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Sample records for protein tyrosine nitration

  1. Protein tyrosine nitration

    PubMed Central

    Chaki, Mounira; Leterrier, Marina; Barroso, Juan B

    2009-01-01

    Nitric oxide metabolism in plant cells has a relative short history. Nitration is a chemical process which consists of introducing a nitro group (-NO2) into a chemical compound. in biological systems, this process has been found in different molecules such as proteins, lipids and nucleic acids that can affect its function. This mini-review offers an overview of this process with special emphasis on protein tyrosine nitration in plants and its involvement in the process of nitrosative stress. PMID:19826215

  2. 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.

  3. Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects.

    PubMed

    Batthyány, Carlos; Bartesaghi, Silvina; Mastrogiovanni, Mauricio; Lima, Analía; Demicheli, Verónica; Radi, Rafael

    2017-03-01

    "Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function. Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation. The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.

  4. Protein tyrosine nitration in pea roots during development and senescence

    PubMed Central

    Corpas, Francisco J.

    2013-01-01

    Protein tyrosine nitration is a post-translational modification mediated by reactive nitrogen species (RNS) that is associated with nitro-oxidative damage. No information about this process is available in relation to higher plants during development and senescence. Using pea plants at different developmental stages (ranging from 8 to 71 days), tyrosine nitration in the main organs (roots, stems, leaves, flowers, and fruits) was analysed using immunological and proteomic approaches. In the roots of 71-day-old senescent plants, nitroproteome analysis enabled the identification a total of 16 nitrotyrosine-immunopositive proteins. Among the proteins identified, NADP-isocitrate dehydrogenase (ICDH), an enzyme involved in the carbon and nitrogen metabolism, redox regulation, and responses to oxidative stress, was selected to evaluate the effect of nitration. NADP-ICDH activity fell by 75% during senescence. Analysis showed that peroxynitrite inhibits recombinant cytosolic NADP-ICDH activity through a process of nitration. Of the 12 tyrosines present in this enzyme, mass spectrometric analysis of nitrated recombinant cytosolic NADP-ICDH enabled this study to identify the Tyr392 as exclusively nitrated by peroxynitrite. The data as a whole reveal that protein tyrosine nitration is a nitric oxide-derived PTM prevalent throughout root development and intensifies during senescence. PMID:23362300

  5. 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

  6. Protein tyrosine nitration in plants: Present knowledge, computational prediction and future perspectives.

    PubMed

    Kolbert, Zsuzsanna; Feigl, Gábor; Bordé, Ádám; Molnár, Árpád; Erdei, László

    2017-04-01

    Nitric oxide (NO) and related molecules (reactive nitrogen species) regulate diverse physiological processes mainly through posttranslational modifications such as protein tyrosine nitration (PTN). PTN is a covalent and specific modification of tyrosine (Tyr) residues resulting in altered protein structure and function. In the last decade, great efforts have been made to reveal candidate proteins, target Tyr residues and functional consequences of nitration in plants. This review intends to evaluate the accumulated knowledge about the biochemical mechanism, the structural and functional consequences and the selectivity of plants' protein nitration and also about the decomposition or conversion of nitrated proteins. At the same time, this review emphasizes yet unanswered or uncertain questions such as the reversibility/irreversibility of tyrosine nitration, the involvement of proteasomes in the removal of nitrated proteins or the effect of nitration on Tyr phosphorylation. The different NO producing systems of algae and higher plants raise the possibility of diversely regulated protein nitration. Therefore studying PTN from an evolutionary point of view would enrich our present understanding with novel aspects. Plant proteomic research can be promoted by the application of computational prediction tools such as GPS-YNO2 and iNitro-Tyr software. Using the reference Arabidopsis proteome, Authors performed in silico analysis of tyrosine nitration in order to characterize plant tyrosine nitroproteome. Nevertheless, based on the common results of the present prediction and previous experiments the most likely nitrated proteins were selected thus recommending candidates for detailed future research. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. Protein Tyrosine Nitration: Biochemical Mechanisms and Structural Basis of its Functional Effects

    PubMed Central

    Radi, Rafael

    2012-01-01

    CONSPECTUS The nitration of protein tyrosine residues to 3-nitrotyrosine represents an oxidative postranslational modification that unveils the disruption of nitric oxide (•NO) signaling and metabolism towards pro-oxidant processes. Indeed, excess levels of reactive oxygen species in the presence of •NO or •NO-derived metabolites lead to the formation of nitrating species such as peroxynitrite. Thus, protein 3-nitrotyrosine has been established as a biomarker of cell, tissue and systemic “nitroxidative stress”. Moreover, tyrosine nitration modifies key properties of the amino acid (i.e. phenol group pKa, redox potential, hydrophobicity and volume). Thus, the incorporation of a nitro group (−NO2) to protein tyrosines can lead to profound structural and functional changes, some of which contribute to altered cell and tissue homeostasis. In this Account, I describe our current efforts to define 1) biologically-relevant mechanisms of protein tyrosine nitration and 2) how this modification can cause changes in protein structure and function at the molecular level. First, the relevance of protein tyrosine nitration via free radical-mediated reactions (in both peroxynitrite-dependent or independent pathways) involving the intermediacy of tyrosyl radical (Tyr•) will be underscored. This feature of the nitration process becomes critical as Tyr• can take variable fates, including the formation of 3-nitrotyrosine. Fast kinetic techniques, electron paramagnetic resonance (EPR) studies, bioanalytical methods and kinetic simulations have altogether assisted to characterize and fingerprint the reactions of tyrosine with peroxynitrite and one-electron oxidants and its further evolution to 3-nitrotyrosine. Recent findings show that nitration of tyrosines in proteins associated to biomembranes is linked to the lipid peroxidation process via a connecting reaction that involves the one-electron oxidation of tyrosine by lipid peroxyl radicals (LOO•). Second

  8. Site selectivity for protein tyrosine nitration: insights from features of structure and topological network.

    PubMed

    Cheng, Shangli; Lian, Baofeng; Liang, Juan; Shi, Ting; Xie, Lu; Zhao, Yi-Lei

    2013-11-01

    Tyrosine nitration is a covalent post-translational modification, which regulates protein functions such as hindering tyrosine phosphorylation and affecting essential signal transductions in cells. Based on up-to-date proteomics data, tyrosine nitration appears to be a highly selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. Quite a few investigations included the protein structural information from the RCSB PDB database, where near 100,000 high-quality three-dimensional structures are available. In this work, we analyzed the local protein structures and amino acid topological networks of the nitrated and non-nitrated tyrosine sites in nitrated proteins, including neighboring atomic distribution, amino acid pair (AAP) and amino acid triangle (AAT). It has been found that aromatic and aliphatic residues, particularly with large volume, aromatic, aliphatic, or acidic side chains, are disfavored for the nitration. After integrating these structural features and topological network features with traditional sequence features, the predictive model achieves a sensitivity of 63.30% and a specificity of 92.24%, resulting in a much better accuracy compared to the previous models with only protein sequence information. Our investigation implies that the site selectivity may stem from a more open, hydrophilic and high-pH chemical environment around the tyrosine residue.

  9. Protein tyrosine nitration in higher plants grown under natural and stress conditions

    PubMed Central

    Corpas, Francisco J.; Palma, José M.; del Río, Luis A.; Barroso, Juan B.

    2013-01-01

    Protein tyrosine nitration is a post-translational modification (PTM) mediated by reactive nitrogen species (RNS) that is linked to nitro-oxidative damages in plant cells. During the last decade, the identification of proteins undergoing this PTM under adverse environmental conditions has increased. However, there is also a basal endogenous nitration which seems to have a regulatory function. The technological advances in proteome analysis have allowed identifying these modified proteins and have shown that the number and identity of the nitrated proteins change among plant species, analysed organs and growing/culture conditions. In this work, the current knowledge of protein tyrosine nitration in higher plants under different situations is reviewed. PMID:23444154

  10. Protein Tyrosine Nitration during Development and Abiotic Stress Response in Plants

    PubMed Central

    Mata-Pérez, Capilla; Begara-Morales, Juan C.; Chaki, Mounira; Sánchez-Calvo, Beatriz; Valderrama, Raquel; Padilla, María N.; Corpas, Francisco J.; Barroso, Juan B.

    2016-01-01

    In recent years, the study of nitric oxide (NO) in plant systems has attracted the attention of many researchers. A growing number of investigations have shown the significance of NO as a signal molecule or as a molecule involved in the response against (a)biotic processes. NO can be responsible of the post-translational modifications (NO-PTM) of target proteins by mechanisms such as the nitration of tyrosine residues. The study of protein tyrosine nitration during development and under biotic and adverse environmental conditions has increased in the last decade; nevertheless, there is also an endogenous nitration which seems to have regulatory functions. Moreover, the advance in proteome techniques has enabled the identification of new nitrated proteins, showing the high variability among plant organs, development stage and species. Finally, it may be important to discern between a widespread protein nitration because of greater RNS content, and the specific nitration of key targets which could affect cell-signaling processes. In view of the above point, we present a mini-review that offers an update about the endogenous protein tyrosine nitration, during plant development and under several abiotic stress conditions. PMID:27895655

  11. 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.

  12. Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls.

    PubMed

    Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana M; Carreras, Alfonso; López-Jaramillo, Javier; Luque, Francisco; Palma, José M; Pedrajas, José R; Begara-Morales, Juan C; Sánchez-Calvo, Beatriz; Gómez-Rodríguez, María V; Corpas, Francisco J; Barroso, Juan B

    2009-01-01

    Tyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO(2)-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3'-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO(2)-Tyr in hypocotyls was 0.56 micromol mg(-1) protein and 0.19 pmol mg(-1) protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S-adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be

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

    PubMed

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

    2015-09-01

    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. 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. 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. The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S

  14. 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

  15. 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.

  16. 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.

  17. Copper reduces striatal protein nitration and tyrosine hydroxylase inactivation induced by MPP+ in rats.

    PubMed

    Rubio-Osornio, M; Montes, S; Pérez-Severiano, F; Aguilera, P; Floriano-Sánchez, E; Monroy-Noyola, A; Rubio, C; Ríos, C

    2009-06-01

    Striatal administration of 1-methyl-4-phenylpyridinium (MPP(+)), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson's disease. Copper acts as a prosthetic group of several antioxidant enzymes and recent data show that copper attenuated MPP(+)-evoked neurotoxicity. We evaluated the effect of copper (as a supplement) upon proteins nitration (60 kDa) and tyrosine hydroxylase (TH) inactivation induced by MPP(+) (10 microg/8 microL) injection into the rat striatum. Copper pretreatment (10 micromol/kg i.p.) prevented both MPP(+)-induced proteins nitration and TH inactivation. Copper treatment also prevented the dopamine-depleting effect of MPP(+) injection. Those results were accompanied by a significant reduction of enzymatic activity of the constitutive nitric oxide synthase (cNOS), whereas, the protein levels of the three isoforms of NOS remained unchanged. Results indicate that the effect of copper against MPP(+)-induced proteins nitration and TH inactivation in the striatum of rat may be mediated by a reduction of cNOS activity.

  18. Protein tyrosine nitration is associated with cold- and drug-resistant microtubules in neuronal-like PC12 cells.

    PubMed

    Cappelletti, Graziella; Maggioni, Maria Grazia; Ronchi, Cristina; Maci, Rosalba; Tedeschi, Gabriella

    2006-06-19

    Among the myriad of cellular functions played by nitric oxide in the brain, there is increasing evidence that nitric oxide might be a primary player in the program of neurogenesis and neuronal differentiation. We have recently reported that tyrosine nitration of proteins is implicated in the signaling pathway triggered by nitric oxide during NGF-induced neuronal differentiation in PC12 cells. The cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and the cytoskeletal proteins alpha-tubulin and tau are two of the targets. Here, we have studied the association of nitrated proteins with the cytoskeletal fraction in differentiating PC12 cells following exposure to microtubule depolymerising treatments and found that nitration of the cytoskeleton correlates with the increased microtubule stability underlying the progression of neuronal differentiation. These results suggest a novel functional role for nitrated cytoskeletal proteins in the stabilisation of neurites occurring in differentiated neuronal cells.

  19. Up-regulation of protein tyrosine nitration in methamphetamine-induced neurotoxicity through DDAH/ADMA/NOS pathway.

    PubMed

    Zhang, Fu; Chen, Ling; Liu, Chao; Qiu, Pingming; Wang, Aifeng; Li, Lizeng; Wang, Huijun

    2013-06-01

    Protein tyrosine nitration is an important post-translational modification mediated by nitric oxide (NO) associated oxidative stress, occurring in a variety of neurodegenerative diseases. In our previous study, an elevated level of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein was observed in different brain regions of acute methamphetamine (METH) treated rats, indicating the possibility of an enhanced expression of protein nitration that is mediated by excess NO through the DDAH1/ADMA (Asymmetric Dimethylated l-arginine)/NOS (Nitric Oxide Synthase) pathway. In the present study, proteomic methods, including stable isotope labeling with amino acids in cell culture (SILAC) and two dimensional electrophoresis, were used to determine the relationship between protein nitration and METH induced neurotoxicity in acute METH treated rats and PC12 cells. We found that acute METH administration evokes a positive activation of DDAH1/ADMA/NOS pathway and results in an over-production of NO in different brain regions of rat and PC12 cells, whereas the whole signaling could be repressed by DDAH1 inhibitor N(ω)-(2-methoxyethyl)-arginine (l-257). In addition, enhanced expressions of 3 nitroproteins were identified in rat striatum and increased levels of 27 nitroproteins were observed in PC12 cells. These nitrated proteins are key factors for Cdk5 activation, cytoskeletal structure, ribosomes function, etc. l-257 also displayed significant protective effects against METH-induced protein nitration, apoptosis and cell death. The overall results illustrate that protein nitration plays a significant role in the acute METH induced neurotoxicity via the activation of DDAH1/ADMA/NOS pathway.

  20. Iron overload-induced rat liver injury: Involvement of protein tyrosine nitration and the effect of baicalin.

    PubMed

    Zhang, Yan; Huang, Yi; Deng, Xiaorong; Xu, Yan; Gao, Zhonghong; Li, Hailing

    2012-04-05

    Baicalin has been reported to protect against liver injury in iron-overload mice, however, the mechanisms underlying the hepatoprotective properties of baicalin are poorly understood. In this study, we systematically studied the protective effect of baicalin on iron overload induced liver injury, as well as the underlying mechanism based on nitrative stress in rat model. We found that when iron overload rats (500mgiron/kg) were fed baicalin-containing diet (0.3% and 1% w/w) for 45days, baicalin dose dependently protected against iron overload induced liver injury, including alleviation of hepatic pathological damage, decrease of SOD activity, iron content, carbonyl content, and the thiobarbituric acid-reactive substances level in hepatic tissues. It also increased serum iron content, SH content and GPx activity, decreased serum ALT and AST activities. Immunohistochemistry and immunoprecipitation analysis revealed that baicalin could also inhibit iron overload induced protein tyrosine nitration in liver. Moreover, in iron overload rat liver, we found that baicalin decreased the iron overload increased level of glutathione-S-transferases (GSTs) expression, oxidation and nitration. These results suggest that not only oxidative stress, but also nitrative stress, is involved in iron overload induced liver injury, and the underlying mechanism might partially relate to the involvement of GSTs expression and post-translational modification. Baicalin can effectively prevent iron overload caused abnormality and can be a candidate medicine for iron overload diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Differential hepatic protein tyrosine nitration of mouse due to aging - effect on mitochondrial energy metabolism, quality control machinery of the endoplasmic reticulum and metabolism of drugs.

    PubMed

    Marshall, Adrienne; Lutfeali, Reshma; Raval, Alpan; Chakravarti, Deb N; Chakravarti, Bulbul

    2013-01-04

    Aging is the inevitable fate of life which leads to the gradual loss of functions of different organs and organelles of all living organisms. The liver is no exception. Oxidative damage to proteins and other macromolecules is widely believed to be the primary cause of aging. One form of oxidative damage is tyrosine nitration of proteins, resulting in the potential loss of their functions. In this study, the effect of age on the nitration of tyrosine in mouse liver proteins was examined. Liver proteins from young (19-22 weeks) and old (24 months) C57/BL6 male mice were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes. Proteins undergoing tyrosine nitration were identified using anti-nitrotyrosine antibody. Three different protein bands were found to contain significantly increased levels of nitrotyrosine in old mice (Wilconxon rank-sum test, p<0.05). Electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC-MS/MS) was used to identify the proteins in these bands, which included aldehyde dehydrogenase 2, Aldehyde dehydrogenase family 1, subfamily A1, ATP synthase, H(+) transporting, mitochondrial F1 complex, β subunit, selenium-binding protein 2, and protein disulfide-isomerase precursor. The possible impairment of their functions can lead to altered hepatic activity and have been discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Nitration of Hsp90 on Tyrosine 33 Regulates Mitochondrial Metabolism*

    PubMed Central

    Franco, Maria C.; Ricart, Karina C.; Gonzalez, Analía S.; Dennys, Cassandra N.; Nelson, Pascal A.; Janes, Michael S.; Mehl, Ryan A.; Landar, Aimee; Estévez, Alvaro G.

    2015-01-01

    Peroxynitrite production and tyrosine nitration are present in several pathological conditions, including neurodegeneration, stroke, aging, and cancer. Nitration of the pro-survival chaperone heat shock protein 90 (Hsp90) in position 33 and 56 induces motor neuron death through a toxic gain-of-function. Here we show that nitrated Hsp90 regulates mitochondrial metabolism independently of the induction of cell death. In PC12 cells, a small fraction of nitrated Hsp90 was located on the mitochondrial outer membrane and down-regulated mitochondrial membrane potential, oxygen consumption, and ATP production. Neither endogenous Hsp90 present in the homogenate nor unmodified and fully active recombinant Hsp90 was able to compete with the nitrated protein for the binding to mitochondria. Moreover, endogenous or recombinant Hsp90 did not prevent the decrease in mitochondrial activity but supported nitrated Hsp90 mitochondrial gain-of-function. Nitrotyrosine in position 33, but not in any of the other four tyrosine residues prone to nitration in Hsp90, was sufficient to down-regulate mitochondrial activity. Thus, in addition to induction of cell death, nitrated Hsp90 can also regulate mitochondrial metabolism, suggesting that depending on the cell type, distinct Hsp90 nitration states regulate different aspects of cellular metabolism. This regulation of mitochondrial homeostasis by nitrated Hsp90 could be of particular relevance in cancer cells. PMID:26085096

  3. Angiotensin-converting enzyme inhibition curbs tyrosine nitration of mitochondrial proteins in the renal cortex during the early stage of diabetes mellitus in rats.

    PubMed

    Ishii, Naohito; Carmines, Pamela K; Yokoba, Masanori; Imaizumi, Hiroyuki; Ichikawa, Tsuyoshi; Ikenagasa, Hideki; Kodera, Yoshio; Oh-Ishi, Masamichi; Aoki, Yoshikazu; Maeda, Tadakazu; Takenaka, Tsuneo; Katagiri, Masato

    2013-04-01

    Experiments were performed to evaluate the hypothesis that ACE (angiotensin-converting enzyme) inhibition (enalapril) suppresses 3-NT (3-nitrotyrosine) production in the renal cortex during the early stage of Type 1 DM (diabetes mellitus) in the rat. Enalapril was administered chronically for 2 weeks to subsets of STZ (streptozotocin)-induced DM and vehicle-treated sham rats. O(2)(-) (superoxide anion) and NO(x) (nitrate+nitrite) levels were measured in the media bathing renal cortical slices after 90 min incubation in vitro. SOD (superoxide dismutase) activity and 3-NT content were measured in the renal cortex homogenate. Renal cortical nitrated protein was identified by proteomic analysis. Renal cortical production of O(2)(-) and 3-NT was increased in DM rats; however, enalapril suppressed these changes. DM rats also exhibited elevated renal cortical NO(x) production and SOD activity, and these changes were magnified by enalapril treatment. 2-DE (two-dimensional gel electrophoresis)-based Western blotting revealed more than 20 spots with positive 3-NT immunoreactivity in the renal cortex of DM rats. Enalapril treatment blunted the DM-induced increase in tyrosine nitration of three proteins ACO2, GDH1 and MMSDH (aconitase 2, glutamate dehydrogenase 1 and methylmalonate-semialdehyde dehydrogenase), each of which resides in mitochondria. These data are consistent with enalapril preventing DM-induced tyrosine nitration of mitochondrial proteins by a mechanism involving suppression of oxidant production and enhancement of antioxidant capacity, including SOD activation.

  4. 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

  5. Tyrosine oxidation and nitration in transmembrane peptides is connected to lipid peroxidation.

    PubMed

    Bartesaghi, Silvina; Herrera, Daniel; Martinez, Débora M; Petruk, Ariel; Demicheli, Verónica; Trujillo, Madia; Martí, Marcelo A; Estrín, Darío A; Radi, Rafael

    2017-05-15

    Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by tyrosine nitration

    PubMed Central

    Palamalai, Vikram; Miyagi, Masaru

    2010-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifaceted protein that is involved in numerous processes including glycolysis, translational silencing, transcriptional regulation of specific genes, and acting as a nitric oxide sensor. The precise mechanism on how GAPDH is targeted to these different roles is unclear but believed to involve specific posttranslational modification to the protein. Numerous studies have demonstrated that GAPDH is a target for tyrosine nitration. However, the site of modification and the molecular consequence have not been defined. Rabbit GAPDH with a reversibly protected catalytic cysteine was nitrated in vitro with tetranitromethane, resulting in complete loss of GAPDH catalytic activity. Nitration was estimated as 0.32 mol of nitrotyrosine residue per mole of GAPDH. Mass spectrometry analysis of nitrated GAPDH indicated that Tyr311 and Tyr317 were the sole sites of nitration. The X-ray crystal structure revealed that the distances between Tyr311 and Tyr317 and the cofactor nicotinamide adenine dinucleotide (NAD+) were less than 7.2 and 3.7 Å, respectively, implying that nitration of these two residues may affect NAD+ binding. This possibility was assessed using an NAD+ binding assay, which showed that nitrated GAPDH was incapable of binding NAD+. Thus, these results strongly suggest that Tyr311 and Tyr317 nitration prohibits NAD+ binding, leading to the loss of catalytic activity. PMID:20014444

  7. Temporal patterns of tyrosine nitration in embryo heart development

    PubMed Central

    Viera, Liliana; Radmilovich, Milka; Vargas, Marcelo R.; Dennys, Cassandra N.; Wilson, Landon; Barnes, Stephen; Franco, Maria Clara; Beckman, Joseph S.; Estévez, Alvaro G.

    2012-01-01

    Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide (NO) production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF shows that nitration occurs in actin, myosin, and the mitochondrial ATP synthase alpha chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development. PMID:23195686

  8. Lipid peroxyl radicals mediate tyrosine dimerization and nitration in membranes

    PubMed Central

    Bartesaghi, Silvina; Wenzel, Jorge; Trujillo, Madia; López, Marcos; Joseph, Joy; Kalyanaraman, Balaraman; Radi, Rafael

    2012-01-01

    Protein tyrosine dimerization and nitration by biologically-relevant oxidants usually depend on the intermediate formation of tyrosyl radical (•Tyr). In the case of tyrosine oxidation in proteins associated to hydrophobic biocompartments, the participation of unsaturated fatty acids in the process must be considered since they typically constitute preferential targets for the initial oxidative attack. Thus, we postulate that lipid-derived radicals mediate the one-electron oxidation of tyrosine to •Tyr, which can afterwards react with another •Tyr or with nitrogen dioxide (•NO2) to yield 3,3´-dityrosine or 3-nitrotyrosine within the hydrophobic structure, respectively. To test this hypothesis, we have studied tyrosine oxidation in saturated and unsaturated fatty acid-containing phosphatidylcholine (PC) liposomes with an incorporated hydrophobic tyrosine analog BTBE (N-t- BOC L-tyrosine tert-butyl ester) and its relationship with lipid peroxidation promoted by three oxidations systems, namely peroxynitrite, hemin and 2,2´-azobis (2-amidinopropane) hydrochloride (ABAP). In all cases, significant tyrosine (BTBE) oxidation was seen in unsaturated PC liposomes, in a way that was largely decreased at low oxygen concentrations. Tyrosine oxidation levels paralleled those of lipid peroxidation (i.e. malondialdehyde and lipid hydroperoxides) and lipid-derived radicals and BTBE phenoxyl radicals were simultaneously detected by ESR-spin trapping, supporting an association between the two processes. Indeed, α-tocopherol, a known reactant with lipid peroxyl radicals (LOO•), inhibited both tyrosine oxidation and lipid peroxidation induced by all three oxidation systems. Moreover, oxidant-stimulated liposomal oxygen consumption was dose-dependently inhibited by BTBE but not by its phenylalanine analog, BPBE (N-t-BOC L-phenylalaline tert-butyl ester), providing a direct evidence for the reaction between LOO• and the phenol moiety in BTBE, with an estimated second

  9. Electron capture dissociation mass spectrometry of tyrosine nitrated peptides.

    PubMed

    Jones, Andrew W; Mikhailov, Victor A; Iniesta, Jesus; Cooper, Helen J

    2010-02-01

    In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification. Here, we have investigated the electron capture dissociation (ECD) and collision-induced dissociation (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains.

  10. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.

    PubMed

    Ischiropoulos, H; Zhu, L; Chen, J; Tsai, M; Martin, J C; Smith, C D; Beckman, J S

    1992-11-01

    Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.

  11. Immunoglobulins against Tyrosine Nitrated Epitopes in Coronary Artery Disease

    PubMed Central

    Thomson, Leonor; Tenopoulou, Margarita; Lightfoot, Richard; Tsika, Epida; Parastatidis, Ioannis; Martinez, Marissa; Greco, Todd M.; Doulias, Paschalis-Thomas; Wu, Yuping; Tang, W. H. Wilson; Hazen, Stanley L.; Ischiropoulos, Harry

    2012-01-01

    Background Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine nitrated proteins, a footprint of oxygen and nitrogen derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of coronary artery disease (CAD) subjects. However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored. Methods and Results Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of CAD subjects. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in CAD patients (n=374) and subjects without CAD (non CAD controls, n=313). A ten-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in the CAD subjects (3.75 ± 1.8 μg antibody Eq/mL plasma vs. 0.36 ± 0.8 μg antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD. Conclusions The results of this cross sectional study suggest that post-translational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neoepitopes for elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD. PMID:23081989

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

    PubMed

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

    2013-02-28

    Protein tyrosine nitration is a post-translational modification (PTM) mediated by reactive nitrogen species (RNS) and it is a new area of research in higher plants. Previously, it was demonstrated that the exposition of sunflower (Helianthus annuus L.) seedlings to high temperature (HT) caused both oxidative and nitrosative stress. The nitroproteome analysis under this stress condition showed the induction of 13 tyrosine-nitrated proteins being the carbonic anhydrase (CA) one of these proteins. The analysis of CA activity under high temperature showed that this stress inhibited the CA activity by a 43%. To evaluate the effect of nitration on the CA activity in sunflower it was used 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent. Thus the CA activity was inhibited by 41%. In silico analysis of the pea CA protein sequence suggests that Tyr(205) is the most likely potential target for nitration. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Protein tyrosine phosphorylation in streptomycetes.

    PubMed

    Waters, B; Vujaklija, D; Gold, M R; Davies, J

    1994-07-01

    Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

  14. Mechanisms of peroxynitrite-mediated nitration of tyrosine.

    PubMed

    Gunaydin, Hakan; Houk, K N

    2009-05-01

    The mechanisms of tyrosine nitration by peroxynitrous acid or nitrosoperoxycarbonate were investigated with the CBS-QB3 method. Either the protonation of peroxynitrite or a reaction with carbon dioxide gives a reactive peroxide intermediate. Peroxynitrous acid-mediated nitration of phenol occurs via unimolecular decomposition to give nitrogen dioxide and hydroxyl radicals. Nitrosoperoxycarbonate also undergoes unimolecular decomposition to give carbonate and nitrogen dioxide radicals. The reactions of tyrosine with the hydroxyl or carbonate radicals give a phenoxy radical intermediate. The reaction of the nitrogen dioxide with this radical intermediate followed by tautomerization gives nitrated tyrosine in both cases. According to CBS-QB3 calculations, the rate-limiting step for the nitration of phenol is the decomposition of peroxynitrous acid or nitrosoperoxycarbonate.

  15. Hypothyroidism attenuates protein tyrosine nitration, oxidative stress and renal damage induced by ischemia and reperfusion: effect unrelated to antioxidant enzymes activities

    PubMed Central

    Tenorio-Velázquez, Verónica M; Barrera, Diana; Franco, Martha; Tapia, Edilia; Hernández-Pando, Rogelio; Medina-Campos, Omar Noel; Pedraza-Chaverri, José

    2005-01-01

    Background It has been established that hypothyroidism protects rats against renal ischemia and reperfusion (IR) oxidative damage. However, it is not clear if hypothyroidism is able to prevent protein tyrosine nitration, an index of nitrosative stress, induced by IR or if antioxidant enzymes have involved in this protective effect. In this work it was explored if hypothyroidism is able to prevent the increase in nitrosative and oxidative stress induced by IR. In addition the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was studied. Control and thyroidectomized (HTX) rats were studied 24 h of reperfusion after 60 min ischemia. Methods Male Wistar rats weighing 380 ± 22 g were subjected to surgical thyroidectomy. Rats were studied 15 days after surgery. Euthyroid sham-operated rats were used as controls (CT). Both groups of rats underwent a right kidney nephrectomy and suffered a 60 min left renal ischemia with 24 h of reperfusion. Rats were divided in four groups: CT, HTX, IR and HTX+IR. Rats were sacrificed and samples of plasma and kidney were obtained. Blood urea nitrogen (BUN) and creatinine were measured in blood plasma. Kidney damage was evaluated by histological analysis. Oxidative stress was measured by immunohistochemical localization of protein carbonyls and 4-hydroxy-2-nonenal modified proteins. The protein carbonyl content was measured using antibodies against dinitrophenol (DNP)-modified proteins. Nitrosative stress was measured by immunohistochemical analysis of 3-nitrotyrosine modified proteins. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was measured by spectrophotometric methods. Multiple comparisons were performed with ANOVA followed by Bonferroni t test. Results The histological damage and the rise in plasma creatinine and BUN induced by IR were significantly lower in HTX+IR group. The increase in protein carbonyls and in 3-nitrotyrosine and 4

  16. Tyrosine phosphorylation of WW proteins

    PubMed Central

    Reuven, Nina; Shanzer, Matan

    2015-01-01

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

  17. 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.

  18. Peroxynitrite-induced nitration of tyrosine-34 does not inhibit Escherichia coli iron superoxide dismutase.

    PubMed Central

    Soulère, L; Claparols, C; Périé, J; Hoffmann, P

    2001-01-01

    The peroxynitrite anion is a potent oxidizing agent, formed by the diffusion-limited combination of nitric oxide and superoxide, and its production under physiological conditions is associated with the pathologies of a number of inflammatory and neurodegenerative diseases. Nitration of Escherichia coli iron superoxide dismutase (Fe-SOD) by peroxynitrite was investigated, and demonstrated by spectral changes and electrospray mass spectroscopic analysis. HPLC and mass studies of the tryptic digests of the mono-nitrated Fe-SOD indicated that tyrosine-34 was the residue most susceptible to nitration by peroxynitrite. Exclusive nitration of this residue occurred when Fe-SOD was exposed to a cumulative dose of 0.4 mM peroxynitrite. Unlike with human Mn-SOD, this single modification did not inactivate E. coli Fe-SOD at pH 7.4. When Fe-SOD was exposed to higher concentrations of peroxynitrite (7 mM), eight tyrosine residues per subunit of the protein, of the nine available, were nitrated without loss of catalytic activity of the enzyme. The pK(a) of nitrated tyrosine-34 was determined to be 7.95+/-0.15, indicating that the peroxynitrite-modified enzyme appreciably maintains its protonation state under physiological conditions. PMID:11736645

  19. Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease.

    PubMed

    Sacksteder, Colette A; Qian, Wei-Jun; Knyushko, Tatyana V; Wang, Haixing; 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 abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins. More than half of the nitrated proteins that have been identified are 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 an increased level of 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, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.

  20. Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit.

    PubMed

    Guittet, O; Decottignies, P; Serani, L; Henry, Y; Le Maréchal, P; Laprévote, O; Lepoivre, M

    2000-04-25

    Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 microM peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.

  1. Deprotonation of tyrosines in bacteriorhodopsin as studied by Fourier transform infrared spectroscopy with deuterium and nitrate labeling.

    PubMed

    Lin, S L; Ormos, P; Eisenstein, L; Govindjee, R; Konno, K; Nakanishi, K

    1987-12-15

    Fourier transform infrared (FTIR) difference spectra are presented for bacteriorhodopsin (BR) at low temperature. Previous FTIR measurements have identified several tyrosine residues that change their absorption characteristics between light-adapted BR and dark-adapted BR, or between intermediates K and M [Dollinger, G., Eisenstein, L., Lin, S.-L., Nakanishi, K., Odashima, K., & Termini, J. (1986) Methods Enzymol. 127, 649-662]. These changes were explained by protonation/deprotonation of tyrosine moieties and perturbation of the protein environment surrounding tyrosines. A tyrosine deprotonation was observed to occur between intermediates K and M. The present studies confine the deprotonation to being between intermediates L and M and show that no tyrosines undergo changes between the K and the L states. Evidence is presented that none of the tyrosines undergoing changes at low temperature can be assigned to tyrosine-64. The environmental changes of these tyrosines are discussed in relation to the proton pumping mechanism. Their spatial relation to the chromophore is also discussed. At least two tyrosines are suggested to reside close to the retinal binding site. The reactive groups of the nitrated tyrosine-64 are speculated to be remote from the Schiff base and the active tyrosines but can possibly interact sterically with the ionone ring of the retinal.

  2. Peroxynitrite-induced nitration of tyrosine hydroxylase: identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis.

    PubMed

    Kuhn, Donald M; Sadidi, Mahdieh; Liu, Xiuli; Kreipke, Christian; Geddes, Timothy; Borges, Chad; Watson, J Throck

    2002-04-19

    Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432) as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine

  3. 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.

  4. 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.

  5. Antioxidant promotion of tyrosine nitration in the presence of copper(II).

    PubMed

    Qiao, Liang; Liu, Baohong; Girault, Hubert H

    2013-06-01

    Copper(II) is known to catalyze the generation of reactive nitrogen species in the presence of hydrogen peroxide, nitrite or nitric oxide, leading to tyrosine nitration, a biomarker for free radical species associated diseases. Here, we find that biological antioxidants such as ascorbic acid can promote tyrosine nitration in the presence of copper(II) and nitrite under aerobic and weak acidic conditions. Tyrosine nitration is demonstrated on both the β-amyloid peptide and angiotensin I. These studies show that (i) ascorbic acid works as a pro-oxidant in the presence of copper(II) to induce oxidation and nitration on peptides, (ii) both free and coordinated copper(II) can catalyze peptide oxidation and nitration, (iii) nitration occurs under mild acidic conditions (pH = 6.0-6.5).

  6. 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.

  7. Protein tyrosine phosphatases as potential therapeutic targets

    PubMed Central

    He, Rong-jun; Yu, Zhi-hong; Zhang, Ruo-yu; Zhang, Zhong-yin

    2014-01-01

    Protein tyrosine phosphorylation is a key regulatory process in virtually all aspects of cellular functions. Dysregulation of protein tyrosine phosphorylation is a major cause of human diseases, such as cancers, diabetes, autoimmune disorders, and neurological diseases. Indeed, protein tyrosine phosphorylation-mediated signaling events offer ample therapeutic targets, and drug discovery efforts to date have brought over two dozen kinase inhibitors to the clinic. Accordingly, protein tyrosine phosphatases (PTPs) are considered next-generation drug targets. For instance, PTP1B is a well-known targets of type 2 diabetes and obesity, and recent studies indicate that it is also a promising target for breast cancer. SHP2 is a bona-fide oncoprotein, mutations of which cause juvenile myelomonocytic leukemia, acute myeloid leukemia, and solid tumors. In addition, LYP is strongly associated with type 1 diabetes and many other autoimmune diseases. This review summarizes recent findings on several highly recognized PTP family drug targets, including PTP1B, Src homology phosphotyrosyl phosphatase 2(SHP2), lymphoid-specific tyrosine phosphatase (LYP), CD45, Fas associated phosphatase-1 (FAP-1), striatal enriched tyrosine phosphatases (STEP), mitogen-activated protein kinase/dual-specificity phosphatase 1 (MKP-1), phosphatases of regenerating liver-1 (PRL), low molecular weight PTPs (LMWPTP), and CDC25. Given that there are over 100 family members, we hope this review will serve as a road map for innovative drug discovery targeting PTPs. PMID:25220640

  8. Protein tyrosine phosphatase: enzymatic assays.

    PubMed

    Montalibet, Jacqueline; Skorey, Kathryn I; Kennedy, Brian P

    2005-01-01

    Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.

  9. Impaired response to interferon-γ in activated macrophages due to tyrosine nitration of STAT1 by endogenous nitric oxide

    PubMed Central

    Llovera, Marta; Pearson, Jeremy D; Moreno, Carlos; Riveros-Moreno, Valentina

    2001-01-01

    Inducible NO synthase (iNOS) expression and activity were measured in the mouse macrophage cell line J774 after exposure to bacterial lipopolysaccharide (LPS) with or without interferon-γ (IFN-γ). Inhibition of NOS activity by concomitant NG-monomethyl-L-arginine (L-NMMA) treatment further increased iNOS protein levels, with a substantial increase in iNOS half-life. Western blotting and ELISA demonstrated that several cell proteins were tyrosine-nitrated when iNOS activity was high. Rapid IFN-γ-induced phosphorylation of STAT1 was reduced by about 40% when cells were pretreated to induce iNOS, unless L-NMMA was present during the pretreatment period. 2D gel electrophoresis demonstrated the presence of nitrotyrosine in STAT1 after iNOS induction, and confirmed the reduction in phospho-STAT1 on subsequent stimulation with IFN-γ for 15 min and its partial restoration when L-NMMA was present during the pretreatment period. We did not detect tyrosine nitration of the upstream kinase JAK2 in LPS+IFN-γ pretreated cells, but JAK2 activity was also impaired, and was partially restored by concomitant L-NMMA pretreatment. We conclude that endogenous production of NO induces feedback inhibition of signalling pathways activated by IFN-γ, at least in part by nitrating tyrosine residues in STAT1 which prevents phosphorylation. PMID:11159690

  10. Haemophilus ducreyi LspA Proteins Are Tyrosine Phosphorylated by Macrophage-Encoded Protein Tyrosine Kinases▿

    PubMed Central

    Deng, Kaiping; Mock, Jason R.; Greenberg, Steven; van Oers, Nicolai S. C.; Hansen, Eric J.

    2008-01-01

    The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins. PMID:18678665

  11. Haemophilus ducreyi LspA proteins are tyrosine phosphorylated by macrophage-encoded protein tyrosine kinases.

    PubMed

    Deng, Kaiping; Mock, Jason R; Greenberg, Steven; van Oers, Nicolai S C; Hansen, Eric J

    2008-10-01

    The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.

  12. Inactivation of PYR/PYL/RCAR ABA receptors by tyrosine nitration may enable rapid inhibition of ABA signaling by nitric oxide in plants.

    PubMed

    Castillo, Mari-Cruz; Lozano-Juste, Jorge; González-Guzmán, Miguel; Rodriguez, Lesia; Rodriguez, Pedro L; León, José

    2015-09-01

    Abscisic acid (ABA) is a phytohormone that inhibits growth and enhances adaptation to stress in plants. ABA perception and signaling rely on its binding to receptors of the pyrabactin resistance1/PYR1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) family, the subsequent inhibition of clade A type 2C protein phosphatases (PP2Cs), and the phosphorylation of ion channels and transcription factors by protein kinases of the SnRK2 family. Nitric oxide (NO) may inhibit ABA signaling because NO-deficient plants are hypersensitive to ABA. Regulation by NO often involves posttranslational modification of proteins. Mass spectrometry analysis of ABA receptors expressed in plants and recombinant receptors modified in vitro revealed that the receptors were nitrated at tyrosine residues and S-nitrosylated at cysteine residues. In an in vitro ABA-induced, PP2C inhibition assay, tyrosine nitration reduced receptor activity, whereas S-nitrosylated receptors were fully capable of ABA-induced inhibition of the phosphatase. PYR/PYL/RCAR proteins with nitrated tyrosine, which is an irreversible covalent modification, were polyubiquitylated and underwent proteasome-mediated degradation. We propose that tyrosine nitration, which requires NO and superoxide anions, is a rapid mechanism by which NO limits ABA signaling under conditions in which NO and reactive oxygen species are both produced.

  13. Interaction between hydrogen sulfide-induced sulfhydration and tyrosine nitration in the KATP channel complex

    PubMed Central

    Kang, Minho; Hashimoto, Atsushi; Gade, Aravind

    2014-01-01

    Hydrogen sulfide (H2S) is an endogenous gaseous mediator affecting many physiological and pathophysiological conditions. Enhanced expression of H2S and reactive nitrogen/oxygen species (RNS/ROS) during inflammation alters cellular excitability via modulation of ion channel function. Sulfhydration of cysteine residues and tyrosine nitration are the posttranslational modifications induced by H2S and RNS, respectively. The objective of this study was to define the interaction between tyrosine nitration and cysteine sulfhydration within the ATP-sensitive K+ (KATP) channel complex, a significant target in experimental colitis. A modified biotin switch assay was performed to determine sulfhydration of the KATP channel subunits, Kir6.1, sulphonylurea 2B (SUR2B), and nitrotyrosine measured by immunoblot. NaHS (a donor of H2S) significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. 3-Morpholinosydnonimine (SIN-1) (a donor of peroxynitrite) induced nitration of Kir6.1 subunit but not SUR2B. Pretreatment with NaHS reduced the nitration of Kir6.1 by SIN-1 in Chinese hamster ovary cells cotransfected with the two subunits, as well as in enteric glia. Two specific mutations within SUR2B, C24S, and C1455S prevented sulfhydration by NaHS, and these mutations prevented NaHS-induced reduction in tyrosine nitration of Kir6.1. NaHS also reversed peroxynitrite-induced inhibition of smooth muscle contraction. These studies suggest that posttranslational modifications of the two subunits of the KATP channel interact to alter channel function. The studies described herein demonstrate a unique mechanism by which sulfhydration of one subunit modifies tyrosine nitration of another subunit within the same channel complex. This interaction provides a mechanistic insight on the protective effects of H2S in inflammation. PMID:25552582

  14. 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

  15. 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

  16. Protein Nitration in Placenta – Functional Significance

    PubMed Central

    Webster, RP; Roberts, VHJ; Myatt, L

    2009-01-01

    Crucial roles of the placenta are disrupted in early and mid-trimester pregnancy loss, preeclampsia, eclampsia and intrauterine growth restriction. The pathophysiology of these disorders includes a relative hypoxia of the placenta, ischemia/reperfusion injury, an inflammatory response and oxidative stress. Reactive oxygen species including nitric oxide (NO), carbon monoxide and superoxide have been shown to participate in trophoblast invasion, regulation of placental vascular reactivity and other events. Superoxide, which regulates expression of redox sensitive genes, has been implicated in up-regulation of transcription factors, antioxidant production, angiogenesis, proliferation and matrix remodeling. When superoxide and nitric oxide are present in abundance, their interaction yields peroxynitrite a potent pro-oxidant, but also alters levels of nitric oxide, which in turn affect physiological functions. The peroxynitrite anion is extremely unstable thus evidence of its formation in vivo has been indirect via the occurrence of nitrated moieties including nitrated lipids and nitrotyrosine residues in proteins. Formation of 3-nitrotyrosine (protein nitration) is a “molecular fingerprint” of peroxynitrite formation. Protein nitration has been widely reported in a number of pathological states associated with inflammation but is reported to occur in normal physiology and is thought of as a prevalent, functionally relevant post-translational modification of proteins. Nitration of proteins can give either no effect, a gain or a loss of function. Nitration of a range of placental proteins is found in normal pregnancy but increased in pathologic pregnancies. Evidence is presented for nitration of placental signal transduction enzymes and transporters. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic settings. PMID:18851882

  17. Protein nitration is predominantly mediated by a peroxynitrite-dependent pathway in cultured human leucocytes.

    PubMed Central

    Galiñanes, Manuel; Matata, Bashir M

    2002-01-01

    Protein nitration is a common characteristic of oxidative injury caused by the invasion of leucocytes into inflammatory lesions. Two distinct pathways of nitration of protein tyrosine residues, namely the peroxynitrite (ONOO(-))-mediated pathway and another catalysed by the haem-containing peroxidases, have been reported under experimental conditions. However, the contribution of these two pathways in human leucocytes is still controversial. The present study demonstrates that the process of phenolic nitration of proteins in cultured human leucocytes is mainly ONOO(-)-mediated and that it differs between granulocytes and mononuclear cells, depending on the cell compartment and the stimuli. We have also shown that NO induces protein nitration via a ONOO(-)-dependent pathway, whereas NO(2)(-), the NO metabolite, does not increase but decreases nitration in PMA-stimulated leucocytes. The inhibition of myeloperoxidase activity did not reduce protein nitration; on the other hand, the myeloperoxidase inhibitor aminobenzoic hydrazide caused increased nitration, which was mediated by ONOO(-). These results suggest that protein nitration is predominantly mediated by a ONOO(-)-dependent pathway in cultured human leucocytes and that the myeloperoxidase-catalysed pathway does not play a significant role in protein nitration. PMID:12099887

  18. Reusable amperometric biosensor for measuring protein tyrosine kinase activity.

    PubMed

    Wang, Chung-Liang; Wei, Lan-Yi; Yuan, Chiun-Jye; Hwang, Kuo Chu

    2012-01-17

    This work presents a simple, low-cost and reusable label-free method for detecting protein tyrosine kinase activity using a tyrosinase-based amperometric biosensor (tyrosine kinase biosensor). This method is based on the observation that phosphorylation can block the tyrosinase-catalyzed oxidation of tyrosine or tyrosyl residue in peptides. Therefore, the activity of p60c-src protein tyrosine kinase (Src) on the developed tyrosine kinase biosensor could be quickly determined when its specific peptide substrate, p60c-src substrate I, was used. The tyrosine kinase biosensor was highly sensitive to the activity of Src with a linear dynamic range of 1.9-237.6 U/mL and the lowest detection limit of 0.23 U/mL. Interestingly, the tyrosine kinase activity can be measured using the developed tyrosine kinase biosensor repetitively without regeneration. The inhibitory effect of various kinase inhibitors on the Src activity could be determined on the tyrosine kinase biosensor. Src-specific inhibitors, PP2 and Src inhibitor I, effectively suppressed Src activity, whereas PD153035, an inhibitor of the epidermal growth factor receptor, was ineffective. Staurosporine, a universal kinase inhibitor, inhibited Src activity in an ATP concentration-dependent manner. These results suggests that the activities of tyrosine kinases and their behaviors toward various reagents can be effectively measured using the developed tyrosine kinase biosensor.

