosis, tumor formation and immune response. In this study, a homolog of nrPTK Fyn was identified for the first time in the lamprey, Lampetra japonica and was named "Lja-Fyn". The cDNA fragment of lamprey lja-fyn contains a 1611-bp open reading frame, which encodes a protein of 537 amino acids. Multiple sequence alignment analysis showed that it shares four conserved domains (Src homology (SH) 4, SH3, SH2 and protein kinases catalytic domains) and a variable unique domain with vertebrates Fyn molecules. Though Lja-Fyn has high sequence similarity with typical Fyn and Yes molecules of jawed vertebrates, the identities among Lja-Fyn and typical Fyn molecules in unique domain are relatively higher than that among Lja-Fyn and typical Yes molecules. The result indicates that Lja-Fyn is a homolog of Fyn rather than Yes. The phylogenetic analysis showed that Fyn, Yes and Src molecules are grouped into three distinct phylogenetic clusters, and Lja-Fyn is grouped as a single branch in Fyn cluster. The real-time quantitative PCR assay revealed the wide distribution of the lja-fyn mRNA in lamprey immune related tissues. After stimulation with mixed antigens, the levels of lja-fyn mRNA were obviously up-regulated in the gill and lymphocyte-like cells, and the similar results were got by western blot analysis of Lja-Fyn protein expression. These results indicated that nrPTK Lja-Fyn was likely to be involved in immune response. Furthermore, our present findings also provide the necessary information for understanding the distinction between lamprey Lja-Fyn and other members of jawed vertebrates in Src family. PMID:26743129

  12. Loss of Protein Tyrosine Phosphatase Receptor J Expression Predicts an Aggressive Clinical Course in Patients with Esophageal Squamous Cell Carcinoma.

    PubMed

    Qiao, Dongfeng; Li, Ming; Pu, Juan; Wang, Wanwei; Zhu, Weiguo; Liu, Haiyan

    2016-07-01

    Protein Tyrosine Phosphatase Receptor J (PTPRJ) has been reported to be a tumor suppressor in various human cancers. The aim of this study was to investigate the clinical significance of PTPRJ in ESCC patients and its effects on biological behaviors of ESCC cells. PTPRJ expression, at mRNA and protein levels, were respectively detected by quantitative real-time PCR, western blot and immunohistochemistry, based on 106 newly diagnosed ESCC patients. The associations between PTPRJ expression and clinicopathological characteristics of ESCC patients were statistically analyzed. Then, the effects of PTPRJ in migration and invasion were determined by wound healing and transwell assays based on ESCC cell line transfected with siRNA or expression vector of PTPRJ. Expression of PTPRJ at mRNA and protein levels were both significantly lower in ESCC tissues than those in normal esophageal mucosa. Immunohistochemistry showed that PTPRJ protein was localized in the cytoplasm of cancer cells in ESCC tissues. In addition, PTPRJ downregulation was found to be closely correlated with advanced tumor stage (P = 0.01) and poor differentiation (P = 0.03). Moreover, knockdown of PTPRJ in KYSE510 cells could significantly promote cell migration and invasion (both P < 0.05), which were reversed by the restoration of PTPRJ expression in vitro (both P < 0.05). Our data offer the convincing evidence that loss of PTPRJ expression may predict an aggressive clinical course in ESCC patients. PTPRJ may function as a tumor suppressor and play an important role in the regulation of ESCC cell motility, suggesting its potentials as a therapeutic agent for human ESCC. PMID:26694178

  13. A 32-kDa tyrosine-phosphorylated protein shows a protease-dependent increase in dead boar spermatozoa.

    PubMed

    Tabuchi, Tomohito; Shidara, Osamu; Harayama, Hiroshi

    2008-12-01

    Boar sperm TyrP32 is a 32-kDa tyrosine-phosphorylated protein that increases during the capacitation and acrosome reaction and during cryocapacitation. However, it is still unclear whether the increase in TyrP32 is an event that is limited to the process of sperm fertilization, including cryocapacitation. The aims of the present study were to demonstrate that TyrP32 is increased in dead spermatozoa after freeze-thawing without a cryoprotectant and to find the causal factors for this increase. Washed spermatozoa were resuspended in a salt solution and then frozen. The frozen samples were rapidly thawed in a warm water bath and then used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)/Western blotting to detect TyrP32, SDS-PAGE/silver staining of sperm proteins and staining of acrosomal contents with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA). In the samples before freezing, TyrP32 was barely detectable, and the distribution of the acrosomal contents was normal in most spermatozoa. One cycle of freeze-thawing induced an increase in TyrP32, a decrease in major sperm proteins and disorder in the acrosomal contents. However, the addition of a protease inhibitor (APMSF, 1 mM) suppressed the increase in TyrP32 and the decrease in the major sperm proteins, although it did not have any influence on the disorder in the acrosomal contents. Additionally, the spermatozoa did not exhibit any flagellar movement after freeze-thawing, which showed that almost all of them were dead. These results indicate that TyrP32 can show a protease-dependent increase in dead spermatozoa after freeze-thawing without a cryoprotectant even though the dead spermatozoa do not undergo cryocapacitation. PMID:18787309

  14. Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme that Catalyzes Post-Translational Protein Tyrosine O-Sulfation, Causes Male Infertility*

    PubMed Central

    Borghei, Atefeh; Ouyang, Ying-Bin; Westmuckett, Andrew D.; Marcello, Matthew R.; Landel, Carlisle P.; Evans, Janice P.; Moore, Kevin L.

    2006-01-01

    Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo, and gain insight into other potential TPST substrates, TPST-2 deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2+/- mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2-/- mice have moderately delayed growth, but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1-/- males that have normal fertility, Tpst2-/- males are infertile. Tpst2-/- sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida (ZP)-intact eggs. Adhesion of Tpst2-/- sperm to the egg plasma membrane is reduced compared to wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility. PMID:16469738

  15. Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein.

    PubMed

    Dunning, Christopher J R; Black, Hannah L; Andrews, Katie L; Davenport, Elizabeth C; Conboy, Michael; Chawla, Sangeeta; Dowle, Adam A; Ashford, David; Thomas, Jerry R; Evans, Gareth J O

    2016-05-01

    Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate β-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease. PMID:26865271

  16. Effects of dietary protein concentration on ammonia volatilization, nitrate leaching, and plant nitrogen uptake from dairy manure applied to lysimeters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This lysimeter experiment was designed to investigate the effects of dietary crude protein (CP) concentration on nitrate-N (NO3-N) and ammonia (NH3) losses from dairy manure applied to soil and manure N use for plant growth. Lactating dairy cows were fed diets with 16.7 (HighCP) or 14.8% (LowCP) cru...

  17. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.

    PubMed

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K; Kline, Douglas W; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  18. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

    PubMed Central

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K.; Kline, Douglas W.; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  19. Mussel-inspired new approach for polymerization and cross-linking of peptides and proteins containing tyrosines by Fremy's salt oxidation.

    PubMed

    Wilchek, Meir; Miron, Talia

    2015-03-18

    Our objective was to develop a method mimicking the natural process of coherence in marine mollusks, by direct chemical conversion of protein tyrosine residues to DOPA-o-quinones, which consequently generates polymerization and cross-linking. Fremy's salt, (ON(SO3K)2, was used to convert tyrosine residues in peptides and proteins to reactive o-quinones. The conversion of tyrosines to DOPA-o-quinones, and their ability to polymerize or cross-link, was tested on tyramine, peptides, and proteins. The peptides tested were as follows: biotin-PEG4-tyramine (PEG-BT), and two decapeptides (identical to the repeating units comprising the mussel's adhesive protein). The proteins tested were as follows: bovine pancreatic ribonuclease A (RNase), lysozyme, IgG, avidin, and streptavidin. The oxidized peptides and proteins were all shown to incorporate oxygen atoms and undergo polymerization and cross-linking, depending on the availability of nucleophiles, mostly lysine amino groups of proteins. All the peptides and the noninteracting proteins such as RNase and lysozyme underwent homopolymerization upon Fremy's salt oxidation. When Fremy's salt oxidaized PEG-BT was mixed with the above proteins, it did not react with any of these proteins because PEG-BT underwent fast self-polymerization. Conversely, streptavidin or avidin cross-linked with PEG-BT after preincubation, thus showing that biorecognition is a prerequisite for cross-linking. Polymerization and cross-linking also occurred, following Fremy's salt oxidation of interacting proteins such as avidin and strepavidin with biotinyilated lysozyme or biotinylated RNase. This indicates that only proteins in very close proximity readily cross-link and polymerize via tyrosine residues. Attempts to convert DOPA-quinone to DOPA by reduction with sodium dithionite (Na2S2O4), was successful as far as small peptides were used. Fremy's salt oxidation can serve as an easy and useful tool to polymerize and cross-link proteins, for

  20. 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

  1. 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

  2. Chronic restraint stress induces sperm acrosome reaction and changes in testicular tyrosine phosphorylated proteins in rats

    PubMed Central

    Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Maneenin, Chanwit; Iamsaard, Sitthichai