  19. Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism.

    PubMed

    Sainz, Martha; Calvo-Begueria, Laura; Pérez-Rontomé, Carmen; Wienkoop, Stefanie; Abián, Joaquín; Staudinger, Christiana; Bartesaghi, Silvina; Radi, Rafael; Becana, Manuel

    2015-03-01

    Protein tyrosine (Tyr) nitration is a post-translational modification yielding 3-nitrotyrosine (NO2 -Tyr). Formation of NO2 -Tyr is generally considered as a marker of nitro-oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2 -Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2 -Tyr25 and NO2 -Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3- and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and NO2- by alternative means to nitrate reductase, probably via a ˙NO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires NO2- + H2 O2 and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to NO3-. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2 -Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23566956

  1. Aniline-induced nitrosative stress in rat spleen: proteomic identification of nitrated proteins.

    PubMed

    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 123.6kDa. 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.

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

    PubMed Central

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

    2011-01-01

    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. PMID:21708182

  3. 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.

  4. Protein tyrosine phosphatases: structure-function relationships.

    PubMed

    Tabernero, Lydia; Aricescu, A Radu; Jones, E Yvonne; Szedlacsek, Stefan E

    2008-03-01

    Structural analysis of protein tyrosine phosphatases (PTPs) has expanded considerably in the last several years, producing more than 200 structures in this class of enzymes (from 35 different proteins and their complexes with ligands). The small-medium size of the catalytic domain of approximately 280 residues plus a very compact fold makes it amenable to cloning and overexpression in bacterial systems thus facilitating crystallographic analysis. The low molecular weight PTPs being even smaller, approximately 150 residues, are also perfect targets for NMR analysis. The availability of different structures and complexes of PTPs with substrates and inhibitors has provided a wealth of information with profound effects in the way we understand their biological functions. Developments in mammalian expression technology recently led to the first crystal structure of a receptor-like PTP extracellular region. Altogether, the PTP structural work significantly advanced our knowledge regarding the architecture, regulation and substrate specificity of these enzymes. In this review, we compile the most prominent structural traits that characterize PTPs and their complexes with ligands. We discuss how the data can be used to design further functional experiments and as a basis for drug design given that many PTPs are now considered strategic therapeutic targets for human diseases such as diabetes and cancer.

  5. Characterization of Tyrosine Nitration and Cysteine Nitrosylation Modifications by Metastable Atom-Activation Dissociation Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Cook, Shannon L.; Jackson, Glen P.

    2011-02-01

    The fragmentation behavior of nitrated and S-nitrosylated peptides were studied using collision induced dissociation (CID) and metastable atom-activated dissociation mass spectrometry (MAD-MS). Various charge states, such as 1+, 2+, 3+, 2-, of modified and unmodified peptides were exposed to a beam of high kinetic energy helium (He) metastable atoms resulting in extensive backbone fragmentation with significant retention of the post-translation modifications (PTMs). Whereas the high electron affinity of the nitrotyrosine moiety quenches radical chemistry and fragmentation in electron capture dissociation (ECD) and electron transfer dissociation (ETD), MAD does produce numerous backbone cleavages in the vicinity of the modification. Fragment ions of nitrosylated cysteine modifications typically exhibit more abundant neutral losses than nitrated tyrosine modifications because of the extremely labile nature of the nitrosylated cysteine residues. However, compared with CID, MAD produced between 66% and 86% more fragment ions, which preserved the labile -NO modification. MAD was also able to differentiate I/L residues in the modified peptides. MAD is able to induce radical ion chemistry even in the presence of strong radical traps and therefore offers unique advantages to ECD, ETD, and CID for determination of PTMs such as nitrated and S-nitrosylated peptides.

  6. Staining proteins in gels with silver nitrate.

    PubMed

    Simpson, Richard J

    2007-07-01

    INTRODUCTIONSilver staining is one of the commonly used procedures for visualizing proteins in acrylamide gels. All silver staining methods rely on the reduction of ionic to metallic silver to provide metallic silver images; the selective reduction at gel sites occupied by proteins compared to nonprotein sites is dependent on differences in the oxidation-reduction potentials at these sites. There are two broad methodologies for silver staining. One approach (nondiamine silver nitrate stains) uses silver nitrate as the silvering agent and formaldehyde in alkaline carbonate solution as the developing agent, whereas the other approach (diamine or ammoniacal stains) uses ammoniacal silver as the silvering agent and formaldehyde in dilute citric acid as the developing agent. Although protocols using ammoniacal silver are arguably more sensitive and give darker hues than those based on silver nitrate, they are more prone to negative staining, resulting in hollow or "doughnut" spots, give unacceptable backgrounds with tricine-based gel systems, and are not very robust because of their reliance on the ammonia-silver ratio. Additionally, ammoniacal silver staining is more sensitive for basic proteins but less so for very acidic proteins. This protocol describes a silver nitrate staining approach. Its sensitivity is in the low-nanogram range, which is 50-100 times more sensitive than classical Coomassie Blue staining, ~10 times better than colloidal Coomassie Blue staining, and at least twice as sensitive as the zinc/imidazole negative staining method.

  7. Inhibition of nitrous acid-dependent tyrosine nitration and DNA base deamination by flavonoids and other phenolic compounds.

    PubMed

    Oldreive, C; Zhao, K; Paganga, G; Halliwell, B; Rice-Evans, C

    1998-12-01

    Exposure of tyrosine or DNA bases to acidic nitrite at low pH results in the nitration of tyrosine and the formation of base deamination products, respectively. At pH 1, hypoxanthine and xanthine are formed from the deamination of adenine and guanine, respectively, whereas under the same conditions, uracil is not detected. The yield of 3-nitrotyrosine derived from interaction of equimolar nitrite and tyrosine at pH 1 is approximately 50% of that obtained from equimolar peroxynitrite-tyrosine interactions at pH 7. 4. The ability of a range of plant phenolic constituents to prevent damage mediated by acidic nitrite was also examined in comparison with the activity of vitamin C. The epicatechin/gallate family of flavonols, constituents of green tea, red wine, etc., demonstrates the most extensive inhibitory properties against both tyrosine nitration and base deamination. The results also show that ascorbic acid is a poor inhibitor of nitration or deamination under acidic conditions such as those of the stomach. The ability of plant phenolics to scavenge reactive nitrogen species derived from acidic nitrite may contribute to the protective effects of tea polyphenols against gastric cancer.

  8. High temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of nitrosative stress which provokes the inhibition of ferredoxin-NADP reductase by tyrosine nitration.

    PubMed

    Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana M; Carreras, Alfonso; Gómez-Rodríguez, Maria V; López-Jaramillo, Javier; Begara-Morales, Juan C; Sánchez-Calvo, Beatriz; Luque, Francisco; Leterrier, Marina; Corpas, Francisco J; Barroso, Juan B

    2011-11-01

    High temperature (HT) is considered a major abiotic stress that negatively affects both vegetative and reproductive growth. Whereas the metabolism of reactive oxygen species (ROS) is well established under HT, less is known about the metabolism of reactive nitrogen species (RNS). In sunflower (Helianthus annuus L.) seedlings exposed to HT, NO content as well as S-nitrosoglutathione reductase (GSNOR) activity and expression were down-regulated with the simultaneous accumulation of total S-nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO). However, the content of tyrosine nitration (NO(2) -Tyr) studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and by confocal laser scanning microscope was induced. Nitroproteome analysis under HT showed that this stress induced the protein expression of 13 tyrosine-nitrated proteins. Among the induced proteins, ferredoxin-NADP reductase (FNR) was selected to evaluate the effect of nitration on its activity after heat stress and in vitro conditions using 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent, the FNR activity being inhibited. Taken together, these results suggest that HT augments SNOs, which appear to mediate protein tyrosine nitration, inhibiting FNR, which is involved in the photosynthesis process.

  9. Huntingtin-Interacting Protein 1 Phosphorylation by Receptor Tyrosine Kinases

    PubMed Central

    Ames, Heather M.; Wang, Anmin A.; Coughran, Alanna; Evaul, Kristen; Huang, Sha; Graves, Chiron W.; Soyombo, Abigail A.

    2013-01-01

    Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor β receptor (PDGFβR) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGFβR (H/P), and TEL/PDGFβR (T/P). We identified a four-tyrosine “HIP1 phosphorylation motif” (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGFβR but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1's only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1's role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival. PMID:23836884

  10. Ozone exposure induces iNOS expression and tyrosine nitration in rat aorta.

    PubMed

    Sánchez-González, Dolores J; Moro, María A; Castillo-Henkel, Carlos; Herrera-González, Norma; Hernández-Pando, Rogelio; Larios-Medina, Francisco J; Cobilt, Rafael; Blanco, José A; Pedraza-Chaverrí, José; Villanueva, Cleva

    2004-05-01

    The aim was to study whether ozone affects vascular endothelium by causing inducible nitric oxide synthase (iNOS) expression and tyrosine nitration. We also studied biomarkers of endothelial function. Male Wistar rats were exposed to ozone (0.25ppm, 4h/day) or filtered air (control, ozone <0.05ppm). After ozone exposure, blood samples were taken to measure 6-keto prostaglandin F1α (6-keto PGF1α), dehydro-thromboxane B(2) (DH-TxB(2)), endothelin-1 and NO(2)(-)/NO(3)(-) (NO(x)(-)). iNOS and nitrotyrosine were detected in aorta by immunohistochemistry. Nitrotyrosine was also detected by immunoelectromicroscopy. Control aortae failed to show either iNOS or nitrotyrosine. Time-dependent positive iNOS and nitrotyrosine cells were observed in exposed animals. Except for NO(x)(-), endothelial markers decreased after 14 days of ozone exposure (P<0.05). After 28 days of ozone, 6-keto PGF1α remained low (P<0.05) while DH-TxB(2) increased (P<0.05). It is concluded that ozone causes endothelial dysfunction manifested early with peroxynitrite formation and lately with changes in endothelial markers.

  11. Beetroot betalain inhibits peroxynitrite-mediated tyrosine nitration and DNA strand cleavage.

    PubMed

    Sakihama, Yasuko; Maeda, Makiko; Hashimoto, Makoto; Tahara, Satoshi; Hashidoko, Yasuyuki

    2012-01-01

    Two major betalains, red-purple betacyanins and yellow betaxanthins, were isolated from red beetroots (Beta vulgaris L.), and their peroxynitrite (ONOO(-)) scavenging capacity was investigated. Apparent colours of the betalains were bleached by the addition of ONOO(-), and the absorbance decreases were suppressed in the presence of glutathione, a ONOO(-) scavenger. After bleaching, a new absorption maximum was observed at 350 nm in the spectrum of the resulting reaction mixture. New peaks were detected from HPLC analysis of the reaction products of betanin, a representative constituent of red beetroot betacyanins, treated with ONOO(-) monitoring at 350 nm, and the intensity of the major peak was positively correlated with ONOO(-) concentration. Betanin inhibited the ONOO(-) (0.5 mM)-dependent nitration of tyrosine (0.1 mM). Additionally, the IC(50) value of betanin (19.2 μM) was lower than that of ascorbate (79.6 μM). The presence of betanin (0.05-1.0 mM) also inhibited ONOO(-) (0.5 mM)-dependent DNA strand cleavage in a concentration-dependent manner. These results suggest that betalains can protect cells from nitrosative stress in addition to protecting them from oxidative stresses.

  12. 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.

  13. Targeting Protein Tyrosine Phosphatases for Anticancer Drug Discovery

    PubMed Central

    Scott, Latanya. M.; Lawrence, Harshani. R.; Sebti, Saïd. M.; Lawrence, Nicholas. J.; Wu, Jie.

    2010-01-01

    Protein tyrosine phosphatases (PTPs) are a diverse family of enzymes encoded by 107 genes in the human genome. Together with protein tyrosine kinases (PTKs), PTPs regulate various cellular activities essential for the initiation and maintenance of malignant phenotypes. While PTK inhibitors are now used routinely for cancer treatment, the PTP inhibitor development field is still in the discovery phase. In this article, the suitability of targeting PTPs for novel anticancer drug discovery is discussed. Examples are presented for PTPs that have been targeted for anticancer drug discovery as well as potential new PTP targets for novel anticancer drug discovery. PMID:20337577

  14. The effects of aging on pulmonary oxidative damage, protein nitration, and extracellular superoxide dismutase down-regulation during systemic inflammation.

    PubMed

    Starr, Marlene E; Ueda, Junji; Yamamoto, Shoji; Evers, B Mark; Saito, Hiroshi

    2011-01-15

    Systemic inflammatory response syndrome (SIRS), a serious clinical condition characterized by whole-body inflammation, is particularly threatening for elderly patients, who suffer much higher mortality rates than the young. A major pathological consequence of SIRS is acute lung injury caused by neutrophil-mediated oxidative damage. Previously, we reported an increase in protein tyrosine nitration (a marker of oxidative/nitrosative damage) and a decrease in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD) in the lungs of young mice during endotoxemia-induced SIRS. Here we demonstrate that during endotoxemia, down-regulation of EC-SOD is significantly more profound and prolonged, whereas up-regulation of iNOS is augmented, in aged compared to young mice. Aged mice also showed 2.5-fold higher protein nitration levels, compared to young mice, with particularly strong nitration in the pulmonary vascular endothelium during SIRS. Additionally, by two-dimensional gel electrophoresis, Western blotting, and mass spectrometry, we identified proteins that show increased tyrosine nitration in age- and SIRS-dependent manners; these proteins (profilin-1, transgelin-2, LASP 1, tropomyosin, and myosin) include components of the actin cytoskeleton responsible for maintaining pulmonary vascular permeability. Reduced EC-SOD in combination with increased oxidative/nitrosative damage and altered cytoskeletal protein function due to tyrosine nitration may contribute to augmented lung injury in the aged with SIRS.

  15. PROTEIN TYROSINE-O-SULFATION IN THE RETINA

    PubMed Central

    Kanan, Yogita; Hoffhines, Adam; Rauhauser, Alysha; Murray, Anne; Al-Ubaidi, Muayyad R.

    2009-01-01

    Tyrosine-O-sulfation, a post-translational modification, is catalyzed by two independent tyrosylprotein sulfotransferases (TPSTs). As an initial step towards understanding the role of TPSTs in retinal function, this study was undertaken to determine the extent to which tyrosine-O-sulfation of proteins is utilized in the retina. A previously characterized anti-sulfotyrosine antibody was used to determine the presence and localization of tyrosine-O-sulfated proteins (TOSPs) in the retina. Using Western blot, RT-PCR and immunohistochemical analyses, we detected TOSPs in the retinas from diverse species, including frog, fish, mouse and human. Some of the variability in the observed sizes of retinal TOSPs in the mouse, at least, may result from differential patterns of glycosylation; however, there seem to be species-specific sulfated retinal proteins as well. TOSPs were detected in most of the retinal layers as well as in the retinal pigment epithelium from human and mouse. Several retinal TOSPs were detected in the inter-photoreceptor matrix, which is consistent with the secreted nature of some sulfated proteins. Transcripts for both TPST-1 and -2 were expressed in both the human and mouse retinas. These data show that retinal protein tyrosine-O-sulfation is highly conserved which suggest a functional significance of these proteins to retinal function and structure. PMID:19523945

  16. In vitro enzymatic assays of protein tyrosine phosphatase 1B.

    PubMed

    Lubben, T; Clampit, J; Stashko, M; Trevillyan, J; Jirousek, M R

    2001-08-01

    Many hormone or growth factor receptors signal via the activation of protein-tyrosine kinases and phosphatases. Alteration of the phosphorylation state of tyrosine residues in certain proteins can directly regulate enzyme activity or cause formation of protein complexes necessary for transducing intracellular signals. Genetic and biochemical evidence also implicates protein-tyrosine phosphatases in several disease processes, including negative regulation of insulin receptor signaling at the level of the insulin receptor and perhaps in signaling at the IRS-1 level. The expression of protein tyrosine phosphatase-1B (PTP1B) is elevated in muscle and adipose tissue in insulin-resistant states both in man and rodents suggesting that PTP1B may play a role in the insulin-resistant state associated with diabetes and obesity. As described in this unit, PTP1B activity can be determined with the small molecule substrate, p-nitrophenyl phosphate (pNPP), in which the cleavage of the phosphate results in production of p-nitrophenol (pNP) and an increase in absorbance at 405 nm. Alternatively, PTP1B activity can be measured as described using model phosphotyrosyl-containing peptide substrates in which the release of free phosphate from the peptide is determined using a malachite green colorimetric assay.

  17. 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¿.

    USDA-ARS?s Scientific Manuscript database

    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...

  18. Specific chemical modification of the readily nitrated tyrosine of the RTEM beta-lactamase and of bacillus cereus beta-lactamase I. The role of the tyrosine in beta-lactamase catalysis.

    PubMed

    Wolozin, B L; Myerowitz, R; Pratt, R F

    1982-02-18

    The function of the hydroxyl group of the tyrosine residue readily nitrated by tetranitromethane (tyrosine-105) in the RTEM plasmid-derived beta-lactamase (penicillinase; penicillin amido beta-lactam-hydrolase, EC 3.5.1.6) from E. coli and in Bacillus cereus beta-lactamase I has been investigated by chemical modification methods. In the case of B. cereus beta-lactamase I the nitrated tyrosine can be acetylated by acetic anhydride without effect on beta-lactamase activity The nitrated tyrosine of the E. coli enzyme can also be acetylated but in this case beta-lactamase activity is lost in a manner which directly correlates with extent of acetylation. However, deacetylation of the nitrotyrosine does not restore activity. The dilemma created by the latter result has been resolved by development of a new method of tyrosine hydroxyl modification at low pH. The nitrated enzyme is reduced by dithionite and then treated with either carbonyldiimidazole or N-(2.2.2-trifluoroethoxycarbonyl)imidazole, both of which convert 3-aminotyrosine into benzoxazolinonylalanine. That the final modification has been achieved is demonstrated both by classical chemical methods and by employment of Fourier transform infrared spectroscopy to detect the characteristic benzoxazolinone carbonyl absorption. Further, it is shown that no significant loss of beta-lactamase activity is associated with this modification. Hence in neither the B. cereus or the E. coli enzyme does the readily nitrated tyrosine residue have a direct chemical function at the beta-lactamase active site.

  19. 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

  20. 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

  1. 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.

  2. Targeting the Reversibly Oxidized Protein Tyrosine Phosphatase Superfamily

    PubMed Central

    Boivin, Benoit; Yang, Ming; Tonks, Nicholas K.

    2010-01-01

    Controlled production of reactive oxygen species leads to reversible oxidation of protein tyrosine phosphatases (PTPs) and has emerged as an important tier of regulation over phosphorylation-dependent signal transduction. We present a modified cysteinyl-labeling assay that detects reversible oxidation of members of each of the different PTP subclasses. Here, we describe the methods for enriching reversibly oxidized PTPs from complex protein extracts, illustrating the procedure in IMR90 fibroblasts. PMID:20807953

  3. 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

  4. 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.

  5. Inhibition of a protein tyrosine phosphatase using mesoporous oxides.

    PubMed

    Kapoor, S; Girish, T S; Mandal, S S; Gopal, B; Bhattacharyya, A J

    2010-03-11

    The feasibility of utilizing mesoporous matrices of alumina and silica for the inhibition of enzymatic activity is presented here. These studies were performed on a protein tyrosine phosphatase by the name chick retinal tyrosine phosphotase-2 (CRYP-2), a protein that is identical in sequence to the human glomerular epithelial protein-1 and involved in hepatic carcinoma. The inhibition of CRYP-2 is of tremendous therapeutic importance. Inhibition of catalytic activity was examined using the sustained delivery of p-nitrocatechol sulfate (pNCS) from bare and amine functionalized mesoporous silica (MCM-48) and mesoporous alumina (Al(2)O(3)). Among the various mesoporous matrices employed, amine functionalized MCM-48 exhibited the best release of pNCS and also inhibition of CRYP-2. The maximum speed of reaction v(max) (=160 +/- 10 micromol/mnt/mg) and inhibition constant K(i) (=85.0 +/- 5.0 micromol) estimated using a competitive inhibition model were found to be very similar to inhibition activities of protein tyrosine phosphatases using other methods.

  6. Pathways for intracellular generation of oxidants and tyrosine nitration by a macrophage cell line.

    PubMed

    Palazzolo-Ballance, Amy M; Suquet, Christine; Hurst, James K

    2007-06-26

    various times post-activation by using fluorescein-conjugated polyacrylamide beads, which efficiently trap MPO-generated HOCl in neutrophils to give stable chlorofluorescein products. However, chlorination of the dye was not detected under any conditions in RAW cells, virtually precluding MPO involvement in their intracellular reactions. This same probe was used to determine changes in intraphagosomal pH, which increased slowly from approximately 6.5 to approximately 8.2 over a 20 h post-phagocytosis period. The cumulative data suggest that activation is followed by sequential induction of an endogenous peroxidase, iNOS, and COX-2, with NADPH oxidase-derived O2*- playing a minimal role in the direct generation of intracellular oxidants. To account for reported observations of intracellular tyrosine nitration late in the life cycles of macrophages, we propose a novel mechanism wherein iNOS-generated NO2- is used by COX-2 to produce NO2* as a terminal microbicidal oxidant and nitrating agent.

  7. Pathways for Intracellular Generation of Oxidants and Tyrosine Nitration by a Macrophage Cell Line†

    PubMed Central

    Palazzolo-Ballance, Amy M.; Suquet, Christine; Hurst, James K.

    2008-01-01

    -activation by using fluorescein-conjugated polyacrylamide beads, which efficiently trap MPO-generated HOCl in neutrophils to give stable chlorofluorescein products. However, chlorination of the dye was not detected under any conditions in RAW cells, virtually precluding MPO involvement in their intracellular reactions. This same probe was used to determine changes in intraphagosomal pH, which increased slowly from ∼6.5 to ∼8.2 over a 20 h post-phagocytosis period. The cumulative data suggest activation is followed by sequential induction of an endogenous peroxidase, iNOS, and COX-2, with NADPH oxidase-derived O2·- playing a minimal role in direct generation of intracellular oxidants. To account for reported observations of intracellular tyrosine nitration late in the life cycles of macrophages, we propose a novel mechanism wherein iNOS-generated NO2- is used by COX-2 to produce NO2· as a terminal microbicidal oxidant and nitrating agent. PMID:17530864

  8. Identification of Tyrosine O Sulfated Proteins in Cow Retina and the 661W Cell Line.

    PubMed

    Kanan, Yogita; Al-Ubaidi, Muayyad R

    2016-01-01

    Lack of tyrosine O Sulfation compromises both rod and cone electroretinographic responses emphasizing the importance of this post-translational modification for vision. To identify tyrosine sulfated proteins in retina, cow retinal lysates were subjected to immunoaffinity purification using an anti-sulfotyrosine antibody. The tyrosine sulfated proteins were eluted from the column using a sulfotyrosine pentapeptide and identified using mass spectrometry. Similarly, tyrosine sulfated proteins secreted by the 661W cell line were identified. Proteins identified were vitronectin, fibronectin, fibulin 2, nidogen, collagen V alpha 2, complement component 3 and C4 and fibrinogen beta. All proteins were subjected to analysis by 'Sulfinator' to determine potential sulfated tyrosines.

  9. Hypervalent Organochalcogenanes as Inhibitors of Protein Tyrosine Phosphatases

    PubMed Central

    Piovan, Leandro; Wu, Li; Zhang, Zhong-Yin; Andrade, Leandro H.

    2011-01-01

    A series of organochalcogenanes was synthesized and evaluated as protein tyrosine phosphatases (PTPs) inhibitors. The results indicate that organochalcogenanes inactivate the PTPs in a time- and concentration-dependent fashion, most likely through covalent modification of the active site sulfur-moiety by the chalcogen atom. Consequently, organochalcogenanes represent a new class of mechanism-based probes to modulate the PTP-mediated cellular processes. PMID:21240419

  10. Nitration of soluble proteins in organotypic culture models of Parkinson's disease.

    PubMed

    Larsen, Trine R; Söderling, Ann-Sofi; Caidahl, Kenneth; Roepstorff, Peter; Gramsbergen, Jan Bert

    2008-02-01

    Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP(+)) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24 h) or MPP(+) treatments (48 h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10 mM SIN-1 or twofold by 10 microM MPP(+), but significant DA depletions occurred at much lower concentrations of MPP(+) (1 microM). Chronic MPP(+) or rotenone treatments (3 weeks) caused maximum protein nitration by 1 microM (twofold) or 10nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 microM) prevented protein nitration by MPP(+), but did not protect against MPP(+)-induced DA depletion or inhibition of TH activity. Acute incubation with 100 microM SIN-1 inhibited TH activity, which could be blocked by co-treatment with the tetrahydrobiopterin precursor l-sepiapterin, but tissue DA depletions required higher doses of SIN-1 (>1 mM, 24 h) and longer survival. In conclusion, protein nitration and TH activity or DA depletion are not directly related in these models.

  11. Contribution of Protein Tyrosine Phosphateses to the Ontogeny and Progression of Chronic Myeloid Leukemia

    DTIC Science & Technology

    2006-04-01

    potential caused of the reduced phoshorylation of the JAK kinases in CML. 15. SUBJECT TERMS TYROSINE PHOSPHATASE SIGNALING JAK STAT CANCER LEUKEMIA ...1-0837 TITLE: Contribution of Protein Tyrosine Phosphateses to the Ontogeny and Progression of Chronic Myeloid Leukemia ...Contribution of Protein Tyrosine Phosphateses to the Ontogeny and Progression of 5a. CONTRACT NUMBER Chronic Myeloid Leukemia 5b. GRANT NUMBER

  12. Endogenous 3,4-dihydroxyphenylalanine and dopaquinone modifications on protein tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical.

    PubMed

    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, Wei-Jun

    2010-06-01

    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 proteome survey of endogenous site-specific modifications, i.e. DOPA and its further oxidation product dopaquinone in mouse brain and heart tissues. Results from LC-MS/MS analyses included 50 and 14 DOPA-modified tyrosine sites identified from brain and heart, respectively, whereas 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, 20 and 12 dopaquinone-modified peptides were observed from brain and heart, respectively; nearly one-fourth 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 mitochondrially 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. Collectively, the results suggest that these modifications are linked with mitochondrially derived oxidative stress and may serve as sensitive markers for disease pathologies.

  13. 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

  14. Lipid peroxidation and tyrosine nitration in traumatic brain injury: Insights into secondary injury from redox proteomics.

    PubMed

    Butterfield, D Allan; Reed, Tanea T

    2016-12-01

    Traumatic brain injury (TBI) is a spontaneous event in which sudden trauma and secondary injury cause brain damage. Symptoms of TBI can range from mild to severe depending on extent of injury. The outcome can span from complete patient recovery to permanent memory loss and neurological decline. Currently, there is no known cure for TBI; however, immediate medical attention after injury is most beneficial for patient recovery. It is a well-established concept that imbalances in the production of reactive oxygen species (ROS), reactive nitrogen species (RNS), and native antioxidant mechanisms have been shown to increase oxidative stress. Over the years, proteomics has been used to identify specific biomarkers in diseases such as cancers and neurological disorders such as Alzheimer disease and Parkinson disease. As TBI is a risk factor for a multitude of neurological diseases, biomarkers for this phenomenon are a likely field of study in order to confirm diagnosis. This review highlights the current proteomics studies that investigated excessively nitrated proteins and those altered by lipid peroxidation in TBI. This review also highlights possible diagnostic measures and provides insights for future treatment strategies.

  15. NMDA receptor stimulation induces temporary alpha-tubulin degradation signaled by nitric oxide-mediated tyrosine nitration in the nervous system of Sepia officinalis.

    PubMed

    Palumbo, Anna; Fiore, Gabriella; Di Cristo, Carlo; Di Cosmo, Anna; d'Ischia, Marco

    2002-05-24

    Biochemical and immunohistochemical evidence is reported, showing basal protein nitration in specific regions of the optic lobes of Sepia officinalis, mainly in the fiber layers of the plexiform zone. SDS-PAGE analysis of optic lobe extracts revealed an intense 3-nitrotyrosine immunoreactive band identified as alpha-tubulin by immunoprecipitation and partial purification. Stimulation of NMDA receptors resulted in a selective decrease in alpha-tubulin levels within 30 min with partial recovery after 4 h. The effect was suppressed by the NO synthase (NOS) inhibitor L-nitroarginine. Incubation of optic lobes with free 3-nitrotyrosine resulted likewise in a selective loss of alpha-tubulin, due apparently to incorporation of the amino acid into the C-terminus of detyrosinated alpha-tubulin to give the nitrated protein purportedly more susceptible to degradation. Overall, these results point to a novel potential physiologic role of NO and free 3-nitrotyrosine in the control of the alpha-tubulin tyrosination/detyrosination cycle and turnover in Sepia nervous tissue.

  16. Evans Blue and other dyes as protein tyrosine phosphatase inhibitors.

    PubMed

    Shrestha, Suja; Shim, Yi Sup; Kim, Ki Chul; Lee, Keun-Hyeung; Cho, Hyeongjin

    2004-04-19

    Commonly used dyes including Evans Blue and Trypan Blue were examined for their inhibitory activities against protein tyrosine phosphatases (PTPases), all of them showed inhibition of PTPases with different potencies. Of the 13 dyes tested, four exhibited IC(50) value of less than 10 microM, Evans Blue lowest IC(50) of 1.3 microM against PTP1B. Care must be taken in the use of dyes for clinical or biochemical experiments to avoid unwanted side effects. Some of the low molecular weight dyes might be useful as lead compounds for the development of potent and selective PTPase inhibitors.

  17. Reduced expression of CD45 Protein-Tyrosine Phosphatase Pr

    DTIC Science & Technology

    2009-05-08

    complex ( MHC ) I (28-14-8), MHC II (M5/114.15.2), CD44 (IM7), and Ly6G (1A8). Cells (1 106) were resuspended in Fc block (anti CD16/CD32 antibody diluted...enzyme (supplemental Fig. 3). Themajority of the phosphatases tested in this panel belong to the class of protein-tyrosine phosphatases (SHP-1, SHP- 2 ...and Sina Bavari‡ 2 From the ‡United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702-5011, §Target Structure

  18. 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.

  19. Genome-wide analysis and experimentation of plant serine/ threonine/tyrosine-specific protein kinases.

    PubMed

    Rudrabhatla, Parvathi; Reddy, Mamatha M; Rajasekharan, Ram

    2006-01-01

    Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.

  20. Key role of succinate dehydrogenase in insulin-induced inactivation of protein tyrosine phosphatases.

    PubMed

    Pomytkin, I A; Kolesova, O E

    2002-06-01

    We studied the role of mitochondria in insulin-induced inactivation of protein tyrosine phosphatases in the liver. The mitochondrial respiratory chain is an insulin-sensitive source of H(2)O(2)that acts as a physiological inhibitor of protein tyrosine phosphatases. Succinate dehydrogenase plays a key role in insulin-stimulated generation of H(2)O(2)and inactivation of liver protein tyrosine phosphatases.

  1. Analysis of nitrated proteins in Saccharomyces cerevisiae involved in mating signal transduction.

    PubMed

    Kang, Jeong Won; Lee, Na Young; Cho, Kyung-Cho; Lee, Min Young; Choi, Do-Young; Park, Sang-Hyun; Kim, Kwang Pyo

    2015-01-01

    Protein tyrosine nitration (PTN) is a PTM that regulates signal transduction and inflammatory responses, and is related to neurodegenerative and cardiovascular diseases. The cellular function of PTN remains unclear because the low stoichiometry of PTN limits the identification and quantification of nitrated peptides. Effective enrichment is an important aspect of PTN analysis. In this study, we analyzed the in vivo nitroproteome elicited by mating signal transduction in Saccharomyces cerevisiae using a novel chemical enrichment method followed by LC-MS/MS. Nitroproteome profiling successfully identified changes in the nitration states of 14 proteins during mating signal transduction in S. cerevisiae, making this the first reported in vivo nitroproteome in yeast. We investigated the biological functions of these nitroproteins and their relationships to mating signal transduction in S. cerevisiae using a protein-protein interaction network. Our results suggest that PTN and denitration may be involved in nonreactive nitrogen species-mediated signal transduction and can provide clues for understanding the functional roles of PTN in vivo.

  2. 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.

  3. Atmospheric protein chemistry influenced by anthropogenic air pollutants: nitration and oligomerization upon exposure to ozone and nitrogen dioxide.

    PubMed

    Liu, Fobang; Lakey, Pascale S J; Berkemeier, Thomas; Tong, Haijie; Kunert, Anna Theresa; Meusel, Hannah; Cheng, Yafang; Su, Hang; Fröhlich-Nowoisky, Janine; Lai, Senchao; Weller, Michael G; Shiraiwa, Manabu; Pöschl, Ulrich; Kampf, Christopher J

    2017-08-24

    The allergenic potential of airborne proteins may be enhanced via post-translational modification induced by air pollutants like ozone (O3) and nitrogen dioxide (NO2). The molecular mechanisms and kinetics of the chemical modifications that enhance the allergenicity of proteins, however, are still not fully understood. Here, protein tyrosine nitration and oligomerization upon simultaneous exposure of O3 and NO2 were studied in coated-wall flow-tube and bulk solution experiments under varying atmospherically relevant conditions (5-200 ppb O3, 5-200 ppb NO2, 45-96% RH), using bovine serum albumin as a model protein. Generally, more tyrosine residues were found to react via the nitration pathway than via the oligomerization pathway. Depending on reaction conditions, oligomer mass fractions and nitration degrees were in the ranges of 2.5-25% and 0.5-7%, respectively. The experimental results were well reproduced by the kinetic multilayer model of aerosol surface and bulk chemistry (KM-SUB). The extent of nitration and oligomerization strongly depends on relative humidity (RH) due to moisture-induced phase transition of proteins, highlighting the importance of cloud processing conditions for accelerated protein chemistry. Dimeric and nitrated species were major products in the liquid phase, while protein oligomerization was observed to a greater extent for the solid and semi-solid phase states of proteins. Our results show that the rate of both processes was sensitive towards ambient ozone concentration, but rather insensitive towards different NO2 levels. An increase of tropospheric ozone concentrations in the Anthropocene may thus promote pro-allergic protein modifications and contribute to the observed increase of allergies over the past decades.

  4. MAS promoter regulation: a role for Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism.

    PubMed

    Prokop, Jeremy W; Rauscher, Frank J; Peng, Hongzhuang; Liu, Yuanjie; Araujo, Fabiano C; Watanabe, Ingrid; Reis, Fernando M; Milsted, Amy

    2014-05-01

    The ACE2 (angiotensin-converting enzyme 2)/Ang-(1-7) [angiotensin-(1-7)]/MAS axis of the RAS (renin-angiotensin system) has emerged as a pathway of interest in treating both cardiovascular disorders and cancer. The MAS protein is known to bind to and be activated by Ang-(1-7); however, the mechanisms of this activation are just starting to be understood. Although there are strong biochemical data regarding the regulation and activation of the AT1R (angiotensin II type 1 receptor) and the AT2R (angiotensin II type 2 receptor), with models of how AngII (angiotensin II) binds each receptor, fewer studies have characterized MAS. In the present study, we characterize the MAS promoter and provide a potential feedback mechanism that could compensate for MAS degradation following activation by Ang-(1-7). Analysis of ENCODE data for the MAS promoter revealed potential epigenetic control by KRAB (Krüppel-associated box)/KAP-1 (KRAB-associated protein-1). A proximal promoter construct for the MAS gene was repressed by the SOX [SRY (sex-determining region on the Y chromosome) box] proteins SRY, SOX2, SOX3 and SOX14, of which SRY is known to interact with the KRAB domain. The KRAB-KAP-1 complex can be tyrosine-nitrated, causing the dissociation of the KAP-1 protein and thus a potential loss of epigenetic control. Activation of MAS can lead to an increase in nitric oxide, suggesting a feedback mechanism for MAS on its own promoter. The results of the present study provide a more complete view of MAS regulation and, for the first time, suggest biochemical outcomes for nitration of the KRAB domain.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  6. Nitration of the egg-allergen ovalbumin enhances protein allergenicity but reduces the risk for oral sensitization in a murine model of food allergy.

    PubMed

    Untersmayr, Eva; Diesner, Susanne C; Oostingh, Gertie Janneke; Selzle, Kathrin; Pfaller, Tobias; Schultz, Cornelia; Zhang, Yingyi; Krishnamurthy, Durga; Starkl, Philipp; Knittelfelder, Regina; Förster-Waldl, Elisabeth; Pollak, Arnold; Scheiner, Otto; Pöschl, Ulrich; Jensen-Jarolim, Erika; Duschl, Albert

    2010-12-02

    Nitration of proteins on tyrosine residues, which can occur due to polluted air under "summer smog" conditions, has been shown to increase the allergic potential of allergens. Since nitration of tyrosine residues is also observed during inflammatory responses, this modification could directly influence protein immunogenicity and might therefore contribute to food allergy induction. In the current study we have analyzed the impact of protein nitration on sensitization via the oral route. BALB/c mice were immunized intragastrically by feeding untreated ovalbumin (OVA), sham-nitrated ovalbumin (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze the impact of the sensitization route, the allergens were also injected intraperitoneally. Animals being fed OVA or snOVA under acid-suppressive medication developed significantly elevated levels of IgE, and increased titers of specific IgG1 and IgG2a antibodies. Interestingly, oral immunizations of nOVA under anti-acid treatment did not result in IgG and IgE formation. In contrast, intraperitoneal immunization induced high levels of OVA specific IgE, which were significantly increased in the group that received nOVA by injection. Furthermore, nOVA triggered significantly enhanced mediator release from RBL cells passively sensitized with sera from allergic mice. Gastric digestion experiments demonstrated protein nitration to interfere with protein stability as nOVA was easily degraded, whereas OVA and snOVA remained stable up to 120 min. Additionally, HPLC-chip-MS/MS analysis showed that one tyrosine residue (Y(107)) being very efficiently nitrated is part of an ovalbumin epitope recognized exclusively after oral sensitization. These data indicated that despite the enhanced triggering capacity in existing allergy, nitration of OVA may be associated with a reduced de novo sensitizing capability via the oral route due to enhanced protein digestibility and/or changes in antibody epitopes.

  7. Pepsin is nitrated in the rat stomach, acquiring antiulcerogenic activity: a novel interaction between dietary nitrate and gut proteins.

    PubMed

    Rocha, Bárbara S; Gago, Bruno; Barbosa, Rui M; Lundberg, Jon O; Mann, Giovanni E; Radi, Rafael; Laranjinha, João

    2013-05-01

    Dietary nitrate is reduced to nitrite and nitric oxide ((•)NO) in the gut, producing reactive species able to nitrate proteins and lipids. We investigated intragastric production of (•)NO and nitrating agents in vivo by examining selective nitration of pepsinogen and pepsin. We further addressed the functional impact of nitration on peptic activity by evaluating the progression of secretagogue-induced ulcers. Pepsinogen nitration was assessed in healthy and diclofenac-induced ulcerated rat stomachs. Both groups were fed nitrite or water by oral gavage. Protein nitration was studied by immunofluorescence and immunoprecipitation. In parallel experiments, pentagastrin was administered to rats and nitrite was then instilled intragastrically. (•)NO levels were measured before and after nitrite administration by chemiluminescence. Macroscopic damage was assessed and nitrated pepsin was examined in the margin of ulcers. Protein nitration was detected physiologically in the stomach of healthy animals. Nitrite had a dual effect on intragastric nitration: overall nitration was decreased under physiological conditions but enhanced by acute inflammation. Pepsin and pepsinogen were also nitrated via a nitrite-dependent pathway. Nitration of both pepsin and its zymogen led to decreased peptic activity in response to classical substrates (e.g., collagen). Under conditions of acute ulceration, nitrite-dependent pepsin nitration prevented the development of gastric ulcers. Dietary nitrite generates nitrating agents in the stomach in vivo, markedly decreasing peptic activity. Under inflammatory and ulcerogenic conditions pepsin nitration attenuates the progression of gastric ulceration. These results suggest that dietary nitrite-dependent nitration of pepsin may have a novel antiulcerogenic effect in vivo.

  8. 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.

  9. Signal processing by protein tyrosine phosphorylation in plants

    PubMed Central

    2011-01-01

    Protein phosphorylation is a reversible post-translational modification controlling many biological processes. Most phosphorylation occurs on serine and threonine, and to a less extend on tyrosine (Tyr). In animals, Tyr phosphorylation is crucial for the regulation of many responses such as growth or differentiation. Only recently with the development of mass spectrometry, it has been reported that Tyr phosphorylation is as important in plants as in animals. The genes encoding protein Tyr kinases and protein Tyr phosphatases have been identified in the Arabidopsis thaliana genome. Putative substrates of these enzymes, and thus Tyr-phosphorylated proteins have been reported by proteomic studies based on accurate mass spectrometry analysis of the phosphopeptides and phosphoproteins. Biochemical approaches, pharmacology and genetic manipulations have indicated that responses to stress and developmental processes involve changes in protein Tyr phosphorylation. The aim of this review is to present an update on Tyr phosphorylation in plants in order to better assess the role of this post-translational modification in plant physiology. PMID:21628997

  10. Oxidative modification of native protein residues using cerium(IV) ammonium nitrate.

    PubMed

    Seim, Kristen L; Obermeyer, Allie C; Francis, Matthew B

    2011-10-26

    A new protein modification strategy has been developed that is based on an oxidative coupling reaction that targets electron-rich amino acids. This strategy relies on cerium(IV) ammonium nitrate (CAN) as an oxidation reagent and results in the coupling of tyrosine and tryptophan residues to phenylene diamine and anisidine derivatives. The methodology was first identified and characterized on peptides and small molecules, and was subsequently adapted for protein modification by determining appropriate buffer conditions. Using the optimized procedure, native and introduced solvent-accessible residues on proteins were selectively modified with polyethylene glycol (PEG) and small peptides. This unprecedented bioconjugation strategy targets these under-utilized amino acids with excellent chemoselectivity and affords good-to-high yields using low concentrations of the oxidant and coupling partners, short reaction times, and mild conditions.

  11. Crystal Structure of a Complex Between Protein Tyrosine Phosphatase 1B and the Insulin Receptor Tyrosine Kinase

    SciTech Connect

    Li,S.; Depetris, R.; Barford, D.; Chernoff, J.; Hubbard, S.

    2005-01-01

    Protein tyrosine phosphatase 1B (PTP1B) is a highly specific negative regulator of insulin receptor signaling in vivo. The determinants of PTP1B specificity for the insulin receptor versus other receptor tyrosine kinases are largely unknown. Here, we report a crystal structure at 2.3 Angstroms resolution of the catalytic domain of PTP1B (trapping mutant) in complex with the phosphorylated tyrosine kinase domain of the insulin receptor (IRK). The crystallographic asymmetric unit contains two PTP1B-IRK complexes that interact through an IRK dimer interface. Rather than binding to a phosphotyrosine in the IRK activation loop, PTP1B binds instead to the opposite side of the kinase domain, with the phosphorylated activation loops sequestered within the IRK dimer. The crystal structure provides evidence for a noncatalytic mode of interaction between PTP1B and IRK, which could be important for the selective recruitment of PTP1B to the insulin receptor.

  12. Solubilized placental membrane protein inhibits insulin receptor tyrosine kinase activity

    SciTech Connect

    Strout, H.V. Jr.; Slater, E.E.

    1987-05-01

    Regulation of insulin receptor (IR) tyrosine kinase (TK) activity may be important in modulating insulin action. Utilizing an assay which measures IR phosphorylation of angiotensin II (AII), the authors investigated whether fractions of TX-100 solubilized human placental membranes inhibited IR dependent AII phosphorylation. Autophosphorylated IR was incubated with membrane fractions before the addition of AII, and kinase inhibition measured by the loss of TSP incorporated in AII. An inhibitory activity was detected which was dose, time, and temperature dependent. The inhibitor was purified 200-fold by sequential chromatography on wheat germ agglutinin, DEAE, and hydroxyapatite. This inhibitory activity was found to correlate with an 80 KD protein which was electroeluted from preparative slab gels and rabbit antiserum raised. Incubation of membrane fractions with antiserum before the IRTK assay immunoprecipitated the inhibitor. Protein immunoblots of crude or purified fractions revealed only the 80 KD protein. Since IR autophosphorylation is crucial to IRTK activity, the authors investigated the state of IR autophosphorylation after treatment with inhibitor; no change was detected by phosphoamino acid analysis.

  13. 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.

  14. Redox Regulation of Protein Tyrosine Phosphatase Activity by Hydroxyl Radical

    PubMed Central

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2012-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H2O2 include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H2O2 abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H2O2 is poorly reactive in chemical terms and the second order rate constants for the H2O2-mediated PTP inactivation are ~10 M−1s−1, which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H2O2. We find that hydroxyl radical is produced from H2O2 solutions in the absence of metal chelating agent by the Fenton reaction. We show that hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H2O2 for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2–3 orders of magnitude higher than those mediated by H2O2 under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. PMID:22819876

  15. Redox regulation of protein tyrosine phosphatase activity by hydroxyl radical.

    PubMed

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2013-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H(2)O(2) include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H(2)O(2) abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H(2)O(2) is poorly reactive in chemical terms and the second order rate constants for the H(2)O(2)-mediated PTP inactivation are ~10M(-1)s(-1), which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H(2)O(2). We find that hydroxyl radical is produced from H(2)O(2) solutions in the absence of metal chelating agent by the Fenton reaction. We show that the hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H(2)O(2) for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2-3 orders of magnitude higher than those mediated by H(2)O(2) under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.

  16. The Sts Proteins Target Tyrosine Phosphorylated, Ubiquitinated Proteins within TCR Signaling Pathways

    SciTech Connect

    Carpino, N.; Chen, Y; Nassar, N; Oh, H

    2009-01-01

    The T cell receptor (TCR) detects the presence of infectious pathogens and activates numerous intracellular signaling pathways. Protein tyrosine phosphorylation and ubiquitination serve as key regulatory mechanisms downstream of the TCR. Negative regulation of TCR signaling pathways is important in controlling the immune response, and the Suppressor of TCR Signaling proteins (Sts-1 and Sts-2) have been shown to function as critical negative regulators of TCR signaling. Although their mechanism of action has yet to be fully uncovered, it is known that the Sts proteins possess intrinsic phosphatase activity. Here, we demonstrate that Sts-1 and Sts-2 are instrumental in down-modulating proteins that are dually modified by both protein tyrosine phosphorylation and ubiquitination. Specifically, both naive and activated T cells derived from genetically engineered mice that lack the Sts proteins display strikingly elevated levels of tyrosine phosphorylated, ubiquitinated proteins following TCR stimulation. The accumulation of the dually modified proteins is transient, and in activated T cells but not naive T cells is significantly enhanced by co-receptor engagement. Our observations hint at a novel regulatory mechanism downstream of the T cell receptor.

  17. Simultaneous determination of nitrated and oligomerized proteins by size exclusion high-performance liquid chromatography coupled to photodiode array detection.

    PubMed

    Liu, Fobang; Reinmuth-Selzle, Kathrin; Lai, Senchao; Weller, Michael G; Pöschl, Ulrich; Kampf, Christopher J

    2017-04-28

    Chemical modifications such as nitration and cross-linking may enhance the allergenic potential of proteins. The kinetics and mechanisms of the underlying chemical processes, however, are not yet well understood. Here, we present a size-exclusion chromatography/spectrophotometry method (SEC-HPLC-DAD) that allows a simultaneous detection of mono-, di-, tri-, and higher protein oligomers, as well as their individual nitration degrees (NDs). The ND results of proteins from this new method agree well with the results from an alternative well-established method, for the analysis of tetranitromethane (TNM)- and nitrogen dioxide and ozone (NO2/O3)-nitrated protein samples. Importantly, the NDs for individual oligomer fractions can be obtained from the new method, and also, we provide a proof of principle for the calculation of the concentrations for individual protein oligomer fractions by their determined NDs, which will facilitate the investigation of the kinetics and mechanism for protein tyrosine nitration and cross-linking. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Cellular biochemistry methods for investigating protein tyrosine phosphatases.

    PubMed

    Stanford, Stephanie M; Ahmed, Vanessa; Barrios, Amy M; Bottini, Nunzio

    2014-05-10

    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. 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. 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. 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.

  19. Protein-tyrosine phosphatase sigma is associated with ulcerative colitis.

    PubMed

    Muise, Aleixo M; Walters, Thomas; Wine, Eytan; Griffiths, Anne M; Turner, Dan; Duerr, Richard H; Regueiro, Miguel D; Ngan, Bo-Yee; Xu, Wei; Sherman, Philip M; Silverberg, Mark S; Rotin, Daniela

    2007-07-17

    Inflammatory bowel disease (IBD), a relatively common chronic debilitating intestinal illness, is composed of two broadly defined groups, Crohn's disease (CD) and ulcerative colitis (UC). Although several susceptibility genes for CD have been recently described, susceptibility genes exclusive for UC have not been forthcoming. Here, we show that receptor protein-tyrosine phosphatase sigma (PTPRS-encoding PTPsigma) knockout mice spontaneously develop mild colitis that becomes severe when challenged with two known inducers of colitis. We also demonstrate that E-cadherin and beta-catenin, two important adherens junction proteins involved in maintenance of barrier defense in the colon, act as colonic substrates for PTPsigma. Furthermore, we show that three SNPs (rs886936, rs17130, and rs8100586) that flank exon 8 in the human PTPRS gene are associated with UC. The presence of these SNPs is associated with novel splicing that removes the third immunoglobulin-like domain (exon 9) from the extracellular portion of PTPsigma, possibly altering dimerization or ligand recognition. We propose that polymorphisms in the human PTPRS gene lead to ulcerative colitis.

  20. Protein tyrosine phosphatase 1B inhibitory activity of alkaloids from Rhizoma Coptidis and their molecular docking studies.

    PubMed

    Choi, Jae Sue; Ali, Md Yousof; Jung, Hyun Ah; Oh, Sang Ho; Choi, Ran Joo; Kim, Eon Ji

    2015-08-02

    Rhizoma Coptidis (the rhizome of Coptis chinensis Franch) has commonly been used for treatment of diabetes mellitus in traditional Chinese medicine due to its blood sugar-lowering properties and therapeutic benefits which highly related to the alkaloids therein. However, a limited number of studies focused on the Coptis alkaloids other than berberine. In the present study, we investigated the anti-diabetic potential of Coptis alkaloids, including berberine (1), epiberberine (2), magnoflorine (3), and coptisine (4), by evaluating the ability of these compounds to inhibit protein tyrosine phosphatase 1B (PTP1B), and ONOO(-)-mediated protein tyrosine nitration. We scrutinized the potentials of Coptis alkaloids as PTP1B inhibitors via enzyme kinetics and molecular docking simulation. The Coptis alkaloids 1-4 exhibited remarkable inhibitory activities against PTP1B with the IC50 values of 16.43, 24.19, 28.14, and 51.04 μM, respectively, when compared to the positive control ursolic acid. These alkaloids also suppressed ONOO(-)-mediated tyrosine nitration effectively in a dose dependent manner. In addition, our kinetic study using the Lineweaver-Burk and Dixon plots revealed that 1 and 2 showed a mixed-type inhibition against PTP1B, while 3 and 4 noncompetitively inhibited PTP1B. Moreover, molecular docking simulation of these compounds demonstrated negative binding energies (Autodock 4.0=-6.7 to -7.8 kcal/mol; Fred 2.0=-59.4 to -68.2 kcal/mol) and a high proximity to PTP1B residues, including Phe182 and Asp181 in the WPD loop, Cys215 in the active sites and Tyr46, Arg47, Asp48, Val49, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221 and Gln262 in the pocket site, indicating a higher affinity and tighter binding capacity of these alkaloids for the active site of the enzyme. Our results clearly indicate the promising anti-diabetic potential of Coptis alkaloids as inhibitors on PTP1B as well as suppressors of ONOO(-)-mediated protein tyrosine nitration, and thus hold

  1. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore.

    PubMed

    Antosiewicz, Jan M; Shugar, David

    Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV-Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.

  2. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore.

    PubMed

    Antosiewicz, Jan M; Shugar, David

    2016-06-01

    Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV-Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.

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

    USDA-ARS?s Scientific Manuscript database

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

  4. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

    PubMed

    Hortelano, S; Alvarez, A M; Boscá, L

    1999-12-01

    Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (DeltaPsi(m)) with specific fluorescent probes showed a rapid and persistent increase of DeltaPsi(m), a potential that usually decreases in cells undergoing apoptosis through mitochondrial-dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of DeltaPsi(m). The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of DeltaPsi(m), and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of DeltaPsi(m).-Hortelano, S., Alvarez, A. M., Boscá, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

  5. Tyrosine phosphorylation on spleen tyrosine kinase (Syk) is differentially regulated in human and murine platelets by protein kinase C isoforms.

    PubMed

    Buitrago, Lorena; Bhavanasi, Dheeraj; Dangelmaier, Carol; Manne, Bhanu Kanth; Badolia, Rachit; Borgognone, Alessandra; Tsygankov, Alexander Y; McKenzie, Steven E; Kunapuli, Satya P

    2013-10-04

    Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.

  6. Glutathione depletion in a midbrain-derived immortalized dopaminergic cell line results in limited tyrosine nitration of mitochondrial complex I subunits: implications for Parkinson's disease.

    PubMed

    Bharath, Srinivas; Andersen, Julie Kay

    2005-01-01

    Oxidative stress and mitochondrial dysfunction signify two important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models and in affected tissues from the disease itself have demonstrated a selective inhibition of mitochondrial complex I activity that appears to affect normal mitochondrial physiology leading to neuronal cell death. Earlier experiments from our laboratory have demonstrated that induced depletion of glutathione (GSH + GSSG) in cultured dopaminergic cells resulted in increased oxidative stress and a decrease in mitochondrial function. Furthermore, this dysfunction was linked to a selective decrease in mitochondrial complex I activity that appears to be due to oxidation of this complex. Glutathione depletion is the earliest detectable biochemical event during PD progression and occurs prior to complex I inhibition. Recent observations have also indicated that oxidative damage to complex I via naturally occurring free radicals such as peroxynitrite leads to modification of tyrosine and/or cysteine residues resulting in complex I inhibition. Using the sucrose gradient method, we detected in complex I-enriched fractions from a glutathione-depleted dopaminergic cell line two bands corresponding to approximately 25-kDa and approximately 30-kDa polypeptides that demonstrate anti-nitrotyrosine immunoreactivity, suggesting the possible involvement of protein nitration by peroxynitrite in glutathione depletion-mediated complex I inhibition.

  7. Tyrosine phosphorylation of an SH2-containing protein tyrosine phosphatase is coupled to platelet thrombin receptor via a pertussis toxin-sensitive heterotrimeric G-protein.

    PubMed Central

    Li, R Y; Gaits, F; Ragab, A; Ragab-Thomas, J M; Chap, H

    1995-01-01

    SH-PTP1 is a protein tyrosine phosphatase (PTP) predominantly expressed in haematopoietic cells and containing two src homology-2 (SH2) domains. Here we report that SH-PTP1 is phosphorylated on both serine and tyrosine residues in response to thrombin or phorbol myristate acetate (PMA), which increased by 60 and 40%, respectively, SH-PTP1 activity. Thrombin-induced phosphorylation of SH-PTP1 is an early signalling event (maximal within 10 s) involving neither integrin signalling, nor calcium, nor release of ADP or thromboxane A2. Moreover, in contrast with PMA, the effect of thrombin on the tyrosine phosphorylation of SH-PTP1 was hardly affected by GF109203X, a specific protein kinase C (PKC) inhibitor. Finally, phosphorylation of SH-PTP1 could be provoked in permeabilized platelets by thrombin or GTP gamma S. This was abolished by pertussis toxin, the specificity of this effect being verified with the megakaryocytic cell line Dami cell. Our data thus identify SH-PTP1 as an in vivo substrate of a putative protein tyrosine kinase linked to the thrombin receptor by a Gi protein. This might offer some clue to unravel the mechanism of thrombin not only in platelets but also in nucleated cells, where its mitogenic effect is known to involve pertussis toxin-sensitive G-proteins, tyrosine phosphorylation and the ras pathway. Images PMID:7781604

  8. Protection against peroxynitrite dependent tyrosine nitration and alpha 1-antiproteinase inactivation by some anti-inflammatory drugs and by the antibiotic tetracycline.

    PubMed

    Whiteman, M; Kaur, H; Halliwell, B

    1996-06-01

    To examine in vitro the ability of several drugs to protect against deleterious effects of peroxynitrite, a cytotoxic agent formed by reaction of nitric oxide with superoxide radical, that may be generated in the rheumatoid joint and could cause joint damage. The ability of several drugs to protect against such possible toxic actions of peroxynitrite as inactivation of alpha 1-antiproteinase and nitration of tyrosine was evaluated. Most non-steroidal anti-inflammatory drugs were moderately (indomethacin, diclofenac, naproxen, tolmetin) or only weakly (sulindac, ibuprofen, aurothioglucose, flurbiprofen, sulphasalazine, salicylate, penicillamine disulphide) effective in preventing tyrosine nitration and alpha 1-antiproteinase inactivation by peroxynitrite, but 5-aminosalicylate and penicillamine were much more effective, as was the antibiotic tetracycline (but not ampicillin). Phenylbutazone and flufenamic acid protected effectively against tyrosine nitration, but could not be tested in the alpha 1-antiproteinase system. The analgesic paracetamol was highly protective in both assay systems. Many drugs used in the treatment of rheumatoid arthritis are unlikely to act by scavenging peroxynitrite. The feasibility of peroxynitrite scavenging as a mechanism of penicillamine, 5-aminosalicylate, and paracetamol action in vivo is discussed.

  9. Inhibition of myeloperoxidase-mediated protein nitration by tempol: Kinetics, mechanism, and implications.

    PubMed

    Vaz, Sandra M; Augusto, Ohara

    2008-06-17

    Despite the therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetra-methyl-1-piperidinyloxy) and related nitroxides as antioxidants, their effects on peroxidase-mediated protein tyrosine nitration remain unexplored. This posttranslational protein modification is a biomarker of nitric oxide-derived oxidants, and, relevantly, it parallels tissue injury in animal models of inflammation and is attenuated by tempol treatment. Here, we examine tempol effects on ribonuclease (RNase) nitration mediated by myeloperoxidase (MPO), a mammalian enzyme that plays a central role in various inflammatory processes. Some experiments were also performed with horseradish peroxidase (HRP). We show that tempol efficiently inhibits peroxidase-mediated RNase nitration. For instance, 10 muM tempol was able to inhibit by 90% the yield of 290 muM 3-nitrotyrosine produced from 370 muM RNase. The effect of tempol was not completely catalytic because part of it was consumed by recombination with RNase-tyrosyl radicals. The second-order rate constant of the reaction of tempol with MPO compound I and II were determined by stopped-flow kinetics as 3.3 x 10(6) and 2.6 x 10(4) M(-1) s(-1), respectively (pH 7.4, 25 degrees C); the corresponding HRP constants were orders of magnitude smaller. Time-dependent hydrogen peroxide and nitrite consumption and oxygen production in the incubations were quantified experimentally and modeled by kinetic simulations. The results indicate that tempol inhibits peroxidase-mediated RNase nitration mainly because of its reaction with nitrogen dioxide to produce the oxammonium cation, which, in turn, recycles back to tempol by reacting with hydrogen peroxide and superoxide radical to produce oxygen and regenerate nitrite. The implications for nitroxide antioxidant mechanisms are discussed.

  10. ROLE OF TYROSINE-SULFATED PROTEINS IN RETINAL STRUCTURE AND FUNCTION

    PubMed Central

    Kanan, Y.; Al-Ubaidi, M.R.

    2014-01-01

    The extracellular matrix (ECM) plays a significant role in cellular and retinal health. The study of retinal tyrosine-sulfated proteins is an important first step toward understanding the role of ECM in retinal health and diseases. These secreted proteins are members of the retinal ECM. Tyrosine sulfation was shown to be necessary for the development of proper retinal structure and function. The importance of tyrosine sulfation is further demonstrated by the evolutionary presence of tyrosylprotein sulfotransferases, enzymes that catalyze proteins’ tyrosine sulfation, and the compensatory abilities of these enzymes. Research has identified four tyrosine-sulfated retinal proteins: fibulin 2, vitronectin, complement factor H (CFH), and opticin. Vitronectin and CFH regulate the activation of the complement system and are involved in the etiology of some cases of age-related macular degeneration. Analysis of the role of tyrosine sulfation in fibulin function showed that sulfation influences the protein's ability to regulate growth and migration. Although opticin was recently shown to exhibit anti-angiogenic properties, it is not yet determined what role sulfation plays in that function. Future studies focusing on identifying all of the tyrosine-sulfated retinal proteins would be instrumental in determining the impact of sulfation on retinal protein function in retinal homeostasis and diseases. PMID:25819460

  11. Involvement of protein tyrosine phosphatases in adipogenesis: New anti-obesity targets?

    PubMed Central

    Bae, Kwang-Hee; Kim, Won Kon; Lee, Sang Chul

    2012-01-01

    Obesity is a worldwide epidemic as well as being a major risk factor for diabetes, cardiovascular diseases and several types of cancers. Obesity is mainly due to the overgrowth of adipose tissue arising from an imbalance between energy intake and energy expenditure. Adipose tissue, primarily composed of adipocytes, plays a key role in maintaining whole body energy homeostasis. In view of the treatment of obesity and obesity-related diseases, it is critical to understand the detailed signal transduction mechanisms of adipogenic differentiation. Adipogenic differentiation is tightly regulated by many key signal cascades, including insulin signaling. These signal cascades generally transfer or amplify the signal by using serial tyrosine phosphorylations. Thus, protein tyrosine kinases and protein tyrosine phosphatases are closely related to adipogenic differentiation. Compared to protein tyrosine kinases, protein tyrosine phosphatases have received little attention in adipogenic differentiation. This review aims to highlight the involvement of protein tyrosine phosphatases in adipogenic differentiation and the possibility of protein tyrosine phosphatases as drugs to target obesity. [BMB Reports 2012; 45(12): 700-706] PMID:23261055

  12. Involvement of protein tyrosine phosphatases in adipogenesis: new anti-obesity targets?

    PubMed

    Bae, Kwang-Hee; Kim, Won Kon; Lee, Sang Chul

    2012-12-01

    Obesity is a worldwide epidemic as well as being a major risk factor for diabetes, cardiovascular diseases and several types of cancers. Obesity is mainly due to the overgrowth of adipose tissue arising from an imbalance between energy intake and energy expenditure. Adipose tissue, primarily composed of adipocytes, plays a key role in maintaining whole body energy homeostasis. In view of the treatment of obesity and obesity-related diseases, it is critical to understand the detailed signal transduction mechanisms of adipogenic differentiation. Adipogenic differentiation is tightly regulated by many key signal cascades, including insulin signaling. These signal cascades generally transfer or amplify the signal by using serial tyrosine phosphorylations. Thus, protein tyrosine kinases and protein tyrosine phosphatases are closely related to adipogenic differentiation. Compared to protein tyrosine kinases, protein tyrosine phosphatases have received little attention in adipogenic differentiation. This review aims to highlight the involvement of protein tyrosine phosphatases in adipogenic differentiation and the possibility of protein tyrosine phosphatases as drugs to target obesity.

  13. Protein tyrosine phosphatase inhibition by metals and metal complexes.

    PubMed

    Lu, Liping; Zhu, Miaoli

    2014-05-10

    Protein tyrosine phosphatases (PTPs) play essential roles in controlling cell proliferation, differentiation, communication, and adhesion. The dysregulated activities of PTPs are involved in the pathogenesis of a number of human diseases such as cancer, diabetes, and autoimmune diseases. Many PTPs have emerged as potential new targets for novel drug discovery. PTP inhibitors have attracted much attention. Many PTP inhibitors have been developed. Some of them have been proven to be efficient in lowering blood glucose levels in vivo or inhibiting tumor xenograft growth. Some metal ions and metal complexes potently inhibit PTPs. The metal atoms within metal complexes play an important role in PTP binding, while ligand structures influence the inhibitory potency and selectivity. Some metal complexes can penetrate the cell membrane and selectively bind to their targeting PTPs, enhancing the phosphorylation of the related substrates and influencing cellular metabolism. PTP inhibition is potentially involved in the pathophysiological and toxicological processes of metals and some PTPs may be cellular targets of certain metal-based therapeutic agents. Investigating the structural basis of the interactions between metal complexes and PTPs would facilitate a comprehensive understanding of the structure-activity relationship and accelerate the development of promising metal-based drugs targeting specific PTPs.

  14. Zinc ions modulate protein tyrosine phosphatase 1B activity.

    PubMed

    Bellomo, Elisa; Massarotti, Alberto; Hogstrand, Christer; Maret, Wolfgang

    2014-07-01

    Protein tyrosine phosphatases (PTPs) are key enzymes in cellular regulation. The 107 human PTPs are regulated by redox signalling, phosphorylation, dimerisation, and proteolysis. Recent findings of very strong inhibition of some PTPs by zinc ions at concentrations relevant in a cellular environment suggest yet another mechanism of regulation. One of the most extensively investigated PTPs is PTP1B (PTPN1). It regulates the insulin and leptin signalling pathway and is implicated in cancer and obesity/diabetes. The development of novel assay conditions to investigate zinc inhibition of PTP1B provides estimates of about 5.6 nM affinity for inhibitory zinc(II) ions. Analysis of three PTP1B 3D structures (PDB id: 2CM2, 3I80 and 1A5Y) identified putative zinc binding sites and supports the kinetic studies in suggesting an inhibitory zinc only in the closed and cysteinyl-phosphate intermediate forms of the enzyme. These observations gain significance with regard to recent findings of regulatory roles of zinc ions released from the endoplasmic reticulum.

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

    PubMed

    Kuznetsov, Vyacheslav I; Hengge, Alvan C

    2013-11-12

    LDP3 (VHZ) is the smallest classical protein tyrosine phosphatase (PTP) known to date and was originally misclassified as an atypical dual-specificity phosphatase. Kinetic isotope effects with steady-state and pre-steady-state kinetics of VHZ and mutants with p-nitrophenol phosphate 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 of 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.

  16. Protein Tyrosine Kinase Signaling During Oocyte Maturation and Fertilization

    PubMed Central

    McGinnis, Lynda K.; Carroll, David J.; Kinsey, William H.

    2011-01-01

    The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans. PMID:21681843

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

    PubMed Central

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

    2016-01-01

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

  18. Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

    PubMed Central

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

    2016-01-01

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

  19. 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.

  20. 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.

  1. Tyrosine Phosphorylation Pattern in Sperm Proteins Isolated from Normospermic and Teratospermic Men

    PubMed Central

    Jabbari, Sepideh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mahdi; Ebrahim Habibi, Azadeh; Amirjanati, Naser; Lakpour, Niknam; Asgharpour, Lima; Ardekani, Ali M.

    2009-01-01

    Introduction In mammalian system, spermatozoa are not able to fertilize the oocyte immediately upon ejaculation, thus they undergo a series of biochemical and molecular changes which is termed capacitation. During sperm capacitation, signal transduction pathways are activated which lead to protein tyrosine phosphorylation. Tyrosine phosphorylated proteins have an important role in sperm capacitation such as hyperactive motility, interaction with zona pellucida and acrosome reaction. Evaluation of tyrosine phosphorylation pattern is important for further understanding of molecular mechanisms of fertilization and the etiology of sperm dysfunctions and abnormalities such as teratospermia. The goal of this study is to characterize tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic infertile men attending Avicenna Infertility Clinic in Tehran. Materials and Methods Semen samples were collected and the spermatozoa were isolated using Percoll gradient centrifugation. Then the spermatozoa were incubated up to 6h at 37°C with 5% CO2 in 3% Bovine Serum Albumin-supplemented Ham's F-10 for capacitation to take place. The total proteins from spermatozoa were extracted and were subjected to SDS-PAGE before and after capacitation. To evaluate protein tyrosine phosphorylation pattern, western blotting with specific antibody against phosphorylated tyrosines was performed. Results The results upon western blotting showed: 1) at least six protein bands were detected before capacitation in the spermatozoa from normospermic samples. However, comparable levels of tyrosine phosphorylation was not observed in the spermatozoa from teratospermic samples. 2) The intensity of protein tyrosine phosphorylation appears to have been increased during capacitation in the normospermic relative to the teratospermic group. Conclusion For the first time, these findings demonstrate and suggest that the differences in the types of proteins and diminished

  2. A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

    PubMed

    Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E

    1996-07-19

    We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

  3. 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

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

    PubMed Central

    Guo, Y L; Roux, S J

    1995-01-01

    A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate. PMID:11536662

  5. Protein tyrosine phosphatase PTP-RR regulates corticosteroid sensitivity.

    PubMed

    Kobayashi, Yoshiki; Ito, Kazuhiro; Kanda, Akira; Tomoda, Koich; Miller-Larsson, Anna; Barnes, Peter J; Mercado, Nicolas

    2016-03-24

    We have recently reported that protein phosphate 2A (PP2A) inactivation resulted in increased phosphorylation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase 1 (JNK1) and glucocorticoid receptors (GR) at Ser(226), thereby reducing GR nuclear translocation and causing corticosteroid insensitivity in severe asthmatics. Protein tyrosine phosphatases (PTPs) are also known to be critically involved in the regulation of MAPKs, such as JNK and therefore potentially associated with GR function. The aim of study was to elucidate the involvement of MAPK-PTPs (PTP-RR, PTP-N5 and PTP-N7), which can dephosphorylate MAPKs, in the regulation of corticosteroid sensitivity. Corticosteroid sensitivity, GR nuclear translocation, phosphorylation levels of GR-Ser(226), JNK1 and PP2A catalytic subunit (PP2AC)-Tyr(307) and protein expression levels and activities of PTP-RR and PP2AC were evaluated in U937 cells and/or peripheral blood mononuclear cells (PBMCs). Knock-down effects of MAPK-PTPs using siRNA were also evaluated. Knock-down of PTP-RR, but not of PTP-N5 or PTP-N7 impaired corticosteroid sensitivity, induced GR-Ser(226) phosphorylation and reduced GR nuclear translocation. Under IL-2/IL-4-induced corticosteroid insensitivity, PTP-RR expression, activity and associations with JNK1 and GR were reduced but PTP-RR activity was restored by formoterol. Also in PBMCs from severe asthmatic patients, PTP-RR and JNK1 expression were reduced and GR-Ser(226) phosphorylation increased. Furthermore, PTP-RR was associated with PP2A. PTP-RR reduction enhanced PP2AC-Tyr(307) phosphorylation leading to impairment of PP2A expression and activity. We demonstrated that with corticosteroid insensitivity PTP-RR fails to reduce phosphorylation of JNK1 and GR-Ser(226), resulting in down-regulation of GR nuclear translocation. Reduced PTP-RR may represent a novel cause of corticosteroid insensitivity in severe asthmatics.

  6. Tyrosine phosphorylation of protein kinase CK2 by Src-related tyrosine kinases correlates with increased catalytic activity.

    PubMed Central

    Donella-Deana, Arianna; Cesaro, Luca; Sarno, Stefania; Ruzzene, Maria; Brunati, Anna Maria; Marin, Oriano; Vilk, Greg; Doherty-Kirby, Amanda; Lajoie, Gilles; Litchfield, David W; Pinna, Lorenzo A

    2003-01-01

    Casein kinase-2 (CK2) is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits, whose regulation is still not well understood. In the present study, we show that the catalytic subunits of human CK2, but not the regulatory beta-subunits, are readily phosphorylated by the Src family protein tyrosine kinases Lyn and c-Fgr to a stoichiometry approaching 2 mol phosphotyrosine/mol CK2alpha with a concomitant 3-fold increase in catalytic activity. We also show that endogenous CK2alpha becomes tyrosine-phosphorylated in pervanadate-treated Jurkat cells. Both tyrosine phosphorylation and stimulation of activity are suppressed by the specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine. By comparison, mutations giving rise to inactive forms of CK2alpha do not abrogate and, in some cases, stimulate Lyn and c-Fgr-dependent tyrosine phosphorylation of CK2. Several radiolabelled phosphopeptides could be resolved by HPLC, following tryptic digestion of CK2alpha that had been phosphoradiolabelled by incubation with [(32)P]ATP and c-Fgr. The most prominent phosphopeptide co-migrates with a synthetic peptide encompassing the 248-268 sequence, phosphorylated previously by c-Fgr at Tyr(255) in vitro. The identification of Tyr(255) as a phosphorylated residue was also supported by MS sequencing of both the phosphorylated and non-phosphorylated 248-268 tryptic fragments from CK2alpha and by on-target phosphatase treatment. A CK2alpha mutant in which Tyr(255) was replaced by phenylalanine proved less susceptible to phosphorylation and refractory to stimulation by c-Fgr. PMID:12628006

  7. 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.

  8. Engineering tyrosine-based electron flow pathways in proteins: the case of aplysia myoglobin.

    PubMed

    Reeder, Brandon J; Svistunenko, Dimitri A; Cooper, Chris E; Wilson, Michael T

    2012-05-09

    Tyrosine residues can act as redox cofactors that provide an electron transfer ("hole-hopping") route that enhances the rate of ferryl heme iron reduction by externally added reductants, for example, ascorbate. Aplysia fasciata myoglobin, having no naturally occurring tyrosines but 15 phenylalanines that can be selectively mutated to tyrosine residues, provides an ideal protein with which to study such through-protein electron transfer pathways and ways to manipulate them. Two surface exposed phenylalanines that are close to the heme have been mutated to tyrosines (F42Y, F98Y). In both of these, the rate of ferryl heme reduction increased by up to 3 orders of magnitude. This result cannot be explained in terms of distance or redox potential change between donor and acceptor but indicates that tyrosines, by virtue of their ability to form radicals, act as redox cofactors in a new pathway. The mechanism is discussed in terms of the Marcus theory and the specific protonation/deprotonation states of the oxoferryl iron and tyrosine. Tyrosine radicals have been observed and quantified by EPR spectroscopy in both mutants, consistent with the proposed mechanism. The location of each radical is unambiguous and allows us to validate theoretical methods that assign radical location on the basis of EPR hyperfine structure. Mutation to tyrosine decreases the lipid peroxidase activity of this myoglobin in the presence of low concentrations of reductant, and the possibility of decreasing the intrinsic toxicity of hemoglobin by introduction of these pathways is discussed.

  9. 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.

  10. Effect of Second-Hand Tobacco Smoke on the Nitration of Brain Proteins: A Systems Biology and Bioinformatics Approach.

    PubMed

    Kobeissy, Firas H; Guingab-Cagmat, Joy; Bruijnzeel, Adriaan W; Gold, Mark S; Wang, Kevin

    2017-01-01

    Second-hand smoke (SHS) exposure leads to the death of approximately 48,000 nonsmokers per year in the United States alone. SHS exposure has been associated with cardiovascular, respiratory, and neurodegenerative diseases. While cardiac function abnormalities and lung cancer due to SHS have been well characterized, brain injury due to SHS has not undergone a full systematic evaluation. Oxidative stress and nitration have been associated with smoking and SHS exposure. Animal studies suggest that exposure to tobacco smoke increases oxidative stress. Oxidative stress is characterized by an increase in reactive oxygen and nitrogen species (ROS/RNS). Among the oxidative mechanisms affecting protein functionality is the posttranslational modification (PTM)-mediated tyrosine nitration. Protein tyrosine nitration, a covalent posttranslational modification, is commonly used as a marker of cellular oxidative stress associated with the pathogenesis of several neurodegenerative diseases. In our previous published work, the utility of a targeted proteomic approach has been evaluated to identify two brain abundant proteins in an in vivo SHS rat model namely the GAPDH and UCH-L1. In this current study, mass spectrometric-based proteomic and complementary biochemical methods were used to characterize the SHS-induced brain nitroproteome followed by bioinformatics/systems biology approach analysis to characterize protein interaction map. Sprague Dawley rats were exposed to SHS for 5 weeks and then cortical tissues were collected. Nitroprotein enrichment was performed via 3-Nitro tyrosine (3-NT) immunoprecipitation of brain lysates proteins. Protein nitration was validated via Western blotting to confirm the presence of nitroproteins complemented by gel-free neuroproteomic analysis by data-dependent LC-MS/MS. We identified 29 differentially expressed proteins in the 3-NT-enriched samples; seven of these proteins were unique to SHS exposure. Network analysis revealed an association of

  11. [Transport of phenylalanine and tyrosine in Brevibacterium linens: specificity and incorporation into proteins].

    PubMed

    Boyaval, P; Moreira, E; Desmazeaud, M J

    1984-04-01

    The specificity of phenylalanine and tyrosine carriers was investigated using actively metabolizing cells of Brevibacterium linens. The cellular protein synthesis of resting cells was very weakly inhibited, even with high concentrations of chloramphenicol or tetracycline. The nonaromatic amino acids were weak inhibitors for these carriers, while fluorinate analogues of phenylalanine and tyrosine were very potent competitive inhibitors. In practice these analogues cannot be used to replace amino acids to evaluate transport without incorporation because they are incorporated in cellular proteins.

  12. Adhesion-Linked Protein Tyrosine Phosphatases, Morphogenesis and Breast Cancer Progression

    DTIC Science & Technology

    2004-07-01

    Award Number: DAMD17-03-1-0496 TITLE: Adhesion-linked Protein Tyrosine Phosphatases, Morphogenesis and Breast Cancer Progression PRINCIPAL...Adhesion-linked Protein Tyrosine Phosphatases, DAMD17-03-1-0496 Morphogenesis and Breast Cancer Progression 6. AUTHOR(S) Valerie M. Weaver, Ph.D. 7...we identified the Band 4.1 PTPs MEG1 and D1 as two candidate PTP metastasis suppressors. Our studies show that during MEC differentiation PTP MEG1

  13. Essential Functions of Protein Tyrosine Phosphatases Ptp2 and Ptp3 and Rim11 Tyrosine Phosphorylation in Saccharomyces cerevisiae Meiosis and Sporulation

    PubMed Central

    Zhan, Xiao-Li; Hong, Yulong; Zhu, Tianqing; Mitchell, Aaron P.; Deschenes, Robert J.; Guan, Kun-Liang

    2000-01-01

    Tyrosine phosphorylation plays a central role in eukaryotic signal transduction. In yeast, MAP kinase pathways are regulated by tyrosine phosphorylation, and it has been speculated that other biochemical processes may also be regulated by tyrosine phosphorylation. Previous genetic and biochemical studies demonstrate that protein tyrosine phosphatases (PTPases) negatively regulate yeast MAP kinases. Here we report that deletion of PTP2 and PTP3 results in a sporulation defect, suggesting that tyrosine phosphorylation is involved in regulation of meiosis and sporulation. Deletion of PTP2 and PTP3 blocks cells at an early stage of sporulation before premeiotic DNA synthesis and induction of meiotic-specific genes. We observed that tyrosine phosphorylation of several proteins, including 52-, 43-, and 42-kDa proteins, was changed in ptp2Δptp3Δ homozygous deletion cells under sporulation conditions. The 42-kDa tyrosine-phosphorylated protein was identified as Mck1, which is a member of the GSK3 family of protein kinases and previously known to be phosphorylated on tyrosine. Mutation of MCK1 decreases sporulation efficiency, whereas mutation of RIM11, another GSK3 member, specifically abolishes sporulation; therefore, we investigated regulation of Rim11 by Tyr phosphorylation during sporulation. We demonstrated that Rim11 is phosphorylated on Tyr-199, and the Tyr phosphorylation is essential for its in vivo function, although Rim11 appears not to be directly regulated by Ptp2 and Ptp3. Biochemical characterizations indicate that tyrosine phosphorylation of Rim11 is essential for the activity of Rim11 to phosphorylate substrates. Our data demonstrate important roles of protein tyrosine phosphorylation in meiosis and sporulation PMID:10679022

  14. Glutamine Synthetase Is a Molecular Target of Nitric Oxide in Root Nodules of Medicago truncatula and Is Regulated by Tyrosine Nitration1[W][OA

    PubMed Central

    Melo, Paula M.; Silva, Liliana S.; Ribeiro, Isa; Seabra, Ana R.; Carvalho, Helena G.

    2011-01-01

    Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis, but its biological role in nodule functioning is still far from being understood. To unravel the signal transduction cascade and ultimately NO function, it is necessary to identify its molecular targets. This study provides evidence that glutamine synthetase (GS), a key enzyme for root nodule metabolism, is a molecular target of NO in root nodules of Medicago truncatula, being regulated by tyrosine (Tyr) nitration in relation to active nitrogen fixation. In vitro studies, using purified recombinant enzymes produced in Escherichia coli, demonstrated that the M. truncatula nodule GS isoenzyme (MtGS1a) is subjected to NO-mediated inactivation through Tyr nitration and identified Tyr-167 as the regulatory nitration site crucial for enzyme inactivation. Using a sandwich enzyme-linked immunosorbent assay, it is shown that GS is nitrated in planta and that its nitration status changes in relation to active nitrogen fixation. In ineffective nodules and in nodules fed with nitrate, two conditions in which nitrogen fixation is impaired and GS activity is reduced, a significant increase in nodule GS nitration levels was observed. Furthermore, treatment of root nodules with the NO donor sodium nitroprusside resulted in increased in vivo GS nitration accompanied by a reduction in GS activity. Our results support a role of NO in the regulation of nitrogen metabolism in root nodules and places GS as an important player in the process. We propose that the NO-mediated GS posttranslational inactivation is related to metabolite channeling to boost the nodule antioxidant defenses in response to NO. PMID:21914816

  15. Glutamine synthetase is a molecular target of nitric oxide in root nodules of Medicago truncatula and is regulated by tyrosine nitration.

    PubMed

    Melo, Paula M; Silva, Liliana S; Ribeiro, Isa; Seabra, Ana R; Carvalho, Helena G

    2011-11-01

    Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis, but its biological role in nodule functioning is still far from being understood. To unravel the signal transduction cascade and ultimately NO function, it is necessary to identify its molecular targets. This study provides evidence that glutamine synthetase (GS), a key enzyme for root nodule metabolism, is a molecular target of NO in root nodules of Medicago truncatula, being regulated by tyrosine (Tyr) nitration in relation to active nitrogen fixation. In vitro studies, using purified recombinant enzymes produced in Escherichia coli, demonstrated that the M. truncatula nodule GS isoenzyme (MtGS1a) is subjected to NO-mediated inactivation through Tyr nitration and identified Tyr-167 as the regulatory nitration site crucial for enzyme inactivation. Using a sandwich enzyme-linked immunosorbent assay, it is shown that GS is nitrated in planta and that its nitration status changes in relation to active nitrogen fixation. In ineffective nodules and in nodules fed with nitrate, two conditions in which nitrogen fixation is impaired and GS activity is reduced, a significant increase in nodule GS nitration levels was observed. Furthermore, treatment of root nodules with the NO donor sodium nitroprusside resulted in increased in vivo GS nitration accompanied by a reduction in GS activity. Our results support a role of NO in the regulation of nitrogen metabolism in root nodules and places GS as an important player in the process. We propose that the NO-mediated GS posttranslational inactivation is related to metabolite channeling to boost the nodule antioxidant defenses in response to NO.

  16. Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.

    PubMed

    Stern, D F; Zheng, P; Beidler, D R; Zerillo, C

    1991-02-01

    A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.

  17. Peptidyl aldehydes as reversible covalent inhibitors of protein tyrosine phosphatases.

    PubMed

    Fu, Hua; Park, Junguk; Pei, Dehua

    2002-08-27

    Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. PTP inhibitors provide potential treatment of human diseases/conditions such as diabetes and obesity as well as useful tools for studying the function of PTPs in signaling pathways. In this work, we have shown that certain aryl-substituted aldehydes act as reversible, slow-binding inhibitors of modest potency against PTP1B, SHP-1, and a dual-specificity phosphatase, VHR. Attachment of the tripeptide Gly-Glu-Glu to the para position of cinnamaldehyde resulted in an inhibitor (Cinn-GEE) of substantially increased potency against all three enzymes (e.g., K(I) = 5.4 microM against PTP1B). The mechanism of inhibition was investigated using Cinn-GEE specifically labeled with (13)C at the aldehyde carbon and (1)H-(13)C heteronuclear single-quantum coherence spectroscopy. While Cinn-GEE alone showed a single cross-peak at delta 9.64 ((1)H) and delta 201 ((13)C), the PTP1B/Cinn-GEE complex showed three distinct cross-peaks at delta 7.6-7.8 ((1)H) and 130-137 ((13)C). Mutation of the catalytic cysteine (Cys-215 in PTP1B) into alanine had no effect on the cross-peaks, whereas mutation of a conserved active-site arginine (Arg-221 in PTP1B) to alanine abolished all three cross-peaks. Similar experiments with Cinn-GEE that had been labeled with (13)C at the benzylic position revealed a change in the hybridization state (from sp(2) to sp(3)) for the benzylic carbon as a result of binding to PTP1B. These results rule out the possibility of a free aldehyde, aldehyde hydrate, or hemithioacetal as the enzyme-bound inhibitor form. Instead, the data are consistent with the formation of an enamine between the aldehyde group of the inhibitor and the guanidine group of Arg-221 in the PTP1B active site. These aldehydes may provide a general core structure that can be further developed into highly potent and specific PTP

  18. 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

  19. A tyrosine phosphorylation switch controls the interaction between the transmembrane modulator protein Wzd and the tyrosine kinase Wze of Lactobacillus rhamnosus.

    PubMed

    Kang, Hye-Ji; Gilbert, Christophe; Badeaux, Frédérique; Atlan, Danièle; LaPointe, Gisèle

    2015-02-21

    One proposed mechanism for assembly of secreted heteropolysaccharides by many Gram positive bacteria relies on the coordinated action of a polymerization complex through reversible phosphorylation events. The role of the tyrosine protein kinase transmembrane modulator is, however, not well understood. The protein sequences deduced from the wzb, wzd and wze genes from Lactobacillus rhamnosus ATCC 9595 and RW-9595 M contain motifs also found in corresponding proteins CpsB, CpsC and CpsD from Streptococcus pneumoniae D39 (serotype 2). Use of an anti-phosphotyrosine antibody demonstrated that both Wzd and Wze can be found in tyrosine phosphorylated form. When tyrosine 266 was mutated to phenylalanine, WzdY266F showed slightly less phosphorylated protein than those produced by using eight other tyrosine mutated Wzd genes, when expressed along with Wze and Wzb in Lactococcus lactis subsp. cremoris MG1363. In order to demonstrate the importance of ATP for the interactions among these proteins, native and fusion Wzb, Wzd and Wze proteins were expressed and purified from Escherichia coli cultures. The modulator protein, Wzd, binds with the phosphotyrosine kinase Wze, irrespective of its phosphorylation status. However, Wze attained a higher phosphorylation level after interacting with phosphorylated Wzd in the presence of 10 mM ATP. This highly phosphorylated Wze did not remain in close association with phosphorylated Wzd. The Wze tyrosine kinase protein of Lactobacillus rhamnosus thus carries out tyrosine phosphorylation of Wzd in addition to auto- and trans- phosphorylation of the kinase itself.

  20. Phosphorylation of phosphatase inhibitor-2 (i-2) by a bovine thymus tyrosine protein kinase, p40

    SciTech Connect

    DePaoli-Roach, A.A.; Votaw, P.; Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

    1987-05-01

    Phosphatase inhibitor-2, a heat stable protein of Mr 22,800, is a regulatory component of the ATP-Mg-dependent phosphatase. It has been shown that in the cell tyrosine kinase activation can result in altered phosphorylation at serine and/or threonine residues, but the mechanism involved is unknown. The authors have found that i-2 is a substrate for a tyrosine specific protein kinase, p40, purified from bovine thymus. The purified enzyme is a monomer of Mr 40,000 that is autophosphorylated at tyrosine residue(s). The stoichiometry of phosphorylation of i-2 by this tyrosine protein kinase is up to 1 mol of phosphate per mol of i-2. Phosphoaminoacid analysis revealed that all the phosphate introduced was associated with tyrosine residues. Mapping of TSP-tryptic peptides by TLE and isoelectric focusing showed one major labeled fragment. Using the ATP-Mg-dependent phosphatase, a lesser extent of phosphorylation of i-2 by p40 was obtained although partial activation of the phosphatase was observed. The effect on the activity was not due to FA/GSK-3 contamination. These results could provide an important link between tyrosine protein kinase activity and modulation of phosphorylation at serine and/or threonine residues.

  1. Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation.

    PubMed

    González-Fernández, L; Ortega-Ferrusola, C; Macias-Garcia, B; Salido, G M; Peña, F J; Tapia, J A

    2009-06-01

    Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was

  2. Crystal structure of SP-PTP, a low molecular weight protein tyrosine phosphatase from Streptococcus pyogenes.

    PubMed

    Ku, Bonsu; Keum, Chae Won; Lee, Hye Seon; Yun, Hye-Yeoung; Shin, Ho-Chul; Kim, Bo Yeon; Kim, Seung Jun

    2016-09-23

    Streptococcus pyogenes, or Group A Streptococcus (GAS), is a pathogenic bacterium that causes a variety of infectious diseases. The GAS genome encodes one protein tyrosine phosphatase, SP-PTP, which plays an essential role in the replication and virulence maintenance of GAS. Herein, we present the crystal structure of SP-PTP at 1.9 Å resolution. Although SP-PTP has been reported to have dual phosphatase specificity for both phosphorylated tyrosine and serine/threonine, three-dimensional structural analysis showed that SP-PTP shares high similarity with typical low molecular weight protein tyrosine phosphatases (LMWPTPs), which are specific for phosphotyrosine, but not with dual-specificity phosphatases, in overall folding and active site composition. In the dephosphorylation activity test, SP-PTP consistently acted on phosphotyrosine substrates, but not or only minimally on phosphoserine/phosphothreonine substrates. Collectively, our structural and biochemical analyses verified SP-PTP as a canonical tyrosine-specific LMWPTP.

  3. Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?

    PubMed

    Petrone, A; Sap, J

    2000-07-01

    Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity of a pathway, can be controlled by multiple tyrosine phosphatases, attesting to the intricate integration of these enzymes in cellular regulation. Lastly, we are starting to appreciate the roles of intracellular topology, tyrosine phosphorylation and oligomerization among the many mechanisms regulating tyrosine phosphatase activity.