    2016-01-01

    Background: Stress is a cause of male infertility. Although sex hormones and sperm quality have been shown to be low in stress, sperm physiology and testicular functional proteins, such as phosphotyrosine proteins, have not been documented. Objective: To investigate the acrosome status and alterations of testicular proteins involved in spermatogenesis and testosterone synthesis in chronic stress in rats. Materials and Methods: In this experimental study, male rats were divided into 2 groups (control and chronic stress (CS), n=7). CS rats were immobilized (4 hr/day) for 42 consecutive days. The blood glucose level (BGL), corticosterone, testosterone, acrosome status, and histopathology were examined. The expressions of testicular steroidogenic acute regulatory (StAR), cytochrome P450 side chain cleavage (CYP11A1), and phosphorylated proteins were analyzed. Results: Results showed that BGL (71.25±2.22 vs. 95.60±3.36 mg/dl), corticosterone level (24.33±4.23 vs. 36.9±2.01 ng/ml), acrosome reacted sperm (3.25±1.55 vs. 17.71±5.03%), and sperm head abnormality (3.29±0.71 vs. 6.21±1.18%) were significantly higher in CS group in comparison with control. In contrast, seminal vesicle (0.41±0.05 vs. 0.24±0.07 g/100g), testosterone level (3.37±0.79 vs. 0.61±0.29 ng/ml), and sperm concentration (115.33±7.70 vs. 79.13±3.65×106 cells/ml) of CS were significantly lower (p<0.05) than controls. Some atrophic seminiferous tubules and low sperm mass were apparent in CS rats. The expression of CYP11A1 except StAR protein was markedly decreased in CS rats. In contrast, a 55 kDa phosphorylated protein was higher in CS testes. Conclusion: CS decreased the expression of CYP11A, resulting in decreased testosterone, and increased acrosome-reacted sperm, assumed to be the result of an increase of 55 kDa phosphorylated protein. PMID:27525328

  3. Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models

    PubMed Central

    Ba, Maowen; Kong, Min; Ma, Guozhao

    2015-01-01

    Context Abnormality in interactions between N-methyl-d-aspartate (NMDA) receptor and its signaling molecules occurs in the lesioned striatum in Parkinson’s disease (PD) and levodopa-induced dyskinesia (LID). It was reported that Fyn-mediated NR2B tyrosine phosphorylation, can enhance NMDA receptor function. Postsynaptic density protein 95 (PSD-95), one of the synapse-associated proteins, regulates interactions between receptor and downstream-signaling molecules. In light of the relationship between PSD-95, NR2B, and Fyn kinases, does PSD-95 contribute to the overactivity of NMDA receptor function induced by dopaminergic treatment? To further prove the possibility, the effects of regulating the PSD-95 expression on the augmented NR2B tyrosine phosphorylation and on the interactions of Fyn and NR2B in LID rat models were evaluated. Methods In the present study, parkinsonian rat models were established by injecting 6-hydroxydopamine. Subsequently, valid PD rats were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily) intraperitoneally for 22 days to create LID rat models. Then, the effect of pretreatment with an intrastriatal injection of the PSD-95mRNA antisense oligonucleotides (PSD-95 ASO) on the rotational response to levodopa challenge was assessed. The effects of pretreatment with an intrastriatal injection of PSD-95 ASO on the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in the LID rat models were detected by immunoblotting and immunoprecipitation. Results Levodopa administration twice daily for 22 days to parkinsonian rats shortened the rotational duration and increased the peak turning responses. The altered rotational responses were attenuated by PSD-95 ASO pretreatment. Meanwhile, PSD-95 ASO pretreatment decreased the level of PSD-95 protein expression and reduced both the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B triggered during the levodopa administration in the

  4. Reversible Post-translational Modification of Proteins by Nitrated Fatty Acids in Vivo*S

    PubMed Central

    Batthyany, Carlos; Schopfer, Francisco J.; Baker, Paul R. S.; Durán, Rosario; Baker, Laura M. S.; Huang, Yingying; Cerveñansky, Carlos; Branchaud, Bruce P.; Freeman, Bruce A.

    2007-01-01

    Nitric oxide (˙NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon β to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC50 of ∼3 μm for both nitroalkenes, an IC50 equivalent to the potent thiol oxidant peroxynitrite (ONOO−) and an IC50 30-fold less than H2O2, indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these

  5. 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.

  6. Protein tyrosine phosphatase 1B negatively regulates S100A9-mediated lung damage during respiratory syncytial virus exacerbations.

    PubMed

    Foronjy, R F; Ochieng, P O; Salathe, M A; Dabo, A J; Eden, E; Baumlin, N; Cummins, N; Barik, S; Campos, M; Thorp, E B; Geraghty, P

    2016-09-01

    Protein tyrosine phosphatase 1B (PTP1B) has anti-inflammatory potential but PTP1B responses are desensitized in the lung by prolonged cigarette smoke exposure. Here we investigate whether PTP1B expression affects lung disease severity during respiratory syncytial viral (RSV) exacerbations of chronic obstructive pulmonary disease (COPD). Ptp1b(-/-) mice infected with RSV exhibit exaggerated immune cell infiltration, damaged epithelial cell barriers, cytokine production, and increased apoptosis. Elevated expression of S100A9, a damage-associated molecular pattern molecule, was observed in the lungs of Ptp1b(-/-) mice during RSV infection. Utilizing a neutralizing anti-S100A9 IgG antibody, it was determined that extracellular S100A9 signaling significantly affects lung damage during RSV infection. Preexposure to cigarette smoke desensitized PTP1B activity that coincided with enhanced S100A9 secretion and inflammation in wild-type animals during RSV infection. S100A9 levels in human bronchoalveolar lavage fluid had an inverse relationship with lung function in healthy subjects, smokers, and COPD subjects. Fully differentiated human bronchial epithelial cells isolated from COPD donors cultured at the air liquid interface secreted more S100A9 than cells from healthy donors or smokers following RSV infection. Together, these findings show that reduced PTP1B responses contribute to disease symptoms in part by enhancing S100A9 expression during viral-associated COPD exacerbations. PMID:26813343

  7. Isopetrosynol, a New Protein Tyrosine Phosphatase 1B Inhibitor, from the Marine Sponge Halichondria cf. panicea Collected at Iriomote Island.

    PubMed

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

    2016-01-01

    A new polyacetylene compound, isopetrosynol (1), was isolated from the Okinawan marine sponge Halichondria cf. panicea together with petrosynol (2), adociacetylene D (3), (5R)-3,15,27-triacontatriene-1,29-diyn-5-ol (4), and petrosterol (5). The structure of 1 was assigned on the basis of spectroscopic data for 1 and 2. Compound 1 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 8.2±0.3 µM, while compound 2, a diastereomer of 1, showed only 28.9±4.5% inhibition at 21.6 µM. The IC50 values of compounds 3 and 4 were 7.8±0.5 and 12.2±0.5 µM, respectively. Oleanolic acid, a positive control, inhibited PTP1B activity at 0.7±0.1 µM (IC50) in the same experiment. The inhibitory activity of 1 was stronger than that of its diastereomer (2). This is the first study to show the inhibitory effects of polyacetylene compounds on PTP1B. PMID:27373628

  8. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells

    SciTech Connect

    Lamprianou, Smaragda; Chatzopoulou, Elli; Thomas, Jean-Léon; Bouyain, Samuel; Harroch, Sheila

    2013-09-23

    The six members of the contactin (CNTN) family of neural cell adhesion molecules are involved in the formation and maintenance of the central nervous system (CNS) and have been linked to mental retardation and neuropsychiatric disorders such as autism. Five of the six CNTNs bind to the homologous receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ), but the biological roles of these interactions remain unclear. We report here the cocrystal structure of the carbonic anhydrase-like domain of PTPRZ bound to tandem Ig repeats of CNTN1 and combine these structural data with binding assays to show that PTPRZ binds specifically to CNTN1 expressed at the surface of oligodendrocyte precursor cells. Furthermore, analyses of glial cell populations in wild-type and PTPRZ-deficient mice show that the binding of PTPRZ to CNTN1 expressed at the surface of oligodendrocyte precursor cells inhibits their proliferation and promotes their development into mature oligodendrocytes. Overall, these results implicate the PTPRZ/CNTN1 complex as a previously unknown modulator of oligodendrogenesis.

  9. The protein tyrosine phosphatases PTPRZ and PTPRG bind to distinct members of the contactin family of neural recognition molecules

    SciTech Connect

    Bouyain, Samuel; Watkins, Dara J.

    2010-04-05

    The receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ) are expressed primarily in the nervous system and mediate cell adhesion and signaling events during development. We report here the crystal structures of the carbonic anhydrase-like domains of PTPRZ and PTPRG and show that these domains interact directly with the second and third immunoglobulin repeats of the members of the contactin (CNTN) family of neural recognition molecules. Interestingly, these receptors exhibit distinct specificities: PTPRZ binds only to CNTN1, whereas PTPRG interacts with CNTN3, 4, 5, and 6. Furthermore, we present crystal structures of the four N-terminal immunoglobulin repeats of mouse CNTN4 both alone and in complex with the carbonic anhydrase-like domain of mouse PTPRG. In these structures, the N-terminal region of CNTN4 adopts a horseshoe-like conformation found also in CNTN2 and most likely in all CNTNs. This restrained conformation of the second and third immunoglobulin domains creates a binding site that is conserved among CNTN3, 4, 5, and 6. This site contacts a discrete region of PTPRG composed primarily of an extended {beta}-hairpin loop found in both PTPRG and PTPRZ. Overall, these findings implicate PTPRG, PTPRZ and CNTNs as a group of receptors and ligands involved in the manifold recognition events that underlie the construction of neural networks.

  10. Directed differentiation of postnatal hippocampal neural stem cells generates nuclear receptor related-1 protein- and tyrosine hydroxylase-expressing cells

    PubMed Central

    Ding, Yinxiu; Zhang, Zixin; Ma, Jiangbo; Xia, Hechun; Wang, Yin; Liu, Yinming; Ma, Quanrui; Sun, Tao; Liu, Juan

    2016-01-01

    Parkinson's disease (PD) is a severe neurodegenerative disorder. Although the detailed underlying molecular mechanism remains to be elucidated, the major pathological feature of PD is the loss of dopaminergic (DA) neurons of the substantia nigra. The use of donor stem cells to replace DA neurons may be a key breakthrough in the treatment of PD. In the present study, the growth kinetics of hippocampal neural stem cells (Hip-NSCs) isolated from postnatal mice and cultured in vitro were observed, specifically the generation of cells expressing DA neuronal markers nuclear receptor related-1 protein (Nurr1) and tyrosine hydroxylase (TH). It was revealed that Hip-NSCs differentiated primarily into astrocytes when cultured in serum-containing medium. However, in low serum conditions, the number of βIII tubulin-positive neurons increased markedly. The proportion of Nurr1-positive cells and TH-positive neurons, significantly increased with increasing duration of directed differentiation of Hip-NSCs (P=0.0187 and 0.0254, respectively). The results of the present study reveal that Hip-NSCs may be induced to differentiate in vitro into neurons expressing Nurr1 and TH, known to be critical regulators of DA neuronal fate. Additionally, their expression may be necessary to facilitate neuronal maturation in vitro. These data suggest that Hip-NSCs may serve as a source of DA neurons for cell therapy in patients diagnosed with PD. PMID:27432537

  11. Structure of the Trypanosoma cruzi protein tyrosine phosphatase TcPTP1, a potential therapeutic target for Chagas' disease

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2013-06-05

    Chagas’ disease, a neglected tropical affliction transmitted by the flagellated protozoan Trypanosoma cruzi, is prevalent in Latin America and affects nearly 18 million people worldwide, yet few approved drugs are available to treat the disease. Moreover, the currently available drugs exhibit severe toxicity or are poorly effective in the chronic phase of the disease. This limitation, along with the large population at risk, underscores the urgent need to discover new molecular targets and novel therapeutic agents. Recently, the T. cruzi protein tyrosine phosphatase TcPTP1 has been implicated in the cellular differentiation and infectivity of the parasite and is therefore a promising target for the design of novel anti-parasitic drugs. Here, we report the X-ray crystal structure of TcPTP1 refined to a resolution of 2.18 Å, which provides structural insights into the active site environment that can be used to initiate structure-based drug design efforts to develop specific TcPTP1 inhibitors. Potential strategies to develop such inhibitors are also discussed.

  12. Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2)

    SciTech Connect

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

    2010-08-13

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

  13. PRL-3 mediates the protein maturation of ULBP2 by regulating the tyrosine phosphorylation of HSP60

    PubMed Central

    Leung, Wai-Hang; Vong, Queenie P.; Lin, Wenwei; Bouck, David; Wendt, Susanne; Sullivan, Erin; Li, Ying; Bari, Rafijul; Chen, Taosheng; Leung, Wing

    2015-01-01

    Many malignant cells release the NKG2D ligand ULBP2 from their cell surface to evade immunosurveillance by natural killer cells and CD8 T cells. Although the shedding mechanism remains unclear, various inhibitors of matrix metalloproteinases have been shown to efficiently block the release of soluble ULBP2. The clinical use of these inhibitors however is limited because of adverse side effects. Using high throughput screening technique, we identified a specific inhibitor of phosphatase of regenerating liver 3 (PRL-3) that could reduce the level of soluble ULBP2 in the culture supernatant of various cancer cell lines. Inhibition or gene knockdown of PRL-3 did not reduce ULBP2 shedding but rather suppressed post-translational maturation of ULBP2, resulting in intracellular retention of immature ULBP2. We then found that ULBP2 was constitutively associated with heat shock protein HSP60. Complete maturation of ULBP2 required tyrosine phosphorylation of HSP60 which was mediated by PRL-3. PMID:25687758

  14. Interactions of Tyrosine Kinase Inhibitors with Organic Cation Transporters, OCTs, and Multidrug and Toxic Compound Extrusion Proteins, MATEs

    PubMed Central

    Minematsu, Tsuyoshi; Giacomini, Kathleen M.

    2011-01-01

    The drug-drug interaction (DDI) potential of tyrosine kinase inhibitors (TKIs) as interacting drugs via transporter inhibition has not been fully assessed. Here, we estimated the half maximal inhibitory concentration (IC50) values for eight small-molecule TKIs (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, sunitinib, lapatinib, and sorafenib) on [14C]metformin transport by human organic cation transporters (OCT1, OCT2, and OCT3), and multidrug and toxic compound extrusion proteins (MATE1 and MATE2-K), using HEK293 cells stably expressing these transporters. We then compared the estimated IC50 values to the maximum clinical concentrations of unbound TKIs in plasma (unbound Cmax,sys,p). Results showed that imatinib, nilotinib, gefitinib, and erlotinib exerted selectively potent inhibitory effects, with unbound Cmax,sys,p/IC50 values ≥ 0.1, on MATE1, OCT3, MATE2-K and OCT1, respectively. In comparison to the common form of OCT1, the OCT1 polymorphism, M420del was more sensitive to drug inhibition by erlotinib. Major metabolites of several TKIs showed IC50 values similar to those for unchanged TKIs. Taken together, these findings suggest the potential of clinical transporter-mediated DDIs between specific TKIs and OCTs and MATEs, which may affect the disposition, efficacy and toxicity of metformin and other drugs that are substrates of these transporters. The study provides the basis for further clinical DDI studies with TKIs. PMID:21252289

  15. Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells

    PubMed Central

    Pandey, Manoj K.; Sung, Bokyung; Aggarwal, Bharat B.

    2009-01-01

    STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulates the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid -induced down regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP-1 and silencing of the SHP-1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescues betulinic acid-induced cell death. Betulinic acid also downregulated the expression of STAT3-regulated gene products such as bcl-xL, bcl-2, cyclin D1, and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub-G1 cell population and an increase in caspase-3–induced PARP cleavage. Consistent with these results, over expression of constitutive active STAT3 significantly reduced the betulinic acid-induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10% to 55%) and bortezomib (from 5% to 70%) in MM cells. Overall, our results suggest that betulinic acid down regulates STAT3 activation through upregulation of SHP-1 and this may have potential in sensitization of STAT3 over expressing tumors to chemotherapeutic agents. PMID:19937797

  16. Structure of the Trypanosoma cruzi protein tyrosine phosphatase TcPTP1, a potential therapeutic target for Chagas’ disease

    PubMed Central

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2014-01-01

    Chagas’ disease, a neglected tropical affliction transmitted by the flagellated protozoan Trypanosoma cruzi, is prevalent in Latin America and affects nearly 18 million people worldwide, yet few approved drugs are available to treat the disease. Moreover, the currently available drugs exhibit severe toxicity or are poorly effective in the chronic phase of the disease. This limitation, along with the large population at risk, underscores the urgent need to discover new molecular targets and novel therapeutic agents. Recently, the T. cruzi protein tyrosine phosphatase TcPTP1 has been implicated in the cellular differentiation and infectivity of the parasite and is therefore a promising target for the design of novel anti-parasitic drugs. Here, we report the X-ray crystal structure of TcPTP1 refined to a resolution of 2.18 Å, which provides structural insights into the active site environment that can be used to initiate structure-based drug design efforts to develop specific TcPTP1 inhibitors. Potential strategies to develop such inhibitors are also discussed. PMID:23137716

  17. The receptor protein tyrosine phosphatase LAR promotes R7 photoreceptor axon targeting by a phosphatase-independent signaling mechanism

    PubMed Central

    Hofmeyer, Kerstin; Treisman, Jessica E.

    2009-01-01

    Receptor protein tyrosine phosphatases (RPTPs) control many aspects of nervous system development. At the Drosophila neuromuscular junction (NMJ), regulation of synapse growth and maturation by the RPTP LAR depends on catalytic phosphatase activity and on the extracellular ligands Syndecan and Dally-like. We show here that the function of LAR in controlling R7 photoreceptor axon targeting in the visual system differs in several respects. The extracellular domain of LAR important for this process is distinct from the domains known to bind Syndecan and Dally-like, suggesting the involvement of a different ligand. R7 targeting does not require LAR phosphatase activity, but instead depends on the phosphatase activity of another RPTP, PTP69D. In addition, a mutation that prevents dimerization of the intracellular domain of LAR interferes with its ability to promote R7 targeting, although it does not disrupt phosphatase activity or neuromuscular synapse growth. We propose that LAR function in R7 is independent of its phosphatase activity, but requires structural features that allow dimerization and may promote the assembly of downstream effectors. PMID:19889974

  18. Strongylophorines, new protein tyrosine phosphatase 1B inhibitors, from the marine sponge Strongylophora strongilata collected at Iriomote Island.