  4. Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism

    PubMed Central

    Sainz, Martha; Calvo-Begueria, Laura; Pérez-Rontomé, Carmen; Wienkoop, Stefanie; Abián, Joaquín; Staudinger, Christiana; Bartesaghi, Silvina; Radi, Rafael; Becana, Manuel

    2015-01-01

    SUMMARY Protein Tyr nitration is a post-translational modification yielding 3-nitrotyrosine (NO2-Tyr). Formation of NO2-Tyr is generally considered as a marker of nitroxidative stress and is involved in some human pathophysiological disorders, but it has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2-Tyr in Lbs. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2-Tyr25 and NO2-Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3− and were found to decrease during senescence. This demonstrates formation of nitric oxide (•NO) and NO2− by alternative means to nitrate reductase, probably via a NO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lbs revealed that Tyr nitration requires NO2− + H2O2 and that peroxynitrite is not an efficient inducer of nitration, possibly by isomerizing it to NO3−. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2-Tyr in Lbs is a consequence of active metabolism in functional nodules, where Lbs may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis. PMID:25603991

  5. Nitrosative Stress and Nitrated Proteins in Trichloroethene-Mediated Autoimmunity

    PubMed Central

    Wang, Gangduo; Wang, Jianling; Luo, Xuemei; Ansari, G. A. Shakeel; Khan, M. Firoze

    2014-01-01

    Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, has been linked to a variety of autoimmune diseases (ADs) including SLE, scleroderma and hepatitis. Mechanisms involved in the pathogenesis of ADs are largely unknown. Earlier studies from our laboratory in MRL+/+ mice suggested the contribution of oxidative/nitrosative stress in TCE-induced autoimmunity, and N-acetylcysteine (NAC) supplementation provided protection by attenuating oxidative stress. This study was undertaken to further evaluate the contribution of nitrosative stress in TCE-mediated autoimmunity and to identify proteins susceptible to nitrosative stress. Groups of female MRL +/+ mice were given TCE, NAC or TCE + NAC for 6 weeks (TCE, 10 mmol/kg, i.p., every 4th day; NAC, ∼250 mg/kg/day via drinking water). TCE exposure led to significant increases in serum anti-nuclear and anti-histone antibodies together with significant induction of iNOS and increased formation of nitrotyrosine (NT) in sera and livers. Proteomic analysis identified 14 additional nitrated proteins in the livers of TCE-treated mice. Furthermore, TCE exposure led to decreased GSH levels and increased activation of NF-κB. Remarkably, NAC supplementation not only ameliorated TCE-induced nitrosative stress as evident from decreased iNOS, NT, nitrated proteins, NF-κB p65 activation and increased GSH levels, but also the markers of autoimmunity, as evident from decreased levels of autoantibodies in the sera. These findings provide support to the role of nitrosative stress in TCE-mediated autoimmune response and identify specific nitrated proteins which could have autoimmune potential. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for designing therapeutic strategies. PMID:24892995

  6. Glucose elevates NITRATE TRANSPORTER2.1 protein levels and nitrate transport activity independently of its HEXOKINASE1-mediated stimulation of NITRATE TRANSPORTER2.1 expression.

    PubMed

    de Jong, Femke; Thodey, Kate; Lejay, Laurence V; Bevan, Michael W

    2014-01-01

    Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth.

  7. 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

    USDA-ARS?s Scientific Manuscript database

    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...

  8. 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

  9. Nitration of the Egg-Allergen Ovalbumin Enhances Protein Allergenicity but Reduces the Risk for Oral Sensitization in a Murine Model of Food Allergy

    PubMed Central

    Oostingh, Gertie Janneke; Selzle, Kathrin; Pfaller, Tobias; Schultz, Cornelia; Zhang, Yingyi; Krishnamurthy, Durga; Starkl, Philipp; Knittelfelder, Regina; Förster-Waldl, Elisabeth; Pollak, Arnold; Scheiner, Otto; Pöschl, Ulrich; Jensen-Jarolim, Erika; Duschl, Albert

    2010-01-01

    Background Nitration of proteins on tyrosine residues, which can occur due to polluted air under “summer smog” conditions, has been shown to increase the allergic potential of allergens. Since nitration of tyrosine residues is also observed during inflammatory responses, this modification could directly influence protein immunogenicity and might therefore contribute to food allergy induction. In the current study we have analyzed the impact of protein nitration on sensitization via the oral route. Methodology/Principal Findings BALB/c mice were immunized intragastrically by feeding untreated ovalbumin (OVA), sham-nitrated ovalbumin (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze the impact of the sensitization route, the allergens were also injected intraperitoneally. Animals being fed OVA or snOVA under acid-suppressive medication developed significantly elevated levels of IgE, and increased titers of specific IgG1 and IgG2a antibodies. Interestingly, oral immunizations of nOVA under anti-acid treatment did not result in IgG and IgE formation. In contrast, intraperitoneal immunization induced high levels of OVA specific IgE, which were significantly increased in the group that received nOVA by injection. Furthermore, nOVA triggered significantly enhanced mediator release from RBL cells passively sensitized with sera from allergic mice. Gastric digestion experiments demonstrated protein nitration to interfere with protein stability as nOVA was easily degraded, whereas OVA and snOVA remained stable up to 120 min. Additionally, HPLC-chip-MS/MS analysis showed that one tyrosine residue (Y107) being very efficiently nitrated is part of an ovalbumin epitope recognized exclusively after oral sensitization. Conclusions/Significance These data indicated that despite the enhanced triggering capacity in existing allergy, nitration of OVA may be associated with a reduced de novo sensitizing capability via the oral route due to

  10. Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide.

    PubMed Central

    Ilan, O; Bloch, Y; Frankel, G; Ullrich, H; Geider, K; Rosenshine, I

    1999-01-01

    In eukaryotes, tyrosine protein phosphorylation has been studied extensively, while in bacteria, it is considered rare and is poorly defined. We demonstrate that Escherichia coli possesses a gene, etk, encoding an inner membrane protein that catalyses tyrosine autophosphorylation and phosphorylation of a synthetic co-polymer poly(Glu:Tyr). This protein tyrosine kinase (PTK) was termed Ep85 or Etk. All the E.coli strains examined possessed etk; however, only a subset of pathogenic strains expressed it. Etk is homologous to several bacterial proteins including the Ptk protein of Acinetobacter johnsonii, which is the only other known prokaryotic PTK. Other Etk homologues are AmsA of the plant pathogen Erwinia amylovora and Orf6 of the human pathogen Klebsiella pneumoniae. These proteins are involved in the production of exopolysaccharide (EPS) required for virulence. We demonstrated that like Etk, AmsA and probably also Orf6 are PTKs. Taken together, these findings suggest that tyrosine protein phosphorylation in prokaryotes is more common than was appreciated previously, and that Etk and its homologues define a distinct protein family of prokaryotic membrane-associated PTKs involved in EPS production and virulence. These prokaryotic PTKs may serve as a new target for the development of new antibiotics. PMID:10369665

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. Tyrosine phosphorylation plays a role in increasing maspin protein levels and its cytoplasmic accumulation

    PubMed Central

    Tamazato Longhi, Mariana; Cella, Nathalie

    2012-01-01

    Maspin is a tumor suppressor with many biological activities, multiple ligands and different subcellular localizations. Its underlying molecular mechanism remains elusive. We hypothesized that phosphorylation might regulate maspin localization and function. Using two-dimensional gel electrophoresis with different focusing power followed by Western blot we identified four different maspin forms with the same molecular weight (42 kDa), but different isoelectric points. Three of these forms were sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated. Sodium peroxidovanadate treatment, a protein-tyrosine phosphatase inhibitor, resulted in a rapid increase in maspin protein levels and cytoplasmic accumulation. These data show that there are three different maspin tyrosine phosphoforms. Inhibition of tyrosine phosphatases increased maspin protein levels and leads to its cytoplasmic accumulation. PMID:23650586

  13. Interaction between G proteins and tyrosine kinases upon T cell receptor.CD3-mediated signaling.

    PubMed

    Stanners, J; Kabouridis, P S; McGuire, K L; Tsoukas, C D

    1995-12-22

    Engagement of the T cell receptor (TCR).CD3 complex results in the induction of multiple intracellular events, with protein tyrosine kinases playing a pivotal role in their initiation. Biochemical studies also exist suggesting the involvement of heterotrimeric GTP-binding proteins (G proteins); however, the functional consequence of this participation in TCR.CD3-mediated signaling is unresolved. Here, we report TCR.CD3-mediated guanine nucleotide exchange among the 42-kDa G protein alpha subunits of the G alpha q/11 family, their physical association with CD3 epsilon, and the G alpha 11-dependent activation of phospholipase C beta. Protein tyrosine kinase inhibitors, however, abrogate TCR.CD3-mediated G protein activation. Quite interesting is the observation that cells transfected with a function-deficient mutant of G alpha 11 display diminished tyrosine phosphorylation of TCR.CD3 zeta and epsilon chains, as well as ZAP-70, upon anti-CD3 antibody triggering. These data indicate the involvement of the G alpha q/11 family in TCR.CD3 signaling at a step proximal to the receptor and suggest a reciprocal regulation between tyrosine kinases and G proteins in T cells.

  14. Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein.

    PubMed

    Sierke, S L; Cheng, K; Kim, H H; Koland, J G

    1997-03-15

    The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidine-tagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptor-derived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-length ErbB3 proteins also yielded no evidence of catalytic activity. A fluorescence assay previously used to characterize the nucleotide-binding properties of the EGF receptor indicated that the ErbB3 protein was unable to bind nucleotide. The hisTKD-B3 protein was subsequently found to be an excellent substrate for the EGF receptor protein tyrosine kinase, which suggested that in vivo phosphorylation of ErbB3 in response to EGF could be attributed to a direct cross-phosphorylation by the EGF receptor protein tyrosine kinase.

  15. Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein.

    PubMed Central

    Sierke, S L; Cheng, K; Kim, H H; Koland, J G

    1997-01-01

    The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidine-tagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptor-derived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-length ErbB3 proteins also yielded no evidence of catalytic activity. A fluorescence assay previously used to characterize the nucleotide-binding properties of the EGF receptor indicated that the ErbB3 protein was unable to bind nucleotide. The hisTKD-B3 protein was subsequently found to be an excellent substrate for the EGF receptor protein tyrosine kinase, which suggested that in vivo phosphorylation of ErbB3 in response to EGF could be attributed to a direct cross-phosphorylation by the EGF receptor protein tyrosine kinase. PMID:9148746

  16. 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.

  17. Oxidation of protein tyrosine or methionine residues: From the amino acid to the peptide

    NASA Astrophysics Data System (ADS)

    Bergès, J.; Trouillas, P.; Houée-Levin, C.

    2011-01-01

    Methionine and tyrosine are competing targets of oxidizing free radicals in peptides or proteins. The first step is the addition of OH radicals either on the sulphur atom of methionine, followed by OH- elimination, or on the aromatic cycle of tyrosine. The next step can be stabilization of methionine radical cation by a two centre-three electron bond, or intramolecular electron transfer from tyrosine to the methionine radical cation. In this latter case a tyrosine radical is formed, which appears deprotonated. In a first step we have compared the stability of the OH radical adducts on Methionine or on Tyrosine. In agreement with experimental results, the thermodynamical data indicate that the OH adduct on Tyrosine and the radical cation are more stable than those on methionine. In a second step we have investigated the stabilization of the radical cations of Methionine by formation of intramolecular S∴X two-center three-electron bond (X=S, N, O). Finally we have compared the spin densities on separated amino acids to that in a radical pentapeptide, methionine enkephalin. One observes a delocalisation of the orbital of the odd electron on the sulfur atom of Met and on the cycle of Tyr. The peptidic chain is also concerned.

  18. Inhibition of formation of filopodia after axotomy by inhibitors of protein tyrosine kinases.

    PubMed

    Goldberg, D J; Wu, D Y

    1995-08-01

    The activity of motile protrusions of the growth cone--filopodia, veils, and lamellipodia--is essential for directed growth of a neuronal process. The regulation of the formation of these protrusions is not well understood. Numerous filopodia and veils or lamellipodia form within minutes of transection of an Aplysia axon in culture, as the initial components of growth cones of regenerating neurites. Axotomy, therefore, provides a robust and reliable protocol for analyzing the formation of these protrusions. We evaluated the involvement of protein phosphorylation in the regulation of protrusive activity. Of the inhibitors of protein kinases assayed, only the inhibitors of protein tyrosine kinases--genistein, lavendustin A, herbimycin A, and erbstatin analogue--suppressed the formation of protrusions, as assessed by high magnification video microscopy. These drugs did not work by preventing resealing of the axon, as evident from visual inspection and by the unimpaired effectiveness of genistein or lavendustin in preventing formation of filopodia when applied after resealing. Inhibition of protein tyrosine kinases not only prevented the formation of actin-based protrusions, but also caused deterioration of the actin network underlying the protrusive area of preexisting growth cones. Consistent with an involvement of protein tyrosine phosphorylation in the generation of protrusive structures, immunocytochemistry revealed that aggregates of phosphotyrosine appeared at the margins of the axon, from which protrusions emerge shortly after axotomy. These results suggest a role for protein tyrosine phosphorylation in the formation and maintenance of actin-based protrusive structures.

  19. DhARO4 induction and tyrosine nitration in response to reactive radicals generated by salt stress in Debaryomyces hansenii.

    PubMed

    Calderón-Torres, Marissa; Castro, Daniela E; Montero, Paloma; Peña, Antonio

    2011-10-01

    It has been previously reported that growth of Debaryomyces hansenii in 2 M NaCl induced the expression of ARO4. This gene codifies for DhAro4p, involved in the synthesis of the amino acid tyrosine. In this work we studied the activity of DhAro4p upon salt stress; a higher activity was observed in cells grown with 2 M NaCl, but tyrosine levels were not increased. On the other hand, the addition of tyrosine to the saline medium significantly enhanced the growth of D. hansenii. It was found that the oxidized form of tyrosine, 3-nitrotyrosine, increased in the presence of salt. Since NaCl protects against oxidative stress in D. hansenii (Navarrete et al., 2009), we propose that a protective pathway is the de novo synthesis of tyrosine and its immediate oxidation to 3-nitrotyrosine to counteract oxidative stress generated by salt stress, so we measured the production of reactive oxygen species (ROS) and nitric oxide (NO⁻) in D. hansenii after growing in 2 M NaCl. Results showed the presence of NO⁻ and the increased production of ROS; this is probably due to an increased respiratory activity in the cells grown in the presence of salt. Our results demonstrate that upon salt stress D hansenii responds to oxidative stress via the transcriptional activation of specific genes such as DhARO4. Copyright © 2011 John Wiley & Sons, Ltd.

  20. Analysis of Protein Tyrosine Kinase Specificity Using Positional Scanning Peptide Microarrays.

    PubMed

    Deng, Yang; Turk, Benjamin E

    2016-01-01

    Protein tyrosine kinases phosphorylate their substrates within the context of specific consensus sequences surrounding the site of modification. We describe a peptide microarray approach to rapidly determine tyrosine kinase phosphorylation site motifs. This method uses a peptide library that systematically substitutes each of the amino acid residues at multiple positions surrounding a central tyrosine residue. Peptide substrates are synthesized as biotin conjugates for immobilization on avidin-coated slides. Following incubation of the slide with protein kinase and radiolabeled ATP, the relative extent of phosphorylation of each of the peptides is quantified by phosphor imaging. This method allows small quantities of kinase to be analyzed rapidly in parallel, facilitating analysis of large numbers of kinases.

  1. Protein renaturation by the liquid organic salt ethylammonium nitrate.

    PubMed Central

    Summers, C. A.; Flowers, R. A.

    2000-01-01

    The room-temperature liquid salt, ethylammonium nitrate (EAN), has been used to enhance the recovery of denatured-reduced hen egg white lysozyme (HEWL). Our results show that EAN has the ability to prevent aggregation of the denatured protein. The use of EAN as a refolding additive is advantageous because the renaturation is a one-step process. When HEWL was denatured reduced using routine procedures and renatured using EAN as an additive, HEWL was found to regain 75% of its activity. When HEWL was denatured and reduced in neat EAN, dilution resulted in over 90% recovery of active protein. An important aspect of this process is that renaturation of HEWL occurs at concentrations of 1.6 mg/mL, whereas other renaturation processes occur at significantly lower protein concentrations. Additionally, the refolded-active protein can be separated from the molten salt by simple desalting methods. Although the use of a low-temperature molten salt in protein renaturation is unconventional, the power of this approach lies in its simplicity and utility. PMID:11106174

  2. Post-anthesis nitrate uptake is critical to yield and grain protein content in Sorghum bicolor.

    PubMed

    Worland, Belinda; Robinson, Nicole; Jordan, David; Schmidt, Susanne; Godwin, Ian

    2017-09-01

    Crops only use ∼50% of applied nitrogen (N) fertilizer creating N losses and pollution. Plants need to efficiently uptake and utilize N to meet growing global food demands. Here we investigate how the supply and timing of nitrate affects N status and yield in Sorghum bicolor (sorghum). Sorghum was grown in pots with either 10mM (High) or 1mM (Low) nitrate supply. Shortly before anthesis the nitrate supply was either maintained, increased 10-fold or eliminated. Leaf sheaths of sorghum grown with High nitrate accumulated nitrate in concentrations >3-times higher than leaves. Removal of nitrate supply pre-anthesis resulted in the rapid reduction of stored nitrate in all organs. Plants receiving a 10-fold increase in nitrate supply pre-anthesis achieved similar grain yield and protein content and 29% larger grains than those maintained on High nitrate, despite receiving 24% less nitrate over the whole growth period. In sorghum, plant available N is important throughout development, particularly anthesis and grain filling, for grain yield and grain protein content. Nitrate accumulation in leaf sheaths presents opportunities for the genetic analysis of mechanisms behind nitrate storage and remobilization in sorghum to improve N use efficiency. Copyright © 2017 Elsevier GmbH. All rights reserved.

  3. Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.

    PubMed

    Andreozzi, Francesco; Melillo, Rosa Marina; Carlomagno, Francesca; Oriente, Francesco; Miele, Claudia; Fiory, Francesca; Santopietro, Stefania; Castellone, Maria Domenica; Beguinot, Francesco; Santoro, Massimo; Formisano, Pietro

    2003-05-15

    We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.

  4. The Role of Bacterial Protein Tyrosine Phosphatases in the Regulation of the Biosynthesis of Secreted Polysaccharides

    PubMed Central

    Morona, Renato

    2014-01-01

    Abstract Significance: Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria. Recent Advances: Deletion and overexpression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases have suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. Critical Issues: Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. Future Directions: Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel antimicrobials. This is particularly the case for the polymerase and histidinol phosphatase family, which is predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism, and critical importance of these phosphatases in a wide range of bacteria. Antioxid. Redox Signal. 20, 2274–2289. PMID:24295407

  5. STriatal-Enriched protein tyrosine Phosphatase (STEP) Regulates the PTPα/Fyn Signaling Pathway

    PubMed Central

    Xu, Jian; Kurup, Pradeep; Foscue, Ethan; Lombroso, Paul J.

    2015-01-01

    The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr420) or inhibit (Tyr531) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr531 and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr420 and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr789. Dephosphorylation of Tyr789 prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction of STEP61 activity increased the phosphorylation of PTPα at Tyr789, as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by ethanol administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by ethanol leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. PMID:25951993

  6. 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.

  7. Cockroach larval-specific protein, a tyrosine-rich serum protein.

    PubMed

    Duhamel, R C; Kunkel, J G

    1983-12-10

    Larval-specific protein (LSP) is the most abundant protein in the hemolymph of cockroaches shortly before molting, but is rapidly cleared from the hemolymph during the molt (Kunkel, J. G., and Lawler, D. M. (1974) Comp. Biochem. Physiol. 47B, 697-710). Blatta orientalis LSP was purified by sedimentation in preparative sucrose gradients followed by 2-hydroxypropylamino-cellulose anion-exchange chromatography and gel filtration on a column of Bio-Gel A-1.5m. The amino acid composition of LSP includes 16.3 mol % tyrosine and 4.9 mol % phenylalanine, but virtually no cysteine and little methionine. The following physical properties were determined for LSP: R8 = 68.3 A, 8(20),w = 17.8, and V = 0.723. From these values an Mr = 507,900 was calculated. In electron micrographs, LSP appears as rectangular particles of 121 by 134 A. In disc polyacrylamide gel electrophoresis, native LSP exhibits a single band, but in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LSP is resolved into a doublet of closely spaced bands of Mr = 88,100 and 84,400 present in a ratio of 1.38:1. These data indicate that native B. orientalis LSP is a hexamer of subunits averaging approximately Mr = 86,000. Crossed immunoelectrophoresis of Blattella germanica larval serum indicates that LSP in that species is a hexamer composed of a random assortment of two subunits of different charge in the ratio 1.25:1. The amino acid composition and physical properties of LSP suggest that LSP may be the hemimetabolous analogue of the tyrosine- and phenylalanine-rich storage proteins of holometabolous insects.

  8. Engineering the catalytic domain of human protein tyrosine phosphatase beta for structure-based drug discovery.

    PubMed

    Evdokimov, Artem G; Pokross, Matthew; Walter, Richard; Mekel, Marlene; Cox, Brooke; Li, Chuiying; Bechard, Randy; Genbauffe, Frank; Andrews, Ryan; Diven, Conrad; Howard, Brian; Rastogi, Vinit; Gray, Jeffrey; Maier, Matthew; Peters, Kevin G

    2006-12-01

    Protein tyrosine phosphatases (PTPs) play roles in many biological processes and are considered to be important targets for drug discovery. As inhibitor development has proven challenging, crystal structure-based design will be very helpful to advance inhibitor potency and selectivity. Successful application of protein crystallography to drug discovery heavily relies on high-quality crystal structures of the protein of interest complexed with pharmaceutically interesting ligands. It is very important to be able to produce protein-ligand crystals rapidly and reproducibly for as many ligands as necessary. This study details our efforts to engineer the catalytic domain of human protein tyrosine phosphatase beta (HPTPbeta-CD) with properties suitable for rapid-turnaround crystallography. Structures of apo HPTPbeta-CD and its complexes with several novel small-molecule inhibitors are presented here for the first time.

  9. 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 ...

  10. 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 ...

  11. Electrochemical and structural properties of a protein system designed to generate tyrosine Pourbaix diagrams

    PubMed Central

    Martínez-Rivera, Melissa C.; Berry, Bruce W.; Valentine, Kathleen G.; Westerlund, Kristina; Hay, Sam

    2014-01-01

    This report describes a model protein specifically tailored to electrochemically study the reduction potential of protein tyrosine radicals as a function of pH. The model system is based on the 67-residue α3Y three-helix bundle. α3Y contains a single buried tyrosine at position 32 and displays structural properties inherent to a protein. The present report presents differential pulse voltammograms obtained from α3Y at both acidic (pH 5.4) and alkaline (pH 8.3) conditions. The observed Faradaic response is uniquely associated with Y32, as shown by site-directed mutagenesis. This is the first time voltammetry is successfully applied to detect a redox-active tyrosine residing in a structured protein environment. Tyrosine is a proton coupled electron-transfer cofactor making voltammetry-based pH titrations a central experimental approach. A second set of experiments was performed to demonstrate that pH-dependent studies can be conducted on the redox-active tyrosine without introducing large-scale structural changes in the protein scaffold. α3Y was re-engineered with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring. α3Y-K29H and α3Y-K36H each contain a histidine residue which protonation perturbs the fluorescence of Y32. We show that these variants are stable and well-folded proteins whose helical content, tertiary structure, solution aggregation state and solvent-sequestered position of Y32 remain pH insensitive across a range of at least 3–4 pH units. These results confirm that the local environment of Y32 can be altered and the resulting radical site studied by voltammetry over a broad pH range without interference from long-range structural effects. PMID:22011192

  12. Electrochemical and structural properties of a protein system designed to generate tyrosine Pourbaix diagrams.

    PubMed

    Martínez-Rivera, Melissa C; Berry, Bruce W; Valentine, Kathleen G; Westerlund, Kristina; Hay, Sam; Tommos, Cecilia

    2011-11-09

    This report describes a model protein specifically tailored to electrochemically study the reduction potential of protein tyrosine radicals as a function of pH. The model system is based on the 67-residue α(3)Y three-helix bundle. α(3)Y contains a single buried tyrosine at position 32 and displays structural properties inherent to a protein. The present report presents differential pulse voltammograms obtained from α(3)Y at both acidic (pH 5.6) and alkaline (pH 8.3) conditions. The observed Faradaic response is uniquely associated with Y32, as shown by site-directed mutagenesis. This is the first time voltammetry is successfully applied to detect a redox-active tyrosine residing in a structured protein environment. Tyrosine is a proton-coupled electron-transfer cofactor making voltammetry-based pH titrations a central experimental approach. A second set of experiments was performed to demonstrate that pH-dependent studies can be conducted on the redox-active tyrosine without introducing large-scale structural changes in the protein scaffold. α(3)Y was re-engineered with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring. α(3)Y-K29H and α(3)Y-K36H each contain a histidine residue whose protonation perturbs the fluorescence of Y32. We show that these variants are stable and well-folded proteins whose helical content, tertiary structure, solution aggregation state, and solvent-sequestered position of Y32 remain pH insensitive across a range of at least 3-4 pH units. These results confirm that the local environment of Y32 can be altered and the resulting radical site studied by voltammetry over a broad pH range without interference from long-range structural effects.

  13. Analysis of tyrosine phosphorylation and phosphotyrosine-binding proteins in germinating seeds from Scots pine.

    PubMed

    Kovaleva, Valentina; Cramer, Rainer; Krynytskyy, Hryhoriy; Gout, Ivan; Gout, Roman

    2013-06-01

    Protein tyrosine phosphorylation in angiosperms has been implicated in various physiological processes, including seed development and germination. In conifers, the role of tyrosine phosphorylation and the mechanisms of its regulation are yet to be investigated. In this study, we examined the profile of protein tyrosine phosphorylation in Scots pine seeds at different stages of germination. We detected extensive protein tyrosine phosphorylation in extracts from Scots pine (Pinus sylvestris L.) dormant seeds. In addition, the pattern of tyrosine phosphorylation was found to change significantly during seed germination, especially at earlier stages of post-imbibition which coincides with the initiation of cell division, and during the period of intensive elongation of hypocotyls. To better understand the molecular mechanisms of phosphotyrosine signaling, we employed affinity purification and mass spectrometry for the identification of pTyr-binding proteins from the extracts of Scots pine seedlings. Using this approach, we purified two proteins of 10 and 43 kDa, which interacted specifically with pTyr-Sepharose and were identified by mass spectrometry as P. sylvestris defensin 1 (PsDef1) and aldose 1-epimerase (EC:5.1.3.3), respectively. Additionally, we demonstrated that both endogenous and recombinant PsDef1 specifically interact with pTyr-Sepharose, but not Tyr-beads. As the affinity purification approach did not reveal the presence of proteins with known pTyr binding domains (SH2, PTB and C2), we suggest that plants may have evolved a different mode of pTyr recognition, which yet remains to be uncovered.

  14. Inhibition of tyrosine phosphorylation of sperm flagellar proteins, outer dense fiber protein-2 and tektin-2, is associated with impaired motility during capacitation of hamster spermatozoa.

    PubMed

    Mariappa, Daniel; Aladakatti, Ravindranath H; Dasari, Santosh K; Sreekumar, Arun; Wolkowicz, Michael; van der Hoorn, Frans; Seshagiri, Polani B

    2010-02-01

    In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

  15. Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

    PubMed Central

    Bogdanova, Olena V; Kot, Larysa I; Lavrova, Kateryna V; Bogdanov, Volodymyr B; Sloan, Erica K; Beregova, Tetyana V; Ostapchenko, Ludmyla I

    2010-01-01

    AIM: To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration. METHODS: Gastric lesions were induced in rats using restraint cold stress. To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair, total activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTP), antioxidant enzymes, nitric oxide synthase (NOS), 2’,5’-oligoadenylate synthetase, hydroxyl radical and zinc levels were assayed in parallel. RESULTS: Ulcer provocation induced an immediate decrease in tyrosine kinase (40% in plasma membranes and 56% in cytosol, P < 0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol), followed by 2.3-2.4-fold decrease (P < 0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity, 30% increase (P < 0.05) in catalase activity, 2.3-fold inhibition (P < 0.05) of glutathione peroxidase, 3.3-fold increase (P < 0.05) in hydroxyl radical content, and 2.3-fold decrease (P < 0.05) in zinc level in gastric mucosa. NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration, PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase, P < 0.05), but remained inhibited (1.6-3-fold decrease on days 3, 4 and 5, P < 0.05) in the cytosol. Tyrosine phosphatases remained inhibited both in membranes and cytosol (1.5-2.4-fold, P < 0.05). NOS activity remained increased on days 1, 2 and 3 (3.8-, 2.6-, 2.2-fold, respectively, P < 0.05). Activity of SOD increased 1.6 times (P < 0.05) days 4 and 5 after stress. Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3- and 2-fold, respectively, P < 0.05) on the last day. Activity of 2’,5’-oligoadenylate synthethase increased 2.8-fold (P < 0.05) at the

  16. 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

  17. Isolation and characterization of a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase from Klebsiella pneumoniae.

    PubMed

    Preneta, R; Jarraud, S; Vincent, C; Doublet, P; Duclos, B; Etienne, J; Cozzone, A J

    2002-01-01

    Two proteins of Klebsiella pneumoniae, termed Yor5 and Yco6, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, protein Yco6 was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive adenosine triphosphate, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, protein Yor5 was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Yor5 was able to dephosphorylate protein Yco6 previously autophosphorylated. Together, these data demonstrate that similarly to other bacterial species including Acinetobacter johnsonii and Escherichia coli, the cells of K. pneumoniae contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. Since Yco6 and Yor5 are both involved in the synthesis of capsular polysaccharide and since capsules are essential to the virulence of K. pneumoniae, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.

  18. 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.

  19. 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

  20. Measurement of protein synthesis: in vitro comparison of (68)Ga-DOTA-puromycin, [ (3)H]tyrosine, and 2-fluoro-[ (3)H]tyrosine.

    PubMed

    Eigner, Sebastian; Beckford Vera, Denis R; Fellner, Marco; Loktionova, Natalia S; Piel, Markus; Melichar, Frantisek; Rösch, Frank; Roß, Tobias L; Lebeda, Ondrej; Henke, Katerina Eigner

    2013-01-01

    Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using (68)Ga-DOTA-puromycin ((68)Ga-DOTA-Pur), [(3)H]tyrosine, and 2-fluoro-[(3)H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine. DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. (68)Ga was obtained from a (68)Ge/(68)Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741-1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [(3)H]Tyrosine and 2-fluoro-[(3)H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). (68)Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20-25 MBq (68

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

    PubMed

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

    2013-04-01

    The major goal of cancer drug discovery is to find an agent that is safe and affordable, yet effective against cancer. Here we show that morin (3,5,7,2',4'-pentahydroxyflavone) has potential against cancer cells through suppression of the signal transducer and activator of transcription 3 (STAT3) pathway, which is closely linked to the transformation, survival, proliferation, and metastasis of cancer. We found that morin completely suppressed inducible and constitutively activated STAT3 and blocked the nuclear translocation of STAT3 and its DNA binding in multiple myeloma and head and neck squamous carcinoma cells. Morin inhibited activated Src, JAK-1, and JAK-2, all of which are linked to STAT3 activation, while up-regulating a protein inhibitor of activated STAT3, PIAS3. Pervanadate reversed the effects of morin on STAT3 phosphorylation, indicating the role of a protein tyrosine phosphatase. Furthermore, morin induced SHP1 expression at both the mRNA and protein levels, and silencing of SHP1 abrogated the effect of morin on STAT3 phosphorylation, indicating that morin mediates its effects on STAT3 through SHP1. Suppression of STAT3 correlated with the down-regulation of various gene products linked to tumor survival, proliferation, and angiogenesis and led to sensitization of tumor cells to thalidomide and bortezomib. Comparing the activities of morin with those of four structurally related flavonols demonstrated the importance of hydroxyl groups in the B ring in inhibiting STAT3 activation. These findings suggest that morin suppresses the STAT3 pathway, leading to the down-regulation of STAT3-dependent gene expression and chemosensitization of tumor cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase.

    PubMed Central

    Nakielny, S; Cohen, P; Wu, J; Sturgill, T

    1992-01-01

    A 'MAP kinase activator' was purified several thousand-fold from insulin-stimulated rabbit skeletal muscle, which resembled the 'activator' from nerve growth factor-stimulated PC12 cells in that it could be inactivated by incubation with protein phosphatase 2A, but not by protein tyrosine phosphatases and its apparent molecular mass was 45-50 kDa. In the presence of MgATP, 'MAP kinase activator' converted the normal 'wild-type' 42 kDa MAP kinase from an inactive dephosphorylated form to the fully active diphosphorylated species. Phosphorylation occurred on the same threonine and tyrosine residues which are phosphorylated in vivo in response to growth factors or phorbol esters. A mutant MAP kinase produced by changing a lysine at the active centre to arginine was phosphorylated in an identical manner by the 'MAP kinase activator', but no activity was generated. The results demonstrate that 'MAP kinase activator' is a protein kinase (MAP kinase kinase) and not a protein that stimulates the autophosphorylation of MAP kinase. MAP kinase kinase is the first established example of a protein kinase that can phosphorylate an exogenous protein on threonine as well as tyrosine residues. Images PMID:1318193

  3. SH2 domain proteins as high-affinity receptor tyrosine kinase substrates.

    PubMed

    Sierke, S L; Koland, J G

    1993-09-28

    Activation of a growth factor receptor tyrosine kinase (RTK) is accompanied by a rapid autophosphorylation of the receptor on tyrosine residues. Receptor activation has been shown to promote the association of signal-transducing proteins containing SH2 domains (second domain of src homology). These receptor-associated proteins can, in turn, be phosphorylated by the RTK, an event which presumably regulates their activities. It has been suggested that SH2 domains in signal-transducing proteins target these proteins as substrates of the activated RTK. To test this hypothesis, recombinant proteins were generated that contained tyrosine phosphorylation sites of the erbB3 receptor and/or the SH2 domain of c-src. Incorporation of the SH2 domain led to a decrease in KM and an increase in Vmax for the substrate. The KM determined for one chimeric SH2/erbB3 substrate was among the lowest reported for epidermal growth factor RTK substrates. Experiments with a truncated kinase lacking C-terminal autophosphorylation sites indicated that the reduction in KM for these substrates was mediated by interactions between the substrate SH2 domain and phosphotyrosine residues of the RTK. These interactions could also inhibit RTK activity. These results demonstrate that the SH2 domain can effectively target substrates to a RTK and that SH2 domain proteins can regulate RTK activity.

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

    PubMed Central

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

    2016-01-01

    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

  5. Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb.

    PubMed

    Vincent, C; Doublet, P; Grangeasse, C; Vaganay, E; Cozzone, A J; Duclos, B

    1999-06-01

    Two proteins of Escherichia coli, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, Wzb was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Wzb was able to dephosphorylate previously autophosphorylated Wzc. Together, these data demonstrate, for the first time, that E. coli cells contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. From comparative analysis of amino acid sequences, Wzc was found to be similar to a number of proteins present in other bacterial species which are all involved in the synthesis or export of exopolysaccharides. Since these polymers are considered important virulence factors, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.

  6. Activation of the Lck tyrosine protein kinase by hydrogen peroxide requires the phosphorylation of Tyr-394.

    PubMed Central

    Hardwick, J S; Sefton, B M

    1995-01-01

    Exposure of cells to H2O2 mimics many of the effects of treatment of cells with extracellular ligands. Among these is the stimulation of tyrosine phosphorylation. In this study, we show that exposure of cells to H2O2 increases the catalytic activity of the lymphocyte-specific tyrosine protein kinase p56lck (Lck) and induces tyrosine phosphorylation of Lck at Tyr-394, the autophosphorylation site. Using mutant forms of Lck, we found that Tyr-394 is required for H2O2-induced activation of Lck, suggesting that phosphorylation of this site may activate Lck. In addition, H2O2 treatment induced phosphorylation at Tyr-394 in a catalytically inactive mutant of Lck in cells that do not express endogenous Lck. This demonstrates that a kinase other than Lck itself is capable of phosphorylating Lck at the so-called autophosphorylation site and raises the possibility that this as yet unidentified tyrosine protein kinase functions as an activator of Lck. Such an activating enzyme could play an important role in signal transduction in T cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7538674

  7. Analysis of Chlorination, Nitration, and Nitrosylation of Tyrosine and Oxidation of Methionine and Cysteine in Hemoglobin from Type 2 Diabetes Mellitus Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry.

    PubMed

    Chen, Hauh-Jyun Candy; Yang, Ya-Fen; Lai, Pang-Yen; Chen, Pin-Fan

    2016-09-20

    The post-translational modification (PTM) of proteins by endogenous reactive chlorine, nitrogen, and oxygen species is implicated in certain pathological conditions, including diabetes mellitus. Evidence showed that the extents of modifications on a number of proteins are elevated in diabetic patients. Measuring modification on hemoglobin has been used to monitor the extent of exposure. This study develops an assay for simultaneous quantification of the extent of chlorination, nitration, and oxidation in human hemoglobin and to examine whether the level of any of these modifications is higher in poorly controlled type 2 diabetic mellitus patients. This mass spectrometry-based assay used the bottom-up proteomic strategy. Due to the low amount of endogenous modification, we first characterized the sites of chlorination at tyrosine in hypochlorous acid-treated hemoglobin by an accurate mass spectrometer. The extents of chlorination, nitration, and oxidation of a total of 12 sites and types of modifications in hemoglobin were measured by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry under the selected reaction monitoring mode. Relative quantification of these PTMs in hemoglobin extracted from blood samples shows that the extents of chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in diabetic patients (n = 19) than in nondiabetic individuals (n = 18). After excluding the factor of smoking, chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in the nonsmoking diabetic patients (n = 12) than in normal nonsmoking subjects (n = 11). Multiple regression analysis performed on the combined effect of age, body-mass index (BMI), and HbA1c showed that the diabetes factor HbA1c contributes significantly to the extent of chlorination at α-Tyr-24 in nonsmokers. In addition, age contributes to oxidation at

  8. Multifaceted Modulation of K+ Channels by Protein-tyrosine Phosphatase ϵ Tunes Neuronal Excitability*

    PubMed Central

    Ebner-Bennatan, Sharon; Patrich, Eti; Peretz, Asher; Kornilov, Polina; Tiran, Zohar; Elson, Ari; Attali, Bernard

    2012-01-01

    Non-receptor-tyrosine kinases (protein-tyrosine kinases) and non-receptor tyrosine phosphatases (PTPs) have been implicated in the regulation of ion channels, neuronal excitability, and synaptic plasticity. We previously showed that protein-tyrosine kinases such as Src kinase and PTPs such as PTPα and PTPϵ modulate the activity of delayed-rectifier K+ channels (IK). Here we show cultured cortical neurons from PTPϵ knock-out (EKO) mice to exhibit increased excitability when compared with wild type (WT) mice, with larger spike discharge frequency, enhanced fast after-hyperpolarization, increased after-depolarization, and reduced spike width. A decrease in IK and a rise in large-conductance Ca2+-activated K+ currents (mBK) were observed in EKO cortical neurons compared with WT. Parallel studies in transfected CHO cells indicate that Kv1.1, Kv1.2, Kv7.2/7.3, and mBK are plausible molecular correlates of this multifaceted modulation of K+ channels by PTPϵ. In CHO cells, Kv1.1, Kv1.2, and Kv7.2/7.3 K+ currents were up-regulated by PTPϵ, whereas mBK channel activity was reduced. The levels of tyrosine phosphorylation of Kv1.1, Kv1.2, Kv7.3, and mBK potassium channels were increased in the brain cortices of neonatal and adult EKO mice compared with WT, suggesting that PTPϵ in the brain modulates these channel proteins. Our data indicate that in EKO mice, the lack of PTPϵ-mediated dephosphorylation of Kv1.1, Kv1.2, and Kv7.3 leads to decreased IK density and enhanced after-depolarization. In addition, the deficient PTPϵ-mediated dephosphorylation of mBK channels likely contributes to enhanced mBK and fast after-hyperpolarization, spike shortening, and consequent increase in neuronal excitability observed in cortical neurons from EKO mice. PMID:22722941

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

    PubMed Central

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

    2011-01-01

    The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the viral-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of HA tag addition varied with other fusion proteins, as parainfluenza virus 5 F-HA showed decreased surface expression and no stimulation in fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in modulation of the membrane fusion reaction promoted by these viral glycoproteins. PMID:21175223

  10. Clickable tyrosine binding bifunctional linkers for preparation of DNA-protein conjugates.

    PubMed

    Bauer, Dennis M; Ahmed, Ishtiaq; Vigovskaya, Antonina; Fruk, Ljiljana

    2013-06-19

    We have prepared bifunctional linkers containing clickable functional groups that enable preparation of protein-DNA conjugates through binding onto tyrosine residues. Mild conjugation strategy was demonstrated using two proteins, streptavidin(STV) and myoglobin (Mb) and it resulted in conjugates with preserved functionality of both the proteins and DNA strands. Furthermore, we show that protein-DNA conjugates can be successfully immobilized onto solid surface containing complementary DNA strands and the enzymatic activity of Mb-DNA conjugates is even higher than that of corresponding conjugates prepared through Lys binding.

  11. The protein-tyrosine phosphatase SHP-1 associates with the phosphorylated immunoreceptor tyrosine-based activation motif of Fc gamma RIIa to modulate signaling events in myeloid cells.

    PubMed

    Ganesan, Latha P; Fang, Huiqing; Marsh, Clay B; Tridandapani, Susheela

    2003-09-12

    Fc gamma RIIa is a low affinity IgG receptor uniquely expressed in human cells that promotes phagocytosis of immune complexes and induces inflammatory cytokine gene transcription. Recent studies have revealed that phagocytosis initiated by Fc gamma RIIa is tightly controlled by the inositol phosphatase SHIP-1, and the protein-tyrosine phosphatase SHP-1. Whereas the molecular nature of SHIP-1 involvement with Fc gamma RIIa has been well studied, it is not clear how SHP-1 is activated by Fc gamma RIIa to mediate its regulatory effect. Here we report that Fc gamma RIIa clustering induces SHP-1 phosphatase activity in THP-1 cells. Using synthetic phosphopeptides, and stable transfectants expressing immunoreceptor tyrosine-based activation motif (ITAM) tyrosine mutants of Fc gamma RIIa, we demonstrate that SHP-1 associates with the phosphorylated amino-terminal ITAM tyrosine of Fc gamma RIIa, whereas the tyrosine kinase Syk associates with the carboxyl-terminal ITAM tyrosine. Association of SHP-1 with Fc gamma RIIa ITAM appears to suppress total cellular tyrosine phosphorylation. Furthermore, Fc gamma RIIa clustering results in the association of SHP-1 with key signaling molecules such as Syk, p85 subunit of PtdIns 3-kinase, and p62dok, suggesting that these molecules may be substrates of SHP-1 in this system. Finally, overexpression of wild-type SHP-1 but not catalytically deficient SHP-1 led to a down-regulation of NF kappa B-dependent gene transcription in THP-1 cells activated by clustering Fc gamma RIIa.

  12. Identification of protein targets underlying dietary nitrate-induced protection against doxorubicin cardiotoxicity.

    PubMed

    Xi, Lei; Zhu, Shu-Guang; Hobbs, Daniel C; Kukreja, Rakesh C

    2011-11-01

    We recently demonstrated protective effect of chronic oral nitrate supplementation against cardiomyopathy caused by doxorubicin (DOX), a highly effective anticancer drug. The present study was designed to identify novel protein targets related to nitrate-induced cardioprotection. Adult male CF-1 mice received cardioprotective regimen of nitrate (1 g NaNO(3) per litre of drinking water) for 7 days before DOX injection (15 mg/kg, i.p.) and continued for 5 days after DOX treatment. Subsequently the heart samples were collected for proteomic analysis with two-dimensional differential in-gel electrophoresis with 3 CyDye labelling. Using 1.5 cut-off ratio, we identified 36 proteins that were up-regulated by DOX in which 32 were completely reversed by nitrate supplementation (89%). Among 19 proteins down-regulated by DOX, 9 were fully normalized by nitrate (47%). The protein spots were further identified with Matrix Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF)/TOF tandem mass spectrometry. Three mitochondrial antioxidant enzymes were altered by DOX, i.e. up-regulation of manganese superoxide dismutase and peroxiredoxin 3 (Prx3), and down-regulation of Prx5, which were reversed by nitrate. These results were further confirmed by Western blots. Nitrate supplementation also significantly improved animal survival rate from 80% in DOX alone group to 93% in Nitrate + DOX group 5 days after the DOX treatment. In conclusion, the proteomic analysis has identified novel protein targets underlying nitrate-induced cardioprotection. Up-regulation of Prx5 by nitrate may explain the observed enhancement of cardiac antioxidant defence by nitrate supplementation.

  13. No Obvious Abnormality in Mice Deficient in Receptor Protein Tyrosine Phosphatase β

    PubMed Central

    Harroch, S.; Palmeri, M.; Rosenbluth, J.; Custer, A.; Okigaki, M.; Shrager, P.; Blum, M.; Buxbaum, J. D.; Schlessinger, J.

    2000-01-01

    The development of neurons and glia is governed by a multitude of extracellular signals that control protein tyrosine phosphorylation, a process regulated by the action of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Receptor PTPβ (RPTPβ; also known as PTPζ) is expressed predominantly in the nervous system and exhibits structural features common to cell adhesion proteins, suggesting that this phosphatase participates in cell-cell communication. It has been proposed that the three isoforms of RPTPβ play a role in regulation of neuronal migration, neurite outgrowth, and gliogenesis. To investigate the biological functions of this PTP, we have generated mice deficient in RPTPβ. RPTPβ-deficient mice are viable, are fertile, and showed no gross anatomical alterations in the nervous system or other organs. In contrast to results of in vitro experiments, our study demonstrates that RPTPβ is not essential for neurite outgrowth and node formation in mice. The ultrastructure of nerves of the central nervous system in RPTPβ-deficient mice suggests a fragility of myelin. However, conduction velocity was not altered in RPTPβ-deficient mice. The normal development of neurons and glia in RPTPβ-deficient mice demonstrates that RPTPβ function is not necessary for these processes in vivo or that loss of RPTPβ can be compensated for by other PTPs expressed in the nervous system. PMID:11003666

  14. Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity*

    PubMed Central

    Xu, Jian; Kurup, Pradeep; Bartos, Jason A.; Patriarchi, Tommaso; Hell, Johannes W.; Lombroso, Paul J.