    PubMed

    Lee, Jong-Soo; Abdjul, Delfly B; Yamazaki, Hiroyuki; Takahashi, Ohgi; Kirikoshi, Ryota; Ukai, Kazuyo; Namikoshi, Michio

    2015-09-15

    A new meroditerpene, 26-O-ethylstrongylophorine-14 (1), was isolated from the Okinawan marine sponge Strongylophora strongilata together with six known strongylophorines: 26-O-methylstrongylophorine-16 (2) and strongylophorines-2 (3), -3 (4), -8 (5), -15 (6), and -17 (7). The structure of 1 was assigned on the basis of its spectroscopic data. Compound 1 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with an IC50 value of 8.7 μM, while known compounds 2-8 gave IC50 values of 8.5, >24.4, 9.0, 21.2, 11.9, and 14.8 μM, respectively. Oleanolic acid, a positive control, inhibited PTP1B activity at 0.7 μM (IC50). The inhibitory activities of strongylophorines possessing the acetal moiety at C-26 (1, 2, and 6) were stronger than those of the lactone derivatives (3 and 5). This is the first study to demonstrate that meroditerpenes inhibit PTP1B activity. PMID:26253631

  19. MetaBlasts: tracing protein tyrosine phosphatase gene family roots from Man to Drosophila melanogaster and Caenorhabditis elegans genomes.

    PubMed

    Wälchli, S; Colinge, J; Hooft van Huijsduijnen, R

    2000-08-01

    At increasing speed, sequencing data are being made public from both complex and simple life forms. Although biomedical interests tend to focus on mammalian genes, only simple organisms allow rapid genetic manipulation and functional analysis. A prerequisite for the meaningful extrapolation of gene functional studies from invertebrates to man is that the orthologs under study are unambiguously linked. However, identifying orthologs is not trivial, especially where large gene families are involved. We present here an automated sequence analysis procedure that allows a rapid visualization of most likely ortholog pairs. We illustrate the utility of this approach for the human gene family of protein tyrosine phosphatases (PTPs) as compared with the full set of Caenorhabditis elegans and Drosophila melanogaster conceptual ORFs. The approach is based on a reciprocal series of BLAST searches, which are automatically stored and represented in an HTML-formatted table. We have used this 'MetaBlast' approach to compile lists of human PTPs and their worm and fly orthologs. Many of these PTP orthologs had not been previously identified as such. PMID:10940550

  20. 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.

  1. Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists.

    PubMed Central

    Nahas, N; Molski, T F; Fernandez, G A; Sha'afi, R I

    1996-01-01

    The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [gamma-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine

  2. CD8+ T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli.

    PubMed Central

    Weaver, C T; Pingel, J T; Nelson, J O; Thomas, M L

    1991-01-01

    CD45 is a high-molecular-weight transmembrane protein tyrosine phosphatase expressed only by nucleated cells of hematopoietic origin. To examine function, mouse CD8+ cytolytic T-cell clones were derived that had a specific defect in the expression of CD45. Northern (RNA) blot analysis indicates that the CD45 deficiency is due to either a transcriptional defect or mRNA instability. The CD45-deficient cells were greatly diminished in their ability to respond to antigen. All functional parameters of T-cell receptor signalling analyzed (cytolysis of targets, proliferation, and cytokine production) were markedly diminished. A CD45+ revertant was isolated, and the ability to respond to antigen was restored. These results support a central and immediate role for this transmembrane protein tyrosine phosphatase in T-cell receptor signalling. Images PMID:1652055

  3. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues.

    PubMed

    Cook, Naomi L; Moeke, Cassidy H; Fantoni, Luca I; Pattison, David I; Davies, Michael J

    2016-01-01

    Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN(-)) oxidation by H2O2 to form hypothiocyanous acid (HOSCN), an oxidant that targets Cys residues. Dysregulated phosphorylation and elevated MPO levels have been associated with chronic inflammatory diseases where HOSCN can be generated. Previous studies have shown that HOSCN inhibits isolated PTP1B and induces cellular dysfunction in cultured macrophage-like cells. The present study extends this previous work and shows that physiologically-relevant concentrations of HOSCN alter the activity and structure of other members of the wider PTP family (including leukocyte antigen-related PTP, PTP-LAR; T-cell PTP, TC-PTP; CD45 and Src homology phosphatase-1, Shp-1) by targeting Cys residues. Isolated PTP activity, and activity in lysates of human monocyte-derived macrophages (HMDM) was inhibited by 0-100 µM HOSCN with this being accompanied by reversible oxidation of Cys residues, formation of sulfenic acids or sulfenyl-thiocyanates (detected by Western blotting, and LC-MS as dimedone adducts), and structural changes. LC-MS/MS peptide mass-mapping has provided data on the modified Cys residues in PTP-LAR. This study indicates that inflammation-induced oxidants, and particularly myeloperoxidase-derived species, can modulate the activity of multiple members of the PTP superfamily via oxidation of Cys residues to sulfenic acids. This alteration of the balance of PTP/kinase activity may perturb protein phosphorylation and disrupt cell signaling with subsequent induction of apoptosis at sites of inflammation. PMID:26616646

  4. Protein and Nitrate Content of Lemna Sp. as a Function of Developmental Stage and Incubation Temperature 1

    PubMed Central

    Lehman, Peggy W.; Silk, Wendy Kuhn; Knight, Allen W.

    1981-01-01

    Lemna protein per frond and per root increases with developmental stage until plants are at least two generations old. Protein per frond, per root, and per unit dry weight is greater in plants grown at 23.9 C than at 18.3 C. More protein is found in fronds than in roots, and more nitrate occurs in roots than in fronds. Nitrate per root increases with developmental stage and is higher (per root) in plants grown at 23.9 C than in those grown at 18.3 C. The distribution of generations within a growing population is constant for at least eight doubling times. Whether populations multiply slowly at 15.6 C or more rapidly at 23.9 C, fronds which have not yet produced progeny form 62% of the population; fronds which are one generation old form 24% of the population; and fronds which are two generations old form 9% of the population. Images PMID:16661855

  5. Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src.

    PubMed Central

    Sabe, H; Hata, A; Okada, M; Nakagawa, H; Hanafusa, H

    1994-01-01

    Csk (C-terminal Src kinase), a protein-tyrosine kinase, bearing the Src homology 2 and 3 (SH2 and SH3) domains, has been implicated in phosphorylation of c-Src Tyr-527, resulting in suppression of c-Src kinase activity. We found that mutations in the SH2 or SH3 domain of Csk, though they did not affect its kinase activity, resulted in a loss of suppression of c-Src activity in fibroblasts. In normal fibroblasts, tyrosine-phosphorylated paxillin and focal adhesion kinase pp125FAK, which colocalize at focal adhesion plaques, were the major proteins to which the Csk SH2 domain bound. Loss of binding to these proteins by the Csk SH2 mutants correlated with loss of the activity to suppress c-Src. Consistent with this observation, the levels of tyrosine phosphorylation of paxillin and pp125FAK were greatly reduced during mitosis, whereas the kinase activity of c-Src was elevated. We suggest that the SH2 domain is required for Csk to suppress c-Src, perhaps in combination with the SH3 domain, by anchoring Csk to a particular subcellular location where c-Src may exist. Our data also indicate that a certain fraction of the Csk and Src family kinases function at the focal adhesion plaques. The activity of the c-Src kinase localized at the focal adhesion plaques appears to be regulated by cell adhesion to the extracellular matrix. Images PMID:7513429

  6. 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

  7. 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

  8. Selective expression of a protein-tyrosine kinase, p56 sup lyn , in hematopoietic cells and association with production of human T-cell lymphotropic virus type I

    SciTech Connect

    Yamanashi, Yuji; Mori, Shigeo; Inoue, Kazushi; Yamamoto, Tadashi; Toyoshima, Kumao ); Yoshida, Mitsuaki ); Kishimoto, Tadamitsu )

    1989-09-01

    This paper reports the identification of the lyn gene product, a member of the src-related family of protein-tyrosine kinases, and its expression in hematopoietic cells. A lyn-specific sequence (Arg-25 to Ala-119 of the protein) was expressed in Escherichia coli as a fusion protein with {beta}-galactosidase. Antiserum raised against the fusion protein immunoprecipitated a 56-kDa protein from human B lymphocytes. Incubation of the immunoprecipitate with ({gamma}-{sup 32}P)ATP resulted in the phosphorylation of this protein at tyrosine residues. Immunohistological and immunoblotting analyses showed that the lyn gene product was expressed in lymphatic tissues (spleen and tonsil) and in adult lung, which contains many macrophages. Furthermore, both the transcripts and the protein products of the lyn gene accumulated in macrophages/monocytes, platelets, and B lymphocytes but were not expressed appreciably in granulocytes, erythrocytes, or T lymphocytes, suggesting that lyn gene products function primarily in certain differentiated cells of lymphoid and myeloid lineages.