    2012-01-01

    Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr1472. Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr402. In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr402. STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr402 and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP. PMID:22544749

  15. Oleanolic acid and its derivatives: new inhibitor of protein tyrosine phosphatase 1B with cellular activities.

    PubMed

    Zhang, Yi-Nan; Zhang, Wei; Hong, Di; Shi, Lei; Shen, Qiang; Li, Jing-Ya; Li, Jia; Hu, Li-Hong

    2008-09-15

    Protein tyrosine phosphatase 1B is a key factor in the negative regulation of insulin pathway and a promising target for treatment of diabetes and obesity. Herein, a series of competitive inhibitors were optimized from oleanolic acid, a natural triterpenoid identified against PTP1B by screening libraries of traditional Chinese medicinal herbs. Modifying at 3 and 28 positions, we obtained compound 13 with a K(i) of 130 nM, which exhibited good selectivity between other phosphatases involved in insulin pathway except T-cell protein tyrosine phosphatase. Further evaluation in cell models illustrated that the derivatives enhanced insulin receptor phosphorylation in CHO/hIR cells and also stimulated glucose uptake in L6 myotubes with or addition of without insulin.

  16. 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

  17. Tyrosine phosphorylation of the herpes simplex virus type 1 regulatory protein ICP22 and a cellular protein which shares antigenic determinants with ICP22.

    PubMed Central

    Blaho, J A; Zong, C S; Mortimer, K A

    1997-01-01

    At least eight herpes simplex virus type 1 (HSV-1) and five HSV-2 proteins were tyrosine phosphorylated in infected cells. The first viral tyrosine phosphoprotein identified was the HSV-1 regulatory protein ICP22. Also, two novel phosphotyrosine proteins were bound by anti-ICP22 antibodies. H(R22) is a cellular protein, while the F(R10) protein is observed only in HSV-1-infected cells. PMID:9371655

  18. Receptor Type Protein Tyrosine Phosphatase ζ-Pleiotrophin Signaling Controls Endocytic Trafficking of DNER That Regulates Neuritogenesis▿ †

    PubMed Central

    Fukazawa, Nobuna; Yokoyama, Seisuke; Eiraku, Mototsugu; Kengaku, Mineko; Maeda, Nobuaki

    2008-01-01

    Protein tyrosine phosphatase ζ (PTPζ) is a receptor type protein tyrosine phosphatase that uses pleiotrophin as a ligand. Pleiotrophin inactivates the phosphatase activity of PTPζ, resulting in the increase of tyrosine phosphorylation levels of its substrates. We studied the functional interaction between PTPζ and DNER, a Notch-related transmembrane protein highly expressed in cerebellar Purkinje cells. PTPζ and DNER displayed patchy colocalization in the dendrites of Purkinje cells, and immunoprecipitation experiments indicated that these proteins formed complexes. Several tyrosine residues in and adjacent to the tyrosine-based and the second C-terminal sorting motifs of DNER were phosphorylated and were dephosphorylated by PTPζ, and phosphorylation of these tyrosine residues resulted in the accumulation of DNER on the plasma membrane. DNER mutants lacking sorting motifs accumulated on the plasma membrane of Purkinje cells and Neuro-2A cells and induced their process extension. While normal DNER was actively endocytosed and inhibited the retinoic-acid-induced neurite outgrowth of Neuro-2A cells, pleiotrophin stimulation increased the tyrosine phosphorylation level of DNER and suppressed the endocytosis of this protein, which led to the reversal of this inhibition, thus allowing neurite extension. These observations suggest that pleiotrophin-PTPζ signaling controls subcellular localization of DNER and thereby regulates neuritogenesis. PMID:18474614

  19. Reversible voltammograms and a Pourbaix diagram for a protein tyrosine radical

    PubMed Central

    Berry, Bruce W.; Martínez-Rivera, Melissa C.; Tommos, Cecilia

    2012-01-01

    Reversible voltammograms and a voltammetry half-wave potential versus solution pH diagram are described for a protein tyrosine radical. This work required a de novo designed tyrosine-radical protein displaying a unique combination of structural and electrochemical properties. The α3Y protein is structurally stable across a broad pH range. The redox-active tyrosine Y32 resides in a desolvated and well-structured environment. Y32 gives rise to reversible square-wave and differential pulse voltammograms at alkaline pH. The formal potential of the Y32-O•/Y32-OH redox couple is determined to 918 ± 2 mV versus the normal hydrogen electrode at pH 8.40 ± 0.01. The observation that Y32 gives rise to fully reversible voltammograms translates into an estimated lifetime of ≥30 ms for the Y32-O• state. This illustrates the range of tyrosine-radical stabilization that a structured protein can offer. Y32 gives rise to quasireversible square-wave and differential pulse voltammograms at acidic pH. These voltammograms represent the Y32 species at the upper edge of the quasirevesible range. The square-wave net potential closely approximates the formal potential of the Y32-O•/Y32-OH redox couple to 1,070 ± 1 mV versus the normal hydrogen electrode at pH 5.52 ± 0.01. The differential pulse voltammetry half-wave potential of the Y32-O•/Y32-OH redox pair is measured between pH 4.7 and 9.0. These results are described and analyzed. PMID:22675121

  20. New cytotoxic bisindole alkaloids with protein tyrosine kinase inhibitory activity from a myxomycete Lycogala epidendrum.

    PubMed

    Hosoya, Takahiro; Yamamoto, Yukinori; Uehara, Yoshimasa; Hayashi, Masahiko; Komiyama, Kanki; Ishibashi, Masami

    2005-06-02

    Two new bisindole alkaloids, 6-hydroxystaurosporinone (1) and 5,6-dihydroxyarcyriaflavin A (2) were isolated from field-collected fruit bodies of a myxomycete Lycogala epidendrum, along with eight known bisindoles (3-10). The structures of these new compounds were determined on the basis of spectroscopic data. Compounds 1 and 2 showed cytotoxicity against HeLa, Jurkat, and vincristine resistant KB/VJ300 cells, and compound 1, particularly, inhibited protein tyrosine kinase activity.

  1. Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs

    PubMed Central

    Hines, John; Gough, Jonathan D.; Corson, Timothy W.; Crews, Craig M.

    2013-01-01

    Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects. PMID:23674677

  2. Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs.

    PubMed

    Hines, John; Gough, Jonathan D; Corson, Timothy W; Crews, Craig M

    2013-05-28

    Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.

  3. A biochemical study on the level of proteins and their percentage of nitration in the hair and nail of autistic children.

    PubMed

    Lakshmi Priya, Malarveni Damodaran; Geetha, Arumugam

    2011-05-12

    Autism is a complex disorder which is heterogeneous in nature with varying degrees of severity for which no specific biological marker has been identified. Several studies are focused on the hair and nail protein pattern as a means to identify specific markers for the diagnosis of many childhood disorders like mental retardation, dyslexia, trichorrhexis nodosa, trichothiodystrophy, etc. The present study is one such approach in investigating the electrophoretic pattern of proteins in hard keratins and their percentage of nitration since nitric oxide production and nitration of tyrosine residues in proteins of autistic children are the emerging topic of research. We extracted and quantified the proteins from hair and nail samples of autistic children with different grades of severity, [low functioning autism (LFA), medium functioning autism (MFA), and high functioning autism (HFA)] and also from age- and sex-matched normal children. Protein pattern was evaluated by one-dimensional SDS-PAGE and the separated proteins were made to cross react with anti-nitro tyrosine antibody by Western blot analysis. Blood levels of TBARS, NO, GSH, vitamins A and C, SOD and GPx were also determined. In the autistic groups, decreased concentration of protein in both hair and nail samples was observed. The SDS-PAGE analysis revealed that there was a significant decrease in both high and low sulfur proteins in the hair and nail extracts of autistic children and the Western blot analysis showed increased percentage of nitration of low sulfur proteins in autistic children when compared with normal children. Decreased levels of enzymatic and non-enzymatic antioxidants and increased concentration of TBARS and NO were also observed in the blood of autistic children. The LFA group showed more significant alteration (p<0.001) in the concentration of proteins (in hair and nail) and percentage of nitration when compared with HFA and controls. Lower protein content and higher percentage of

  4. Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.

    PubMed Central

    Schmidt, A; Rutledge, S J; Endo, N; Opas, E E; Tanaka, H; Wesolowski, G; Leu, C T; Huang, Z; Ramachandaran, C; Rodan, S B; Rodan, G A

    1996-01-01

    Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action. Images Fig. 2 Fig. 3 PMID:8610169

  5. Role of Protein-Tyrosine Phosphatases in Regulation of Osteoclastic Activity

    PubMed Central

    Sheng, Matilda H.-C.; Lau, K.-H. William

    2009-01-01

    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-ε, PTP-PEST, and PTP-oc) in osteoclasts. Cyt-PTP-ε, 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

  6. Potent inhibition of protein tyrosine phosphatases by copper complexes with multi-benzimidazole derivatives.

    PubMed

    Li, Ying; Lu, Liping; Zhu, Miaoli; Wang, Qingming; Yuan, Caixia; Xing, Shu; Fu, Xueqi; Mei, Yuhua

    2011-12-01

    A series of copper complexes with multi-benzimidazole derivatives, including mono- and di-nuclear, were synthesized and characterized by Fourier transform IR spectroscopy, UV-Vis spectroscopy, elemental analysis, electrospray ionization mass spectrometry. The speciation of Cu/NTB in aqueous solution was investigated by potentiometric pH titrations. Their inhibitory effects against human protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2), srchomology phosphatase 1 (SHP-1) and srchomology phosphatase 2 (SHP-2) were evaluated in vitro. The five copper complexes exhibit potent inhibition against PTP1B, TCPTP and PTP-MEG2 with almost same inhibitory effects with IC(50) at submicro molar level and about tenfold weaker inhibition versus SHP-1, but almost no inhibition against SHP-2. Kinetic analysis indicates that they are reversible competitive inhibitors of PTP1B. Fluorescence study on the interaction between PTP1B and complex 2 or 4 suggests that the complexes bind to PTP1B with the formation of a 1:1 complex. The binding constant are about 1.14 × 10(6) and 1.87 × 10(6) M(-1) at 310 K for 2 and 4, respectively.

  7. Structural Stability of Human Protein Tyrosine Phosphatase ρ Catalytic Domain: Effect of Point Mutations

    PubMed Central

    Knapp, Stefan; Alfano, Ivan; Ardini, Matteo; Stefanini, Simonetta; Chiaraluce, Roberta

    2012-01-01

    Protein tyrosine phosphatase ρ (PTPρ) belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ∼4.0 M urea. PMID:22389709

  8. Functional interaction of vascular endothelial-protein-tyrosine phosphatase with the angiopoietin receptor Tie-2.

    PubMed

    Fachinger, G; Deutsch, U; Risau, W

    1999-10-21

    During development of the vertebrate vascular system essential signals are transduced via protein-tyrosine phosphorylation. Null-mutations of receptor-tyrosine kinase (RTK) genes expressed in endothelial cells (ECs) display early lethal vascular phenotypes. We aimed to identify endothelial protein-tyrosine phosphatases (PTPs), which should have similar importance in EC-biology. A murine receptor-type PTP was identified by a degenerated PCR cloning approach from endothelial cells (VE-PTP). By in situ hybridization this phosphatase was found to be specifically expressed in vascular ECs throughout mouse development. In experiments using GST-fusion proteins, as well as in transient transfections, trapping mutants of VE-PTP co-precipitated with the Angiopoietin receptor Tie-2, but not with the Vascular Endothelial Growth Factor receptor 2 (VEGFR-2/Flk-1). In addition, VE-PTP dephosphorylates Tie-2 but not VEGFR-2. We conclude that VE-PTP is a Tie-2 specific phosphatase expressed in ECs, and VE-PTP phosphatase activity serves to specifically modulate Angiopoietin/Tie-2 function. Based on its potential role as a regulator of blood vessel morphogenesis and maintainance, VE-PTP is a candidate gene for inherited vascular malformations similar to the Tie-2 gene.

  9. Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro.

    PubMed

    Lehr, S; Kotzka, J; Herkner, A; Klein, E; Siethoff, C; Knebel, B; Noelle, V; Brüning, J C; Klein, H W; Meyer, H E; Krone, W; Müller-Wieland, D

    1999-01-05

    Grb2-associated binder-1 (Gab-1) has been identified recently in a cDNA library of glioblastoma tumors and appears to play a central role in cellular growth response, transformation, and apoptosis. Structural and functional features indicate that Gab-1 is a multisubstrate docking protein downstream in the signaling pathways of different receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Therefore, the aim of the study was to characterize the phosphorylation of recombinant human Gab-1 (hGab-1) protein by EGFR in vitro. Using the pGEX system to express the entire protein and different domains of hGab-1 as glutathione S-transferase proteins, kinetic data for phosphorylation of these proteins by wheat germ agglutinine-purified EGFR and the recombinant EGFR (rEGFR) receptor kinase domain were determined. Our data revealed similar affinities of hGab-1-C for both receptor preparations (KM = 2.7 microM for rEGFR vs 3.2 microM for WGA EGFR) as well as for the different recombinant hGab-1 domains. To identify the specific EGFR phosphorylation sites, hGab-1-C was sequenced by Edman degradation and mass spectrometry. The entire protein was phosphorylated by rEGFR at eight tyrosine residues (Y285, Y373, Y406, Y447, Y472, Y619, Y657, and Y689). Fifty percent of the identified radioactivity was incorporated in tyrosine Y657 as the predominant peak in HPLC analysis, a site exhibiting features of a potential Syp (PTP1D) binding site. Accordingly, GST-pull down assays with A431 and HepG2 cell lysates showed that phosphorylated intact hGab-1 was able to bind Syp. This binding appears to be specific, because it was abolished by changing the Y657 of hGab-1 to F657. These results demonstrate that hGab-1 is a high-affinity substrate for the EGFR and the major tyrosine phosphorylation site Y657 in the C terminus is a specific binding site for the tyrosine phosphatase Syp.

  10. Substrate recognition by the Lyn protein-tyrosine kinase. NMR structure of the immunoreceptor tyrosine-based activation motif signaling region of the B cell antigen receptor.

    PubMed

    Gaul, B S; Harrison, M L; Geahlen, R L; Burton, R A; Post, C B

    2000-05-26

    The immunoreceptor tyrosine-based activation motif (ITAM) plays a central role in transmembrane signal transduction in hematopoietic cells by mediating responses leading to proliferation and differentiation. An initial signaling event following activation of the B cell antigen receptor is phosphorylation of the CD79a (Ig-alpha) ITAM by Lyn, a Src family protein-tyrosine kinase. To elucidate the structural basis for recognition between the ITAM substrate and activated Lyn kinase, the structure of an ITAM-derived peptide bound to Lyn was determined using exchange-transferred nuclear Overhauser NMR spectroscopy. The bound substrate structure has an irregular helix-like character. Docking based on the NMR data into the active site of the closely related Lck kinase strongly favors ITAM binding in an orientation similar to binding of cyclic AMP-dependent protein kinase rather than that of insulin receptor tyrosine kinase. The model of the complex provides a rationale for conserved ITAM residues, substrate specificity, and suggests that substrate binds only the active conformation of the Src family tyrosine kinase, unlike the ATP cofactor, which can bind the inactive form.

  11. 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...

  12. 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...

  13. Activation of lysophosphatidic acid receptor by gintonin inhibits Kv1.2 channel activity: involvement of tyrosine kinase and receptor protein tyrosine phosphatase α.

    PubMed

    Lee, Jun-Ho; Choi, Sun-Hye; Lee, Byung-Hwan; Hwang, Sung-Hee; Kim, Hyeon-Joong; Rhee, Jeehae; Chung, Chihye; Nah, Seung-Yeol

    2013-08-26

    Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. The primary action of gintonin is to elicit a transient increase in [Ca(2+)]i via activation of LPA receptor subtypes. Voltage-gated potassium (Kv) channels play important roles in synaptic transmission in nervous systems. The previous reports have shown that Kv channels can be regulated by Gαq/11 protein-coupled receptor ligands. In the present study, we examined the effects of gintonin on Kv1.2 channel activity expressed in Xenopus oocytes after injection of RNA encoding the human Kv1.2 α subunit. Gintonin treatment inhibited Kv1.2 channel activity in reversible and concentration-dependent manners. The inhibitory effect of gintonin on Kv1.2 channel activity was blocked by active phospholipase C inhibitor, inositol 1,4,5-triphosphate receptor antagonist, and intracellular Ca(2+) chelator. The co-expression of active receptor protein tyrosine phosphatase α (RPTPα) with Kv1.2 channel greatly attenuated gintonin-mediated inhibition of Kv1.2 channel activity, but attenuation was not observed with catalytically inactive RPTPα. Furthermore, neither genistein, a tyrosine kinase inhibitor, nor site-directed mutation of a tyrosine residue (Y132 to Y132F), which is phosphorylated by tyrosine kinase of the N-terminal of the Kv1.2 channel α subunit, significantly attenuated gintonin-mediated inhibition of Kv1.2 channel activity. These results indicate that the gintonin-mediated Kv1.2 channel regulation involves the dual coordination of both tyrosine kinase and RPTPα coupled to this receptor. Finally, gintonin-mediated regulation of Kv1.2 channel activity might explain one of the modulations of gintonin-mediated neuronal activities in nervous systems.

  14. Formylbenzene diazonium hexafluorophosphate reagent for tyrosine-selective modification of proteins and the introduction of a bioorthogonal aldehyde

    PubMed Central

    Gavrilyuk, Julia; Ban, Hitoshi; Nagano, Masanobu; Hakamata, Wataru; Barbas, Carlos F.

    2012-01-01

    4-Formylbenzene diazonium hexafluorophosphate (FBDP) is a novel bench-stable crystalline diazonium salt that reacts selectively with tyrosine to install a bioorthogonal aldehyde functionality. Model studies with N-acyl-tyrosine methylamide allowed us to identify conditions optimal for tyrosine ligation reactions with small peptides and proteins. FBDP-based conjugation was used for the facile introduction of small molecule tags, poly(ethylene) glycol chains (PEGylation), and functional small molecules onto model proteins and to label the surface of living cells. PMID:23181702

  15. Differential regulation of protein tyrosine kinase signalling by Dock and the PTP61F variants.

    PubMed

    Willoughby, Lee F; Manent, Jan; Allan, Kirsten; Lee, Han; Portela, Marta; Wiede, Florian; Warr, Coral; Meng, Tzu-Ching; Tiganis, Tony; Richardson, Helena E

    2017-07-01

    Tyrosine phosphorylation-dependent signalling is coordinated by the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). There is a growing list of adaptor proteins that interact with PTPs and facilitate the dephosphorylation of substrates. The extent to which any given adaptor confers selectivity for any given substrate in vivo remains unclear. Here we have taken advantage of Drosophila melanogaster as a model organism to explore the influence of the SH3/SH2 adaptor protein Dock on the abilities of the membrane (PTP61Fm)- and nuclear (PTP61Fn)-targeted variants of PTP61F (the Drosophila othologue of the mammalian enzymes PTP1B and TCPTP respectively) to repress PTK signalling pathways in vivo. PTP61Fn effectively repressed the eye overgrowth associated with activation of the epidermal growth factor receptor (EGFR), PTK, or the expression of the platelet-derived growth factor/vascular endothelial growth factor receptor (PVR) or insulin receptor (InR) PTKs. PTP61Fn repressed EGFR and PVR-induced mitogen-activated protein kinase signalling and attenuated PVR-induced STAT92E signalling. By contrast, PTP61Fm effectively repressed EGFR- and PVR-, but not InR-induced tissue overgrowth. Importantly, coexpression of Dock with PTP61F allowed for the efficient repression of the InR-induced eye overgrowth, but did not enhance the PTP61Fm-mediated inhibition of EGFR and PVR-induced signalling. Instead, Dock expression increased, and PTP61Fm coexpression further exacerbated the PVR-induced eye overgrowth. These results demonstrate that Dock selectively enhances the PTP61Fm-mediated attenuation of InR signalling and underscores the specificity of PTPs and the importance of adaptor proteins in regulating PTP function in vivo. © 2017 Federation of European Biochemical Societies.

  16. Weak oligomerization of low-molecular-weight protein tyrosine phosphatase is conserved from mammals to bacteria.

    PubMed

    Blobel, Jascha; Bernadó, Pau; Xu, Huimin; Jin, Changwen; Pons, Miquel

    2009-08-01

    The well-characterized self-association of a mammalian low-molecular-weight protein tyrosine phosphatase (lmwPTP) produces inactive oligomers that are in equilibrium with active monomers. A role of the inactive oligomers as supramolecular proenzymes has been suggested. The oligomerization equilibrium of YwlE, a lmwPTP from Bacillus subtilis, was studied by NMR. Chemical shift data and NMR relaxation confirm that dimerization takes place through the enzyme's active site, and is fully equivalent to the dimerization previously characterized in a eukaryotic low-molecular-weight phosphatase, with similarly large dissociation constants. The similarity between the oligomerization of prokaryotic and eukaryotic phosphatases extends beyond the dimer and involves higher order oligomers detected by NMR relaxation analysis at high protein concentrations. The conservation across different kingdoms of life suggests a physiological role for lmwPTP oligomerization in spite of the weak association observed in vitro. Structural data suggest that substrate modulation of the oligomerization equilibrium could be a regulatory mechanism leading to the generation of signaling pulses. The presence of a phenylalanine residue in the dimerization site of YwlE, replacing a tyrosine residue conserved in all eukaryotic lmwPTPs, demonstrates that lmwPTP regulation by oligomerization can be independent from tyrosine phosphorylation.

  17. Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

    PubMed Central

    Andersen, J N; Elson, A; Lammers, R; Rømer, J; Clausen, J T; Møller, K B; Møller, N P

    2001-01-01

    To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function. PMID:11237862

  18. Protein Tyrosine Kinase 6 Negatively Regulates Growth and Promotes Enterocyte Differentiation in the Small Intestine

    PubMed Central

    Haegebarth, Andrea; Bie, Wenjun; Yang, Ruyan; Crawford, Susan E.; Vasioukhin, Valeri; Fuchs, Elaine; Tyner, Angela L.

    2006-01-01

    Protein tyrosine kinase 6 (PTK6) (also called Brk or Sik) is an intracellular tyrosine kinase that is expressed in breast cancer and normal epithelial linings. In adult mice, PTK6 expression is high in villus epithelial cells of the small intestine. To explore functions of PTK6, we disrupted the mouse Ptk6 gene. We detected longer villi, an expanded zone of PCNA expression, and increased bromodeoxyuridine incorporation in the PTK6-deficient small intestine. Although differentiation of major epithelial cell types occurred, there was a marked delay in expression of intestinal fatty acid binding protein, suggesting a role for PTK6 in enterocyte differentiation. However, fat absorption was comparable in wild-type and Ptk6−/− mice. It was previously shown that the serine threonine kinase Akt is a substrate of PTK6 and that PTK6-mediated phosphorylation of Akt on tyrosine resulted in inhibition of Akt activity. Consistent with these findings, we detected increased Akt activity and nuclear β-catenin in intestines of PTK6-deficient mice and decreased nuclear localization of the Akt substrate FoxO1 in villus epithelial cells. PTK6 contributes to maintenance of tissue homeostasis through negative regulation of Akt in the small intestine and is associated with cell cycle exit and differentiation in normal intestinal epithelial cells. PMID:16782882

  19. Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

    PubMed

    Kobayashi, Michihiro; Nabinger, Sarah C; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro-Ohtani, Yumi; Pear, Warren S; Carlesso, Nadia; Yoder, Mervin C; Kapur, Reuben; Kaplan, Mark H; Daniel Lacorazza, Hugo; Zhang, Zhong-Yin; Liu, Yan

    2017-04-01

    The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064.

  20. Tyrosine-Selective Functionalization for Bio-Orthogonal Cross-Linking of Engineered Protein Hydrogels.

    PubMed

    Madl, Christopher M; Heilshorn, Sarah C

    2017-03-15

    Engineered protein hydrogels have shown promise as artificial extracellular matrix materials for the 3D culture of stem cells due to the ability to decouple hydrogel biochemistry and mechanics. The modular design of these proteins allows for incorporation of various bioactive sequences to regulate cellular behavior. However, the chemistry used to cross-link the proteins into hydrogels can limit what bioactive sequences can be incorporated, in order to prevent nonspecific cross-linking within the bioactive region. Bio-orthogonal cross-linking chemistries may allow for the incorporation of any arbitrary bioactive sequence, but site-selective and scalable incorporation of bio-orthogonal reactive groups such as azides that do not rely on commonly used amine-reactive chemistry is often challenging. In response, we have optimized the reaction of an azide-bearing 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) with engineered elastin-like proteins (ELPs) to selectively azide-functionalize tyrosine residues within the proteins. The PTAD-azide functionalized ELPs cross-link with bicyclononyne (BCN) functionalized ELPs via the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to form hydrogels. Human mesenchymal stem cells and murine neural progenitor cells encapsulated within these hydrogels remain highly viable and maintain their phenotypes in culture. Tyrosine-specific modification may expand the number of bioactive sequences that can be designed into protein-engineered materials by permitting incorporation of lysine-containing sequences without concern for nonspecific cross-linking.

  1. Haemophilus ducreyi targets Src family protein tyrosine kinases to inhibit phagocytic signaling.

    PubMed

    Mock, Jason R; Vakevainen, Merja; Deng, Kaiping; Latimer, Jo L; Young, Jennifer A; van Oers, Nicolai S C; Greenberg, Steven; Hansen, Eric J

    2005-12-01

    Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcgamma receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacterial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.

  2. Inhibition of protein tyrosine phosphatase 1B by lignans from Myristica fragrans.

    PubMed

    Yang, Senugmi; Na, Min Kyun; Jang, Jun Pil; Kim, Kyung Ah; Kim, Bo Yeon; Sung, Nak Ju; Oh, Won Keun; Ahn, Jong Seog

    2006-08-01

    Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been proposed as one of the drug targets for treating type 2 diabetes and obesity. Bioassay-guided fractionation of a MeOH extract of the semen of Myristica fragrans Houtt. (Myristicaceae) afforded PTP1B inhibitory compounds, meso-dihydroguaiaretic acid (1) and otobaphenol (2). Compounds 1 and 2 inhibited PTP1B with IC(50) values of 19.6 +/- 0.3 and 48.9 +/- 0.5 microM, respectively, in the manner of non-competitive inhibitors. Treatment with compound 1 on 32D cells overexpressing the insulin receptor (IR) resulted in a dose-dependent increase in the tyrosine phosphorylation of IR. These results indicate that compound 1 can act as an enhancing agent in intracellular insulin signaling, possibly through the inhibition of PTP1B activity.

  3. Free tyrosine and tyrosine-rich peptide-dependent superoxide generation catalyzed by a copper-binding, threonine-rich neurotoxic peptide derived from prion protein.

    PubMed

    Yokawa, Ken; Kagenishi, Tomoko; Goto, Kaishi; Kawano, Tomonori

    2009-01-01

    Previously, generation of superoxide anion (O(2)(*-)) catalyzed by Cu-binding peptides derived from human prion protein (model sequence for helical Cu-binding motif VNITKQHTVTTTT was most active) in the presence of catecholamines and related aromatic monoamines such as phenylethylamine and tyramine, has been reported [Kawano, T., Int J Biol Sci 2007; 3: 57-63]. The peptide sequence (corresponding to helix 2) tested here is known as threonine-rich neurotoxic peptide. In the present article, the redox behaviors of aromatic monoamines, 20 amino acids and prion-derived tyrosine-rich peptide sequences were compared as putative targets of the oxidative reactions mediated with the threonine-rich prion-peptide. For detection of O(2)(*-), an O(2)(*-)-specific chemiluminescence probe, Cypridina luciferin analog was used. We found that an aromatic amino acid, tyrosine (structurally similar to tyramine) behaves as one of the best substrates for the O(2)(*-) generating reaction (conversion from hydrogen peroxide) catalyzed by Cu-bound prion helical peptide. Data suggested that phenolic moiety is required to be an active substrate while the presence of neither carboxyl group nor amino group was necessarily required. In addition to the action of free tyrosine, effect of two tyrosine-rich peptide sequences YYR and DYEDRYYRENMHR found in human prion corresponding to the tyrosine-rich region was tested as putative substrates for the threonine-rich neurotoxic peptide. YYR motif (found twice in the Y-rich region) showed 2- to 3-fold higher activity compared to free tyrosine. Comparison of Y-rich sequence consisted of 13 amino acids and its Y-to-F substitution mutant sequence revealed that the tyrosine-residues on Y-rich peptide derived from prion may contribute to the higher production of O(2)(*-). These data suggest that the tyrosine residues on prion molecules could be additional targets of the prion-mediated reactions through intra- or inter-molecular interactions. Lastly, possible

  4. Identification of an oligodeoxynucleotide sequence motif that specifically inhibits phosphorylation by protein tyrosine kinases.

    PubMed

    Krieg, A M; Matson, S; Cheng, K; Fisher, E; Koretzky, G A; Koland, J G

    1997-04-01

    Protein tyrosine kinases (PTKs) have central roles in cellular signal transduction. We have identified a sequence motif (CGT[C]GA) in phosphorothioate-modified oligodeoxynucleotides (ODNs) that specifically inhibits the enzymatic activity of recombinant or immunoprecipitated PTK in vitro. Hexamer ODNs containing this motif block both substrate and autophosphorylation of at least four different PTKs but have no apparent effect on the enzymatic activity of a serine/threonine protein kinase. These data suggest possible new applications for ODNs and have implications for the design and interpretation of experiments using antisense or triplex ODNs.

  5. Valosin containing protein (VCP/p97) is a novel substrate for the protein tyrosine phosphatase PTPL1

    PubMed Central

    Abaan, Ogan D.; Hendriks, Wiljan; Üren, Aykut; Toretsky, Jeffrey A.; Erkizan, Hayriye V.

    2013-01-01

    Identification of Protein Tyrosine Phosphatase (PTP) substrates is critical in understanding cellular role in normal cells as well as cancer cells. We have previously shown that reduction of PTPL1 protein levels in Ewings sarcoma (ES) inhibit cell growth and tumorigenesis. Therefore, we sought to identify novel PTPL1 substrates that may be important for tumorigenesis. In this current work, we demonstrated that mouse embryonic fibroblasts without PTPL1 catalytic activity fail to form foci when transfected with oncogenes. We proved that catalytic activity of PTPL1 is important for ES cell growth. Using a substrate-trapping mutant of PTPL1 we identified putative PTPL1 substrates by mass-spectrometry. One of these putative substrates was characterized as Valosin Containing Protein (VCP/p97). Using multiple biochemical assays we validated VCP as a novel substrate of PTPL1. We also provide evidence that tyrosine phosphorylation of VCP might be important for its midbody localization during cytokinesis. In conclusion, our work identifies VCP as a new substrate for PTPL1, which may be important in cellular transformation. Our investigation link an oncogenic transcription factor EWS-FLI1, with a key transcriptional target protein tyrosine phosphatase PTPL1, and its substrate VCP. Given our observation that PTPL1 catalytic activity is important for cell transformation, our results may also suggest that VCP regulation by PTPL1 might be important for tumorigenesis. PMID:23018179

  6. Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes

    PubMed Central

    1992-01-01

    After the intraportal injection of EGF, the EGF receptor (EGFR) is rapidly internalized into hepatic endosomes where it remains largely receptor bound (Lai et al., 1989. J. Cell Biol. 109:2751-2760). In the present study, we evaluated the phosphotyrosine content of EGFRs at the cell surface and in endosomes in order to assess the consequences of internalization. Quantitative estimates of specific radioactivity of the EGFR in these two compartments revealed that tyrosine phosphorylation of the EGFR was observed at the cell surface within 30 s of ligand administration. However, the EGFR was also highly phosphorylated in endosomes reaching levels of tyrosine phosphorylation significantly higher than those of the cell surface receptor at 5 and 15 min after EGF injection. A 55-kD tyrosine phosphorylated polypeptide (pyp55) was observed in association with the EGFR at the cell surface within 30 s of EGF injection. The protein was also found in association with the EGFR in endosomes as evidenced by coprecipitation studies using a mAb to the EGFR as well as by coelution with the EGR in gel permeation chromatography. Limited proteolysis of isolated endosomes indicated that the tyrosine phosphorylated domains of the EGFR and associated pyp55 were cytosolically oriented while internalized EGF was intraluminal. The identification of pyp55 in association with EGFR in both hepatic plasma membranes and endosomes may be relevant to EGFR function and/or trafficking of the EGFR. PMID:1370492

  7. Mitigation of a nitrate reducing Pseudomonas aeruginosa biofilm and anaerobic biocorrosion using ciprofloxacin enhanced by D-tyrosine.

    PubMed

    Jia, Ru; Yang, Dongqing; Xu, Dake; Gu, Tingyue

    2017-07-31

    Pseudomonas aeruginosa (PA) is a ubiquitous microbe. It can form recalcitrant biofilms in clinical and industrial settings. PA biofilms cause infections in patients. They also cause biocorrosion of medical implants. In this work, D-tyrosine (D-tyr) was investigated as an antimicrobial enhancer for ciprofloxacin (CIP) against a wild-type PA biofilm (strain PAO1) on C1018 carbon steel in a strictly anaerobic condition. Seven-day biofilm prevention test results demonstrated that 2 ppm (w/w) D-tyr enhanced 30 ppm CIP by achieving extra 2-log sessile cell reduction compared with the 30 ppm CIP alone treatment. The cocktail of 30 ppm CIP + 2 ppm D-tyr achieved similar efficacy as the 80 ppm CIP alone treatment in the biofilm prevention test. Results also indicated that the enhanced antimicrobial treatment reduced weight loss and pitting corrosion. In the 3-hour biofilm removal test, the cocktail of 80 ppm CIP + 5 ppm D-tyr achieved extra 1.5-log reduction in sessile cell count compared with the 80 ppm CIP alone treatment. The cocktail of 80 ppm CIP + 5 ppm D-tyr achieved better efficacy than the 150 ppm CIP alone treatment in the biofilm removal test.

  8. Entry of Neisseria meningitidis into Mammalian Cells Requires the Src Family Protein Tyrosine Kinases ▿

    PubMed Central

    Slanina, Heiko; König, Alexandra; Hebling, Sabrina; Hauck, Christof R.; Frosch, Matthias; Schubert-Unkmeir, Alexandra

    2010-01-01

    Neisseria meningitidis, the causative agent of meningitis and septicemia, is able to attach to and invade a variety of cell types. In a previous study we showed that entry of N. meningitidis into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin bound to the outer membrane protein Opc, which forms a molecular bridge to α5β1-integrins. This interaction results in cytoskeletal remodeling and uptake of the bacteria. In this study we identified and characterized the intracellular signals involved in integrin-initiated uptake of N. meningitidis. We determined that the Src protein tyrosine kinases (PTKs) are activated in response to contact with N. meningitidis. Inhibition of Src PTK activity by the general tyrosine kinase inhibitor genistein and the specific Src inhibitor PP2 reduced Opc-mediated invasion of HBMEC and human embryonic kidney (HEK) 293T cells up to 90%. Moreover, overexpression of the cellular Src antagonist C-terminal Src kinase (CSK) also significantly reduced N. meningitidis invasion. Src PTK-deficient fibroblasts were impaired in the ability to internalize N. meningitidis and showed reduced phosphorylation of the cytoskeleton and decreased development of stress fibers. These data indicate that the Src family PTKs, particularly the Src protein, along with other proteins, are important signal proteins that are responsible for the transfer of signals from activated integrins to the cytoskeleton and thus mediate the endocytosis of N. meningitidis into brain endothelial cells. PMID:20176789

  9. 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

    2015-08-04

    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.

  10. 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

  11. Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

    PubMed Central

    Peris, Leticia; Thery, Manuel; Fauré, Julien; Saoudi, Yasmina; Lafanechère, Laurence; Chilton, John K.; Gordon-Weeks, Phillip; Galjart, Niels; Bornens, Michel; Wordeman, Linda; Wehland, Juergen; Andrieux, Annie; Job, Didier

    2006-01-01

    Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to α-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization. PMID:16954346

  12. 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

  13. Multifaceted modulation of K+ channels by protein-tyrosine phosphatase ε tunes neuronal excitability.

    PubMed

    Ebner-Bennatan, Sharon; Patrich, Eti; Peretz, Asher; Kornilov, Polina; Tiran, Zohar; Elson, Ari; Attali, Bernard

    2012-08-10

    Non-receptor-tyrosine kinases (protein-tyrosine kinases) and non-receptor tyrosine phosphatases (PTPs) have been implicated in the regulation of ion channels, neuronal excitability, and synaptic plasticity. We previously showed that protein-tyrosine kinases such as Src kinase and PTPs such as PTPα and PTPε modulate the activity of delayed-rectifier K(+) channels (I(K)). Here we show cultured cortical neurons from PTPε knock-out (EKO) mice to exhibit increased excitability when compared with wild type (WT) mice, with larger spike discharge frequency, enhanced fast after-hyperpolarization, increased after-depolarization, and reduced spike width. A decrease in I(K) and a rise in large-conductance Ca(2+)-activated K(+) currents (mBK) were observed in EKO cortical neurons compared with WT. Parallel studies in transfected CHO cells indicate that Kv1.1, Kv1.2, Kv7.2/7.3, and mBK are plausible molecular correlates of this multifaceted modulation of K(+) channels by PTPε. In CHO cells, Kv1.1, Kv1.2, and Kv7.2/7.3 K(+) currents were up-regulated by PTPε, whereas mBK channel activity was reduced. The levels of tyrosine phosphorylation of Kv1.1, Kv1.2, Kv7.3, and mBK potassium channels were increased in the brain cortices of neonatal and adult EKO mice compared with WT, suggesting that PTPε in the brain modulates these channel proteins. Our data indicate that in EKO mice, the lack of PTPε-mediated dephosphorylation of Kv1.1, Kv1.2, and Kv7.3 leads to decreased I(K) density and enhanced after-depolarization. In addition, the deficient PTPε-mediated dephosphorylation of mBK channels likely contributes to enhanced mBK and fast after-hyperpolarization, spike shortening, and consequent increase in neuronal excitability observed in cortical neurons from EKO mice.

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

    USDA-ARS?s Scientific Manuscript database

    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. Site-specific Nitration of Apolipoprotein A-I at Tyrosine 166 Is Both Abundant within Human Atherosclerotic Plaque and Dysfunctional*

    PubMed Central

    DiDonato, Joseph A.; Aulak, Kulwant; Huang, Ying; Wagner, Matthew; Gerstenecker, Gary; Topbas, Celalettin; Gogonea, Valentin; DiDonato, Anthony J.; Tang, W. H. Wilson; Mehl, Ryan A.; Fox, Paul L.; Plow, Edward F.; Smith, Jonathan D.; Fisher, Edward A.; Hazen, Stanley L.

    2014-01-01

    We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr166), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr166 nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr166-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr166-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ∼8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr166-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063–1.21). NO2-Tyr166-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr166-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr166 is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall. PMID:24558038

  16. Denaturation of protein by chlorine dioxide: oxidative modification of tryptophan and tyrosine residues.

    PubMed

    Ogata, Norio

    2007-04-24

    Oxychlorine compounds, such as hypochlorous acid (HOCl) and chlorine dioxide (ClO2), have potent antimicrobial activity. Although the biochemical mechanism of the antimicrobial activity of HOCl has been extensively investigated, little is known about that of ClO2. Using bovine serum albumin and glucose-6-phosphate dehydrogenase of Saccharomyces cerevisiae as model proteins, here I demonstrate that the antimicrobial activity of ClO2 is attributable primarily to its protein-denaturing activity. By solubility analysis, circular dichroism spectroscopy, differential scanning calorimetry, and measurement of enzymatic activity, I demonstrate that protein is rapidly denatured by ClO2 with a concomitant decrease in the concentration of ClO2 in the reaction mixture. Circular dichroism spectra of the ClO2-treated proteins show a change in ellipticity at 220 nm, indicating a decrease in alpha-helical content. Differential scanning calorimetry shows that transition temperature and endothermic transition enthalpy of heat-induced unfolding decrease in the ClO2-treated protein. The enzymatic activity of glucose-6-phosphate dehydrogenase decreases to 10% within 15 s of treatment with 10 microM ClO2. Elemental analyses show that oxygen, but not chlorine, atoms are incorporated in the ClO2-treated protein, providing direct evidence that protein is oxidized by ClO2. Furthermore, mass spectrometry and nuclear magnetic resonance spectroscopy show that tryptophan residues become N-formylkynurenine and tyrosine residues become 3,4-dihydroxyphenylalanine (DOPA) or 2,4,5-trihydroxyphenylalanine (TOPA) in the ClO2-treated proteins. Taking these results together, I conclude that microbes are inactivated by ClO2 owing to denaturation of constituent proteins critical to their integrity and/or function, and that this denaturation is caused primarily by covalent oxidative modification of their tryptophan and tyrosine residues.

  17. Protective effects of resveratrol against oxidative/nitrative modifications of plasma proteins and lipids exposed to peroxynitrite.

    PubMed

    Olas, Beata; Nowak, Pawel; Kolodziejczyk, Joanna; Ponczek, Michal; Wachowicz, Barbara

    2006-02-01

    The protective effects of resveratrol (3, 4', 5-trihydroxystilbene; present naturally in different plants) against the oxidative/nitrative damage of human plasma proteins induced by peroxynitrite (ONOO-) were studied and compared with those of deferoxamine (DFO; a natural siderophore isolated from Streptomyces pilosus), which is a typical and well-known antioxidant. We also studied the effect of ONOO- on plasma lipid peroxidation and the role of tested antioxidants in this process. ONOO- at the used concentrations (0.01-1 mM) showed toxicity to human plasma components. Exposure of plasma to ONOO- (0.1 mM) resulted in an increase of the level of carbonyl groups and nitrotyrosine residues in plasma proteins (approximately 4-fold and 76-fold, respectively) and in a distinct augmentation of lipid peroxidation (approximately 2-fold). In the presence of 0.1-mM resveratrol, a distinct decrease of carbonyl group formation and tyrosine nitration in plasma proteins caused by 0.1-mM ONOO- was observed (by approximately 70% and 65%, respectively). Addition of 0.1-mM DFO to plasma also distinctly reduced the level of carbonyl groups and nitrotyrosines caused by 0.1-mM ONOO- (by approximately 50% and 60%, respectively). Moreover, these antioxidants also inhibited plasma lipid peroxidation induced by ONOO- (0.1 mM). The obtained results indicate that in vitro resveratrol, like well-known antioxidant DFO, has inhibitory effects on ONOO- -mediated oxidation of proteins and lipids in human plasma.

  18. 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.

  19. 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.

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

    SciTech Connect

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

    2010-01-01

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

  1. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

    PubMed Central

    Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

    1996-01-01

    The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. Protein tyrosine phosphatase receptor R and Z1 expression as independent prognostic indicators in oral squamous cell carcinoma.

    PubMed

    Duś-Szachniewicz, Kamila; Woźniak, Marta; Nelke, Kamil; Gamian, Elżbieta; Gerber, Hanna; Ziółkowski, Piotr

    2015-12-01

    The actions of tyrosine phosphorylation and dephosphorylation are controlled by tyrosine kinases and phosphatases. Although substantial previous data have revealed the role of several protein tyrosine phosphatases (PTPs) in various cancers, the function of protein tyrosine phosphatase receptor R (PTPRR) and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1) proteins in oral cavity squamous cell carcinoma (SCC) has not been studied to date. The PTPRR and PTPRZ1 immunoreactivity in 67 formalin-fixed and paraffin-embedded oral cancer tissues at different stages were analyzed with the technique of immunohistochemistry (IHC). The presence of PTPRR in cancerous tissue was confirmed by Western blotting. The occurrence of PTPRR and PTPRZ1 proteins in the cancer specimens was more frequent in lower grade tumors. In addition, the association between the immunoreactivity of both examined proteins and improved patients survival was detected. Moreover, the PTPRR expression was found to be related to the absence of synchronous lymph node involvement. The above results indicate that the PTPRR and PTPRZ1 protein expression should be monitored in oral cancer for patients' prognostic stratification. © 2015 Wiley Periodicals, Inc.

  7. Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

    PubMed

    Guidon, P T; Salvatori, R; Bockman, R S

    1993-01-01

    Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

  8. Oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion spermatozoa.

    PubMed

    Leemans, Bart; Gadella, Bart M; Sostaric, Edita; Nelis, Hilde; Stout, Tom A E; Hoogewijs, Maarten; Van Soom, Ann

    2014-07-01

    Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca(2+), and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10(6) spermatozoa/ml), during which it transpired that the highest density (per mm(2)) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviduct-bound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pHi) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pHi, presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pHi changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm. © 2014 by the Society for the Study of Reproduction, Inc.

  9. 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.

  10. 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.

  11. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-08-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.

  12. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed Central

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-01-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein. Images PMID:2901092

  13. Extracellular regulation of type IIa receptor protein tyrosine phosphatases: mechanistic insights from structural analyses

    PubMed Central

    Coles, Charlotte H.; Jones, E. Yvonne; Aricescu, A. Radu

    2016-01-01

    The receptor protein tyrosine phosphatases (RPTPs) exhibit a wide repertoire of cellular signalling functions. In particular, type IIa RPTP family members have recently been highlighted as hubs for extracellular interactions in neurons, regulating neuronal extension and guidance, as well as synaptic organisation. In this review, we will discuss the recent progress of structural biology investigations into the architecture of type IIa RPTP ectodomains and their interactions with extracellular ligands. Structural insights, in combination with biophysical and cellular studies, allow us to begin to piece together molecular mechanisms for the transduction and integration of type IIa RPTP signals and to propose hypotheses for future experimental validation. PMID:25234613

  14. 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

  15. Modulation of EGF receptor autophosphorylation by alpha-hemolysin of Staphylococcus aureus via protein tyrosine phosphatase.

    PubMed

    Vandana, Sharma; Navneet, Sangha; Surinder, Kaur; Krishnasastry, M V

    2003-01-30

    In the presence of assembled alpha-hemolysin (alpha-HL) of Staphylococcus aureus, the epidermal growth factor receptor (EGFr) is rapidly dephosphorylated. Several obvious possibilities that otherwise would have contributed to the dephosphorylation were ruled out. Instead, an elevation in the activity of a protein tyrosine phosphatase appears to be responsible for the observed loss of phosphorylation signal of EGFr. For this dephosphorylation, the assembly of alpha-HL is necessary while lytic pore formation is not required. In summary, the EGFr is unable to retain its phosphorylation signal in the presence of alpha-HL and the process is irreversible.

  16. Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa.

    PubMed

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

    2016-01-01

    Hexavalent chromium reportedly induces reproductive toxicity and further inhibits male fertility in mammals. In this study, we investigated the molecular mechanism by which hexavalent chromium affects motility signaling in boar spermatozoa in vitro. The results indicated that Cr(VI) decreased sperm motility, protein phosphorylation, mitochondrial membrane potential (ΔΨm) and metabolic enzyme activity starting at 4μmol/mL following incubation for 1.5h. Notably, all parameters were potently inhibited by 10μmol/mL Cr, while supplementation with the dibutyryl-cAMP (dbcAMP) and the 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibition of protein phosphorylation. Interestingly, high concentrations of Cr (>10μmol/mL) increased the tyrosine phosphorylation of some high-molecular-weight proteins in the principle piece but decreased that in the middle piece associated with an extreme reduction of sperm motility. These results suggest that chromium affects boar sperm motility by impairing tyrosine phosphorylation in the midpiece of sperm by blocking the cAMP/PKA pathway in boar sperm in vitro.

  17. Implication of a protein-tyrosine-phosphatase in human lung cancer.

    PubMed

    Gaits, F; Li, R Y; Ragab, A; Selves, J; Ragab-Thomas, J M; Chap, H

    1994-07-01

    Protein tyrosyl phosphorylation plays an essential role in regulating cellular events such as proliferation, differentiation and oncogenesis. The recent characterization of the family of protein tyrosine phosphatases (PTPases) suggests that dephosphorylation might be a crucial event in these phenomena. One of the functions of PTPases is to reverse the effect of protein tyrosine kinases (PTKases), many of which are oncogenes, suggesting that they may act as tumor suppressors as described for HPTP gamma. In order to investigate the implication in lung cancer of HPTP beta, a receptor PTPase, we have developed a semi-quantitative method derived from primer-directed reverse transcription (RT) and subsequent polymerase chain reaction (PCR) with 32P-labelled nucleotide. We have demonstrated that the expression of HPTP beta mRNA was dramatically decreased in lung adenocarcinomas and lung malpighian carcinomas as compared to normal lung tissue. In addition, HPTP beta was not expressed in the pulmonar adenocarcinoma cell line A427, which proliferates in a deregulated way. These results suggest that the loss of expression of HPTP beta might play a role in neoplasic transformation and thus this molecule could act as a tumor suppressor factor.

  18. Effect of cooling (4°C) and cryopreservation on cytoskeleton actin and protein tyrosine phosphorylation in buffalo spermatozoa.

    PubMed

    Naresh, Sai

    2016-02-01

    Semen cryopreservation is broadly utilized as a part of the bovine reproducing industry, a large portion of the spermatozoa does not survive and the majority of those that do survive experience various molecular and physiological changes that influence their fertilizing capacity. The main aim of this study is to determine the effect of cooling (4 °C) and cryopreservation on cytoskeleton actin, tyrosine phosphorylation and quality of buffalo spermatozoa, and to determine the similarity between in vitro capacitation and cryopreservation induced capacitation like changes. To achieve this, Western blot was used to examine the changes in actin expression and protein tyrosine phosphorylation, whereas changes in actin polymerization, localization of actin and protein tyrosine phosphorylation during capacitation and cryopreservation were evaluated by indirect immunofluorescence technique. Localization studies revealed that the actin localized to flagella and acrosome membrane regions and following, capacitation it migrated towards the acrosome region of sperm. Time dependent increase in actin polymerization and protein tyrosine phosphorylation was observed during in vitro capacitation. The cooling phase (4 °C) and cryopreservation processes resulted in the loss/damage of cytoskeleton actin. In addition, we performed the actin polymerization and protein tyrosine phosphorylation in cooled and cryopreserved buffalo spermatozoa. Interestingly, cooling and cryopreservation induces actin polymerization and protein tyrosine phosphorylation, which were similar to in vitro capacitation (cryo-capacitation). These changes showed 1.3 folds reduction in the sperm quality parameters which includes motility, viability and plasma membrane integrity. Furthermore, our findings indicate that cooling and cryopreservation damages the cytoskeleton actin and also induces capacitation like changes such as protein tyrosine phosphorylation and actin polymerization. This could be one of the

  19. A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II.

    PubMed

    Ruan, Kangcheng; Li, Jiong; Liang, Ruqiang; Xu, Chunhe; Yu, Yong; Lange, Reinhard; Balny, Claude

    2002-04-26

    An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.

  20. Kinetic Isotope Effects In The Characterization Of Catalysis by Protein Tyrosine Phosphatases

    PubMed Central

    Hengge, Alvan C.

    2015-01-01

    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. PMID:25840000

  1. 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

  2. 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

  3. STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE (STEP) KNOCKOUT MICE HAVE ENHANCED HIPPOCAMPAL MEMORY

    PubMed Central

    Venkitaramani, Deepa V.; Moura, Paula J.; Picciotto, Marina R.; Lombroso, Paul J.

    2011-01-01

    STEP is a brain-specific phosphatase that opposes synaptic strengthening by the regulation of key synaptic signaling proteins. Previous studies suggest a possible role for STriatal-Enriched protein tyrosine Phosphatase (STEP) in learning and memory. To demonstrate the functional importance of STEP in learning and memory, we generated STEP knockout (KO) mice and examined the effect of deletion of STEP on behavioral performance, as well as the phosphorylation and expression of its substrates. Here we report that loss of STEP leads to significantly enhanced performance in hippocampal-dependent learning and memory tasks. In addition, STEP KO mice displayed greater dominance behavior, although they were normal in their motivation, motor coordination, visual acuity and social interactions. STEP KO mice displayed enhanced tyrosine phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2), the NR2B subunit of the N-methyl-D-aspartate receptor (NMDAR), Proline-rich tyrosine kinase (Pyk2), as well as an increased phosphorylation of ERK1/2 substrates. Concomitant to the increased phosphorylation of NR2B, synaptosomal expression of NR1/NR2B NMDARs was increased in STEP KO mice, as was the GluR1/GluR2 containing α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPAR), providing a potential molecular mechanism for the improved cognitive performance. The data support a role for STEP in the regulation of synaptic strengthening. The absence of STEP improves cognitive performance, and may do so by the regulation of downstream effectors necessary for synaptic transmission. PMID:21501258

  4. Protein tyrosine kinase regulation by ubiquitination: Critical roles of Cbl-family ubiquitin ligases

    PubMed Central

    Mohapatra, Bhopal; Ahmad, Gulzar; Nadeau, Scott; Zutshi, Neha; An, Wei; Scheffe, Sarah; Dong, Lin; Feng, Dan; Goetz, Benjamin; Arya, Priyanka; Bailey, Tameka A.; Palermo, Nicholas; Borgstahl, Gloria E.O.; Natarajan, Amarnath; Raja, Srikumar M.; Naramura, Mayumi; Band, Vimla; Band, Hamid

    2012-01-01

    Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell–cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant “activated PTK-selective” ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader

  5. Signal transduction activated by the cancer chemopreventive isothiocyanates: cleavage of BID protein, tyrosine phosphorylation and activation of JNK

    PubMed Central

    Xu, K; Thornalley, P J

    2001-01-01

    Phenethyl isothiocyanate and allyl isothiocyanate induce apoptosis of human leukaemia HL60 cells in vitro. Apoptosis was associated with cleavage of p22 BID protein to p15, p13 and p11 fragments and activation of JNK and tyrosine phosphorylation (18 kDa and 45 kDa proteins). All these effects and apoptosis were prevented by exogenous glutathione (15 mM). Protein tyrosine phosphatase activity was unchanged. The general caspase inhibitor Z-VAD-fmk prevented apoptosis but not JNK activation – excluding a role for caspases in JNK activation, whereas curcumin prevented JNK activation but only delayed apoptosis. This suggests that in isothiocyanate-induced apoptosis, the caspase pathway has an essential role, the JNK pathway a supporting role, and inhibition of protein tyrosine phosphatases is not involved. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11237388

  6. 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

  7. Evolutionary conservation of mammalian sperm proteins associates with overall, not tyrosine, phosphorylation in human spermatozoa.

    PubMed

    Schumacher, Julia; Ramljak, Sanja; Asif, Abdul R; Schaffrath, Michael; Zischler, Hans; Herlyn, Holger

    2013-12-06

    We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein-protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility.

  8. Luteinizing hormone modulates intracellular calcium, protein tyrosine phosphorylation and motility during human sperm capacitation.

    PubMed

    López-Torres, Aideé S; González-González, María E; Mata-Martínez, Esperanza; Larrea, Fernando; Treviño, Claudia L; Chirinos, Mayel

    2017-02-05

    In order to fertilize, spermatozoa must undergo physiological and biochemical changes during their transit along the female reproductive tract before reaching and fusing with the oocyte, process known as capacitation. Sperm modifications associated with capacitation are modulated by their interaction with molecules present in the female reproductive tract. During the woman fertile window, some reproductive hormones reach their maximum concentrations in serum, such as the luteinizing hormone (LH). Since spermatozoa preparing to fertilize may be exposed to LH, the purpose of this work was to study the effects of this hormone on intracellular Ca(2+) concentrations ([Ca(2+)]i), protein tyrosine phosphorylation, sperm motility and acrosome reaction under capacitating conditions. The results showed that LH increases the duration and amplitude of Ca(2+) oscillations. Furthermore, motility analysis indicated that LH decreases rapid progressive motility and that sperm hyperactivation as well as several kinetic parameters augment in the presence of 0.5 and 1 μg/ml of the hormone. In addition, these two hormone concentrations also consistently promoted protein tyrosine phosphorylation. However, no effects on acrosome reaction were observed. In conclusion, the evidence indicates that LH modulates several sperm function variables involved in capacitation, suggesting that may have an important and unexplored role during human fertilization. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. syk protein tyrosine kinase regulates Fc receptor gamma-chain-mediated transport to lysosomes.

    PubMed Central

    Bonnerot, C; Briken, V; Brachet, V; Lankar, D; Cassard, S; Jabri, B; Amigorena, S

    1998-01-01

    B- and T-cell receptors, as well as most Fc receptors (FcR), are part of a large family of membrane proteins named immunoreceptors and are expressed on all cells of the immune system. Immunoreceptors' biological functions rely on two of their fundamental attributes: signal transduction and internalization. The signals required for these two functions are present in the chains associated with immunoreceptors, within conserved amino acid motifs called immunoreceptor tyrosine-based activation motifs (ITAMs). We have examined the role of the protein tyrosine kinase (PTK) syk, a critical effector of immunoreceptor-mediated cell signalling through ITAMs, in FcR-associated gamma-chain internalization and lysosomal targeting. A point mutation in the immunoreceptor-associated gamma-chain ITAM affecting syk activation, as well as overexpression of a syk dominant negative mutant, inhibited signal transduction without affecting receptor coated-pit localization or internalization. In contrast, blocking of gamma-chain-mediated syk activation impaired FcR transport from endosomes to lysosomes and selectively inhibited the presentation of certain T-cell epitopes. Therefore, activation of the PTK syk is dispensable for receptor internalization, but necessary for cell signalling and for gamma-chain-mediated FcR delivery to lysosomes. PMID:9707420

  10. Therapeutic Implications for Striatal-Enriched Protein Tyrosine Phosphatase (STEP) in Neuropsychiatric Disorders

    PubMed Central

    Goebel-Goody, Susan M.; Baum, Matthew; Paspalas, Constantinos D.; Fernandez, Stephanie M.; Carty, Niki C.; Kurup, Pradeep

    2012-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase that modulates key signaling molecules involved in synaptic plasticity and neuronal function. Targets include extracellular-regulated kinase 1 and 2 (ERK1/2), stress-activated protein kinase p38 (p38), the Src family tyrosine kinase Fyn, N-methyl-d-aspartate receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). STEP-mediated dephosphorylation of ERK1/2, p38, and Fyn leads to inactivation of these enzymes, whereas STEP-mediated dephosphorylation of surface NMDARs and AMPARs promotes their endocytosis. Accordingly, the current model of STEP function posits that it opposes long-term potentiation and promotes long-term depression. Phosphorylation, cleavage, dimerization, ubiquitination, and local translation all converge to maintain an appropriate balance of STEP in the central nervous system. Accumulating evidence over the past decade indicates that STEP dysregulation contributes to the pathophysiology of several neuropsychiatric disorders, including Alzheimer's disease, schizophrenia, fragile X syndrome, epileptogenesis, alcohol-induced memory loss, Huntington's disease, drug abuse, stroke/ischemia, and inflammatory pain. This comprehensive review discusses STEP expression and regulation and highlights how disrupted STEP function contributes to the pathophysiology of diverse neuropsychiatric disorders. PMID:22090472

  11. Characterization and genomic mapping of genes and pseudogenes of a new human protein tyrosine phosphatase

    SciTech Connect

    Zhao, Zhaoyang; Lee, Cheng-Chi; Monckton, D.G.

    1996-07-01

    Previously described protein tyrosine phosphatases (PTPs) are classified into three types according to their sequence homology and structural features. Here we describe the characterization of genes and pseudogenes of a member of a fourth type of PTP, designated protein tyrosine phosphatase 4A (PTP4A). The 167-amino-acid human PTPs, but does not show any other sequence homology to any of the previously described PTPs. Two cDNA encoding PTP4A that differed in their noncoding regions were isolated. Another cDNA that has a high level of sequence identity with these two cDNAs and a deletion in the coding region was also isolated. Northern analysis using a probe from a common 3{prime}-untranslated region of the cDNAs recognized mRNAs of about 2 and 4 kb. Both species of mRNA were seen in all human adult and fetal tissues tested. Fluorescence in situ hybridization mapping of the corresponding yeast artificial chromosome clones and sequence-tagged site analysis suggested that one of the PTP4A coding genes is located at 1p35 and the other is on chromosome 11. A processed pseudogene for PTP4A was found in the BRCA1 region of 17q21 and shares 96% sequence identity to one of the PTP4A coding cDNAs. Our studies also suggest the existence of another processed pseudogene on chromosome 11. 31 refs., 6 figs., 1 tab.

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

    PubMed

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

    2016-02-11

    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.

  13. 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

  14. Protein tyrosine phosphatase PTPRB regulates Src phosphorylation and tumour progression in NSCLC.

    PubMed

    Qi, Yinliang; Dai, Yuanchang; Gui, Shuyu

    2016-10-01

    Protein tyrosine-phosphatases (PTPs) play important roles in various biological processes. Deregulation in PTP function has been implicated in carcinogenesis and tumour progression in many cancer types. However, the role of protein tyrosine phosphatase receptor type B (PTPRB) in non-small-cell lung cancer (NSCLC) tumorigenesis has not been investigated. Lentiviral vector expressing PTPRB cDNA or shRNA was infected into A549 and H1299 cell lines, followed by cell proliferation, colony formation, soft agar and invasion assays. A549 xenograft mouse model was used to evaluate in vivo function of PTPRB. Quantitative polymerase chain reaction (PCR) was used to measure PTPRB expression in NSCLC patient samples. Kaplan Meier analysis was performed to assess association between PTPRB expression and patient overall survival (OS). Multivariate analysis was performed to evaluate prognostic significance of PTPRB. Overexpression of PTPRB reduced cell proliferation rate, colony formation efficiency, soft agar growth and cell invasion in A549 and H1299 cells, as well as tumour growth rate in A549 xenograft. Knockdown of PTPRB increased Src phosphorylation and cell invasion, which was reversed by Src inhibitor PP2. Additionally, PTPRB was down-regulated in NSCLC patient and was associated with patient OS. PTPRB regulates Src phosphorylation and tumorigenesis in NSCLC. PTPRB may serve as an independent prognostic biomarker for NSCLC patients.

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

    PubMed Central

    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

  16. 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

    2015-06-04

    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.

  17. Expression of epidermal growth factor receptor sequences as E. coli fusion proteins: applications in the study of tyrosine kinase function.

    PubMed

    Koland, J G; O'Brien, K M; Cerione, R A

    1990-01-15

    To investigate the functions of key domains of the epidermal growth factor receptor (EGFR), various EGFR-derived peptide sequences were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The purified fusion proteins (GST-TK0-8) were tested as substrates for the tyrosine kinase activities of the EGFR and c-src. Both the GST-TK4 fusion protein, which contains the major C-terminal tyrosine autophosphorylation sites of the EGFR, and GST-TK7, which contains the connecting sequence between the EGFR kinase domain and the C-terminal autophosphorylation domain, were strongly phosphorylated by the EGFR and c-src. Hence the candidate tyrosine phosphorylation sites present in the connecting sequences of the EGFR, as well as the known autophosphorylation sites of the EGFR, can be phosphorylated by the two tyrosine kinases. The protein GST-TK7 was phosphorylated by c-src with a KM of 5-10 microM, which indicated a potential interaction between the connecting segment of the EGFR and the c-src kinase. The GST fusion proteins were also used to map the sites recognized by two anti-EGFR monoclonal antibodies and a polyclonal serum raised against an EGFR tyrosine kinase domain fragment. The recognition site of one monoclonal antibody was determined to be in a short sequence surrounding tyr1068, a primary site of autophosphorylation in the C-terminal domain of the receptor. The anti-peptide polyclonal serum recognized only sequences in the GST-TK7 fusion protein, and hence binds to the connecting sequence between the kinase core and the C-terminal domain. These antibodies will therefore be useful reagents for studying the function of two key structural elements of the EGFR tyrosine kinase. The GST-TK fusion proteins should have many other applications in the study of EGFR catalysis and mitogenic signalling.

  18. Perivascular iron deposits are associated with protein nitration in cerebral experimental autoimmune encephalomyelitis.

    PubMed

    Sands, Scott A; Williams, Rachel; Marshall, Sylvester; LeVine, Steven M

    2014-10-17

    Nitration of proteins, which is thought to be mediated by peroxynitrite, is a mechanism of tissue damage in multiple sclerosis (MS). However, protein nitration can also be catalyzed by iron, heme or heme-associated molecules independent of peroxynitrite. Since microhemorrhages and perivascular iron deposits are present in the CNS of MS patients, we sought to determine if iron is associated with protein nitration. A cerebral model of experimental autoimmune encephalomyelitis (cEAE) was utilized since this model has been shown to have perivascular iron deposits similar to those present in MS. Histochemical staining for iron was used together with immunohistochemistry for nitrotyrosine, eNOS, or iNOS on cerebral sections. Leakage of the blood-brain barrier (BBB) was studied by albumin immunohistochemistry. Iron deposits were colocalized with nitrotyrosine staining around vessels in cEAE mice while control animals revealed minimal staining. This finding supports the likelihood that nitrotyrosine formation was catalyzed by iron or iron containing molecules. Examples of iron deposits were also observed in association with eNOS and iNOS, which could be one source of substrates for this reaction. Extravasation of albumin was present in cEAE mice, but not in control animals. Extravasated albumin may act to limit tissue injury by binding iron and/or heme as well as being a target of nitration, but the protection is incomplete. In summary, iron-catalyzed nitration of proteins is a likely mechanism of tissue damage in MS.

  19. 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

  20. Protein tyrosine phosphatases PTPδ, PTPσ, and LAR: presynaptic hubs for synapse organization

    PubMed Central

    Takahashi, Hideto; Craig, Ann Marie

    2013-01-01

    Synapse development requires differentiation of presynaptic neurotransmitter release sites and postsynaptic receptive apparatus coordinated by synapse organizing proteins. In addition to the well-characterized neurexins, recent studies identified presynaptic type IIa receptor-type protein tyrosine phosphatases (RPTPs) as mediators of presynaptic differentiation and triggers of postsynaptic differentiation, thus extending the roles of RPTPs from axon outgrowth and guidance. Similarly to neurexins, RPTPs exist in multiple isoforms generated by alternative splicing that interact in a splice-selective code with diverse postsynaptic partners. The parallel RPTP and neurexin hub design facilitates synapse self-assembly through cooperation, pairs presynaptic similarity with postsynaptic diversity, and balances excitation with inhibition. Upon mutation of individual genes in neuropsychiatric disorders, imbalance of this synaptic organizing network may contribute to impaired cognitive function. PMID:23835198

  1. 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.

  2. Inhibitors of the Yersinia protein tyrosine phosphatase through high throughput and virtual screening approaches.

    PubMed

    Hu, Xin; Vujanac, Milos; Southall, Noel; Stebbins, C Erec

    2013-02-15

    The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Inhibitors of the Yersinia Protein Tyrosine Phosphatase Through High Throughput and Virtual Screening Approaches

    PubMed Central

    Hu, Xin; Vujanac, Milos; Southall, Noel; Stebbins, C. Erec

    2013-01-01

    The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design. PMID:23294700

  4. Protein tyrosine phosphatases PTPδ, PTPσ, and LAR: presynaptic hubs for synapse organization.

    PubMed

    Takahashi, Hideto; Craig, Ann Marie

    2013-09-01

    Synapse development requires differentiation of presynaptic neurotransmitter release sites and postsynaptic receptive apparatus coordinated by synapse organizing proteins. In addition to the well-characterized neurexins, recent studies identified presynaptic type IIa receptor-type protein tyrosine phosphatases (RPTPs) as mediators of presynaptic differentiation and triggers of postsynaptic differentiation, thus extending the roles of RPTPs from axon outgrowth and guidance. Similarly to neurexins, RPTPs exist in multiple isoforms generated by alternative splicing that interact in a splice-selective code with diverse postsynaptic partners. The parallel RPTP and neurexin hub design facilitates synapse self-assembly through cooperation, pairs presynaptic similarity with postsynaptic diversity, and balances excitation with inhibition. Upon mutation of individual genes in neuropsychiatric disorders, imbalance of this synaptic organizing network may contribute to impaired cognitive function.

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

    PubMed

    Oh, Man-Ho; Wu, Xia; Kim, Hyoung Seok; Harper, Jeffrey F; Zielinski, Raymond E; Clouse, Steven D; Huber, Steven C

    2012-11-30

    Although calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases, autophosphorylation on tyrosine residues was observed for soybean CDPKβ and several Arabidopsis isoforms (AtCPK4 and AtCPK34). We identified Ser-8, Thr-17, Tyr-24 (in the kinase domain), Ser-304, and Ser-358 as autophosphorylation sites of His(6)-GmCDPKβ. Overall autophosphorylation increased kinase activity with synthetic peptides, but autophosphorylation of Tyr-24 appears to attenuate kinase activity based on studies with the Y24F directed mutant. While much remains to be done, it is clear that several CDPKs are dual-specificity kinases, which raises the possibility that phosphotyrosine signaling may play a role in Ca(2+)/CDPK-mediated processes. Published by Elsevier B.V.

  6. 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.

  7. Modification of Hypoxic Respiratory Response by Protein Tyrosine Kinase in Brainstem Ventral Respiratory Neuron Group

    PubMed Central

    Wang, Hui; Huai, Ruituo; Yang, Junqing; Li, Yanchun

    2016-01-01

    Protein tyrosine kinase (PTK) mediated the tyrosine phosphorylation modification of neuronal receptors and ion channels. Whether such modification resulted in changes of physiological functions was not sufficiently studied. In this study we examined whether the hypoxic respiratory response—which is the enhancement of breathing in hypoxic environment could be affected by the inhibition of PTK at brainstem ventral respiratory neuron column (VRC). Experiments were performed on urethane anesthetized adult rabbits. Phrenic nerve discharge was recorded as the central respiratory motor output. Hypoxic respiratory response was produced by ventilating the rabbit with 10% O2-balance 90% N2 for 5 minutes. The responses of phrenic nerve discharge to hypoxia were observed before and after microinjecting PTK inhibitor genistein, AMPA receptor antagonist CNQX, or inactive PTK inhibitor analogue daidzein at the region of ambiguus nucleus (NA) at levels 0–2 mm rostral to obex where the inspiratory subgroup of VRC were recorded. Results were as follows: 1. the hypoxic respiratory response was significantly attenuated after microinjection of genistein and/or CNQX, and no additive effect (i.e., further attenuation of hypoxic respiratory response) was observed when genistein and CNQX were microinjected one after another at the same injection site. Microinjection of daidzein had no effect on hypoxic respiratory response. 2. Fluorescent immunostaining showed that hypoxia significantly increased the number of phosphotyrosine immunopositive neurons in areas surrounding NA and most of these neurons were also immunopositive to glutamate AMPA receptor subunit GluR1. These results suggested that PTK played an important role in regulating the hypoxic respiratory response, possibly through the tyrosine phosphorylation modification of glutamate AMPA receptors on the respiratory neurons of ventral respiratory neuron column. PMID:27798679

  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. A novel protein tyrosine kinase Tec identified in lamprey, Lampetra japonica.

    PubMed

    Li, Ranran; Su, Peng; Liu, Chang; Zhang, Qiong; Zhu, Ting; Pang, Yue; Liu, Xin; Li, Qingwei

    2015-08-01

    Protein tyrosine kinase Tec, a kind of non-receptor tyrosine kinase, is primarily found to be expressed in T cells, B cells, hematopoietic cells, and liver cells as a cytoplasmic protein. Tec has been proved to be a critical modulator of T cell receptor signaling pathway. In the present study, a homolog of Tec was identified in the lamprey, Lampetra japonica. The full-length Tec cDNA of L. japonica (Lja-Tec) contains a 1923 bp open reading frame that encodes a 641-amino acid protein. The multi-alignment of the deduced amino acid sequence of Lja-Tec with typical vertebrate Tecs showed that it possesses all conserved domains of the Tec family proteins, indicating that an ortholog of Tec exists in the extant jawless vertebrate. In the phylogenetic tree that was reconstructed with 24 homologs of jawless and jawed vertebrates, the Tecs from lampreys and hagfish were clustered as a single clade. The genetic distance between the outgroup and agnathan Tecs' group is closer than that between outgroup and gnathostome Tecs' group, indicating that its origin was far earlier than any of the jawed vertebrates. The mRNA levels of Lja-Tec in lymphocyte-like cells and gills were detected by real-time quantitative polymerase chain reaction. Results showed that it was significantly upregulated under stimulation with mixed pathogens. This result was further confirmed by western blot analysis. All these results indicated that Lja-Tec plays an important role in immune response. Our data will provide a reference for the further study of lamprey Tec and its immunological function in jawless vertebrates.

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

    PubMed Central

    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

    2016-01-01

    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. STEP61 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 pGluN2BTyr1472 and pERK1/2Thr202/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 STEP61 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 STEP61 inactivation and increased pGluN2BTyr1472 and pERK1/2Thr202/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

  11. Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation

    PubMed Central

    Vasu, Dudipeta; Kumar, Pasupuleti Santhosh; Prasad, Uppu Venkateswara; Swarupa, Vimjam; Yeswanth, Sthanikam; Srikanth, Lokanathan; Sunitha, Manne Mudhu; Choudhary, Abhijith; Krishna Sarma, Potukuchi Venkata Gurunadha

    2017-01-01

    Background: When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. Methods: Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK, and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. Results: Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. Conclusion: The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and the progress of infection. PMID:27695030

  12. Kinetics and Mechanism of Protein Tyrosine Phosphatase 1B (PTP1B) Inactivation by Acrolein

    PubMed Central

    Seiner, Derrick R.; LaButti, Jason N.; Gates, Kent S.

    2010-01-01

    Human cells are exposed to the electrophilic α,β-unsaturated aldehyde acrolein from a variety of sources. Reaction of acrolein with functionally critical protein thiol residues can yield important biological consequences. Protein tyrosine phosphatases (PTPs) are an important class of cysteine-dependent enzymes whose reactivity with acrolein previously has not been well characterized. These enzymes catalyze the dephosphorylation of phosphotyrosine residues on proteins via a phosphocysteine intermediate. PTPs work in tandem with protein tyrosine kinases to regulate a number of critically important mammalian signal transduction pathways. We find that acrolein is a potent time-dependent inactivator of the enzyme PTP1B (kinact = 0.02 ± 0.005 s−1, KI = 2.3 ± 0.6 × 10−4 M). Enzyme activity does not return upon gel filtration of the inactivated enzyme and addition of the competitive phosphatase inhibitor vanadate slows inactivation of PTP1B by acrolein. Together these observations suggest that acrolein covalently modifies the active site of PTP1B. Mass spectrometric analysis reveals that acrolein modifies the catalytic cysteine residue at the active site of the enzyme. Aliphatic aldehydes such as glyoxal, acetaldehyde, and propanal are relatively weak inactivators of PTP1B under the conditions employed here. Similarly, unsaturated aldehydes such as crotonaldehyde and 3-methyl-2-butenal bearing substitution at the alkene terminus are poor inactivators of the enzyme. Overall, the data suggest that enzyme inactivation occurs via conjugate addition of the catalytic cysteine residue to the carbon-carbon double bond of acrolein. The results indicate that inactivation of PTPs should be considered as a possible contributor to the diverse biological activities of acrolein and structurally-related α,β-unsaturated aldehydes. PMID:17655273

  13. Kinetics and mechanism of protein tyrosine phosphatase 1B inactivation by acrolein.

    PubMed

    Seiner, Derrick R; LaButti, Jason N; Gates, Kent S

    2007-09-01

    Human cells are exposed to the electrophilic alpha,beta-unsaturated aldehyde acrolein from a variety of sources. The reaction of acrolein with functionally critical protein thiol residues can yield important biological consequences. Protein tyrosine phosphatases (PTPs) are an important class of cysteine-dependent enzymes whose reactivity with acrolein previously has not been well-characterized. These enzymes catalyze the dephosphorylation of phosphotyrosine residues on proteins via a phosphocysteine intermediate. PTPs work in tandem with protein tyrosine kinases to regulate a number of critically important mammalian signal transduction pathways. We find that acrolein is a potent time-dependent inactivator of the enzyme PTP1B ( k inact = 0.02 +/- 0.005 s (-1) and K I = 2.3 +/- 0.6 x 10 (-4) M). The enzyme activity does not return upon gel filtration of the inactivated enzyme, and addition of the competitive phosphatase inhibitor vanadate slows inactivation of PTP1B by acrolein. Together, these observations suggest that acrolein covalently modifies the active site of PTP1B. Mass spectrometric analysis reveals that acrolein modifies the catalytic cysteine residue at the active site of the enzyme. Aliphatic aldehydes such as glyoxal, acetaldehyde, and propanal are relatively weak inactivators of PTP1B under the conditions employed here. Similarly, unsaturated aldehydes such as crotonaldehyde and 3-methyl-2-butenal bearing substitution at the alkene terminus are poor inactivators of the enzyme. Overall, the data suggest that enzyme inactivation occurs via conjugate addition of the catalytic cysteine residue to the carbon-carbon double bond of acrolein. The results indicate that inactivation of PTPs should be considered as a possible contributor to the diverse biological activities of acrolein and structurally related alpha,beta-unsaturated aldehydes.

  14. 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.

  15. Identification of a fungi-specific lineage of protein kinases closely related to tyrosine kinases.

    PubMed

    Zhao, Zhongtao; Jin, Qiaojun; Xu, Jin-Rong; Liu, Huiquan

    2014-01-01

    Tyrosine kinases (TKs) specifically catalyze the phosphorylation of tyrosine residues in proteins and play essential roles in many cellular processes. Although TKs mainly exist in animals, recent studies revealed that some organisms outside the Opisthokont clade also contain TKs. The fungi, as the sister group to animals, are thought to lack TKs. To better understand the origin and evolution of TKs, it is important to investigate if fungi have TK or TK-related genes. We therefore systematically identified possible TKs across the fungal kingdom by using the profile hidden Markov Models searches and phylogenetic analyses. Our results confirmed that fungi lack the orthologs of animal TKs. We identified a fungi-specific lineage of protein kinases (FslK) that appears to be a sister group closely related to TKs. Sequence analysis revealed that members of the FslK clade contain all the conserved protein kinase sub-domains and thus are likely enzymatically active. However, they lack key amino acid residues that determine TK-specific activities, indicating that they are not true TKs. Phylogenetic analysis indicated that the last common ancestor of fungi may have possessed numerous members of FslK. The ancestral FslK genes were lost in Ascomycota and Ustilaginomycotina and Pucciniomycotina of Basidiomycota during evolution. Most of these ancestral genes, however, were retained and expanded in Agaricomycetes. The discovery of the fungi-specific lineage of protein kinases closely related to TKs helps shed light on the origin and evolution of TKs and also has potential implications for the importance of these kinases in mushroom fungi.

  16. 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

  17. Amyloid Precursor Protein Mediates a Tyrosine-kinase Dependent Activation Response in Endothelial Cells

    PubMed Central

    Austin, S.A.; Sens, M.A.; Combs, C.K.

    2010-01-01

    Amyloid precursor protein (APP) is a ubiquitously expressed type one integral membrane protein. It has the ability to bind numerous extracellular matrix components and propagate signaling responses via its cytoplasmic phosphotyrosine, 682YENPTY687, binding motif. We recently demonstrated increased protein levels of APP, phosphorylated APP (Tyr682), and beta-amyloid (Aβ) in brain vasculature of atherosclerotic and Alzheimer’s disease (AD) tissue co-localizing primarily within the endothelial layer. This study demonstrates similar APP changes in peripheral vasculature from human and mouse apoE−/− aorta suggesting APP-related changes are not restricted to brain vasculature. Therefore, primary mouse aortic endothelial cells (PAEC) and human umbilical vein endothelial cells (HUVEC) were used as a model system to examine the function of APP in endothelial cells. APP multimerization with an anti-N-terminal APP antibody, 22C11, to simulate ligand binding stimulated a Src kinase family dependent increase in protein phosphotyrosine levels, APP phosphorylation, and Aβ secretion. Furthermore, APP multimerization stimulated increased protein levels of the proinflammatory proteins, cyclooxygenase (COX)-2 and vascular cell adhesion molecule (VCAM)-1 also in a Src kinase family dependent fashion. Endothelial APP was also involved in mediating monocytic cell adhesion. Collectively, these data demonstrate that endothelial APP regulates immune cell adhesion and stimulates a tyrosine kinase-dependent response driving acquisition of a reactive endothelial phenotype. These APP-mediated events may serve as therapeutic targets for intervention in progressive vascular changes common to cerebrovascular disease and AD. PMID:19923279

  18. 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

  19. Photo-oxidation of tyrosine in a bio-engineered bacterioferritin 'reaction centre'-a protein model for artificial photosynthesis.

    PubMed

    Hingorani, Kastoori; Pace, Ron; Whitney, Spencer; Murray, James W; Smith, Paul; Cheah, Mun Hon; Wydrzynski, Tom; Hillier, Warwick

    2014-10-01

    The photosynthetic reaction centre (RC) is central to the conversion of solar energy into chemical energy and is a model for bio-mimetic engineering approaches to this end. We describe bio-engineering of a Photosystem II (PSII) RC inspired peptide model, building on our earlier studies. A non-photosynthetic haem containing bacterioferritin (BFR) from Escherichia coli that expresses as a homodimer was used as a protein scaffold, incorporating redox-active cofactors mimicking those of PSII. Desirable properties include: a di-nuclear metal binding site which provides ligands for bivalent metals, a hydrophobic pocket at the dimer interface which can bind a photosensitive porphyrin and presence of tyrosine residues proximal to the bound cofactors, which can be utilised as efficient electron-tunnelling intermediates. Light-induced electron transfer from proximal tyrosine residues to the photo-oxidised ZnCe6(•+), in the modified BFR reconstituted with both ZnCe6 and Mn(II), is presented. Three site-specific tyrosine variants (Y25F, Y58F and Y45F) were made to localise the redox-active tyrosine in the engineered system. The results indicate that: presence of bound Mn(II) is necessary to observe tyrosine oxidation in all BFR variants; Y45 the most important tyrosine as an immediate electron donor to the oxidised ZnCe6(•+) and that Y25 and Y58 are both redox-active in this system, but appear to function interchangebaly. High-resolution (2.1Å) crystal structures of the tyrosine variants show that there are no mutation-induced effects on the overall 3-D structure of the protein. Small effects are observed in the Y45F variant. Here, the BFR-RC represents a protein model for artificial photosynthesis. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

  20. Cloning of three human tyrosine phosphatases reveals a multigene family of receptor-linked protein-tyrosine-phosphatases expressed in brain.

    PubMed Central

    Kaplan, R; Morse, B; Huebner, K; Croce, C; Howk, R; Ravera, M; Ricca, G; Jaye, M; Schlessinger, J

    1990-01-01

    A human brainstem cDNA library in bacteriophage lambda gt11 was screened under conditions of reduced hybridization stringency with a leukocyte common antigen (LCA) probe that spanned both conserved cytoplasmic domains. cDNA encoding a receptor-linked protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), RPTPase alpha, has been cloned and sequenced. Human RPTPase alpha consists of 802 amino acids. The extracellular domain of 150 residues includes a hydrophobic signal peptide and eight potential N-glycosylation sites. This is followed by a transmembrane region and two tandemly repeated conserved domains characteristic of all RPTPases identified thus far. The gene for RPTPase alpha has been localized to human chromosome region 20pter-20q12 by analysis of its segregation pattern in rodent-human somatic cell hybrids. Northern blot analysis revealed the presence of two major transcripts of 4.3 and 6.3 kilobases. In addition to RPTPase alpha, two other RPTPases (beta and gamma), identified in the same screen, have been partially cloned and sequenced. Analysis of sequence comparisons among LCA, the LCA-related protein LAR, and RPTPases alpha, beta, and gamma reveals the existence of a multigene family encoding different RPTPases, each containing a distinct extracellular domain, a single hydrophobic transmembrane region, and two tandemly repeated conserved cytoplasmic domains. Images PMID:2169617

  1. Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors.

    PubMed

    Wang, X Y; Bergdahl, K; Heijbel, A; Liljebris, C; Bleasdale, J E

    2001-02-28

    One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.

  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. Stress-evoked tyrosine phosphorylation of signal regulatory protein α regulates behavioral immobility in the forced swim test.

    PubMed

    Ohnishi, Hiroshi; Murata, Takaaki; Kusakari, Shinya; Hayashi, Yuriko; Takao, Keizo; Maruyama, Toshi; Ago, Yukio; Koda, Ken; Jin, Feng-Jie; Okawa, Katsuya; Oldenborg, Per-Arne; Okazawa, Hideki; Murata, Yoji; Furuya, Nobuhiko; Matsuda, Toshio; Miyakawa, Tsuyoshi; Matozaki, Takashi

    2010-08-04

    Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein alpha (SIRPalpha) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPalpha that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPalpha in the brain of wild-type mice through activation of Src family kinases. The SIRPalpha ligand CD47 was important for such SIRPalpha phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvbeta2 was regulated by SIRPalpha. Thus, tyrosine phosphorylation of SIRPalpha is important for regulation of depression-like behavior in the response of the brain to stress.

  5. 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. Copyright © 2016 by the American Society of Nephrology.

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

    PubMed Central

    Zhou, Yuehan; Skelton, Lara A.; Xu, Lumei; Chandler, Margaret P.; Berthiaume, Jessica M.

    2016-01-01

    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. Matrix metalloproteinases and protein tyrosine kinases: potential novel targets in acute lung injury and ARDS.

    PubMed

    Aschner, Yael; Zemans, Rachel L; Yamashita, Cory M; Downey, Gregory P

    2014-10-01

    Acute lung injury (ALI) and ARDS fall within a spectrum of pulmonary disease that is characterized by hypoxemia, noncardiogenic pulmonary edema, and dysregulated and excessive inflammation. While mortality rates have improved with the advent of specialized ICUs and lung protective mechanical ventilation strategies, few other therapies have proven effective in the management of ARDS, which remains a significant clinical problem. Further development of biomarkers of disease severity, response to therapy, and prognosis is urgently needed. Several novel pathways have been identified and studied with respect to the pathogenesis of ALI and ARDS that show promise in bridging some of these gaps. This review will focus on the roles of matrix metalloproteinases and protein tyrosine kinases in the pathobiology of ALI in humans, and in animal models and in vitro studies. These molecules can act independently, as well as coordinately, in a feed-forward manner via activation of tyrosine kinase-regulated pathways that are pivotal in the development of ARDS. Specific signaling events involving proteolytic processing by matrix metalloproteinases that contribute to ALI, including cytokine and chemokine activation and release, neutrophil recruitment, transmigration and activation, and disruption of the intact alveolar-capillary barrier, will be explored in the context of these novel molecular pathways.

  8. Active Lyn protein tyrosine kinase is selectively enriched within membrane microdomains of resting platelets.

    PubMed Central

    Dorahy, D J; Burns, G F

    1998-01-01

    Circulating platelets are primed to respond very rapidly to thrombogenic stimuli, but most platelets complete their lifespan without ever becoming activated. Platelet activation is accompanied by waves of sequential tyrosine phosphorylation thought to involve members of the Src family of protein tyrosine kinases (PTKs). We show here that resting platelets contain highly active pp53/56(Lyn) PTK within membrane microdomains (rafts) isolated biochemically with or without the use of detergent. This fraction is also greatly enriched in the transmembrane glycoprotein CD36, known to associate with Lyn PTK, but in transfection studies we could find no evidence to suggest that CD36 affects the distribution or function of Lyn. Upon platelet activation Lyn activity remains constant or diminishes and pp60(c-src) PTK within this fraction becomes highly activated, indicating the dynamic nature of the membrane microdomains. It is suggested that the function of active Lyn PTK in the resting platelet is to allow prolonged survival of this anucleate cell. PMID:9657978

  9. Receptor protein tyrosine phosphatase sigma regulates synapse structure, function and plasticity.

    PubMed

    Horn, Katherine E; Xu, Bin; Gobert, Delphine; Hamam, Bassam N; Thompson, Katherine M; Wu, Chia-Lun; Bouchard, Jean-François; Uetani, Noriko; Racine, Ronald J; Tremblay, Michel L; Ruthazer, Edward S; Chapman, C Andrew; Kennedy, Timothy E

    2012-07-01

    The mechanisms that regulate synapse formation and maintenance are incompletely understood. In particular, relatively few inhibitors of synapse formation have been identified. Receptor protein tyrosine phosphatase σ (RPTPσ), a transmembrane tyrosine phosphatase, is widely expressed by neurons in developing and mature mammalian brain, and functions as a receptor for chondroitin sulfate proteoglycans that inhibits axon regeneration following injury. In this study, we address RPTPσ function in the mature brain. We demonstrate increased axon collateral branching in the hippocampus of RPTPσ null mice during normal aging or following chemically induced seizure, indicating that RPTPσ maintains neural circuitry by inhibiting axonal branching. Previous studies demonstrated a role for pre-synaptic RPTPσ promoting synaptic differentiation during development; however, subcellular fractionation revealed enrichment of RPTPσ in post-synaptic densities. We report that neurons lacking RPTPσ have an increased density of pre-synaptic varicosities in vitro and increased dendritic spine density and length in vivo. RPTPσ knockouts exhibit an increased frequency of miniature excitatory post-synaptic currents, and greater paired-pulse facilitation, consistent with increased synapse density but reduced synaptic efficiency. Furthermore, RPTPσ nulls exhibit reduced long-term potentiation and enhanced novel object recognition memory. We conclude that RPTPσ limits synapse number and regulates synapse structure and function in the mature CNS.

  10. Tyrosine Phosphorylation-independent Regulation of LPS-mediated Response by the Transmembrane Adaptor Protein LAB

    PubMed Central

    Zhu, Minghua; Fuller, Deirdre M; Ou-Yang, Chih-wen; Sullivan, Sarah; Zhang, Weiguo

    2012-01-01

    LAB (linker for activation of B cells)/NTAL (non-T cell activation linker) is a transmembrane adaptor protein that functions in immunoreceptor-mediated signaling. Published studies have shown that LAB has both positive and negative roles in regulating T cell receptor and high-affinity Fc receptor-mediated signaling and cellular function. In this study, we showed that LAB was also expressed in dendritic cells and that LAB deficiency affected LPS-mediated signaling and cytokine production. LPS-mediated MAPK activation was enhanced in LAB−/− bone marrow-derived dendritic cells (BMDCs). These BMDCs also produced more TNF-α, IL-6, and IL-10 than WT cells. Moreover, LAB−/− mice were hyper responsive to LPS-induced septic shock. These data indicated that LAB has a negative role in LPS-mediated responses. By using LAB knock-in mice, which harbor mutations at five membrane-distal tyrosines, we further showed that, in contrast to its role in immunoreceptor-mediated signaling, LAB function in LPS-mediated signaling pathway did not depend on its tyrosine phosphorylation. Our study suggested a novel mechanism by which LAB functions in the regulation of innate immunity. PMID:22308309

  11. Tyrosine phosphorylation-independent regulation of lipopolysaccharide-mediated response by the transmembrane adaptor protein LAB.