  9. The trk Tyrosine Protein Kinase Mediates the Mitogenic Properties of Nerve Growth Factor and Neurotrophin-3

    PubMed Central

    Cordon-Cardo, Carlos; Tapley, Peter; Jing, Shuqian; Nanduri, Venkata; O’Rourke, Edward; Lamballe, Fabienne; Kovary, Karla; Klein, Rüdiger; Jones, Kevin R.; Reichardt, Louis F.; Barbacid, Mariano

    2009-01-01

    Summary The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells. PMID:1649007

  10. Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosine-26 and -64

    SciTech Connect

    Roepe, P.; Scherrer, P.; Ahl, P.L.; Gupta, S.K.D.; Bogomolni, R.A.; Herzfeld, J.; Rothschild, K.J.

    1987-10-20

    Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR/sub 570/ ..-->.. K/sub 630/ or BR/sub 570/ ..-->.. M/sub 412/ transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K/sub 630/ and the deprotonation of a tyrosine by M/sub 412/. Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M/sub 412/ as concluded previously. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm/sup -1/ in the BR/sub 570/ ..-->.. M/sub 412/ FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group.

  11. 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

  12. Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity.

    PubMed

    Chi, Jen-Chih; Roeper, Juliane; Schwarz, Guenter; Fischer-Schrader, Katrin

    2015-03-01

    14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions. PMID:25578809

  13. Nitric oxide (NO) in alleviation of heavy metal induced phytotoxicity and its role in protein nitration.

    PubMed

    Saxena, Ina; Shekhawat, G S

    2013-08-01

    Nitric oxide (NO) is recognized as a biological messenger in various tissues to regulate diverse range of physiological process including growth, development and response to abiotic and biotic factors. The NO emission from plants is known since the 1970s, and there is copious information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. Heavy metal toxicity is one of the major abiotic stresses leading to hazardous effects in plants and its toxicity is based on chemical and physical property. A common consequence of heavy metal toxicity is the uncontrolled and excessive accumulation of reactive oxygen species (ROS) which leads to peroxidation of lipids, oxidation of protein, inactivation of enzymes, DNA damage and/or interact with other vital constituents of plant cells. Recently, an increasing number of articles have reported the effects of exogenous NO on alleviating heavy metal toxicity in plants but knowledge of physiological mechanisms of NO in alleviating heavy metal toxicity is quite limited, and some results contradict one another. Therefore, to help clarify the roles of NO in heavy metal tolerance, it is important to review and discuss the recent advances on this area of research. NO can provoke both beneficial and harmful effects, which depend on the concentration and location of NO in the plant cells. NO alleviates the harmfulness of the ROS, and reacts with other target molecules, and regulates the expression of stress responsive genes under various stress conditions. This manuscript includes, the latest advances in understanding the effects of endogenous NO on heavy metal toxicity and the mechanisms and role of NO as an antioxidant as well as in protein nitration are highlighted. PMID:23545403

  14. Crystal structure of the Yersinia protein-tyrosine phosphatase YopH complexed with a specific small molecule inhibitor.

    PubMed

    Sun, Jin-Peng; Wu, Li; Fedorov, Alexander A; Almo, Steven C; Zhang, Zhong-Yin

    2003-08-29

    The pathogenic bacteria Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to bubonic plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. Because the phosphatase activity of the Yersinia protein tyrosine phosphatase, YopH, is essential for virulence in the Yersinia pathogen, potent and selective YopH inhibitors are expected to serve as novel anti-plague agents. We have identified a specific YopH small molecule inhibitor, p-nitrocatechol sulfate (pNCS), which exhibits a Ki value of 25 microM for YopH and displays a 13-60-fold selectivity in favor of YopH against a panel of mammalian PTPs. To facilitate the understanding of the underlying molecular basis for tight binding and specificity, we have determined the crystal structure of YopH in complex with pNCS at a 2.0-A resolution. The structural data are corroborated by results from kinetic analyses of the interactions of YopH and its site-directed mutants with pNCS. The results show that while the interactions of the sulfuryl moiety and the phenyl ring with the YopH active site contribute to pNCS binding affinity, additional interactions of the hydroxyl and nitro groups in pNCS with Asp-356, Gln-357, Arg-404, and Gln-446 are responsible for the increased potency and selectivity. In particular, we note that residues Arg-404, Glu-290, Asp-356, and a bound water (WAT185) participate in a unique H-bonding network with the hydroxyl group ortho to the sulfuryl moiety, which may be exploited to design more potent and specific YopH inhibitors. PMID:12810712

  15. Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes*

    PubMed Central

    Mobasher, Maysa Ahmed; de Toro-Martín, Juan; González-Rodríguez, Águeda; Ramos, Sonia; Letzig, Lynda G.; James, Laura P.; Muntané, Jordi; Álvarez, Carmen; Valverde, Ángela M.

    2014-01-01

    Many drugs are associated with the development of glucose intolerance or deterioration in glycemic control in patients with pre-existing diabetes. We have evaluated the cross-talk between signaling pathways activated by acetaminophen (APAP) and insulin signaling in hepatocytes with or without expression of the protein-tyrosine phosphatase 1B (PTP1B) and in wild-type and PTP1B-deficient mice chronically treated with APAP. Human primary hepatocytes, Huh7 hepatoma cells with silenced PTP1B, mouse hepatocytes from wild-type and PTP1B-deficient mice, and a mouse model of chronic APAP treatment were used to examine the mechanisms involving PTP1B in the effects of APAP on glucose homeostasis and hepatic insulin signaling. In APAP-treated human hepatocytes at concentrations that did not induce death, phosphorylation of JNK and PTP1B expression and enzymatic activity were increased. APAP pretreatment inhibited activation of the early steps of insulin signaling and decreased Akt phosphorylation. The effects of APAP in insulin signaling were prevented by suramin, a PTP1B inhibitor, or rosiglitazone that decreased PTP1B levels. Likewise, PTP1B deficiency in human or mouse hepatocytes protected against APAP-mediated impairment in insulin signaling. These signaling pathways were modulated in mice with chronic APAP treatment, resulting in protection against APAP-mediated hepatic insulin resistance and alterations in islet alpha/beta cell ratio in PTP1B−/− mice. Our results demonstrate negative cross-talk between signaling pathways triggered by APAP and insulin signaling in hepatocytes, which is in part mediated by PTP1B. Moreover, our in vivo data suggest that chronic use of APAP may be associated with insulin resistance in the liver. PMID:25204659

  16. Ultrasensitive electrochemical detection of protein tyrosine kinase-7 by gold nanoparticles and methylene blue assisted signal amplification.

    PubMed

    Miao, Xiangmin; Li, Zongbing; Zhu, Aihua; Feng, Zhaozhong; Tian, Jun; Peng, Xue

    2016-09-15

    We present here an ultrasensitive and simple strategy for protein tyrosine kinase-7 (PTK7) detection based on the recognition-induced structure change of sgc8 aptamer, and the signal change of methylene blue (MB) that interacted with sandwiched DNA complex. To construct such a sensor, an homogeneous nano-surface was formed firstly on the glass carbon electrode (GCE) by using negatively charged Nafion (Nf) as the inner layer and positively charged gold nanoparticles ((+)AuNPs) as the outer layer, followed by the immobilization of sgc8 aptamer based on Au-S bond. In the presence of helper probe (HP), sandwiched DNA complex was formed between the sgc8 aptamer and the DNA modified gold nanoparticle probe (DNA-AuNPs). Then, a strong current signal was produced due to the capture of abundant MB molecules by both the sandwiched DNA complex and the multiple DNAs that modified on AuNPs surface. However, the specific binding of sgc8 aptamer with PNK7 would trigger a structure transition of it, and directly prevented the following formation of sandwiched structure and the capture of MB. Thus, PTK7 detection could be realized based on monitoring the signal reduction of MB upon incubation of sgc8 aptamer with PTK7. Under optimal conditions, a low detection limit of 372 fM was obtained for PNK7 detection. Due to the employment of sgc8 aptamer, the proposed biosensor exhibited high selectivity to PNK7. Moreover, satisfactory results were obtained when the proposed method was applied for PNK7 detection in cellular debris. PMID:27101533

  17. Src family protein tyrosine kinase (PTK) modulates the effect of SGK1 and WNK4 on ROMK channels.

    PubMed

    Yue, Peng; Lin, Dao-Hong; Pan, Chun-Yang; Leng, Qiang; Giebisch, Gerhard; Lifton, Richard P; Wang, Wen-Hui

    2009-09-01

    WNK4 (with no lysine kinase 4) inhibits ROMK channel activity in the distal nephron by stimulating clathrin-dependent endocytosis, an effect attenuated by SGK1 (serum-glucocorticoids-induced kinase)-mediated phosphorylation. It has been suggested that increased ROMK activity because of SGK1-mediated inhibition of WNK4 plays a role in promoting renal K secretion in response to elevated serum K or high K (HK) intake. In contrast, intravascular volume depletion also increases SGK1 activity but fails to stimulate ROMK channels and K secretion. Because HK intake decreases Src family protein tyrosine kinase (PTK) activity an inhibitor of ROMK channels, it is possible that Src family PTK may modulate the effects of SGK1 on WNK4. Here, we show that c-Src prevents SGK1 from attenuating WNK4's inhibition of ROMK activity. This effect of c-Src was WNK4-dependent because c-Src had no effect on ROMK harboring mutation at the site of c-Src phosphorylation (R1Y337A) in the absence of WNK4. Moreover, expression c-Src diminished the SGK1-mediated increase in serine phosphorylation of WNK4, suggesting that c-Src enhances WNK4-mediated inhibition of ROMK channels by suppressing the SGK1-induced phosphorylation. This notion is also supported by the observation that c-Src was not able to modulate the interaction between SGK1 and WNK4 mutants (WNK4(S1169A) or WNK4(S1169D)) in which an SGK1-phosphorylation site (serine 1169) was mutated by alanine or aspartate. We conclude that c-Src inhibits SGK1-mediated phosphorylation hereby restoring the WNK4-mediated inhibition of ROMK channels thus suppressing K secretion. PMID:19706464

  18. Protein tyrosine phosphatase 1B inhibitors: a molecular level legitimate approach for the management of diabetes mellitus.