    PubMed

    Zhu, Minghua; Fuller, Deirdre M; Ou-Yang, Chih-wen; Sullivan, Sarah A; Zhang, Weiguo

    2012-03-15

    Linker for activation of B cells (LAB)/non-T cell activation linker is a transmembrane adaptor protein that functions in immunoreceptor-mediated signaling. Published studies have shown that LAB has both positive and negative roles in regulating TCR and high-affinity Fc receptor-mediated signaling and cellular function. In this study, we showed that LAB was also expressed in dendritic cells and that LAB deficiency affected LPS-mediated signaling and cytokine production. LPS-mediated MAPK activation was enhanced in LAB(-/-) bone marrow-derived dendritic cells. These bone marrow-derived dendritic cells also produced more TNF-α, IL-6, and IL-10 than wild-type cells. Moreover, LAB(-/-) mice were hyperresponsive to LPS-induced septic shock. These data indicated that LAB has a negative role in LPS-mediated responses. By using LAB knockin mice, which harbor mutations at five membrane-distal tyrosines, we further showed that, in contrast to its role in immunoreceptor-mediated signaling, LAB function in LPS-mediated signaling pathway did not depend on its tyrosine phosphorylation. Our study suggested a novel mechanism by which LAB functions in the regulation of innate immunity.

  12. Store-operated Ca2+ entry in hippocampal neurons: Regulation by protein tyrosine phosphatase PTP1B.

    PubMed

    Koss, David J; Riedel, Gernot; Bence, Kendra; Platt, Bettina

    2013-02-01

    Store operated Ca(2+) entry (SOCE) replenishes intracellular Ca(2+) stores and activates a number of intracellular signalling pathways. Whilst several molecular components forming store operated Ca(2+) channels (SOCC) have been identified, their modulation in neurons remains poorly understood. Here, we extend on our previous findings and show that neuronal SOCE is modulated by tyrosine phosphorylation. Cyclopiazonic acid induced SOCE was characterised in hippocampal cultures derived from forebrain specific protein tyrosine phosphatase 1B knockout (PTP1B KO) mice and wild type (WT) litter mates using Fura-2 Ca(2+) imaging. PTP1B KO cultures expressed elevated SOCE relative to WT cultures without changes in cytoplasmic Ca(2+) homeostasis or depolarisation-induced Ca(2+) influx. WT and PTP1B KO cultures displayed similar pharmacological sensitivities towards the SOCE inhibitors gadolinium and 2-aminoethoxydiphenyl borate, as well as the tyrosine kinase inhibitor Ag126 indicating an augmentation of native SOCCs by PTP1B. Following store depletion WT culture homogenates showed heightened phospho-tyrosine levels, an increase in Src tyrosine kinase activation and two minor PTP1B species. These data suggest tyrosine phosphorylation gating SOCE, and implicate PTP1B as a key regulatory enzyme. The involvement of PTP1B in SOCE and its relation to SOCC components and mechanism of regulation are discussed.

  13. 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.

  14. 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.

  15. Protein-tyrosine-kinase-dependent expression of cyclo-oxygenase-1 and -2 mRNAs in human endothelial cells.

    PubMed Central

    Hirai, K; Takayama, H; Tomo, K; Okuma, M

    1997-01-01

    Endothelial cells possess constitutive or inducible cyclo-oxygenase (COX) isoenzymes for prostacyclin production, but the mechanisms for their expression are largely unknown. We found that vanadate, an inhibitor of protein-tyrosine phosphatases, induced the expression of two COX isoenzyme mRNAs in human umbilical vein endothelial cells (HUVEC) in a time- and dose-dependent manner. Vanadate also stimulated an increase in COX-2 protein levels, but did not affect significantly the levels of constitutively expressed COX-1 protein. Synergistic enhancement of expression of the two COX isoenzyme mRNAs was observed on stimulation of HUVEC with vanadate plus interleukin-1alpha. Tyrphostin-47, which as an inhibitor of protein-tyrosine kinases abolished vanadate-induced protein-tyrosine phosphorylation, inhibited expression of the two COX isoenzyme mRNAs in HUVEC stimulated with vanadate or interleukin-1alpha. These data provide conclusive evidence that activation of protein-tyrosine kinases is causally linked to expression of the mRNAs for the two COX isoenzymes in HUVEC. PMID:9065752

  16. Ibrutinib inhibition of Bruton protein-tyrosine kinase (BTK) in the treatment of B cell neoplasms.

    PubMed

    Roskoski, Robert

    2016-11-01

    The Bruton non-receptor protein-tyrosine kinase (BTK), a deficiency of which leads to X-linked agammaglobulinemia, plays a central role in B cell antigen receptor signaling. Owing to the exclusivity of this enzyme in B cells, the acronym could represent B cell tyrosine kinase. BTK is activated by the Lyn and SYK protein kinases following activation of the B cell receptor. BTK in turn catalyzes the phosphorylation and activation of phospholipase Cγ2 leading to the downstream activation of the Ras/RAF/MEK/ERK pathway and the NF-κB pathways. Both pathways participate in the maturation of antibody-producing B cells. The BTK domains include a PH (pleckstrin homology) domain that interacts with membrane-associated phosphatidyl inositol trisphosphate, a TH (TEC homology) domain, which is followed by an SH3, SH2, and finally a protein kinase domain. Dysregulation of B cell receptor signaling occurs in several B cell neoplasms including mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström macroglobulinemia. Ibrutinib is FDA-approved as first-line or second line treatment for these diseases. The drug binds tightly in the ATP-binding pocket of BTK making salt bridges with residues within the hinge that connects the two lobes of the enzyme; then its unsaturated acrylamide group forms a covalent bond with BTK cysteine 481 to form an inactive adduct. In addition to the treatment of various B cell lymphomas, ibrutinib is under clinical trials for the treatment of numerous solid tumors owing to the role of tumor-promoting inflammation in the pathogenesis of neoplastic diseases.

  17. Low-Molecular-Weight Protein Tyrosine Phosphatase Predicts Prostate Cancer Outcome by Increasing the Metastatic Potential.

    PubMed

    Ruela-de-Sousa, Roberta R; Hoekstra, Elmer; Hoogland, A Marije; Queiroz, Karla C Souza; Peppelenbosch, Maikel P; Stubbs, Andrew P; Pelizzaro-Rocha, Karin; van Leenders, Geert J L H; Jenster, Guido; Aoyama, Hiroshi; Ferreira, Carmen V; Fuhler, Gwenny M

    2016-04-01

    Low-risk patients suffering from prostate cancer (PCa) are currently placed under active surveillance rather than undergoing radical prostatectomy. However, clear parameters for selecting the right patient for each strategy are not available, and new biomarkers and treatment modalities are needed. Low-molecular-weight protein tyrosine phosphatase (LMWPTP) could present such a target. To correlate expression levels of LMWPTP in primary PCa to clinical outcome, and determine the role of LMWPTP in prostate tumor cell biology. Acid phosphatase 1, soluble (ACP1) expression was analyzed on microarray data sets, which were subsequently used in Ingenuity Pathway Analysis. Immunohistochemistry was performed on a tissue microarray containing material of 481 PCa patients whose clinicopathologic data were recorded. PCa cell line models were used to investigate the role of LMWPTP in cell proliferation, migration, adhesion, and anoikis resistance. The association between LMWPTP expression and clinical and pathologic outcomes was calculated using chi-square correlations and multivariable Cox regression analysis. Functional consequences of LMWPTP overexpression or downregulation were determined using migration and adhesion assays, confocal microscopy, Western blotting, and proliferation assays. LMWPTP expression was significantly increased in human PCa and correlated with earlier recurrence of disease (hazard ratio [HR]:1.99; p<0.001) and reduced patient survival (HR: 1.53; p=0.04). Unbiased Ingenuity analysis comparing cancer and normal prostate suggests migratory propensities in PCa. Indeed, overexpression of LMWPTP increases PCa cell migration, anoikis resistance, and reduces activation of focal adhesion kinase/paxillin, corresponding to decreased adherence. Overexpression of LMWPTP in PCa confers a malignant phenotype with worse clinical outcome. Prospective follow-up should determine the clinical potential of LMWPTP overexpression. These findings implicate low

  18. Effect of reactive oxygen species on capacitation and associated protein tyrosine phosphorylation in buffalo (Bubalus bubalis) spermatozoa.

    PubMed

    Roy, S C; Atreja, S K

    2008-08-01

    In the present study, the effect of two particular reactive oxygen species (ROS), superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) on buffalo (Bubalus bubalis) sperm capacitation and associated protein tyrosine phosphorylation was studied. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50 x 10(6)/mL and incubated at 38.5 degrees C for 6h with or without heparin (10(g/mL; a positive control), or xanthine (X; 0.5mM)-xanthine oxidase (XO; 0.05 U/mL)-catalase (C; 2100 U/mL) system that generates O(2)(-) or NADPH (5mM) that stimulates the endogenous O(2)(-) production or H(2)O(2) (50 microM). The specific effect of O(2)(-), H(2)O(2) and NADPH on buffalo sperm capacitation and protein tyrosine phosphorylation was assessed by the addition of superoxide dismutase (SOD), catalase and diphenylene iodonium (DPI), respectively, to the incubation medium. Each of X+XO+C system, NADPH and H(2)O(2) induced a significantly higher percentage (P<0.05) of capacitation in buffalo spermatozoa compared to control. However, DPI inhibited this NADPH-induced capacitation and protein tyrosine phosphorylation and suggested for existence of an oxidase in buffalo spermatozoa. Using immunoblotting technique, at least seven tyrosine-phosphorylated proteins (20, 32, 38, 45, 49, 78 and 95 kDa) were detected in capacitated buffalo spermatozoa. Out of these, the tyrosine phosphorylation of p95 was induced extensively by both O(2)(-) as well as exogenous source of H(2)O(2) and using specific activators and inhibitors of signaling pathways, it was found this induction was regulated through a cAMP-dependent PKA pathway. Further, immunofluorescent localization study revealed that these ROS-induced tyrosine-phosphorylated proteins are mostly distributed in the midpiece and principal piece regions of the flagellum of capacitated spermatozoa and suggested for increased molecular activity in flagellum during capacitation. Thus, the study revealed that both O(2)(-) and H(2)O(2

  19. The phylogenetic origin of the bifunctional tyrosine-pathway protein in the enteric lineage of bacteria.

    PubMed

    Ahmad, S; Jensen, R A

    1988-05-01

    Because bifunctional enzymes are distinctive and highly conserved products of relatively infrequent gene-fusion events, they are particularly useful markers to identify clusters of organisms at different hierarchical levels of a phylogenetic tree. Within the subdivision of gram-negative bacteria known as superfamily B, there are two distinctive types of tyrosine-pathway dehydrogenases: (1) a broad-specificity dehydrogenase (recently termed cyclohexadienyl dehydrogenase [CDH]) that can utilize either prephenate or L-arogenate as alternative substrates and (2) a bifunctional CDH that also posseses chorismate mutase activity. (T-proteins). The bifunctional T-protein, thought to be encoded by fused ancestral genes for chorismate mutase and CDH, was found to be present in enteric bacteria (Escherichia, Shigella, Salmonella, Citrobacter, Klebsiella, Erwinia, Serratia, Morganella, Cedecea, Kluyvera, Hafnia, Edwardsiella, Yersinia, and Proteus) and in Aeromonas and Alteromonas. Outside of the latter "enteric lineage," the T-protein is absent in other major superfamily-B genera, such as Pseudomonas (rRNA homology group I), Xanthomonas, Acinetobacter, and Oceanospirillum. Hence, the T-protein must have evolved after the divergence of the enteric and Oceanospirillum lineages. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase-phe, an early-pathway isozyme sensitive to feedback inhibition by L-phenylalanine, has been found in each member of the enteric lineage examined. The absence of both the T-protein and DAHP synthase-phe elsewhere in superfamily B indicates the emergence of these character states at approximately the same evolutionary time.

  20. Characterisation of photo-oxidation products within photoyellowed wool proteins: tryptophan and tyrosine derived chromophores.

    PubMed

    Dyer, J M; Bringans, S D; Bryson, W G

    2006-07-01

    Understanding the photodegradation of complex protein systems represents a significant goal in protein science. The photo-oxidation and resultant photoyellowing of wool in sunlight is a severe impediment to its marketability. However, although some photomodifications have been found in irradiated model amino acid systems, direct identification of the chromophoric photoproducts responsible for photoyellowing in irradiated wool itself has proved elusive. We here describe the direct characterisation and location of yellow chromophores and related photomodifications within the proteins of photoyellowed wool fabric, utilising a quasi-proteomic approach. In total, eight distinct photoproducts were characterised. Of these, five were derived from tryptophan; namely hydroxytryptophan, N-formylkynurenine, kynurenine, residues consistent with the dehydration of kynurenine, and hydroxykynurenine, while three were derived from tyrosine; namely dihydroxyphenylalanine, dityrosine, and a cross-linked residue consistent with a hydroxylated dityrosine residue. Fourteen modified peptide sequences were identified and the positions of modification for thirteen of these were located within the primary structure of known wool proteins. The nature of the photoproducts characterised offer valuable insight into the reaction pathways followed in the UV-induced photoyellowing of wool proteins.

  1. Protection of epithelial barrier function by the Crohn's disease associated gene protein tyrosine phosphatase n2.

    PubMed

    Scharl, Michael; Paul, Gisela; Weber, Achim; Jung, Barbara C; Docherty, Michael J; Hausmann, Martin; Rogler, Gerhard; Barrett, Kim E; McCole, Declan F

    2009-12-01

    Protein tyrosine phosphatase N2 (PTPN2) has been identified as a Crohn's disease (CD) candidate gene. However, a role for PTPN2 in the pathogenesis of CD has not been identified. Increased permeability of the intestinal epithelium is believed to contribute prominently to CD. The aim of this study was to determine a possible role for PTPN2 in CD pathogenesis. Intestinal epithelial cell (IEC) lines T(84) and HT29cl.19a were used in all studies. Protein analysis was performed by Western blotting, and protein knockdown was induced by small interfering RNA. Primary samples were from control and CD patients. Here, we demonstrate increased PTPN2 expression in CD intestinal biopsy specimens and that the proinflammatory cytokine interferon (IFN)-gamma increases PTPN2 expression and activity in IEC. Moreover, IFN-gamma-induced STAT1 and STAT3 phosphorylation in IEC is enhanced by PTPN2 knockdown. The cellular energy sensor adenosine monophosphate-activated protein kinase partially regulates the IFN-gamma-induced effects on PTPN2. Additionally, PTPN2 knockdown potentiates IFN-gamma-induced increases in epithelial permeability, accompanied by elevated expression of the pore-forming protein claudin-2. PTPN2 is activated by IFN-gamma and limits IFN-gamma-induced signalling and consequent barrier defects. These data suggest a functional role for PTPN2 in maintaining the intestinal epithelial barrier and in the pathophysiology of CD.

  2. 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

  3. Phenylketonuria: reduced tyrosine brain influx relates to reduced cerebral protein synthesis

    PubMed Central

    2013-01-01

    Background In phenylketonuria (PKU), elevated blood phenylalanine (Phe) concentrations are considered to impair transport of large neutral amino acids (LNAAs) from blood to brain. This impairment is believed to underlie cognitive deficits in PKU via different mechanisms, including reduced cerebral protein synthesis. In this study, we investigated the hypothesis that impaired LNAA influx relates to reduced cerebral protein synthesis. Methods Using positron emission tomography, L-[1-11C]-tyrosine (11C-Tyr) brain influx and incorporation into cerebral protein were studied in 16 PKU patients (median age 24, range 16 – 47 years), most of whom were early and continuously treated. Data were analyzed by regression analyses, using either 11C-Tyr brain influx or 11C-Tyr cerebral protein incorporation as outcome variable. Predictor variables were baseline plasma Phe concentration, Phe tolerance, age, and 11C-Tyr brain efflux. For the modelling of cerebral protein incorporation, 11C-Tyr brain influx was added as a predictor variable. Results 11C-Tyr brain influx was inversely associated with plasma Phe concentrations (median 512, range 233 – 1362 μmol/L; delta adjusted R2=0.571, p=0.013). In addition, 11C-Tyr brain influx was positively associated with 11C-Tyr brain efflux (delta adjusted R2=0.098, p=0.041). Cerebral protein incorporation was positively associated with 11C-Tyr brain influx (adjusted R2=0.567, p<0.001). All additional associations between predictor and outcome variables were statistically nonsignificant. Conclusions Our data favour the hypothesis that an elevated concentration of Phe in blood reduces cerebral protein synthesis by impairing LNAA transport from blood to brain. Considering the importance of cerebral protein synthesis for adequate brain development and functioning, our results support the notion that PKU treatment be continued in adulthood. Future studies investigating the effects of impaired LNAA transport on cerebral protein synthesis in

  4. Knowledge-based Characterization of Similarity Relationships in the Human Protein-Tyrosine Phosphatase Family for Rational Inhibitor Design

    PubMed Central

    Vidović, Dušica; Schürer, Stephan C.

    2009-01-01

    Tyrosine phosphorylation, controlled by the coordinated action of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs), is a fundamental regulatory mechanism of numerous physiological processes. PTPs are implicated in a number of human diseases and their potential as prospective drug targets is increasingly being recognized. Despite their biological importance, until now no comprehensive overview has been reported describing how all members of the human PTP family are related. Here we review the entire human PTP family and present a systematic knowledge-based characterization of global and local similarity relationships, which are relevant for the development of small molecule inhibitors. We use parallel homology modeling to expand the current PTP structure space and analyze the human PTPs based on local three-dimensional catalytic sites and domain sequences. Furthermore, we demonstrate the importance of binding site similarities in understanding cross-reactivity and inhibitor selectivity in the design of small molecule inhibitors. PMID:19810703

  5. Lipoarabinomannan of Mycobacterium tuberculosis promotes protein tyrosine dephosphorylation and inhibition of mitogen-activated protein kinase in human mononuclear phagocytes. Role of the Src homology 2 containing tyrosine phosphatase 1.

    PubMed

    Knutson, K L; Hmama, Z; Herrera-Velit, P; Rochford, R; Reiner, N E

    1998-01-02

    Lipoarabinomannan (LAM) is a putative virulence factor of Mycobacterium tuberculosis that inhibits monocyte functions, and this may involve antagonism of cell signaling pathways. The effects of LAM on protein tyrosine phosphorylation in cells of the human monocytic cell line THP-1 were examined. LAM promoted tyrosine dephosphorylation of multiple cell proteins and attenuated phorbol 12-myristate 13-acetate-induced activation of mitogen-activated protein kinase. To examine whether these effects of LAM could be related to activation of a phosphatase, fractions from LAM-treated cells were analyzed for dephosphorylation of para-nitrophenol phosphate. The data show that LAM induced increased phosphatase activity associated with the membrane fraction. The Src homology 2 containing tyrosine phosphatase 1 (SHP-1) is important for signal termination and was examined as a potential target of LAM. Exposure of cells to LAM brought about (i) an increase in tyrosine phosphorylation of SHP-1, and (ii) translocation of the phosphatase to the membrane. Phosphatase assay of SHP-1 immunoprecipitated from LAM-treated cells, using phosphorylated mitogen-activated protein kinase as substrate, indicated that LAM promoted increased activity of SHP-1 in vivo. LAM also activated SHP-1 directly in vitro. Exposure of cells to LAM also attenuated the expression of tumor necrosis factor-alpha, interleukin-12, and major histocompatibility class II molecules. These results suggest that one mechanism by which LAM deactivates monocytes involves activation of SHP-1.

  6. 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

  7. Asperentin B, a New Inhibitor of the Protein Tyrosine Phosphatase 1B

    PubMed Central

    Wiese, Jutta; Aldemir, Hülya; Schmaljohann, Rolf; Gulder, Tobias A. M.; Imhoff, Johannes F.

    2017-01-01

    In the frame of studies on secondary metabolites produced by fungi from deep-sea environments we have investigated inhibitors of enzymes playing key roles in signaling cascades of biochemical pathways relevant for the treatment of diseases. Here we report on a new inhibitor of the human protein tyrosine phosphatase 1B (PTP1B), a target in the signaling pathway of insulin. A new asperentin analog is produced by an Aspergillus sydowii strain isolated from the sediment of the deep Mediterranean Sea. Asperentin B (1) contains an additional phenolic hydroxy function at C-6 and exhibits an IC50 value against PTP1B of 2 μM in vitro, which is six times stronger than the positive control, suramin. Interestingly, asperentin (2) did not show any inhibition of this enzymatic activity. Asperentin B (1) is discussed as possible therapeutic agents for type 2 diabetes and sleeping sickness. PMID:28635658

  8. Protein tyrosine phosphatase receptor type O regulates development and function of the sensory nervous system.

    PubMed

    Gonzalez-Brito, Manuel R; Bixby, John L

    2009-12-01

    The roles of protein tyrosine phosphatases (PTPs) in differentiation and axon targeting by dorsal root ganglion (DRG) neurons are essentially unknown. The type III transmembrane PTP, PTPRO, is expressed in DRG neurons, and is implicated in the guidance of motor and retinal axons. We examined the role of PTPRO in DRG development and function using PTPRO(-/-) mice. The number of peptidergic nociceptive neurons in the DRG of PTPRO(-/-) mice was significantly decreased, while the total number of sensory neurons appeared unchanged. In addition, spinal pathfinding by both peptidergic and proprioceptive neurons was abnormal in PTPRO(-/-) mice. Lastly, PTPRO(-/-) mice performed abnormally on tests of thermal pain and sensorimotor coordination, suggesting that both nociception and proprioception were perturbed. Our data indicate that PTPRO is required for peptidergic differentiation and process outgrowth of sensory neurons, as well as mature sensory function, and provide the first evidence that RPTPs regulate DRG development.

  9. Structure–Activity Relationship of Halophenols as a New Class of Protein Tyrosine Kinase Inhibitors

    PubMed Central

    Feng, Xiu E.; Zhao, Wan Yi; Ban, Shu Rong; Zhao, Cheng Xiao; Li, Qing Shan; Lin, Wen Han

    2011-01-01

    A series of new benzophenone and diphenylmethane halophenol derivatives were prepared. Their structures were established based on 1H NMR, 13C NMR and HRMS data. All prepared compounds were screened for their in vitro protein tyrosine kinase (PTK) inhibitory activities. The effects of modification of the linker, functional groups and substituted positions at the phenyl ring on PTK inhibitory activity were investigated. Twelve halophenols showed significant PTK inhibitory activity. Among them, compounds 6c, 6d, 7d, 9d, 10d, 11d and 13d exhibited stronger activities than that of genistein, the positive reference compound. The results gave a relatively full and definite description of the structure–activity relationship and provided a foundation for further design and structure optimization of the halophenols. PMID:22016647

  10. Asperentin B, a New Inhibitor of the Protein Tyrosine Phosphatase 1B.

    PubMed

    Wiese, Jutta; Aldemir, Hülya; Schmaljohann, Rolf; Gulder, Tobias A M; Imhoff, Johannes F

    2017-06-21

    In the frame of studies on secondary metabolites produced by fungi from deep-sea environments we have investigated inhibitors of enzymes playing key roles in signaling cascades of biochemical pathways relevant for the treatment of diseases. Here we report on a new inhibitor of the human protein tyrosine phosphatase 1B (PTP1B), a target in the signaling pathway of insulin. A new asperentin analog is produced by an Aspergillussydowii strain isolated from the sediment of the deep Mediterranean Sea. Asperentin B (1) contains an additional phenolic hydroxy function at C-6 and exhibits an IC50 value against PTP1B of 2 μM in vitro, which is six times stronger than the positive control, suramin. Interestingly, asperentin (2) did not show any inhibition of this enzymatic activity. Asperentin B (1) is discussed as possible therapeutic agents for type 2 diabetes and sleeping sickness.

  11. Protein tyrosine kinase 7 has a conserved role in Wnt/β-catenin canonical signalling

    PubMed Central

    Puppo, Francesca; Thomé, Virginie; Lhoumeau, Anne-Catherine; Cibois, Marie; Gangar, Akanksha; Lembo, Frédérique; Belotti, Edwige; Marchetto, Sylvie; Lécine, Patrick; Prébet, Thomas; Sebbagh, Michael; Shin, Won-Sik; Lee, Seung-Taek; Kodjabachian, Laurent; Borg, Jean-Paul

    2011-01-01

    The receptor protein tyrosine kinase 7 (PTK7) was recently shown to participate in noncanonical Wnt/planar cell polarity signalling during mouse and frog embryonic development. In this study, we report that PTK7 interacts with β-catenin in a yeast two-hybrid assay and mammalian cells. PTK7-deficient cells exhibit weakened β-catenin/T-cell factor transcriptional activity on Wnt3a stimulation. Furthermore, Xenopus PTK7 is required for the formation of Spemann's organizer and for Siamois promoter activation, events that require β-catenin transcriptional activity. Using epistatic assays, we demonstrate that PTK7 functions upstream from glycogen synthase kinase 3. Taken together, our data reveal a new and conserved role for PTK7 in the Wnt canonical signalling pathway. PMID:21132015

  12. Tyrosine phosphorylation of the chlamydial effector protein Tarp is species specific and not required for recruitment of actin.

    PubMed

    Clifton, Dawn R; Dooley, Cheryl A; Grieshaber, Scott S; Carabeo, Reynaldo A; Fields, Kenneth A; Hackstadt, Ted

    2005-07-01

    Chlamydiae are obligate intracellular pathogens that efficiently induce their endocytosis by susceptible eukaryotic host cells. Recently, a Chlamydia trachomatis type III secreted effector protein, Tarp, was found to be translocated and tyrosine phosphorylated at the site of entry and associated with the recruitment of actin that coincides with endocytosis. C. trachomatis Tarp possesses up to six direct repeats of approximately 50 amino acids each. The majority of the tyrosine residues are found within this repeat region. Here we have ectopically expressed distinct domains of Tarp in HeLa 229 cells and demonstrated that tyrosine phosphorylation occurs primarily within the repeat region, while recruitment of actin is mediated by the C-terminal domain of the protein. A comparison of other sequenced chlamydial genomes revealed that each contains an ortholog of Tarp, although Chlamydia muridarum, Chlamydophila caviae, and Chlamydophila pneumoniae Tarp lack the large repeat region. Immunofluorescence and immunoblotting using an antiphosphotyrosine antibody show no evidence of phosphotyrosine at the site of entry of C. muridarum, C. caviae, and C. pneumoniae, although each species similarly recruits actin. Ectopic expression of full-length C. trachomatis and C. caviae Tarp confirmed that both recruit actin but only C. trachomatis Tarp is tyrosine phosphorylated. The data indicate that the C-terminal domain of Tarp is essential for actin recruitment and that tyrosine phosphorylation may not be an absolute requirement for actin recruitment. The results further suggest the potential for additional, unknown signal transduction pathways associated specifically with C. trachomatis.

  13. Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation.

    PubMed

    Linares, E; Giorgio, S; Mortara, R A; Santos, C X; Yamada, A T; Augusto, O

    2001-06-01

    The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.

  14. A second-generation expression system for tyrosine-sulfated proteins and its application in crop protection

    SciTech Connect

    Schwessinger, Benjamin; Li, Xiang; Ellinghaus, Thomas L.; Chan, Leanne Jade G.; Wei, Tong; Joe, Anna; Thomas, Nicholas; Pruitt, Rory; Adams, Paul D.; Chern, Maw Sheng; Petzold, Christopher J.; Liu, Chang C.; Ronald, Pamela C.

    2015-11-27

    Posttranslational modification (PTM) of proteins and peptides is important for diverse biological processes in plants and animals. The paucity of heterologous expression systems for PTMs and the technical challenges associated with chemical synthesis of these modified proteins has limited detailed molecular characterization and therapeutic applications. Here we describe an optimized system for expression of tyrosine-sulfated proteins in Escherichia coli and its application in a bio-based crop protection strategy in rice.

  15. A second-generation expression system for tyrosine-sulfated proteins and its application in crop protection

    DOE PAGES

    Schwessinger, Benjamin; Li, Xiang; Ellinghaus, Thomas L.; ...

    2015-11-27

    Posttranslational modification (PTM) of proteins and peptides is important for diverse biological processes in plants and animals. The paucity of heterologous expression systems for PTMs and the technical challenges associated with chemical synthesis of these modified proteins has limited detailed molecular characterization and therapeutic applications. Here we describe an optimized system for expression of tyrosine-sulfated proteins in Escherichia coli and its application in a bio-based crop protection strategy in rice.

  16. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    PubMed Central

    Lin, Xiaofeng; Ayrapetov, Marina K; Sun, Gongqin

    2005-01-01

    Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1) binds to ATP, and the other (M2) acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator. PMID:16305747

  17. Macrophage Fusion Is Controlled by the Cytoplasmic Protein Tyrosine Phosphatase PTP-PEST/PTPN12

    PubMed Central

    Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean

    2013-01-01

    Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading. PMID:23589331

  18. Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

    PubMed

    Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

    2013-06-01

    Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

  19. Target-specific control of lymphoid-specific protein tyrosine phosphatase (Lyp) activity

    PubMed Central

    Walton, Zandra E.; Bishop, Anthony C.

    2010-01-01

    Lymphoid-specific protein tyrosine phosphatase (Lyp), a member of the protein tyrosine phosphatase (PTP) superfamily of enzymes, is an important mediator of human-leukocyte signaling. Lyp has also emerged as a potential anti-autoimmune therapeutic target, owing to the association of a Lyp-activating mutation with an array of autoimmune disorders. Toward the goal of generating a selective inhibitor of Lyp activity that could be used for investigating Lyp’s roles in cell signaling and autoimmune-disease progression, here we report that Lyp’s PTP domain can be readily sensitized to target-specific inhibition by a cell-permeable small molecule. Insertion of a tetracysteine-motif-containing peptide at a conserved position in Lyp’s catalytic domain generated a mutant enzyme (Lyp-CCPGCC) that retains activity comparable to that of wild-type Lyp in the absence of added ligand. Upon addition of a tetracysteine-targeting biarsenical compound (FlAsH), however, the activity of the Lyp-CCPGCC drops dramatically, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. We show that FlAsH-induced Lyp-CCPGCC inhibition is potent, specific, rapid, and independent of the nature of the PTP substrate used in the inhibition assay. Moreover, we show that FlAsH can be used to specifically target overexpressed Lyp-CCPGCC in a complex proteomic mixture. Since the mammalian-cell permeability of FlAsH is well established, it is likely that FlAsH-mediated inhibition of Lyp-CCPGCC will be useful for specifically targeting Lyp activity in engineered leukocytes and autoimmune-disease models. PMID:20594861

  20. Effect of Oxidative Stress on Protein Tyrosine Phosphatase-1B in Scleroderma Dermal Fibroblasts

    PubMed Central

    Tsou, Pei-Suen; Talia, Nadine N.; Pinney, Adam J.; Kendzicky, Ann; Piera-Velazquez, Sonsoles; Jimenez, Sergio A.; Seibold, James R.; Phillips, Kristine; Koch, Alisa E.

    2011-01-01

    Objective Platelet-derived growth factor (PDGF) and its receptor (PDGFR) promote fibrosis in scleroderma (SSc) dermal fibroblasts, which produce excessive reactive oxygen species (ROS). PDGFR is phosphorylated upon PDGF stimulation, and dephosphorylated by protein tyrosine phosphatases (PTPs), including PTP1B. In this study we determine whether the thiol-sensitive PTP1B is affected by ROS, thus enhancing PDGFR phosphorylation (p-PDGFR) and collagen I (Col I) synthesis. The effect of a thiol antioxidant, n-acetylcysteine (NAC), was also investigated. Methods Fibroblasts were isolated from skin. A phosphate release assay was used for PTP1B activity. Results ROS and Col I were significantly higher in SSc fibroblasts, accompanied by significantly lower amounts of free thiols compared to normal fibroblasts. After PDGF stimulation, not only were the PDGFR and ERK1/2 phosphorylated to a greater extent, but the ability to produce PTP1B was also hampered in SSc fibroblasts. PTP1B activity was significantly inactivated in SSc fibroblasts, which resulted from cysteine oxidation by higher levels of ROS, since oxidation of multiple PTPs, including PTP1B, was observed. Decreased PTP1B expression in normal fibroblasts led to increased Col I. NAC restored the low PTP1B activity, improved the profile of p-PDGFR, decreased the numbers of tyrosine-phosphorylated proteins and Col I, and scavenged ROS in SSc fibroblasts. Conclusion We introduce a new mechanism by which ROS promote a profibrotic phenotype in SSc fibroblasts through oxidative inactivation of PTP1B leading to pronounced PDGFR activation. Our study also provides a novel molecular mechanism by which NAC therapy may act on ROS and PTP1B to benefit SSc patients. PMID:22161819

  1. 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

  2. Dual roles of protein tyrosine phosphatase kappa in coordinating angiogenesis induced by pro-angiogenic factors

    PubMed Central

    Sun, Ping-Hui; Chen, Gang; Mason, Malcolm; Jiang, Wen G.; Ye, Lin

    2017-01-01

    A potential role may be played by receptor-type protein tyrosine phosphatase kappa (PTPRK) in angiogenesis due to its critical function in coordinating intracellular signal transduction from various receptors reliant on tyrosine phosphorylation. In the present study, we investigated the involvement of PTPRK in the cellular functions of vascular endothelial cells (HECV) and its role in angiogenesis using in vitro assays and a PTPRK knockdown vascular endothelial cell model. PTPRK knockdown in HECV cells (HECVPTPRKkd) resulted in a decrease of cell proliferation and cell-matrix adhesion; however, increased cell spreading and motility were seen. Reduced focal adhesion kinase (FAK) and paxillin protein levels were seen in the PTPRK knockdown cells which may contribute to the inhibitory effect on adhesion. HECVPTPRKkd cells were more responsive to the treatment of fibroblast growth factor (FGF) in their migration compared with the untreated control and cells treated with VEGF. Moreover, elevated c-Src and Akt1 were seen in the PTPRK knockdown cells. The FGF-promoted cell migration was remarkably suppressed by an addition of PLCγ inhibitor compared with other small inhibitors. Knockdown of PTPRK suppressed the ability of HECV cells to form tubules and also impaired the tubule formation that was induced by FGF and conditioned medium of cancer cells. Taken together, it suggests that PTPRK plays dual roles in coordinating angiogenesis. It plays a positive role in cell proliferation, adhesion and tubule formation, but suppresses cell migration, in particular, the FGF-promoted migration. PTPRK bears potential to be targeted for the prevention of tumour associated angiogenesis. PMID:28259897

  3. Receptor protein tyrosine phosphatase σ binds to neurons in the adult mouse brain

    PubMed Central

    Yi, Jae-Hyuk; Katagiri, Yasuhiro; Yu, Panpan; Lourie, Jacob; Bangayan, Nathanael J.; Symes, Aviva J.; Geller, Herbert M.

    2014-01-01

    The role of type IIA receptor protein tyrosine phosphatases (RPTPs), which includes LAR, RPTPσ and RPTPδ, in the nervous system is becoming increasingly recognized. Evidence supports a significant role for these RPTPs during the development of the nervous system as well as after injury, and mutations in RPTPs are associated with human disease. However, a major open question is the nature of the ligands that interact with type IIA RPTPs in the adult brain. Candidates include several different proteins as well as the glycosaminoglycan chains of proteoglycans. In order to investigate this problem, we used a receptor affinity probe assay with RPTPσ-AP fusion proteins on sections of adult mouse brain and to cultured neurons. Our results demonstrate that the major binding sites for RPTPσ in adult mouse brain are on neurons and are not proteoglycan GAG chains, as RPTPσ binding overlaps with the neuronal marker NeuN and was not significantly altered by treatments which eliminate chondroitin sulfate, heparan sulfate, or both. We also demonstrate no overlap of binding of RPTPσ with perineuronal nets, and a unique modulation of RPTPσ binding to brain by divalent cations. Our data therefore point to neuronal proteins, rather than CSPGs, as being the ligands for RPTPσ in the adult, uninjured brain. PMID:24530640

  4. 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

  5. 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.

  6. 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

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

    PubMed

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-03-08

    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.

  8. Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases.

    PubMed

    Muda, Marco; Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Dixon, Jack E

    2002-08-15

    Despite the wealth of information generated by genome-sequencing projects, the identification of in vivo substrates of specific protein kinases and phosphatases is hampered by the large number of candidate enzymes, overlapping enzyme specificity and sequence similarity. In the present study, we demonstrate the power of RNA interference (RNAi) to dissect signal transduction cascades involving specific kinases and phosphatases. RNAi is used to identify the cellular tyrosine kinases upstream of the phosphorylation of Down-Syndrome cell-adhesion molecule (Dscam), a novel cell-surface molecule of the immunoglobulin-fibronectin super family, which has been shown to be important for axonal path-finding in Drosophila. Tyrosine phosphorylation of Dscam recruits the Src homology 2 domain of the adaptor protein Dock to the receptor. Dock, the ortho- logue of mammalian Nck, is also essential for correct axonal path-finding in Drosophila. We further determined that Dock is tyrosine-phosphorylated in vivo and identified DPTP61F as the protein tyrosine phosphatase responsible for maintaining Dock in its non-phosphorylated state. The present study illustrates the versatility of RNAi in the identification of the physiological substrates for protein kinases and phosphatases.

  9. Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases.

    PubMed Central

    Muda, Marco; Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Dixon, Jack E

    2002-01-01

    Despite the wealth of information generated by genome-sequencing projects, the identification of in vivo substrates of specific protein kinases and phosphatases is hampered by the large number of candidate enzymes, overlapping enzyme specificity and sequence similarity. In the present study, we demonstrate the power of RNA interference (RNAi) to dissect signal transduction cascades involving specific kinases and phosphatases. RNAi is used to identify the cellular tyrosine kinases upstream of the phosphorylation of Down-Syndrome cell-adhesion molecule (Dscam), a novel cell-surface molecule of the immunoglobulin-fibronectin super family, which has been shown to be important for axonal path-finding in Drosophila. Tyrosine phosphorylation of Dscam recruits the Src homology 2 domain of the adaptor protein Dock to the receptor. Dock, the ortho- logue of mammalian Nck, is also essential for correct axonal path-finding in Drosophila. We further determined that Dock is tyrosine-phosphorylated in vivo and identified DPTP61F as the protein tyrosine phosphatase responsible for maintaining Dock in its non-phosphorylated state. The present study illustrates the versatility of RNAi in the identification of the physiological substrates for protein kinases and phosphatases. PMID:12014990

  10. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 2: selected applications.

    PubMed

    Antosiewicz, Jan M; Shugar, David

    In Part 2 we discuss application of several different types of UV-Vis spectroscopy, such as normal, difference, and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, of the side-chain of tyrosine residues in different molecular environments. We review the ways these spectroscopies can be used to probe complex protein structures.

  11. Assignment of the human protein tyrosine phosphatase epsilon (PTPRE) gene to chromosome 10q26 by fluorescence in situ hybridization

    SciTech Connect

    Maagdenberg, A.M.J.M. van den; Hurk, H.H. van den; Wieringa, B.

    1995-11-01

    Phosphorylation of cellular protein tyrosine residues is an important mechanism for the transduction of external signals to the intracellular compartment. Protein tyrosine phosphatases (PTPases) act in concert with protein tyrosine kinases (PTKs) to regulate the level of tyrosine phosphorylation in these proteins. PTKs have been studied in detail, and many have been shown to be proto-oncogenes. Because PTPases can be considered functional antagonists of PTKs it has been postulated that these PTPases might act as tumor suppressors. Over 30 different PTPase genes have been isolated so far, and the chromosomal localization has been determined for many of them. Comparison of such mapping data with temporal and spatial expression patterns of individual PTPase genes and losses of heterozygosity (LOH) in relevant tumor types could be indicative of their proposed tumor suppressive activity. Until now, chromosomal deletions have been reported only for the PTPRG gene in primary renal and lung carcinomas and cancer-derived cell lines, but a causal role for a loss of PTPase activity in tumor formation remains to be determined. 15 refs., 1 fig.

  12. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 2: selected applications.

    PubMed

    Antosiewicz, Jan M; Shugar, David

    2016-06-01

    In Part 2 we discuss application of several different types of UV-Vis spectroscopy, such as normal, difference, and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, of the side-chain of tyrosine residues in different molecular environments. We review the ways these spectroscopies can be used to probe complex protein structures.

  13. Isolation of protein-tyrosine phosphatase-like member-a variant from cementum.

    PubMed

    Valdés De Hoyos, A; Hoz-Rodríguez, L; Arzate, H; Narayanan, A S

    2012-02-01

    Cementum has been shown to contain unique polypeptides that participate in cell recruitment and differentiation during cementum formation. We report the isolation of a cDNA variant for protein-tyrosine phosphatase-like (proline instead of catalytic arginine) member-a (PTPLA) from cementum. A cementifying fibroma-derived λ-ZAP expression library was screened by panning with a monoclonal antibody to cementum attachment protein (CAP), and 1435 bp cDNA (gb AC093525.3) was isolated. This cDNA encodes a 140-amino-acid polypeptide, and its N-terminal 125 amino acids are identical to those of PTPLA. This isoform, designated as PTPLA-CAP, results from a read-through of the PTPLA exon 2 splice donor site, truncating after the second putative transmembrane domain. It contains 15 amino acids encoded within the intron between PTPLA exons 2 and 3, which replace the active site for PTPLA phosphatase activity. The recombinant protein, rhPTPLA-CAP, has Mr 19 kDa and cross-reacts with anti-CAP antibody. Anti-rhPTPLA-CAP antibody immunostained cementum cells, cementum, heart, and liver. Quantitative RT-PCR showed that PTPLA was expressed in all periodontal cells; however, PTPLA-CAP expression was limited to cementum cells. The rhPTPLA-CAP promoted gingival fibroblast attachment. We conclude that PTPLA-CAP is a splice variant of PTPLA, and that, in the periodontium, cementum and cementum cells express this variant.

  14. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Using mass spectrometry to study the photo-affinity labeling of protein tyrosine phosphatase 1B

    NASA Astrophysics Data System (ADS)

    Leriche, Tammy; Skorey, Kathryn; Roy, Patrick; McKay, Dan; Bateman, Kevin P.

    2004-11-01

    Protein tyrosine phosphatase 1B (PTP1B) is a potential target for the treatment of Type II diabetes and several companies are developing small molecule inhibitors of this enzyme. Part of the characterization of these compounds as PTP1B inhibitors is the understanding of how they bind in the enzyme active site. The use of photo-activated inhibitors that target the active site can provide such insight. This paper describes the characterization of a photoprobe directed at the active site of PTP1B. Mass spectrometry revealed the specific binding of the probe to the intact protein. Digestion of the labeled protein followed by LC-MS and LC-MS/MS was used to show that the photoprobe binds to a specific active site amino acid. This was confirmed by comparison with the X-ray structure of PTP1B with a PTP1B inhibitor. The probe labels a conserved acidic residue (Asp) that is required for catalytic activity. This photoprobe may prove to be a useful tool for the development of a PTP1B inhibitor or for the study of PTPs in general.

  16. Isolation of Protein-Tyrosine Phosphatase-like Member-a Variant from Cementum

    PubMed Central

    Valdés De Hoyos, A.; Hoz-Rodríguez, L.; Arzate, H.; Narayanan, A.S.

    2012-01-01

    Cementum has been shown to contain unique polypeptides that participate in cell recruitment and differentiation during cementum formation. We report the isolation of a cDNA variant for protein-tyrosine phosphatase-like (proline instead of catalytic arginine) member-a (PTPLA) from cementum. A cementifying fibroma-derived λ-ZAP expression library was screened by panning with a monoclonal antibody to cementum attachment protein (CAP), and 1435 bp cDNA (gb AC093525.3) was isolated. This cDNA encodes a 140-amino-acid polypeptide, and its N-terminal 125 amino acids are identical to those of PTPLA. This isoform, designated as PTPLA-CAP, results from a read-through of the PTPLA exon 2 splice donor site, truncating after the second putative transmembrane domain. It contains 15 amino acids encoded within the intron between PTPLA exons 2 and 3, which replace the active site for PTPLA phosphatase activity. The recombinant protein, rhPTPLA-CAP, has Mr 19 kDa and cross-reacts with anti-CAP antibody. Anti-rhPTPLA-CAP antibody immunostained cementum cells, cementum, heart, and liver. Quantitative RT-PCR showed that PTPLA was expressed in all periodontal cells; however, PTPLA-CAP expression was limited to cementum cells. The rhPTPLA-CAP promoted gingival fibroblast attachment. We conclude that PTPLA-CAP is a splice variant of PTPLA, and that, in the periodontium, cementum and cementum cells express this variant. PMID:22067203

  17. Protein tyrosine phosphatase non-receptor 22 and C-Src tyrosine kinase genes are down-regulated in patients with rheumatoid arthritis.

    PubMed

    Remuzgo-Martínez, Sara; Genre, Fernanda; Castañeda, Santos; Corrales, Alfonso; Moreno-Fresneda, Pablo; Ubilla, Begoña; Mijares, Verónica; Portilla, Virginia; González-Vela, Jesús; Pina, Trinitario; Ocejo-Vinyals, Gonzalo; Irure-Ventura, Juan; Blanco, Ricardo; Martín, Javier; Llorca, Javier; López-Mejías, Raquel; González-Gay, Miguel A

    2017-09-05

    Several protein tyrosine phosphatase non-receptor 22 (PTPN22) single-nucleotide polymorphisms (SNPs) have been significantly related with rheumatoid arthritis (RA) susceptibility. Nevertheless, its potential influence on PTPN22 expression in RA has not been completely elucidated. Furthermore, PTPN22 binds to C-Src tyrosine kinase (CSK) forming a key complex in autoimmunity. However, the information of CSK gene in RA is scarce. In this study, we analyzed the relative PTPN22 and CSK expression in peripheral blood from 89 RA patients and 43 controls to determine if the most relevant PTPN22 (rs2488457, rs2476601 and rs33996649) and CSK (rs34933034 and rs1378942) polymorphisms may influence on PTPN22 and CSK expression in RA. The association between PTPN22 and CSK expression in RA patients and their clinical characteristics was also evaluated. Our study shows for the first time a marked down-regulation of PTPN22 expression in RA patients carrying the risk alleles of PTPN22 rs2488457 and rs2476601 compared to controls (p = 0.004 and p = 0.007, respectively). Furthermore, CSK expression was significantly lower in RA patients than in controls (p < 0.0001). Interestingly, a reduced PTPN22 expression was disclosed in RA patients with ischemic heart disease (p = 0.009). The transcriptional suppression of this PTPN22/CSK complex may have a noteworthy clinical relevance in RA patients.

  18. 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.

  19. 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

  20. Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning.

    PubMed

    Early, A; Gamper, M; Moniakis, J; Kim, E; Hunter, T; Williams, J G; Firtel, R A

    2001-04-01

    The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.

  1. Differential abilities of nitrogen dioxide and nitrite to nitrate proteins in thylakoid membranes isolated from Arabidopsis leaves.

    PubMed

    Takahashi, Misa; Shigeto, Jun; Shibata, Tatsuo; Sakamoto, Atsushi; Morikawa, Hiromichi

    2016-10-02

    Exposure of Arabidopsis leaves to nitrogen dioxide (NO2) results in nitration of specific chloroplast proteins. To determine whether NO2 itself and/or nitrite derived from NO2 can nitrate proteins, Arabidopsis thylakoid membranes were isolated and treated with NO2-bubbled or potassium nitrite (KNO2) buffer, followed by protein extraction, electrophoresis, and immunoblotting using an anti-3-nitrotyrosine (NT) antibody. NO2 concentrations in the NO2-bubbled buffer were calculated by numerically solving NO2 dissociation kinetic equations. The two buffers were adjusted to have identical nitrite concentrations. Both treatments yielded an NT-immunopositive band that LC/MS identified as PSBO1. The difference in the band intensity between the 2 treatments was designated nitration by NO2. Both NO2 and nitrite mediated nitration of proteins, and the nitration ability per unit NO2 concentration was ∼100-fold greater than that of nitrite.

  2. Nitration of pollen aeroallergens by nitrate ion in conditions simulating the liquid water phase of atmospheric particles.

    PubMed

    Ghiani, Alessandra; Bruschi, Maurizio; Citterio, Sandra; Bolzacchini, Ezio; Ferrero, Luca; Sangiorgi, Giorgia; Asero, Riccardo; Perrone, Maria Grazia

    2016-12-15

    Pollen aeroallergens are present in atmospheric particulate matter (PM) where they can be found in coarse biological particles such as pollen grains (aerodynamic diameter dae>10μm), as well as fragments in the finest respirable particles (PM2.5; dae<2.5μm). Nitration of tyrosine residues in pollen allergenic proteins can occur in polluted air, and inhalation and deposition of these nitrated proteins in the human respiratory tract may lead to adverse health effects by enhancing the allergic response in population. Previous studies investigated protein nitration by atmospheric gaseous pollutants such as nitrogen dioxide and ozone. In this work we report, for the first time, a study on protein nitration by nitrate ion in aqueous solution, at nitrate concentrations and pH conditions simulating those occurring in the atmospheric aerosol liquid water phase. Experiments have been carried out on the Bovine serum albumin (BSA) protein and the recombinant Phleum pratense allergen (Phl p 2) both in the dark and under UV-A irradiation (range 4-90Wm(-2)) to take into account thermal and/or photochemical nitration processes. For the latter protein, modifications in the allergic response after treatment with nitrate solutions have been evaluated by immunoblot analyses using sera from grass-allergic patients. Experimental results in bulk solutions showed that protein nitration in the dark occurs only in dilute nitrate solutions and under very acidic conditions (pH<3 for BSA; pH<2.2 for Phl p 2), while nitration is always observed (at pH0.5-5) under UV-A irradiation, both in dilute and concentrated nitrate solutions, being significantly enhanced at the lowest pH values. In some cases, protein nitration resulted in an increase of the allergic response. Copyright © 2016. Published by Elsevier B.V.

  3. Bruton's tyrosine kinase activity is negatively regulated by Sab, the Btk-SH3 domain-binding protein.

    PubMed

    Yamadori, T; Baba, Y; Matsushita, M; Hashimoto, S; Kurosaki, M; Kurosaki, T; Kishimoto, T; Tsukada, S

    1999-05-25

    Bruton's tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase that is crucial for human and murine B cell development, and its deficiency causes human X-linked agammaglobulinemia and murine X-linked immunodeficiency. In this report, we describe the function of the Btk-binding protein Sab (SH3-domain binding protein that preferentially associates with Btk), which we reported previously as a newly identified Src homology 3 domain-binding protein. Sab was shown to inhibit the auto- and transphosphorylation activity of Btk, which prompted us to propose that Sab functions as a transregulator of Btk. Forced overexpression of Sab in B cells led to the reduction of B cell antigen receptor-induced tyrosine phosphorylation of Btk and significantly reduced both early and late B cell antigen receptor-mediated events, including calcium mobilization, inositol 1, 4,5-trisphosphate production, and apoptotic cell death, where the involvement of Btk activity has been demonstrated previously. Together, these results indicate the negative regulatory role of Sab in the B cell cytoplasmic tyrosine kinase pathway.

  4. Bruton’s tyrosine kinase activity is negatively regulated by Sab, the Btk-SH3 domain-binding protein

    PubMed Central

    Yamadori, Tomoki; Baba, Yoshihiro; Matsushita, Masato; Hashimoto, Shoji; Kurosaki, Mari; Kurosaki, Tomohiro; Kishimoto, Tadamitsu; Tsukada, Satoshi

    1999-01-01

    Bruton’s tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase that is crucial for human and murine B cell development, and its deficiency causes human X-linked agammaglobulinemia and murine X-linked immunodeficiency. In this report, we describe the function of the Btk-binding protein Sab (SH3-domain binding protein that preferentially associates with Btk), which we reported previously as a newly identified Src homology 3 domain-binding protein. Sab was shown to inhibit the auto- and transphosphorylation activity of Btk, which prompted us to propose that Sab functions as a transregulator of Btk. Forced overexpression of Sab in B cells led to the reduction of B cell antigen receptor-induced tyrosine phosphorylation of Btk and significantly reduced both early and late B cell antigen receptor-mediated events, including calcium mobilization, inositol 1,4,5-trisphosphate production, and apoptotic cell death, where the involvement of Btk activity has been demonstrated previously. Together, these results indicate the negative regulatory role of Sab in the B cell cytoplasmic tyrosine kinase pathway. PMID:10339589

  5. Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew.

    PubMed Central

    Freeman, R M; Plutzky, J; Neel, B G

    1992-01-01

    src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw) and their similar patterns of expression suggest that SH-PTP2 is the human corkscrew homolog. Sequence comparisons between SH-PTP2, SH-PTP1, corkscrew, and other SH2-containing proteins suggest the existence of a subfamily of SH2 domains found specifically in PTPs, whereas comparison of the PTP domains of the SH2-containing PTPs with other tyrosine phosphatases suggests the existence of a subfamily of PTPs containing SH2 domains. Since corkscrew, a member of the terminal class signal transduction pathway, acts in concert with D-raf to positively transduce the signal generated by the receptor tyrosine kinase torso, these findings suggest several mechanisms by which SH-PTP2 may participate in mammalian signal transduction. Images PMID:1280823

  6. A Role for the Protein Tyrosine Phosphatase CD45 in Macrophage Adhesion through the Regulation of Paxillin Degradation

    PubMed Central

    St-Pierre, Joëlle; Ostergaard, Hanne L.

    2013-01-01

    CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility. These defects are accompanied by substantially decreased levels of the cytoskeletal-associated protein paxillin, without affecting the levels of other proteins. Degradation of paxillin in CD45-deficient macrophages is calpain-mediated, as treatment with a calpain inhibitor restores paxillin levels in these cells and enhances cell spreading. Inhibition of the tyrosine kinases proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK), kinases that are capable of mediating tyrosine phosphorylation of paxillin, also restored paxillin levels, indicating a role for these kinases in the CD45-dependent regulation of paxillin. These data demonstrate that CD45 functions to regulate Pyk2/FAK activity, likely through the activity of Src family kinases, which in turn regulates the levels of paxillin to modulate macrophage adhesion and migration. PMID:23936270

  7. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.

    PubMed

    Takeda, Mitsuhiro; Ono, Akira M; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.

  8. 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

  9. Tyrosine phosphorylation of p34cdc2 is regulated by protein phosphatase 2A in growing immature Xenopus oocytes.

    PubMed

    Rime, H; Jessus, C; Ozon, R

    1995-07-01

    Growing stage IV Xenopus oocytes are unresponsive to progesterone treatment. They contain a store of preMPF composed of tyrosine phosphorylated p34cdc2 and cyclin B2. The endogenous store of preMPF cannot be recruited by cdc25 protein phosphatase or cyclin protein microinjections. This is in contrast with full-grown stage VI oocytes where microinjections of these proteins are known to activate the autoamplification of MPF. When cyclins are microinjected into stage IV oocytes, they associate with endogenous free p34cdc2 and the illegitimate complexes undergo phosphorylation on tyrosine 15. High doses of human cyclin A allow, however, part of the neoformed complexes to be activated as an histone-H1 kinase; this partial activation of p34cdc2 is sufficient to induce germinal vesicle breakdown in these small oocytes. Co-injections of cyclin A or cyclin B together with okadaic acid (10 microM in the microinjection solution), an inhibitor of protein phosphatase 2A (PP2A), lead to the full activation of neoformed p34cdc2/cyclin complexes. These results indicate that small oocytes possess an active tyrosine kinase that inactivates new p34cdc2/cyclin complexes. Inhibition of PP2A by okadaic acid prevents this inactivation reaction and conversely allows the illegitimate complex to be activated. Neither the activating phosphorylation on threonine 161 nor the inactivating phosphorylation on tyrosine 15 take place in stage IV enucleated oocytes. Altogether, our results show that the accumulation of inactive p34cdc2/cyclin B2 during the long-lasting prophase of the oocyte is positively controlled by PP2A through the tyrosine phosphorylation of p34cdc2.

  10. Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

    PubMed Central

    Sakamoto, O; Iwama, A; Amitani, R; Takehara, T; Yamaguchi, N; Yamamoto, T; Masuyama, K; Yamanaka, T; Ando, M; Suda, T

    1997-01-01

    Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization. PMID:9045873

  11. Tyrosine kinase receptor transactivation associated to G protein-coupled receptors.

    PubMed

    Almendro, Vanessa; García-Recio, Susana; Gascón, Pedro

    2010-09-01

    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, cell differentiation and oncogenesis among others. Some of the effects of GPCRs are known to be mediated by the activation of MAPK pathways. Several GPCRs are also able to transactivate receptors with tyrosine kinase activity (TKR) such as EGFR and HER2 and thus to control DNA synthesis and cell proliferation. The interaction between these receptors not only plays an important physiological role but its disregulation can induce pathological states such as cancer. For this reason, the crosstalk between these two types of receptors can be considered a possible mechanism for cell transformation, tumor progression, reactivation of the metastatic disease, and the acquisition of resistance to therapies targeting TKR receptors. The transactivation of some TKRs by GPCRs is related to the lost of response of TKRs to inhibitors of TK activity, mainly by the activation of the c-Src protein which can directly phosphorylate and activate the cytoplasmic domain of a TKR. For these reason, the dual inhibition of GPCRs and TKRs in some types of cancer has been proposed as a better strategy to kill tumor cells. Increased understanding of the mechanisms that interconnect the two pathways regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

  12. PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth.

    PubMed

    Diamond, R H; Cressman, D E; Laz, T M; Abrams, C S; Taub, R

    1994-06-01

    PRL-1 is a particularly interesting immediate-early gene because it is induced in mitogen-stimulated cells and regenerating liver but is constitutively expressed in insulin-treated rat H35 hepatoma cells, which otherwise show normal regulation of immediate-early genes. PRL-1 is expressed throughout the course of hepatic regeneration, and its expression is elevated in a number of tumor cell lines. Sequence analysis reveals that PRL-1 encodes a 20-kDa protein with an eight-amino-acid consensus protein tyrosine phosphatase (PTPase) active site. PRL-1 is able to dephosphorylate phosphotyrosine substrates, and mutation of the active-site cysteine residue abolishes this activity. As PRL-1 has no homology to other PTPases outside the active site, it is a new type of PTPase. PRL-1 is located primarily in the cell nucleus. Stably transfected cells which overexpress PRL-1 demonstrate altered cellular growth and morphology and a transformed phenotype. It appears that PRL-1 is important in normal cellular growth control and could contribute to the tumorigenicity of some cancer cells.

  13. Leveraging Reciprocity to Identify and Characterize Unknown Allosteric Sites in Protein Tyrosine Phosphatases.

    PubMed

    Cui, Danica S; Beaumont, Victor; Ginther, Patrick S; Lipchock, James M; Loria, J Patrick

    2017-07-21

    Drug-like molecules targeting allosteric sites in proteins are of great therapeutic interest; however, identification of potential sites is not trivial. A straightforward approach to identify hidden allosteric sites is demonstrated in protein tyrosine phosphatases (PTP) by creation of single alanine mutations in the catalytic acid loop of PTP1B and VHR. This approach relies on the reciprocal interactions between an allosteric site and its coupled orthosteric site. The resulting NMR chemical shift perturbations (CSPs) of each mutant reveal clusters of distal residues affected by acid loop mutation. In PTP1B and VHR, two new allosteric clusters were identified in each enzyme. Mutations in these allosteric clusters altered phosphatase activity with changes in kcat/KM ranging from 30% to nearly 100-fold. This work outlines a simple method for identification of new allosteric sites in PTP, and given the basis of this method in thermodynamics, it is expected to be generally useful in other systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. 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

  15. LIG family receptor tyrosine kinase-associated proteins modulate growth factor signals during neural development.

    PubMed

    Mandai, Kenji; Guo, Ting; St Hillaire, Coryse; Meabon, James S; Kanning, Kevin C; Bothwell, Mark; Ginty, David D

    2009-09-10

    Genome-wide screens were performed to identify transmembrane proteins that mediate axonal growth, guidance and target field innervation of somatosensory neurons. One gene, Linx (alias Islr2), encoding a leucine-rich repeat and immunoglobulin (LIG) family protein, is expressed in a subset of developing sensory and motor neurons. Domain and genomic structures of Linx and other LIG family members suggest that they are evolutionarily related to Trk receptor tyrosine kinases (RTKs). Several LIGs, including Linx, are expressed in subsets of somatosensory and motor neurons, and select members interact with TrkA and Ret RTKs. Moreover, axonal projection defects in mice harboring a null mutation in Linx resemble those in mice lacking Ngf, TrkA, and Ret. In addition, Linx modulates NGF-TrkA- and GDNF-GFRalpha1/Ret-mediated axonal extension in cultured sensory and motor neurons, respectively. These findings show that LIGs physically interact with RTKs and modulate their activities to control axonal extension, guidance and branching.

  16. Development and validation of an intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses.

    PubMed

    Cromlish, W A; Payette, P; Kennedy, B P

    1999-11-15

    We have developed an intact cell assay to be used in the direct quantitation of protein tyrosine phosphatase (PTP) activity. Utilizing the baculovirus expression system, the assay readily allows for a direct activity readout for PTPs such as PTP1B or CD45. Infected Sf9 cells expressing either full-length PTP1B, full-length CD45, CD45 catalytic domain, or hCOX-1 (mock-infected) are harvested 29 hr post-infection, at which time cells are viable and the expressed proteins are processed, as well as localized to their predicted subcellular compartments. Assays are carried out in a 96-well format, with cells expressing the PTP of interest. Cells are preincubated with or without inhibitor and challenged with substrate, and the phosphatase activity is determined spectrophotometrically by monitoring the conversion of p-nitrophenyl phosphate to p-nitrophenol at OD405. Documented PTP inhibitors have been used to validate this assay system. This study demonstrates that a direct readout of PTP activity in intact cells can be achieved, thus providing a useful cell-based screen for determining selective inhibitors of PTPs.

  17. LIG Family Receptor Tyrosine Kinase-Associated Proteins Modulate Growth Factor Signals During Neural Development

    PubMed Central

    Mandai, Kenji; Guo, Ting; Hillaire, Coryse St.; Meabon, James S.; Kanning, Kevin C.; Bothwell, Mark; Ginty, David D.

    2009-01-01

    SUMMARY Genome-wide screens were performed to identify transmembrane proteins that mediate axonal growth, guidance and target field innervation of somatosensory neurons. One gene, Linx (alias Islr2), encoding a leucine-rich repeat and immunoglobulin (LIG) family protein, is expressed in a subset of developing sensory and motor neurons. Domain and genomic structures of Linx and other LIG family members suggest that they are evolutionarily related to Trk receptor tyrosine kinases (RTKs). Several LIGs, including Linx are expressed in subsets of somatosensory and motor neurons and select members interact with TrkA and Ret RTKs. Moreover, axonal projection defects in mice harboring a null mutation in Linx resemble those in mice lacking Ngf, TrkA and Ret. In addition, Linx modulates NGF–TrkA- and GDNF–GFRα1/Ret-mediated axonal extension in cultured sensory and motor neurons, respectively. These findings show that LIGs physically interact with RTKs and modulate their activities to control axonal extension, guidance and branching. PMID:19755105

  18. Association of LAR-like receptor protein tyrosine phosphatases with an enabled homolog in Hirudo medicinalis.

    PubMed

    Biswas, Subhas C; Dutt, Anindita; Baker, Michael W; Macagno, Eduardo R

    2002-12-01

    Receptor protein tyrosine phosphatases (RPTPs) are thought to play important functions in pathfinding and target recognition by growing neuronal processes. The leech RPTPs HmLAR1 and HmLAR2 are expressed selectively by central neurons, Comb cells, and peripheral muscle tissues in the Hirudo medicinalis embryo. To explore the functions of HmLARs, we have sought to determine their physiological substrates. We report here the cloning and embryonic expression of Lena, the leech homolog of Enabled, a cytosolic protein implicated in actin-based cell motility. Lena is expressed in embryonic central neurons and in the Comb cell. We present experimental evidences indicating that Lena associates selectively with the intracellular domain of HmLAR1 and HmLAR2. Additionally, RNA interference (RNAi) of HmLAR1 in intact leech embryos leads to the hyperphosphorylation of Lena. We propose, therefore, that Lena is an in vivo substrate of HmLAR1 in neurons and perhaps of HmLAR2 in the Comb cells.

  19. Immunohistochemical analysis of IA-2 family of protein tyrosine phosphatases in rat gastrointestinal endocrine cells.

    PubMed

    Gomi, Hiroshi; Kubota-Murata, Chisato; Yasui, Tadashi; Tsukise, Azuma; Torii, Seiji

    2013-02-01

    Islet-associated protein-2 (IA-2) and IA-2β (also known as phogrin) are unique neuroendocrine-specific protein tyrosine phosphatases (PTPs). The IA-2 family of PTPs was originally identified from insulinoma cells and discovered to be major autoantigens in type 1 diabetes. Despite its expression in the neural and canonical endocrine tissues, data on expression of the IA-2 family of PTPs in gastrointestinal endocrine cells (GECs) are limited. Therefore, we immunohistochemically investigated the expression of the IA-2 family of PTPs in the rat gastrointestinal tract. In the stomach, IA-2 and IA-2β were expressed in GECs that secrete serotonin, somatostatin, and cholecystokinin/gastrin-1. In addition to these hormones, secretin, gastric inhibitory polypeptide (also known as the glucose-dependent insulinotropic peptide), glucagon-like peptide-1, and glucagon, but not ghrelin were coexpressed with IA-2 or IA-2β in duodenal GECs. Pancreatic islet cells that secrete gut hormones expressed the IA-2 family of PTPs. The expression patterns of IA-2 and IA-2β were comparable. These results reveal that the IA-2 family of PTPs is expressed in a cell type-specific manner in rat GECs. The extensive expression of the IA-2 family of PTPs in pancreo-gastrointestinal endocrine cells and in the enteric plexus suggests their systemic contribution to nutritional control through a neuroendocrine signaling network.

  20. 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

  1. Non-receptor cytosolic protein tyrosine kinases from various rat tissues.

    PubMed

    Elberg, G; Li, J; Leibovitch, A; Shechter, Y

    1995-11-30

    Adipocytic-cytosolic non-receptor protein tyrosine kinase (CytPTK) when activated can substitute for the insulin receptor tyrosine kinase (InsRTK), in manifesting several insulin effects in insulin-receptor independent fashion. Our aims here were to utilize PolyGlu4Tyr, a good experimental exogenous substrate for protein tyrosine kinases (PTKs) in general, for studying qualitative and quantitative parameters of CytPTKs extracted from different tissue cytosols. At the same time, we would search for a unique specific marker specifically characterizing CytPTKs. High speed supernatants of spleen, thymus, smooth muscle, lung and kidney were found to be rich in CytPTK activities. Their specific activities being 6- to 13-fold that of liver or adipose cytosols. Brain, testis and adrenal cytosols were an intermediate source of CytPTK activity, whereas CytPTK activity of heart and skeletal muscle was low. It was also evaluated that the capacity of the cytosol to phosphorylate PolyGlu4Tyr is 15-50% that of the non-stimulated Triton X-100 extractable plasma membrane PTKs. Fractionation of the cytosols on superose 12 column revealed several CytPTKs within the same tissue, their peaks ranging between 30 and 450 kDa. Immunoblotting analysis showed Fyn and Lyn were present in most tissue cytosols. Upon immunoprecipitation, however, with anti-Fyn or anti-Lyn, negligible amounts (< 2%) of the total cellular CytPTK were precipitated. Thus, these general markers of CytPTKs comprise only a minor proportion of the total intracellular PolyGlu4Tyr phosphorylating capacity. To see whether a specific marker for CytPTK could be detected, we also examined the requirement of CytPTKs for divalent ions, their preferred phosphate donor and their sensitivity to inhibition by known PTK inhibitors. We found that the order of reactivity with divalent cations was Co2+ > Mn2+ > Mg2+, while Zn2+ and Ca2+ did not support CytPTK activity. The best phosphate donor was ATP (ED50 = 5 microM), but other

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

    SciTech Connect

    Liu, Xiao-Yan; Li, Lan-Fen; Su, Xiao-Dong

    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.

  3. Expression and function of striatal enriched protein tyrosine phosphatase is profoundly altered in cerebral ischemia.

    PubMed

    Braithwaite, Steven P; Xu, Jian; Leung, John; Urfer, Roman; Nikolich, Karoly; Oksenberg, Donna; Lombroso, Paul J; Shamloo, Mehrdad

    2008-05-01

    Striatal enriched protein tyrosine phosphatase (STEP) acts in the central nervous system to dephosphorylate a number of important proteins involved in synaptic function including ERK and NMDA receptor subunits. These proteins are also linked to stroke, in which cerebral ischemia triggers a complex cascade of events. Here we demonstrate that STEP is regulated at both the transcriptional and the post-transcriptional levels in rat models of cerebral ischemia and that its regulation may play a role in the outcome of ischemic insults. After transient middle cerebral artery occlusion, there are profound decreases in the levels of STEP mRNA, whilst in global ischemia STEP mRNA is selectively down-regulated in areas susceptible to ischemic damage. In a neuroprotective preconditioning paradigm, and in regions of the brain that are relatively resistant to ischemic damage, STEP mRNA levels are increased. Furthermore, there is a significant processing of STEP after ischemia to generate a novel species, STEP(33), resulting in a redistribution of STEP from membrane-bound to soluble compartments. Concomitant with the cleavage of mature forms of STEP, there are changes in the phosphorylation state of ERK. We show that the cleavage of STEP leads to a catalytically active form, but this cleaved form no longer binds to and dephosphorylates its substrate pERK. Therefore, in response to ischemic insults, there are profound reductions in both the amount and the activity of STEP, its localization, as well as the activity of one of its key substrates, pERK. These changes in STEP may reflect a critical role in the outcomes of ischemic brain injury.

  4. ATF-7, a novel bZIP protein, interacts with the PRL-1 protein-tyrosine phosphatase.

    PubMed

    Peters, C S; Liang, X; Li, S; Kannan, S; Peng, Y; Taub, R; Diamond, R H

    2001-04-27

    We have identified a novel basic leucine zipper (bZIP) protein, designated ATF-7, that physically interacts with the PRL-1 protein-tyrosine phosphatase (PTPase). PRL-1 is a predominantly nuclear, farnesylated PTPase that has been linked to the control of cellular growth and differentiation. This interaction was initially found using the yeast two-hybrid system. ATF-7 is most closely related to members of the ATF/CREB family of bZIP proteins, with highest homology to ATF-4. ATF-7 homodimers can bind specifically to CRE elements. ATF-7 is expressed in a number of different tissues and is expressed in association with differentiation in the Caco-2 cell model of intestinal differentiation. We have confirmed the PRL-1.ATF-7 interaction and mapped the regions of ATF-7 and PRL-1 important for interaction to ATF-7's bZIP region and PRL-1's phosphatase domain. Finally, we have determined that PRL-1 is able to dephosphorylate ATF-7 in vitro. Further insight into ATF-7's precise cellular roles, transcriptional function, and downstream targets are likely be of importance in understanding the mechanisms underlying the complex processes of maintenance, differentiation, and turnover of epithelial tissues.

  5. Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis.

    PubMed

    Pedersen, Anja K; Guo, Xiao-Ling; Møller, Karin B; Peters, Günther H; Andersen, Henrik S; Kastrup, Jette S; Mortensen, Steen B; Iversen, Lars F; Zhang, Zhong-Yin; Møller, Niels Peter H

    2004-03-01

    Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPa and PTPe. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.

  6. Protein tyrosine phosphatase α in the dorsomedial striatum promotes excessive ethanol-drinking behaviors.

    PubMed

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

    2013-09-04

    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.

  7. Inhibition of T Cell Protein Tyrosine Phosphatase Enhances Interleukin-18-Dependent Hematopoietic Stem Cell Expansion

    PubMed Central

    Bourdeau, Annie; Trop, Sébastien; Doody, Karen M; Dumont, Daniel J; Tremblayef, Michel L

    2013-01-01

    The clinical application of hematopoietic progenitor cell-based therapies for the treatment of hematological diseases is hindered by current protocols, which are cumbersome and have limited efficacy to augment the progenitor cell pool. We report that inhibition of T-cell protein tyrosine phosphatase (TC-PTP), an enzyme involved in the regulation of cytokine signaling, through gene knockout results in a ninefold increase in the number of hematopoietic progenitors in murine bone marrow (BM). This effect could be reproduced using a short (48 hours) treatment with a pharmacological inhibitor of TC-PTP in murine BM, as well as in human BM, peripheral blood, and cord blood. We also demonstrate that the ex vivo use of TC-PTP inhibitor only provides a temporary effect on stem cells and did not alter their capacity to reconstitute all hematopoietic components in vivo. We establish that one of the mechanisms whereby inhibition of TC-PTP mediates its effects involves the interleukin-18 (IL-18) signaling pathway, leading to increased production of IL-12 and interferon-gamma by progenitor cells. Together, our results reveal a previously unrecognized role for IL-18 in contributing to the augmentation of the stem cell pool and provide a novel and simple method to rapidly expand progenitor cells from a variety of sources using a pharmacological compound. Stem Cells 2013;31:293–304 PMID:23135963

  8. The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis.

    PubMed

    Crosier, P S; Freeman, S A; Orlic, D; Bodine, D M; Crosier, K E

    1996-02-01

    Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

  9. Oxidation and inactivation of low molecular weight protein tyrosine phosphatase by the anticancer drug Aplidin.

    PubMed

    Taddei, Maria Letizia; Chiarugi, Paola; Cuevas, Carmen; Ramponi, Giampietro; Raugei, Giovanni

    2006-04-15

    The marine plitidepsin Aplidin derived from the Mediterranean tunicate Aplidium albicans is a strong apoptotic inducer with promising antitumor activity. However, little is known about the mechanism of action of the molecule. In this article, we report that Aplidin is cytotoxic for NIH3T3 cells and that its action is exerted through the production of reactive oxygen species (ROS). Rotenone, but not other selective inhibitors of ROS production, blocks the induction of ROS, suggesting the involvement of the mitochondrial respiratory chain in Aplidin action. The intracellular rise of redox potential caused by Aplidin inactivates several molecular targets. Among these targets, we focused our attention on protein tyrosine phosphatases (PTPs). In agreement with the well-characterized effect of ROS-mediated PTP oxidation, due to the presence of a cysteine residue in their catalytic site, we found that Aplidin induces a strong decrease in PTP activity. In particular, since the expression of low molecular weight-PTP (LMW-PTP) is strongly associated with tumor onset and progression, we investigated the effect of Aplidin on this enzyme. Our data show that LMW-PTP is oxidized and inactivated during Aplidin treatment, thus causing a hyper-phosphorylation of its substrate beta-catenin. These findings demonstrate that, at least in part, the antitumoral activity of Aplidin could be due to the direct inhibition of LMW-PTP and its related oncogenic potential. Copyright (c) 2005 Wiley-Liss, Inc.

  10. Effects of protein tyrosine phosphatase-PEST are reversed by Akt in T cells.

    PubMed

    Arimura, Yutaka; Shimizu, Kazuhiko; Koyanagi, Madoka; Yagi, Junji

    2014-12-01

    T cell activation is regulated by a balance between phosphorylation and dephosphorylation that is under the control of kinases and phosphatases. Here, we examined the role of a non-receptor-type protein tyrosine phosphatase, PTP-PEST, using retrovirus-mediated gene transduction into murine T cells. Based on observations of vector markers (GFP or Thy1.1), exogenous PTP-PEST-positive CD4(+) T cells appeared within 2 days after gene transduction; the percentage of PTP-PEST-positive cells tended to decrease during a resting period in the presence of IL-2 over the next 2 days. These vector markers also showed much lower expression intensities, compared with control cells, suggesting a correlation between the percent reduction and the low marker expression intensity. A catalytically inactive PTP-PEST mutant also showed the same tendency, and stepwise deletion mutants gradually lost their ability to induce the above phenomenon. On the other hand, these PTP-PEST-transduced cells did not have an apoptotic phenotype. No difference in the total cell numbers was found in the wells of a culture plate containing VEC- and PTP-PEST-transduced T cells. Moreover, serine/threonine kinase Akt, but not the anti-apoptotic molecules Bcl-2 and Bcl-XL, reversed the phenotype induced by PTP-PEST. We discuss the novel mechanism by which Akt interferes with PTP-PEST. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Integrating Virtual and Biochemical Screening for Protein Tyrosine Phosphatase Inhibitor Discovery

    PubMed Central

    Martin, Katie R.; Narang, Pooja; Franco, Jose L. Medina; Meurice, Nathalie; MacKeigan, Jeffrey P.

    2013-01-01

    Protein tyrosine phosphatases (PTPs) represent an important class of enzymes that mediate signal transduction and control diverse aspects of cell behavior. The importance of their activity is exemplified by their significant contribution to disease etiology with over half of all human PTP genes implicated in at least one disease. Small molecule inhibitors targeting individual PTPs are important biological tools, and are needed to fully characterize the function of these enzymes. Moreover, potent and selective PTP inhibitors hold the promise to transform the treatment of many diseases. While numerous methods exist to develop PTP-directed small molecules, we have found that complimentary use of both virtual (in silico) and biochemical (in vitro) screening approaches expedite compound identification and drug development. Here, we summarize methods pertinent to our work and others. Focusing on specific challenges and successes we have experienced, we discuss the considerable caution that must be taken to avoid enrichment of inhibitors that function by non-selective oxidation. We also discuss the utility of using “open” PTP structures to identify active-site directed compounds, a rather unconventional choice for virtual screening. When integrated closely, virtual and biochemical screening can be used in a productive workflow to identify small molecules targeting PTPs. PMID:23969317

  12. Overexpression of the protein tyrosine phosphatase PRL-2 correlates with breast tumor formation and progression.

    PubMed

    Hardy, Serge; Wong, Nau Nau; Muller, William J; Park, Morag; Tremblay, Michel L

    2010-11-01

    The PRL-1, PRL-2, and PRL-3 phosphatases are prenylated protein tyrosine phosphatases with oncogenic activity that are proposed to drive tumor metastasis. We found that PRL-2 mRNA is elevated in primary breast tumors relative to matched normal tissue, and also dramatically elevated in metastatic lymph nodes compared with primary tumors. PRL-2 knockdown in metastatic MDA-MB-231 breast cancer cells decreased anchorage-independent growth and cell migration, suggesting that the malignant phenotype of these cells is mediated at least in part through PRL-2 signaling. In different mouse mammary tumor-derived cell lines overexpressing PRL-2, we confirmed its role in anchorage-independent growth and cell migration. Furthermore, injection of PRL-2-overexpressing cells into the mouse mammary fat pad promoted extracellular signal-regulated kinase 1/2 activation and tumor formation. MMTV-PRL-2 transgenic mice engineered to overexpress the enzyme in mammary tissue did not exhibit spontaneous tumorigenesis, but they exhibited an accelerated development of mammary tumors initiated by introduction of an MMTV-ErbB2 transgene. Together, our results argue that PRL-2 plays a role in breast cancer progression.

  13. Delayed skin wound repair in proline-rich protein tyrosine kinase 2 knockout mice.

    PubMed

    Koppel, Aaron C; Kiss, Alexi; Hindes, Anna; Burns, Carole J; Marmer, Barry L; Goldberg, Gregory; Blumenberg, Miroslav; Efimova, Tatiana

    2014-05-15

    Proline-rich protein tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family. We used Pyk2 knockout (Pyk2-KO) mice to study the role of Pyk2 in cutaneous wound repair. We report that the rate of wound closure was delayed in Pyk2-KO compared with control mice. To examine whether impaired wound healing of Pyk2-KO mice was caused by a keratinocyte cell-autonomous defect, the capacities of primary keratinocytes from Pyk2-KO and wild-type (WT) littermates to heal scratch wounds in vitro were compared. The rate of scratch wound repair was decreased in Pyk2-KO keratinocytes compared with WT cells. Moreover, cultured human epidermal keratinocytes overexpressing the dominant-negative mutant of Pyk2 failed to heal scratch wounds. Conversely, stimulation of Pyk2-dependent signaling via WT Pyk2 overexpression induced accelerated scratch wound closure and was associated with increased expression of matrix metalloproteinase (MMP)-1, MMP-9, and MMP-10. The Pyk2-stimulated increase in the rate of scratch wound repair was abolished by coexpression of the dominant-negative mutant of PKCδ and by GM-6001, a broad-spectrum inhibitor of MMP activity. These results suggest that Pyk2 is essential for skin wound reepithelialization in vivo and in vitro and that it regulates epidermal keratinocyte migration via a pathway that requires PKCδ and MMP functions.

  14. Pleiotrophin and its receptor protein tyrosine phosphatase beta/zeta as regulators of angiogenesis and cancer.

    PubMed

    Papadimitriou, Evangelia; Pantazaka, Evangelia; Castana, Penelope; Tsalios, Thomas; Polyzos, Alexandros; Beis, Dimitris

    2016-12-01

    Pleiotrophin (PTN) is a secreted heparin-binding growth factor that through its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) has a significant regulatory effect on angiogenesis and cancer. PTN and RPTPβ/ζ are over-expressed in several types of human cancers and regulate important cancer cell functions in vitro and cancer growth in vivo. This review begins with a brief introduction of PTN and the regulation of its expression. PTN receptors are described with special emphasis on RPTPβ/ζ, which also interacts with and/or affects the function of other important targets for cancer therapy, such as vascular endothelial growth factor A, ανβ3 and cell surface nucleolin. PTN biological activities related to angiogenesis and cancer are extensively discussed. Finally, up to date approaches of targeting PTN or RPTPβ/ζ for cancer treatment are presented. Insights into the regulatory role of PTN/RPTPβ/ζ on angiogenesis will be extremely beneficial for future development of alternative anti-angiogenic approaches in cancer therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. 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.

  16. 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

  17. Protein Tyrosine Phosphatase 1B (PTP1B): A Potential Target for Alzheimer’s Therapy?

    PubMed Central

    Vieira, Marcelo N. N.; Lyra e Silva, Natalia M.; Ferreira, Sergio T.; De Felice, Fernanda G.

    2017-01-01

    Despite significant advances in current understanding of mechanisms of pathogenesis in Alzheimer’s disease (AD), attempts at drug development based on those discoveries have failed to translate into effective, disease-modifying therapies. AD is a complex and multifactorial disease comprising a range of aberrant cellular/molecular processes taking part in different cell types and brain regions. As a consequence, therapeutics for AD should be able to block or compensate multiple abnormal pathological events. Here, we examine recent evidence that inhibition of protein tyrosine phosphatase 1B (PTP1B) may represent a promising strategy to combat a variety of AD-related detrimental processes. Besides its well described role as a negative regulator of insulin and leptin signaling, PTB1B recently emerged as a modulator of various other processes in the central nervous system (CNS) that are also implicated in AD. These include signaling pathways germane to learning and memory, regulation of synapse dynamics, endoplasmic reticulum (ER) stress and microglia-mediated neuroinflammation. We propose that PTP1B inhibition may represent an attractive and yet unexplored therapeutic approach to correct aberrant signaling pathways linked to AD. PMID:28197094

  18. 4-Quinolone-3-carboxylic acids as cell-permeable inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Zhi, Ying; Gao, Li-Xin; Jin, Yi; Tang, Chun-Lan; Li, Jing-Ya; Li, Jia; Long, Ya-Qiu

    2014-07-15

    Protein tyrosine phosphatase 1B is a negative regulator in the insulin and leptin signaling pathways, and has emerged as an attractive target for the treatment of type 2 diabetes and obesity. However, the essential pharmacophore of charged phosphotyrosine or its mimetic confer low selectivity and poor cell permeability. Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent PTP1B inhibitors with low micromolar IC50 values. These 4-quinolone-3-carboxylate based PTP1B inhibitors displayed a 2-10 fold selectivity over a panel of PTP's. Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that the 4-quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B active site in the WPD loop closed conformation. Taken together, our study shows that the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological properties over previously described PTB1B inhibitors and warrant further preclinical studies.

  19. Protein tyrosine phosphatase activity in the neural crest is essential for normal heart and skull development

    PubMed Central

    Nakamura, Tomoki; Gulick, James; Colbert, Melissa C.; Robbins, Jeffrey

    2009-01-01

    Mutations within the protein tyrosine phosphatase, SHP2, which is encoded by PTPN11, cause a significant proportion of Noonan syndrome (NS) cases, typically presenting with both cardiac disease and craniofacial abnormalities. Neural crest cells (NCCs) participate in both heart and skull formation, but the role of SHP2 signaling in NCC has not yet been determined. To gain insight into the role of SHP2 in NCC function, we ablated PTPN11 specifically in premigratory NCCs. SHP2-deficient NCCs initially exhibited normal migratory and proliferative patterns, but in the developing heart failed to migrate into the developing outflow tract. The embryos displayed persistent truncus arteriosus and abnormalities of the great vessels. The craniofacial deficits were even more pronounced, with large portions of the face and cranium affected, including the mandible and frontal and nasal bones. The data show that SHP2 activity in the NCC is essential for normal migration and differentiation into the diverse lineages found in the heart and skull and demonstrate the importance of NCC-based normal SHP2 activity in both heart and skull development, providing insight into the syndromic presentation characteristic of NS. PMID:19541608

  20. Receptor protein tyrosine phosphatase PTPRB negatively regulates FGF2-dependent branching morphogenesis.

    PubMed

    Soady, Kelly J; Tornillo, Giusy; Kendrick, Howard; Meniel, Valerie; Olijnyk-Dallis, Daria; Morris, Joanna S; Stein, Torsten; Gusterson, Barry A; Isacke, Clare M; Smalley, Matthew J

    2017-09-04

    PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here we demonstrate that PTPRB negatively regulates branching morphogenesis in the mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in estrogen receptor positive luminal cells. During mammary development Ptprb expression is down-regulated during puberty, a period of extensive of ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of FGFR activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology. © 2017. Published by The Company of Biologists Ltd.

  1. Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid.

    PubMed

    Lipchock, James M; Hendrickson, Heidi P; Douglas, Bonnie B; Bird, Kelly E; Ginther, Patrick S; Rivalta, Ivan; Ten, Nicholas S; Batista, Victor S; Loria, J Patrick

    2017-01-10

    Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B. CGA, while a noncompetitive inhibitor, binds in the second aryl phosphate binding site, rather than the predicted benzfuran binding pocket. The molecular dynamics simulations of the apo enzyme and cysteine-phosphoryl intermediate states with and without bound CGA suggest CGA binding inhibits PTP1B by altering hydrogen bonding patterns at the active site. This study provides a mechanistic understanding of the allosteric inhibition of PTP1B.

  2. Expression of protein tyrosine kinases and stem cell factor in chicken blastodermal cells.

    PubMed

    Sasaki, E; Etches, R J

    2001-02-01

    Chicken blastodermal cells (CBC) from Stage X embryos, which were isolated from newly laid, fertile, unincubated eggs, are pluripotent cells and can produce somatic and germline chimeras when injected into recipient stage X embryos. The CBC retain their pluripotential ability for up to 7 d in vitro. The molecular mechanisms that control proliferation and differentiation of CBC are largely unknown, although protein tyrosine kinases (PTK) are known to play important roles in these processes in similar cells. To understand better the molecular mechanisms of proliferation and differentiation in CBC, expression profiles of PTK and stem cell factor (SCF) were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using gene-specific and degenerate oligonucleotide primers. Seventeen distinct PTK, including 14 receptor-type and 3 nonreceptor-type PTK and SCF were identified by RT-PCR. Expression of all of the genes was confirmed by northern blot analysis. The northern blot analysis showed that all probes hybridized with one or more transcripts at various expression levels. The expression of the 17 PTK and SCF genes in CBC suggests that they might play a role in signal transduction pathways that control the proliferation or differentiation in CBC.

  3. 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

  4. Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA.

    PubMed

    Vega, Carolina; Chou, Seemay; Engel, Katherine; Harrell, Maria E; Rajagopal, Lakshmi; Grundner, Christoph

    2011-10-14

    Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.

  5. Oleanane triterpenes as protein tyrosine phosphatase 1B (PTP1B) inhibitors from Camellia japonica.

    PubMed

    Uddin, Mohammad Nasir; Sharma, Govinda; Yang, Jun-Li; Choi, Hong Seok; Lim, Seong-Il; Kang, Keon Wook; Oh, Won Keun

    2014-07-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role in metabolic signaling, thereby making it an exciting drug target for type 2 diabetes and obesity. Besides, there is substantial evidence that shows its overexpression is involved in breast cancer, which suggests that selective PTP1B inhibition might be effective in breast cancer treatment. As part of our continuous research on PTP1B inhibitors from medicinal plants, four oleanane-type triterpenes were isolated from an EtOAc-soluble extract of fruit peels of Camellia japonica (Theaceae), together with 6 previously known compounds of this class. Their structures were determined on the basis of spectroscopic data analysis (UV, IR, (1)H and (13)CNMR, HMBC, HSQC, NOESY, and MS). All isolates were evaluated for their inhibitory effects on PTP1B, as well as their cytotoxic effects against human breast cancer cell lines MCF7, MCF7/ADR, and MDA-MB-231. Several compounds with OH-3 or/and COOH-28 functionalities showed strong PTP1B inhibitory activity (IC50 values ranging from 3.77±0.11 to 6.40±0.81 μM) as well as significant cytotoxicity (IC50 values ranging from 0.51±0.05 to 13.55±1.44 μM).

  6. Paeciloquinones A, B, C, D, E and F: new potent inhibitors of protein tyrosine kinases produced by Paecilomyces carneus. I. Taxonomy, fermentation, isolation and biological activity.

    PubMed

    Petersen, F; Fredenhagen, A; Mett, H; Lydon, N B; Delmendo, R; Jenny, H B; Peter, H H

    1995-03-01

    Paeciloquinones A to F as well as versiconol have been isolated as inhibitors of protein tyrosine kinase from the culture broth of the fungus Paecilomyces carneus P-177. The novel anthraquinones inhibit epidermal growth factor receptor protein tyrosine kinase in the micromolar range. Two compounds, paeciloquinones A and C, are potent and selective inhibitors of the v-abl protein tyrosine kinase with an IC50 of 0.4 microM. Dependent on the fermentation conditions, partially different sets of paeciloquinones may be produced. An HPLC method allows separation of all major active components.

  7. A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients.

    PubMed

    Vezzalini, Marzia; Mafficini, Andrea; Tomasello, Luisa; Lorenzetto, Erika; Moratti, Elisabetta; Fiorini, Zeno; Holyoake, Tessa L; Pellicano, Francesca; Krampera, Mauro; Tecchio, Cristina; Yassin, Mohamed; Al-Dewik, Nader; Ismail, Mohamed A; Al Sayab, Ali; Monne, Maria; Sorio, Claudio

    2017-06-21

    Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients. TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34(+)/CD38(bright/dim) cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells. The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically

  8. The KIM-family protein-tyrosine phosphatases use distinct reversible oxidation intermediates: Intramolecular or intermolecular disulfide bond formation.

    PubMed

    Machado, Luciana E S F; Shen, Tun-Li; Page, Rebecca; Peti, Wolfgang

    2017-05-26

    The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H2O2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Identification of an immunoreceptor tyrosine-based activation motif of K1 transforming protein of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Lee, H; Guo, J; Li, M; Choi, J K; DeMaria, M; Rosenzweig, M; Jung, J U

    1998-09-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is consistently identified in Kaposi's sarcoma and body cavity-based lymphoma. KSHV encodes a transforming protein called K1 which is structurally similar to lymphocyte receptors. We have found that a highly conserved region of the cytoplasmic domain of K1 resembles the sequence of immunoreceptor tyrosine-based activation motifs (ITAMs). To demonstrate the signal-transducing activity of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K1. Expression of the CD8-K1 chimera in B cells induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. Mutational analyses showed that the putative ITAM of K1 was required for its signal-transducing activity. Furthermore, tyrosine residues of the putative ITAM of K1 were phosphorylated upon stimulation, and this allowed subsequent binding of SH2-containing proteins. These results demonstrate that the KSHV transforming protein K1 contains a functional ITAM in its cytoplasmic domain and that it can transduce signals to induce cellular activation.

  10. Nitric oxide attenuates hydrogen peroxide-induced barrier disruption and protein tyrosine phosphorylation in