    PubMed

    Thareja, Suresh; Aggarwal, Saurabh; Bhardwaj, T R; Kumar, Manoj

    2012-05-01

    Diabetes mellitus is a systemic disease responsible for morbidity in the western world and is gradually becoming prevalent in developing countries too. The prevalence of diabetes is rapidly increasing in industrialized countries and type 2 diabetes accounts for 90% of the disease. Insulin resistance is a major pathophysiological factor in the development of type 2 diabetes, occurring mainly in muscle, adipose tissues, and liver leading to reduced glucose uptake and utilization and increased glucose production. The prevalence and rising incidence of diabetes emphasized the need to explore new molecular targets and strategies to develop novel antihyperglycemic agents. Protein Tyrosine Phosphatase 1B (PTP 1B) has recently emerged as a promising molecular level legitimate therapeutic target in the effective management of type 2 diabetes. PTP 1B, a cytosolic nonreceptor PTPase, has been implicated as a negative regulator of insulin signal transduction. Therefore, PTP 1B inhibitors would increase insulin sensitivity by blocking the PTP 1B-mediated negative insulin signaling pathway and might be an attractive target for type 2 diabetes mellitus and obesity. With X-ray crystallography and NMR-based fragment screening, the binding interactions of several classes of inhibitors have been elucidated, which could help the design of future PTP 1B inhibitors. The drug discovery research in PTP 1B is a challenging area to work with and many pharmaceutical organizations and academic research laboratories are focusing their research toward the development of potential PTP 1B inhibitors which would prove to be a milestone for the management of diabetes. PMID:20814956

  19. 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. PMID:23084926

  20. Delta Opioid activation of the Mitogen-activated protein kinase cascade does not require transphosphorylation of Receptor Tyrosine Kinases

    PubMed Central

    Kramer, H Kenneth; Onoprishvili, Irma; Andria, Matthew L; Hanna, Kayane; Sheinkman, Karina; Haddad, Lisa B; Simon, Eric J

    2002-01-01

    Background In this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned δ-opioid receptor (δ-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive. Results Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed δ-ORs are stimulated by the δ-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the δ-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In δ-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation. Conclusion Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated δ-OR, itself, is likely to act as its own signalling scaffold. PMID:11897012

  1. 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

  2. Mechanisms of tyrosine hydroxylase regulation in striatal synaptosomes: effects of activation of cyclic AMP-dependent protein kinase

    SciTech Connect

    Colby, K.A.

    1987-01-01

    The regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis, was examined in synaptosomes prepared from rat corpus striatum. Exposure of striatal synaptosomes to dibutyryl-cyclic AMP (dbcAMP) causes an increase in the maximal velocity of TH, but does not change the K/sub m/ of the enzyme for the synthetic cofactor, 2-amino-4-hydroxy-6-methyl-tetrahydropterin. Activation of TH by synaptosomal exposure to dbcAMP also causes a decrease in the pH sensitivity and an increase in the thermolability of the enzyme. Striatal synaptosomes were used to examine the in vitro phosphorylation of TH. Under the protocol developed as part of this work, TH in synaptosomes can be labelled with /sup 32/P. This is the first report of in vitro labelling of TH in a biochemically intact CNS preparation. Under certain protocols, treatment of synaptosomes with dbcAMP causes an increase in the /sup 32/P labelling of TH. These results are consistent with the notion that dbcAMP produces changes in the physical properties of TH by activating cAMP-dependent protein kinase which subsequently phosphorylates TH. In vivo electrical stimulation of the rat medial forebrain bundle causes an activation of striatal TH as well as an decrease in the pH sensitivity of the enzyme. Since similar changes are produced upon activation of snaptosomal TH by dbcAMP, it is likely that phosphorylation of TH is involved in the increase in TH activity that is associated with neuronal depolarization.

  3. Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B

    PubMed Central

    Haj, Fawaz G.; Sabet, Ola; Kinkhabwala, Ali; Wimmer-Kleikamp, Sabine; Roukos, Vassilis; Han, Hong-Mei; Grabenbauer, Markus; Bierbaum, Martin; Antony, Claude; Neel, Benjamin G.; Bastiaens, Philippe I.

    2012-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B. PMID:22655028

  4. The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity

    PubMed Central

    Hobeika, Elias; Biesen, Robert; Kollert, Florian; Taddeo, Adriano; Voll, Reinhard E.; Hiepe, Falk

    2014-01-01

    Tyrosine phosphorylation of signaling molecules that mediate B cell activation in response to various stimuli is tightly regulated by protein tyrosine phosphatases (PTPs). PTP1B is a ubiquitously expressed tyrosine phosphatase with well-characterized functions in metabolic signaling pathways. We show here that PTP1B negatively regulates CD40, B cell activating factor receptor (BAFF-R), and TLR4 signaling in B cells. Specifically, PTP1B counteracts p38 mitogen-activated protein kinase (MAPK) activation by directly dephosphorylating Tyr182 of this kinase. Mice with a B cell–specific PTP1B deficiency show increased T cell–dependent immune responses and elevated total serum IgG. Furthermore, aged animals develop systemic autoimmunity with elevated serum anti-dsDNA, spontaneous germinal centers in the spleen, and deposition of IgG immune complexes and C3 in the kidney. In a clinical setting, we observed that B cells of rheumatoid arthritis patients have significantly reduced PTP1B expression. Our data suggest that PTP1B plays an important role in the control of B cell activation and the maintenance of immunological tolerance. PMID:24590766

  5. 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.

  6. A novel tyrosine-heme C−O covalent linkage in F43Y myoglobin: a new post-translational modification of heme proteins.

    PubMed

    Yan, Dao-Jing; Li, Wei; Xiang, Yu; Wen, Ge-Bo; Lin, Ying-Wu; Tan, Xiangshi

    2015-01-01

    Heme post-translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine-heme covalent C−O bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4-vinyl group. This highlights the diverse chemistry of heme post-translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently-bound heme proteins. PMID:25392956

  7. 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. PMID:27246854

  8. The major vault protein is a novel substrate for the tyrosine phosphatase SHP-2 and scaffold protein in epidermal growth factor signaling.

    PubMed

    Kolli, Sivanagarani; Zito, Christina I; Mossink, Marieke H; Wiemer, Erik A C; Bennett, Anton M

    2004-07-01

    The catalytic activity of the Src homology 2 (SH2) domain-containing tyrosine phosphatase, SHP-2, is required for virtually all of its signaling effects. Elucidating the molecular mechanisms of SHP-2 signaling, therefore, rests upon the identification of its target substrates. In this report, we have used SHP-2 substrate-trapping mutants to identify the major vault protein (MVP) as a putative SHP-2 substrate. MVP is the predominant component of vaults that are cytoplasmic ribonucleoprotein complexes of unknown function. We show that MVP is dephosphorylated by SHP-2 in vitro and it forms an enzyme-substrate complex with SHP-2 in vivo. In response to epidermal growth factor (EGF), SHP-2 associates via its SH2 domains with tyrosyl-phosphorylated MVP. MVP also interacts with the activated form of the extracellular-regulated kinases (Erks) in response to EGF and a constitutive complex between tyrosyl-phosphorylated MVP, SHP-2, and the Erks was detected in MCF-7 breast cancer cells. Using MVP-deficient fibroblasts, we demonstrate that MVP cooperates with Ras for optimal EGF-induced Elk-1 activation and is required for cell survival. We propose that MVP functions as a novel scaffold protein for both SHP-2 and Erk. The regulation of MVP tyrosyl phosphorylation by SHP-2 may play an important role in cell survival signaling. PMID:15133037

  9. Visualisation of brain tumors and quantitation of the protein synthesis rate with L-1-[C-11]-tyrosine

    SciTech Connect

    Pruim, J.; Willemsen, A.T.M.; Waarde, A. van

    1994-05-01

    We have developed the tracer L-1-[C-11]-tyrosine (TYR) for the quantitation of the protein synthesis rate (PSR) in tumours. Here the first results with TYR in a group of patients with (suspected) primary or recurrent brain tumours are reported. Twenty-six patients were studied: 12 male, 14 female, age 45{plus_minus}16 (mean{plus_minus}S.D.) years. At the time of the study the diagnosis of a primary tumour or recurrent tumour was considered on the basis of clinical symptoms and CT/MRI. Patients received 237{plus_minus}111 MBq TYR i.v. Seventeen patients were studied in a dynamic mode (frame sequence: 10 x 0.5, 3 x 5, 3 x 10 minutes). During the studies, arterial blood samples were taken for measurement of the input function, and the assessment of metabolites ([C-11]CO{sub 2}, [C-11]proteins). ROIs were placed over the tumour and using a modified Patlak-analysis the PSR was calculated. In the other 9 patients a static emission scan was made, 20-40 min after injection. All images were corrected for attenuation via a transmission scan. Histology or cytology of the tumour was obtained shortly after the TYR-PET in 20 patients. The calculated PSR of the tumours was 1.0{plus_minus}0.6 nmol/ml/min. This is in range with our animal experiments. The PSR in brain tissue of the contralateral hemisphere was 0.7{plus_minus}0.4 nmol/ml/min. Sixteen of the turnouts were correctly identified with TYR-PET. Also, 2 benign lesions were correctly identified. TYR-PET gave 1 false-positive (infarction) and 1 false-negative (astrocytoma of intermediate malignancy) result. In a few patients with extensive peri-tumoural edema on MRI/CT, additional tumour locations were found with TYR-PET, proven to be malignant on biopsy. In conclusion: TYR is applicable for the visualisation of brain tumours. The possibility of calculating a PSR allows its use in the evaluation of therapy.

  10. 18F-labeled Single-Stranded DNA Aptamer for PET Imaging of Protein Tyrosine Kinase-7 Expression

    PubMed Central

    Wang, Lu; Wang, Zhe; Yang, Xiangyu; Dewhurst, Andrew; Ma, Ying; Zhu, Guizhi; Niu, Gang; Kiesewetter, Dale O.; Vasdev, Neil; Liang, Steven H.; Chen, Xiaoyuan

    2016-01-01

    Protein tyrosine kinase-7 (PTK7), a member of receptor tyrosine kinase superfamily initially identified as colon carcinoma kinase-4 (CCK-4), is highly expressed in various human malignancies. Its expression was found to correlate with aggressive biological behaviors such as increased cell proliferation, invasiveness and migration. Despite the importance and unmet need of imaging PTK7 in vivo, there is currently no clinically-relevant method to visualize tumoral PTK7 expression noninvasively such as PET or SPECT. This study aims to develop a specific, selective and high affinity PET radioligand based on single-stranded DNA (ssDNA) aptamer to address this challenge. Methods Sgc8, a 41-oligonucleotide that targets to PTK7, was labeled with F-18 using a two-step radiochemical synthesis, which featured a direct one-step radiofluorination on the distinctive spirocyclic hypervalent iodine(III) precursor to give 18F-fluorobenzyl azide followed by copper mediated “click” conjugation with Sgc8-alkyne. 18F-Sgc8 was evaluated in vitro and in vivo in two cell lines, HCT116 and U87MG, which express high and low amounts of PTK7, respectively. Results Sgc8 was labeled efficiently with F-18 in an isolated radiochemical yield of 62 ± 2%, non-decay-corrected (ndc) based on 18F-fluorobenzyl azide. 18F-Tr-Sgc8 was found to possess high affinity binding to both cell lines, with IC50 values for HCT116 as 2.7 ± 0.6 nM and U87MG as 16.9 ± 2.1 nM. In vivo PET imaging clearly visualized PTK7 expression in HCT116 xenografted mice with tumor uptake of 0.76 ± 0.09 %ID/g at 30 min post-injection (p.i.) for the subcutaneous tumor model and greater than 1.5 %ID/g for the liver metastasis model. U87MG xenograft tumors had much lower tracer accumulation (0.13 ± 0.06 %ID/g at 30 min p.i.), which was consistent with the lower expression of PTK7 in this tumor model. The labeled aptamer was rapidly cleared from the blood through the kidneys and bladder to give high tumor-to-blood and tumor

  11. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family

    SciTech Connect

    Cool, D.E.; Tonks, N.K.; Charbonneau, H.; Walsh, K.A.; Fischer, E.H.; Krebs, E.G. )

    1989-07-01

    A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase. One positive clone was isolated and the nucleotide sequence was determined. It contained 1,305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3{prime} untranslated end, although a poly(A){sup +} tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues. This was supported by the synthesis of a M{sub r} 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low M{sub r} PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes.

  12. Anti-Group B Streptococcus Glycan-Conjugate Vaccines Using Pilus Protein GBS80 As Carrier and Antigen: Comparing Lysine and Tyrosine-directed Conjugation.

    PubMed

    Nilo, Alberto; Morelli, Laura; Passalacqua, Irene; Brogioni, Barbara; Allan, Martin; Carboni, Filippo; Pezzicoli, Alfredo; Zerbini, Francesca; Maione, Domenico; Fabbrini, Monica; Romano, Maria Rosaria; Hu, Qi-Ying; Margarit, Immaculada; Berti, Francesco; Adamo, Roberto

    2015-07-17

    Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design

  13. Modulation of protein tyrosine phosphatase activity alters the subunit assembly in native N-methyl-D-aspartate receptor complex.

    PubMed

    Ferrani-Kile, Karima; Leslie, Steven W

    2005-07-01

    The N-methyl-D-aspartate (NMDA) receptor is crucial for development and neuroplasticity as well as excitotoxicity. The biochemical basis of the disassembly and reassembly of NMDA receptor has never been reported. Using coimmunoprecipitation, Western blotting, and mass spectrometry, we show that inhibition of tyrosine phosphatases triggers disassembly of NR1, NR2A, and NR2B in cortical NMDA receptor complexes. Furthermore, the disassembly of the NMDA receptor subunits is immediate, dose-dependent, and reversible and seems to occur through mechanisms linked to Src kinases. Together, these results define a novel role for tyrosine phosphatases in the complex mechanism of NMDA receptor regulation. PMID:15837820

  14. Comparative proteomics of root plasma membrane proteins reveals the involvement of calcium signalling in NaCl-facilitated nitrate uptake in Salicornia europaea

    PubMed Central

    Nie, Lingling; Feng, Juanjuan; Fan, Pengxiang; Chen, Xianyang; Guo, Jie; Lv, Sulian; Bao, Hexigeduleng; Jia, Weitao; Tai, Fang; Jiang, Ping; Wang, Jinhui; Li, Yinxin

    2015-01-01

    Improving crop nitrogen (N) use efficiency under salinity is essential for the development of sustainable agriculture in marginal lands. Salicornia europaea is a succulent euhalophyte that can survive under high salinity and N-deficient habitat conditions, implying that a special N assimilation mechanism may exist in this plant. In this study, phenotypic and physiological changes of S. europaea were investigated under different nitrate and NaCl levels. The results showed that NaCl had a synergetic effect with nitrate on the growth of S. europaea. In addition, the shoot nitrate concentration and nitrate uptake rate of S. europaea were increased by NaCl treatment under both low N and high N conditions, suggesting that nitrate uptake in S. europaea was NaCl facilitated. Comparative proteomic analysis of root plasma membrane (PM) proteins revealed 81 proteins, whose abundance changed significantly in response to NaCl and nitrate. These proteins are involved in metabolism, cell signalling, transport, protein folding, membrane trafficking, and cell structure. Among them, eight proteins were calcium signalling components, and the accumulation of seven of the above-mentioned proteins was significantly elevated by NaCl treatment. Furthermore, cytosolic Ca2+ concentration ([Ca2+]cyt) was significantly elevated in S. europaea under NaCl treatment. The application of the Ca2+ channel blocker LaCl3 not only caused a decrease in nitrate uptake rate, but also attenuated the promoting effects of NaCl on nitrate uptake rates. Based on these results, a possible regulatory network of NaCl-facilitated nitrate uptake in S. europaea focusing on the involvement of Ca2+ signalling was proposed. PMID:25956883

  15. Comparative proteomics of root plasma membrane proteins reveals the involvement of calcium signalling in NaCl-facilitated nitrate uptake in Salicornia europaea.

    PubMed

    Nie, Lingling; Feng, Juanjuan; Fan, Pengxiang; Chen, Xianyang; Guo, Jie; Lv, Sulian; Bao, Hexigeduleng; Jia, Weitao; Tai, Fang; Jiang, Ping; Wang, Jinhui; Li, Yinxin

    2015-08-01

    Improving crop nitrogen (N) use efficiency under salinity is essential for the development of sustainable agriculture in marginal lands. Salicornia europaea is a succulent euhalophyte that can survive under high salinity and N-deficient habitat conditions, implying that a special N assimilation mechanism may exist in this plant. In this study, phenotypic and physiological changes of S. europaea were investigated under different nitrate and NaCl levels. The results showed that NaCl had a synergetic effect with nitrate on the growth of S. europaea. In addition, the shoot nitrate concentration and nitrate uptake rate of S. europaea were increased by NaCl treatment under both low N and high N conditions, suggesting that nitrate uptake in S. europaea was NaCl facilitated. Comparative proteomic analysis of root plasma membrane (PM) proteins revealed 81 proteins, whose abundance changed significantly in response to NaCl and nitrate. These proteins are involved in metabolism, cell signalling, transport, protein folding, membrane trafficking, and cell structure. Among them, eight proteins were calcium signalling components, and the accumulation of seven of the above-mentioned proteins was significantly elevated by NaCl treatment. Furthermore, cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was significantly elevated in S. europaea under NaCl treatment. The application of the Ca(2+) channel blocker LaCl3 not only caused a decrease in nitrate uptake rate, but also attenuated the promoting effects of NaCl on nitrate uptake rates. Based on these results, a possible regulatory network of NaCl-facilitated nitrate uptake in S. europaea focusing on the involvement of Ca(2+) signalling was proposed. PMID:25956883

  16. Poly(colloid)s: "Polymerization" of Poly(l-tyrosine)-silica Composite Particles through the Photoinduced Cross-Linking of Unmodified Proteins Method.

    PubMed

    Rosu, Cornelia; Cueto, Rafael; Russo, Paul S

    2016-08-23

    Photoinduced cross-linking of unmodified proteins, PICUP, was extended to core-shell silica-polypeptide composite particles to produce poly(colloid)s. Silica particles coated with poly(l-tyrosine), PTYR-SiO2, served as the monomer units. The PICUP reaction accomplished the formation of dityrosil linkages between the tyrosine units by illumination of photo-oxidizing ruthenium(II) bipyridyl catalyst under physiological conditions. The PICUP method was compared with an enzymatic route intermediated by horseradish peroxidase as catalyst. The PTYR-SiO2 particles feature high PTYR content in the shell, which facilitated the formation of heavily cross-linked but unstructured aggregates. After magnetic alignment of superparamagnetic PTYR-SiO2-cobalt composite particles, only the PICUP approach enabled the preparation of isolated chain-like poly(colloid)s. The cross-linking products were confirmed by FTIR. The native secondary structure of poly(l-tyrosine) is preserved in these poly(colloid)s. Because the PICUP reaction does not require the modification of the polypeptide structure, the cross-linked PTYR will retain its characteristic functions as a poly(amino acid). The PICUP method opens the door to a variety of PTYR-based poly(colloid) architectures. PMID:27504929

  17. New potential eukaryotic substrates of the mycobacterial protein tyrosine phosphatase PtpA: hints of a bacterial modulation of macrophage bioenergetics state.

    PubMed

    Margenat, Mariana; Labandera, Anne-Marie; Gil, Magdalena; Carrion, Federico; Purificação, Marcela; Razzera, Guilherme; Portela, María Magdalena; Obal, Gonzalo; Terenzi, Hernán; Pritsch, Otto; Durán, Rosario; Ferreira, Ana María; Villarino, Andrea

    2015-01-01

    The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution. PMID:25743628

  18. Role of Protein Tyrosine Phosphatase Non-Receptor Type 7 in the Regulation of TNF-α Production in RAW 264.7 Macrophages

    PubMed Central

    Seo, Huiyun; Lee, In-Seon; Park, Jae Eun; Park, Sung Goo; Lee, Do Hee; Park, Byoung Chul; Cho, Sayeon

    2013-01-01

    Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7), a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs). Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS) that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA) analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2) and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages. PMID:24265715

  19. Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study.

    PubMed

    Sarhan, Adil R; Patel, Trushar R; Creese, Andrew J; Tomlinson, Michael G; Hellberg, Carina; Heath, John K; Hotchin, Neil A; Cunningham, Debbie L

    2016-06-01

    Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling. PMID:27074791

  20. Syk protein tyrosine kinase involves PECAM-1 signaling through tandem immunotyrosine inhibitory motifs in human THP-1 macrophages.

    PubMed

    Wang, Junchen; Wu, Yanling; Hu, Hai; Wang, Weimin; Lu, Ying; Mao, Huiming; Liu, Xiaoqing; Liu, Zhongmin; Chen, Bing-guan

    2011-01-01

    Although recent evidence supports a functional relationship between platelet endothelial cell adhesion molecule (PECAM-1) and Syk tyrosine kinase, little is known about the interaction of Syk with PECAM-1. We report that down-regulation of Syk inhibits the spreading of human THP-1 macrophage cells. Moreover, our data indicate that Syk binds PECAM-1 through its immune tyrosine-based inhibitory motif (ITIM), and dual phosphorylation of the ITIM domain of PECAM-1 leads to activation of Syk. Our results indicate that the distance between the phosphotyrosines could be up to 22 amino acids in length, depending on the conformational flexibility, and that the dual ITIM tyrosine motifs of PECAM-1 facilitate immunoreceptor tyrosine-based activation motif-like signaling. The preferential binding of PECAM-1 to Src homology region 2 domain-containing phosphatase-2 or Syk may depend on their relative affinities, and could provide a mechanism by which signal transduction from PECAM-1 is internally regulated by both positive and negative signaling enzymes. PMID:22000807

  1. Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets*

    PubMed Central

    Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A.; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P.

    2015-01-01

    Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. PMID:25767114

  2. PTPN13, a Fas-associated protein tyrosine phosphatase, is located on the long arm of chromosome 4 at band q21.3

    SciTech Connect

    Inazawa, Johji; Ariyama, Takeshi; Abe, Tatsuo

    1996-01-15

    PTPN13 is a protein tyrosine phosphatase that associates with the C-terminal negative regulatory domain in the Fas (APO-1/CD95) receptor. The PTPN13 protein contains six GLGF repeats that have been found in the rat postsynaptic density protein (PSD-95) and the Drosophila tumor suppressor protein, lethal-(1)-disclarge-1 (dlg-1). The localization of the PTPN13 gene to human chromosome 4q21.3 was determined by both FISH and PCR analysis of somatic cell hybrids. This 4q21.3 chromosomal region contains a gene for autosomal dominant polycystic kidney disease as well as the region frequently deleted in liver and ovarian cancers, suggesting that PTPN13 is a candidate for one of the putative tumor suppressor genes on the long arm of chromosome 4. 21 refs., 1 fig.

  3. The LIM domain only 4 protein is a metabolic responsive inhibitor of protein tyrosine phosphatase 1B that controls hypothalamic leptin signaling.

    PubMed

    Pandey, Nihar R; Zhou, Xun; Qin, Zhaohong; Zaman, Tariq; Gomez-Smith, Mariana; Keyhanian, Kianoosh; Anisman, Hymie; Brunel, Jean Michel; Stewart, Alexandre F R; Chen, Hsiao-Huei

    2013-07-31

    Protein tyrosine phosphatase 1B (PTP1B) counteracts leptin signaling and is a therapeutic target for obesity and diabetes. Here we found that LIM domain only 4 (LMO4) inhibits PTP1B activity by increasing the oxidized inactive form of PTP1B. Mice with neuronal ablation of LMO4 have elevated PTP1B activity and impaired hypothalamic leptin signaling, and a PTP1B inhibitor normalized PTP1B activity and restored leptin control of circulating insulin levels. LMO4 is palmitoylated at its C-terminal cysteine, and deletion of this residue prevented palmitoylation and retention of LMO4 at the endoplasmic reticulum and abolished its inhibitory effect on PTP1B. Importantly, LMO4 palmitoylation is sensitive to metabolic stress; mice challenged with a brief high-fat diet or acute intracerebroventricular infusion of saturated fatty acid had less palmitoylated LMO4, less oxidized PTP1B, and increased PTP1B activity in the hypothalamus. Thus, unleashed PTP1B activity attributable to loss of LMO4 palmitoylation may account for rapid loss of central leptin signaling after acute exposure to saturated fat. PMID:23904601

  4. Effect of the tyrosine kinase inhibitor lapatinib on CUB-domain containing protein (CDCP1)-mediated breast cancer cell survival and migration

    SciTech Connect

    Seidel, Jeanette; Kunc, Klaudia; Possinger, Kurt; Jehn, Christian; Lueftner, Diana

    2011-10-14

    Highlights: {yields} CDCP1 downregulation reduces anchorage free survival of breast cancer cells. {yields} Anoikis of CDCP1-positive breast cancer cells is increased after CDCP1 downregulation. {yields} CDCP1 knockdown decreases migration and extensively reduces invasiveness in vitro. {yields} Proliferation rate does not correlate with CDCP1 expression. {yields} Lapatinib does not influence tyrosine kinases of CDCP1 signal transduction. -- Abstract: The surface receptor CUB domain-containing protein 1 (CDCP1) is highly expressed in several adenocarcinomas and speculated to participate in anchorage-independent cell survival and cell motility. Tyrosine kinase phosphorylation seems to be crucial for intracellular signaling of CDCP1. Lapatinib, a tyrosine kinase inhibitor (TKI), is approved for treatment of HER-2/neu overexpressing metastatic breast cancer and functions by preventing autophosphorylation following HER-2/neu receptor activation. This study aimed to investigate the effect of CDCP1 expression on anchorage-independent growth and cell motility of breast cancer cells. Moreover, studies were performed to examine if lapatinib provided any beneficial effect on HER-2/neu{sup (+)/-}/CDCP1{sup +} breast cancer cell lines. In our studies, we affirmed that CDCP1 prevents cells from undergoing apoptosis when cultured in the absence of cell-substratum anchorage and that migratory and invasive properties of these cells were decreased when CDCP1 was down-regulated. However, only HER-2/neu{sup +}, but not HER-2/neu{sup (+)/-} cells showed decreased proliferation and invasion and an enhanced level of apoptosis towards loss of anchorage when treated with lapatinib. Therefore, we conclude that CDCP1 might be involved in regulating